The role of hemocytes in A. gambiae antiplasmodial immunity

PubMed Central

Ramirez, Jose Luis; Garver, Lindsey S.; Brayner, Fábio André; Alves, Luiz Carlos; Rodrigues, Janneth; Molina-Cruz, Alvaro; Barillas-Mury, Carolina

2013-01-01

Hemocytes synthesize key components of the mosquito complement-like system, but their role in the activation of antiplasmodial responses has not been established. The effect of activating Toll signaling in hemocytes on Plasmodium survival was investigated by transferring hemocytes or cell-free hemolymph from donor mosquitoes in which the suppressor cactus was silenced. These transfers greatly enhanced antiplasmodial immunity, indicating that hemocytes are active players in the activation of the complement-like system, through an effector(s) regulated by the Toll pathway. A comparative analysis of hemocyte populations between susceptible (S) G3 and the refractory (R) L3-5 A. gambiae mosquito strains did not reveal significant differences under basal conditions or in response to Plasmodium berghei infection. The response of S mosquitoes to different Plasmodium species revealed similar kinetics following infection with P. berghei, P. yoelii or P. falciparum, but the strength of the priming response was stronger in less compatible mosquito-parasite pairs. The Toll, Imd, STAT or JNK signaling cascades were not essential for the production of hemocyte differentiation factor (HDF) in response to P. berghei infection, but disruption of Toll, STAT or JNK abolished hemocyte differentiation in response to HDF. We conclude that hemocytes are key mediators of A. gambiae antiplasmodial responses. PMID:23886925

Specificity of anti-Vibrio immune response through p38 MAPK and PKC activation in the hemocytes of the mussel Mytilus galloprovincialis.

PubMed

Ciacci, Caterina; Betti, Michele; Canonico, Barbara; Citterio, Barbara; Roch, Philippe; Canesi, Laura

2010-09-01

In mussel (Mytilus sp.) hemocytes, differential functional responses to injection with different types of live and heat-killed Vibrio species have been recently demonstrated. In this work, responses of Mytilus hemocytes to heat-killed Vibrio splendidus LGP32 and the mechanisms involved were investigated in vitro and the results were compared with those obtained with Vibrio anguillarum (ATCC 19264). Adhesion of hemocytes after incubation with bacteria was evaluated by flow cytometry: both total hemocyte counts (THC) and percentage of hemocyte sub-populations were determined in non-adherent cells. Functional parameters such as lysosomal membrane stability, lysozyme release, extracellular ROS production and NO production were evaluated, as well as the phosphorylation state of the stress-activated p38 MAPK and PKC. Neither Vibrio affected total hemocyte adhesion, while both induced similar lysosomal destabilization and NO production. However, V. splendidus decreased adhesion of large granulocytes, induced rapid and persistent lysozyme release and stimulated extracellular ROS production: these effects were associated with persistent activation of p38 MAPK and PKC. In contrast, V. anguillarum decreased adhesion of large semigranular hemocytes and increased that of hyalinocytes, had no effect on the extracellular ROS production, and induced significantly lower lysozyme release and phosphorylation of p-38 MAPK and PKC than V. splendidus. These data reinforced the existence of specific interactions between mussel hemocytes and V. splendidus LGP32 and suggest that this Vibrio strain affects bivalve hemocytes through disregulation of immune signaling. The results support the hypothesis that responses of bivalve hemocytes to different bacterial stimuli may depend not only on the nature of the stimulus, but also on the cell subtype, thus leading to differential activation of signaling components. Copyright 2010 Elsevier Inc. All rights reserved.

Effect of lipopolysaccharide on the hemocyte apoptosis of Eriocheir sinensis *

PubMed Central

Xu, Hai-sheng; Lyu, Sun-jian; Xu, Jie-hao; Lu, Bin-jie; Zhao, Jing; Li, Song; Li, Yi-qun; Chen, Yu-yin

2015-01-01

In the present study, we investigated the possible toxicity mechanism of lipopolysaccharide (LPS) extracted from Gram-negative bacteria in Eriocheir sinensis hemocytes. Apoptotic hemocytes and reactive oxygen species (ROS) production induced by the LPS were monitored by the combination of flow cytometry and microscope observation. It was shown that LPS induced serious damage on the DNA and morphological changes in hemocytes, including cell shrinkage, fracture of nucleus membrane, margination, condensation and fragmentation of chromatin, and formation of apoptotic bodies indicating obvious hemocyte apoptosis. As compared with the control group, the apoptotic cell ratio increased to 30.61% and 39.01% after 1-h exposure and 57.72% and 75.01% after 2-h exposure to 1 and 10 μg/ml LPS, respectively (P<0.05). Significant outburst of ROS production was observed in LPS-treated hemocytes with approximately 176.6% of relative dichlorofluorescein mean fluorescence at 1-h exposure, followed by a drastic decline (P<0.05). These results indicated that LPS would induce oxidative stress on hemocytes from E. sinensis and cause ROS burst, DNA damage, and subsequently apoptosis. The process of ROS-mediated apoptosis might be one of the potential toxicity mechanisms of LPS on crustacean hemocytes. PMID:26642180

Natural variation in differentiated hemocytes is related to parasitoid resistance in Drosophila melanogaster.

PubMed

Gerritsma, Sylvia; Haan, Ammerins de; Zande, Louis van de; Wertheim, Bregje

2013-02-01

As a measure of parasitoid resistance, hemocyte load and encapsulation ability were measured in lines collected from natural populations of Drosophila melanogaster in Europe. Results show large geographic variation in resistance against the parasitoid wasp Asobara tabida among the field lines, but there was no clear correlation between resistance and total hemocyte load, neither before nor after parasitization. This was in contrast to the patterns that had been found in a comparison among species of Drosophila, where total hemocyte counts were positively correlated to encapsulation rates. This suggests that the mechanisms underlying between-species variation in parasitoid resistance do not extend to the natural variation that exists within a species. Although hemocyte counts did not correspond to encapsulation ability within D. melanogaster, the ratios of lamellocytes and crystal cells were very similar in lines with successful encapsulation responses. Apart from variation in the hemocytic response of the different hemocyte types, within-species variation was also observed for accurate targeting of the foreign body by the hemocytes. These results are discussed in the context of possible causes of variation in immune functions among natural populations. Copyright © 2012 Elsevier Ltd. All rights reserved.

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis.

PubMed

Hiroyasu, Aoi; DeWitt, David C; Goodman, Alan G

2018-05-24

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the goal of this protocol is to develop a method to visualize the pathogen invasion in a specific immune compartment of flies, namely hemocytes. Using the method presented here, up to 3 × 10 6 live hemocytes can be obtained from 200 Drosophila 3 rd instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3 rd instar larvae followed by hemocyte extraction up to 24 h post-infection. These infected primary cells were fixed, stained, and imaged using confocal microscopy. Then, 3D representations were generated from the images to definitively show pathogen invasion. Additionally, high-quality RNA for qRT-PCR can be obtained for the detection of pathogen mRNA following infection, and sufficient protein can be extracted from these cells for Western blot analysis. Taken together, we present a method for definite reconciliation of pathogen invasion and confirmation of infection using bacterial and viral pathogen types and an efficient method for hemocyte extraction to obtain enough live hemocytes from Drosophila larvae for ex vivo and in vivo infection experiments.

High sequence variability among hemocyte-specific Kazal-type proteinase inhibitors in decapod crustaceans.

PubMed

Cerenius, Lage; Liu, Haipeng; Zhang, Yanjiao; Rimphanitchayakit, Vichien; Tassanakajon, Anchalee; Gunnar Andersson, M; Söderhäll, Kenneth; Söderhäll, Irene

2010-01-01

Crustacean hemocytes were found to produce a large number of transcripts coding for Kazal-type proteinase inhibitors (KPIs). A detailed study performed with the crayfish Pacifastacus leniusculus and the shrimp Penaeus monodon revealed the presence of at least 26 and 20 different Kazal domains from the hemocyte KPIs, respectively. Comparisons with KPIs from other taxa indicate that the sequences of these domains evolve rapidly. A few conserved positions, e.g. six invariant cysteines were present in all domain sequences whereas the position of P1 amino acid, a determinant for substrate specificity, varied highly. A study with a single crayfish animal suggested that even at the individual level considerable sequence variability among hemocyte KPIs produced exist. Expression analysis of four crayfish KPI transcripts in hematopoietic tissue cells and different hemocyte types suggest that some of these KPIs are likely to be involved in hematopoiesis or hemocyte release as they were produced in particular hemocyte types or maturation stages only.

Ontogeny of tick hemocytes: a comparative analysis of Ixodes ricinus and Ornithodoros moubata.

PubMed

Borovicková, Barbara; Hypsa, Václav

2005-01-01

Hemocytes of two tick species, Ixodes ricinus and Ornithodoros moubata, were investigated with the aim to determine their ultrastructural characteristics and developmental relationships. Only a limited number of ultrastructural features was shown to be unequivocally homological across all hemocyte types. The two species, representing distant groups of ticks, differ in the composition of their circular cell populations. In I. ricinus, three groups of distinct morphological types of hemocytes could be determined according to well-defined ultrastructural features: a typical non-phagocytic granular cell with electron-dense granula and homogeneous cytoplasm (Gr II), and two different types of phagocytic hemocytes, namely plasmatocytes with a low number of granula and phagocytic granolocytes, designated as Gr I. In contrast, an additional cell type resembling insect spherulocytes was determined in O. moubata. This cell type does not seem to be homologous to any I. ricinus hemocyte and may represent a cell type typical of soft ticks only. Possible ontogenetic lineages of the hemocytes of both tick-species were inferred.

White spot syndrome virus (WSSV) infects specific hemocytes of the shrimp Penaeus merguiensis.

PubMed

Wang, Y T; Liu, W; Seah, J N; Lam, C S; Xiang, J H; Korzh, V; Kwang, J

2002-12-10

White spot syndrome virus (WSSV) was specifically detected by PCR in Penaeus merguiensis hemocytes, hemolymph and plasma. This suggested a close association between the shrimp hemolymph and the virus. Three types of hemocyte from shrimp were isolated using flow cytometry. Dynamic changes of the hemocyte subpopulations in P. merguiensis at different times after infection were observed, indicating that the WSSV infection selectively affected specific subpopulations. Immunofluorescence assay (IFA) and a Wright-Giemsa double staining study of hemocyte types further confirmed the cellular localization of the virus in the infected hemocytes. Electron microscopy revealed virus particles in both vacuoles and the nucleus of the semigranular cells (SGC), as well as in the vacuoles of the granular cells (GC). However, no virus could be detected in the hyaline cells (HC). Our results suggest that the virus infects 2 types of shrimp hemocytes--GCs and SGCs. The SGC type contains higher virus loads and exhibits faster infection rates, and is apparently more susceptible to WSSV infection.

Characterization of phagocytic hemocytes in Ornithodoros moubata (Acari: Ixodidae).

PubMed

Inoue, N; Hanada, K; Tsuji, N; Igarashi, I; Nagasawa, H; Mikami, T; Fujisaki, K

2001-07-01

Effects of fetal bovine serum (FBS) and complement on phagocytic activity in Ornithodaros moubata (Murray 1877) hemocytes and protease activity in the hemocytes were examined. At least three morphologically different cell types, granulocytes, plasmatocytes, and prohemocytes, were detected in hemolymph of O. moubata, and granulocytes and plasmatocytes showed phagocytic activity. FBS altered phagocytic activity of granulocytes, and complement affected phagocytic activity of plasmatocytes. Ticks were inoculated with fluorescent polystyrene beads in combination with FBS or complement. The average number of beads in granulocytes was significantly higher in the FBS injected group than the control (P < 0.01). The percentage of bead-ingesting plasmatocytes in complement inoculated ticks was significantly lower than that in heat-inactivated complement inoculated and control ticks (P < 0.05). Proteases of tick hemocytes localized in small granules in the cytoplasm not only in phagocytic hemocytes but also in prohemocytes. Results suggested modulation of tick hemocyte function through serum components, and digestion of phagocytosed foreign bodies in the hemocytes.

Hematological changes in white spot syndrome virus-infected shrimp, Fenneropenaeus chinensis (Osbeck)

NASA Astrophysics Data System (ADS)

Feng, Shouming; Zhan, Wenbin; Xing, Jing; Li, Jun; Yang, Kai; Wang, Jing

2008-08-01

The pathological changes of hemocytes in the haemolymph and hepatopancreas were examined in experimentally and naturally WSSV (white spot syndrome virus) infected Fenneropenaeus chinensis. The results showed that the pathological manifestations of hemocytes were similar among moribund shrimps infected via injection, feeding and by nature. Firstly, the total hemocyte counts (THCs) in WSSV-infected shrimp were significantly lower than those in healthy shrimp. Secondly, necrotic, broken and disintegrated cells were often observed, and a typical hematolysis was present in the haemolymph smear of WSSV-infected shrimp. Thirdly, necrosis and typical apoptosis of hemocytes were detected with TEM in the peripheral haemolymph of WSSV-infected shrimp. Hyalinocytes and semi-granulocytes with masses of WSSVs in their nuclei often appeared, whereas no granular hemocytes with WSSV were found in the hepatopancreas of moribund infected shrimps. All our results supported that hemocytes were the main target cells of WSSV, and hyalinocytes and semigranular hemocytes seemed to be more favorable for WSSV infection in F. chinensis.

Circulating Biomphalaria glabrata hemocyte subpopulations possess shared schistosome glycans and receptors capable of binding larval glycoconjugates

PubMed Central

Yoshino, Timothy P.; Wu, Xiao-Jun; Gonzalez, Laura A.; Hokke, Cornelis H.

2013-01-01

Host lectin-like recognition molecules may play an important role in innate resistance in Biomphalaria glabrata snails to larval schistosome infection, thus implicating parasite-expressed glycans as putative ligands for these lectin receptors. While host lectins may utilize specific glycan structures for parasite recognition, it also has been hypothesized that the parasite may use this system to evade immune detection by mimicking naturally-expressed host glycans, resulting in reduced immunorecognition capacity. By employing immunocytochemical (ICC) and Western blot assays using schistosome glycan-specific monoclonal antibodies (mABs) we sought to identify specific glycan epitopes (glycotopes) shared in common between larval S. mansoni and B. glabrata hemocytes, the primary immune effector cells in snails. Results confirmed the presence of selected larval glycotopes on subpopulations of hemocytes by ICC and association with numerous hemocyte proteins by Western blot analyses, including a trimannosyl core N-glycan (TriMan), and two fucosylated lacdiNAc (LDN) variants, F-LDN and F-LDN-F. Snail strain differences were seen in the prevalence of constitutively expressed F-LDN on hemocytes, and in the patterns of protein immunoreactivity with these mABs. In contrast, there was little to no hemocyte reactivity with mABs for Lewis X (LeX), LDN, LDN-F or LDN-DF. When intact hemocytes were exposed to larval transformation products (LTPs), distinct cell subpopulations displayed weak (LeX, LDN-DF) to moderate (LDN, LDN-F) glycotope reactivity by ICC, including snail strain differences in the prevalence of LDN-reactive cellular subsets. Far-Western blot analyses of the hemocytes following exposure to larval transformation proteins (LTPs) also revealed multiple mAB-reactive hemocyte protein bands for LeX, LDN, LDN-F, and LDN-DF. These results demonstrate the existence of complex patterns of shared larval glycan constitutively expressed on hemocytes and their proteins, as well as the ability or hemocytes to acquire shared glycans by the selective binding of parasite-released LTP. Unraveling the functional significance of these naturally expressed and acquired shared glycans on specific hemocyte populations represents an important challenge for future investigations. PMID:23085445

Losing the battle against fungal infection: suppression of termite immune defenses during mycosis.

PubMed

Avulova, Svetlana; Rosengaus, Rebeca B

2011-07-01

The dampwood termite, Zootermopsis angusticollis is known to generate humoral immune responses to the entomopathogenic fungus Metarhizium anisopliae. However, little is known about how the termite's cellular immune system reacts to fungal infection. To test the effect of conidia exposure on cellular immunity, we quantified the number and types of hemocytes in the hemolymph of naïve nymphs and compared their circulating counts with those of nestmates exposed to 0, 2×10(3), 2×10(6) or 2×10(8) conidia/ml doses. These termites were then bled and their hemocytes counted on days 1, 2, 3, 4, 7 post-exposure. Our results show, first, that naïve Z. angusticollis nymphs have three different blood cell types tentatively identified as granular hemocytes, prohemocytes and plasmatocytes. In these individuals, plasmatocytes were on average 13.5 and 3.3 times more numerous than granular hemocytes and prohemocytes, respectively. Second, a full factorial general linear analysis indicated that hemocyte type, time elapsed since conidia exposure and conidia dosage as well as all their interactions explained 43% of the variability in hemocyte density. The numbers of prohemocytes and particularly plasmatocytes, but not granular hemocytes, appear to be affected by the progression of disease. The decline in hemocyte numbers coincided with the appearance of hyphal bodies and the onset of "sluggish" termite behavior that culminated in the insect's death. Hemocyte counts of infected males and females were affected to the same extent. Hence, M. anisopliae overtakes the cellular immune responses of Z. angusticollis mainly by destroying the host's most abundant hemocyte types. Copyright © 2011 Elsevier Ltd. All rights reserved.

The effect of the lectin from Cherax quadricarinatus on its granular hemocytes.

PubMed

Sánchez-Salgado, José Luis; Pereyra, Mohamed Alí; Agundis, Concepción; Vivanco-Rojas, Oscar; Rosales, Carlos; Pascual, Cristina; Alpuche-Osorno, Juan José; Zenteno, Edgar

2018-06-01

In crustaceans, lectins and hemocytes of the innate immune system provide the first line of defense. Although evidence points to the potential role of lectins in regulating hemocyte activity, the processes underlying the lectin activation have not been evaluated. In the present study, the receptor for CqL, a humoral lectin from Cherax quadricarinatus specific for galactose/sialic acid, was identified in a granular subset of hemocytes. The CqL receptor (CqLR) is a 490-kDa glycoprotein, composed of four identical 120-kDa subunits. As shown by immunohistochemistry, CqL at 7.5 μg/mL as optimal dose, after 2 min, induced, specifically on granular hemocytes, increased phosphorylation of serine (152%), threonine (192%), and tyrosine (242%) as compared with non-treated hemocytes; moreover, CqL induced increased generation of reactive oxygen species (ROS). Specific kinase inhibitors showed inhibition (P < 0.001) of ROS production induced by CqL. These results strongly suggest that CqL actively participated in the generation of ROS through kinases induced by a CqLR in a subset of granular hemocytes of the crayfish C. quadricarinatus. The results provide strong evidence that CqL activates, through specific granular hemocytes, receptors that modulate cellular functions in C. quadricarinatus. Copyright © 2018 Elsevier Ltd. All rights reserved.

Multiple alpha subunits of integrin are involved in cell-mediated responses of the Manduca immune system.

PubMed

Zhuang, Shufei; Kelo, Lisha; Nardi, James B; Kanost, Michael R

2008-01-01

The cell-mediated responses of the insect innate immune system-phagocytosis, nodulation, encapsulation-involve multiple cell adhesion molecules of hemocyte surfaces. A hemocyte-specific (HS) integrin and a member of the immunoglobulin (Ig) superfamily (neuroglian) are involved in the encapsulation response of hemocytes in Manduca sexta. In addition, two new integrin alpha (alpha) subunits have been found on these hemocytes. The alpha2 subunit is mainly expressed in epidermis and Malphigian tubules, whereas the alpha3 subunit is primarily expressed on hemocytes and fat body cells. Of the three known alpha subunits, the alpha1 subunit found in HS integrin is the predominant subunit of hemocytes. Cell adhesion assays indicate that alpha2 belongs to the integrin family with RGD-binding motifs, confirming the phylogenetic analysis of alpha subunits based on the amino-acid sequence alignment of different alpha subunits. Double-stranded RNAs (dsRNAs) targeting each of these three integrin alpha subunits not only specifically decreased transcript expression of each alpha subunit in hemocytes, but also abolished the cell-mediated encapsulation response of hemocytes to foreign surfaces. The individual alpha subunits of M. sexta integrins, like their integrin counterparts in mammalian immune systems, have critical, individual roles in cell-substrate and cell-cell interactions during immune responses.

The relative abundance of hemocyte types in a polyphagous moth larva depends on diet.

PubMed

Vogelweith, Fanny; Moret, Yannick; Monceau, Karine; Thiéry, Denis; Moreau, Jérôme

2016-05-01

Hemocytes are crucial cells of the insect immune system because of their involvement in multiple immune responses including coagulation, phagocytosis and encapsulation. There are various types of hemocytes, each having a particular role in immunity, such that variation in their relative abundance affects the outcome of the immune response. This study aims to characterize these various types of hemocytes in larvae of the grapevine pest insect Eupoecilia ambiguella, and to assess variation in their concentration as a function of larval diet and immune challenge. Four types of hemocytes were found in the hemolymph of 5th instar larvae: granulocytes, oenocytoids, plasmatocytes and spherulocytes. We found that the total concentration of hemocytes and the concentration of each hemocyte type varied among diets and in response to the immune challenge. Irrespective of the diet, the concentration of granulocytes increased following a bacterial immune challenge, while the concentration of plasmatocytes and spherulocytes differentially varied between larval diets. The concentration of oenocytoids did not vary among diets before the immune challenge but varied between larval diets in response to the challenge. These results suggest that the resistance of insect larvae to different natural enemies critically depends on the effect of larval diet on the larvae's investment into the different types of hemocytes. Copyright © 2016 Elsevier Ltd. All rights reserved.

CHEMICAL EFFECTS ON OYSTER (CRASSOSTREA VIRGINICA) HEMOCYTE MICROBICIDAL ACTIVITY

EPA Science Inventory

Oyster (Crassostrea virginica) hemocytes, or blood cells, perform important internal defense functions such as phagocytosis and intracellular destruction of pathogens and bacteria. Using techniques such as phagocytosis and chemiluminescence assays, potential impairment of hemocyt...

A flow cytometric approach to the study of crustacean cellular immunity

USGS Publications Warehouse

Cardenas, W.; Jenkins, J.A.; Dankert, J.R.

2000-01-01

Responses of hemocytes from the crayfish Procambarus zonangulus to stimulation by fungal cell walls (Zymosan A) were measured by flow cytometry. Changes in hemocyte physical characteristics were assessed flow cytometrically using forward- and sidescatter light parameters, and viability was measured by two-color fluorescent staining with calcein-AM and ethidium homodimer 1. The main effects of zymosan A on crayfish hemocytes were reduction in cell size and viability compared to control mixtures (hemocytes in buffer only). Adding diethyldithiocarbamic acid, an inhibitor of phenoloxidase, to hemocyte to zymosan mixtures delayed the time course of cell size reduction and cell death compared to zymosan-positive controls. The inclusion of trypsin inhibitor in reaction mixtures further delayed the reduction in hemocyte size and cell death, thereby indicating that a proteolytic cascade, along with prophenoloxidase activation, played a key role in generating signal molecules which mediate these cellular responses. In addition to traditional methods such as microscopy and protein chemistry, flow cytometry can provide a simple, reproducible, and sensitve method for evaluating invertebrate hemocyte responses to immunological stimuli.

The Nimrod transmembrane receptor Eater is required for hemocyte attachment to the sessile compartment in Drosophila melanogaster

PubMed Central

Bretscher, Andrew J.; Honti, Viktor; Binggeli, Olivier; Burri, Olivier; Poidevin, Mickael; Kurucz, Éva; Zsámboki, János; Andó, István; Lemaitre, Bruno

2015-01-01

ABSTRACT Eater is an EGF-like repeat transmembrane receptor of the Nimrod family and is expressed in Drosophila hemocytes. Eater was initially identified for its role in phagocytosis of both Gram-positive and Gram-negative bacteria. We have deleted eater and show that it appears to be required for efficient phagocytosis of Gram-positive but not Gram-negative bacteria. However, the most striking phenotype of eater deficient larvae is the near absence of sessile hemocytes, both plasmatocyte and crystal cell types. The eater deletion is the first loss of function mutation identified that causes absence of the sessile hemocyte state. Our study shows that Eater is required cell-autonomously in plasmatocytes for sessility. However, the presence of crystal cells in the sessile compartment requires Eater in plasmatocytes. We also show that eater deficient hemocytes exhibit a cell adhesion defect. Collectively, our data uncovers a new requirement of Eater in enabling hemocyte attachment at the sessile compartment and points to a possible role of Nimrod family members in hemocyte adhesion. PMID:25681394

The Nimrod transmembrane receptor Eater is required for hemocyte attachment to the sessile compartment in Drosophila melanogaster.

PubMed

Bretscher, Andrew J; Honti, Viktor; Binggeli, Olivier; Burri, Olivier; Poidevin, Mickael; Kurucz, Éva; Zsámboki, János; Andó, István; Lemaitre, Bruno

2015-02-13

Eater is an EGF-like repeat transmembrane receptor of the Nimrod family and is expressed in Drosophila hemocytes. Eater was initially identified for its role in phagocytosis of both Gram-positive and Gram-negative bacteria. We have deleted eater and show that it appears to be required for efficient phagocytosis of Gram-positive but not Gram-negative bacteria. However, the most striking phenotype of eater deficient larvae is the near absence of sessile hemocytes, both plasmatocyte and crystal cell types. The eater deletion is the first loss of function mutation identified that causes absence of the sessile hemocyte state. Our study shows that Eater is required cell-autonomously in plasmatocytes for sessility. However, the presence of crystal cells in the sessile compartment requires Eater in plasmatocytes. We also show that eater deficient hemocytes exhibit a cell adhesion defect. Collectively, our data uncovers a new requirement of Eater in enabling hemocyte attachment at the sessile compartment and points to a possible role of Nimrod family members in hemocyte adhesion. © 2015. Published by The Company of Biologists Ltd.

First characterisation of the populations and immune-related activities of hemocytes from two edible gastropod species, the disk abalone, Haliotis discus discus and the spiny top shell, Turbo cornutus.

PubMed

Donaghy, Ludovic; Hong, Hyun-Ki; Lambert, Christophe; Park, Heung-Sik; Shim, Won Joon; Choi, Kwang-Sik

2010-01-01

The disk abalone Haliotis discus discus and the spiny top shell Turbo cornutus are edible gastropod species of high economic value, mainly in Asia. Mortality outbreaks and variations in worldwide stock abundance have been reported and suggested to be associated, at least in part, with pathogenic infections. Ecology, biology and immunology of both species are currently not well documented. The characterisation of the immune systems of these species is necessary to further assess the responses of H. discus discus and T. cornutus to environmental, chemical and disease stresses. In the present study, we investigated the morphology and immune-related activities of hemocytes in both species using light microscopy and flow cytometry. Two types of hemocytes were identified in the disk abalone hemolymph, blast-like cells and hyalinocytes; whereas four main hemocyte types were distinguished in the spiny top shell, blast-like cells, type I and II hyalinocytes, and granulocytes. Flow cytometric analysis also revealed differences between cell types in immune-related activities. Three subsets of hemocytes, defined by differing lysosomal characteristics, were observed in the hemolymph of the spiny top shell, and only one in the disk abalone. Phagocytic activity was higher in H. discus discus hemocytes than in T. cornutus hemocytes, and the kinetics of PMA-stimulated oxidative activity was different between hemocytes of the disk abalone and the spiny top shell. Finally our results suggest for the first time a predominant mitochondrial origin of oxidative activity in gastropod hemocytes. Copyright 2009 Elsevier Ltd. All rights reserved.

The Involvement of Hemocyte Prophenoloxidase in the Shell-Hardening Process of the Blue Crab, Callinectes sapidus

PubMed Central

Alvarez, Javier V.; Chung, J. Sook

2015-01-01

Cuticular structures of arthropods undergo dramatic molt-related changes from being soft to becoming hard. The shell-hardening process of decapod crustaceans includes sclerotization and mineralization. Hemocyte PPO plays a central role in melanization and sclerotization particularly in wound healing in crustaceans. However, little is known about its role in the crustacean initial shell-hardening process. The earlier findings of the aggregation of heavily granulated hemocytes beneath the hypodermis during ecdysis imply that the hemocytes may be involved in the shell-hardening process. In order to determine if hemocytes and hemocyte PPO have a role in the shell-hardening of crustaceans, a knockdown study using specific CasPPO-hemo-dsRNA was carried out with juvenile blue crabs, Callinectes sapidus. Multiple injections of CasPPO-hemo-dsRNA reduce specifically the levels of CasPPO-hemo expression by 57% and PO activity by 54% in hemocyte lysate at the postmolt, while they have no effect on the total hemocyte numbers. Immunocytochemistry and flow cytometry analysis using a specific antiserum generated against CasPPO show granulocytes, semigranulocytes and hyaline cells as the cellular sources for PPO at the postmolt. Interestingly, the type of hemocytes, as the cellular sources of PPO, varies by molt stage. The granulocytes always contain PPO throughout the molt cycle. However, semigranulocytes and hyaline cells become CasPPO immune-positive only at early premolt and postmolt, indicating that PPO expression in these cells may be involved in the shell-hardening process of C. sapidus. PMID:26393802

Hemocyte Density Increases with Developmental Stage in an Immune-Challenged Forest Caterpillar

PubMed Central

Stoepler, Teresa M.; Castillo, Julio C.; Lill, John T.; Eleftherianos, Ioannis

2013-01-01

The cellular arm of the insect immune response is mediated by the activity of hemocytes. While hemocytes have been well-characterized morphologically and functionally in model insects, few studies have characterized the hemocytes of non-model insects. Further, the role of ontogeny in mediating immune response is not well understood in non-model invertebrate systems. The goals of the current study were to (1) determine the effects of caterpillar size (and age) on hemocyte density in naïve caterpillars and caterpillars challenged with non-pathogenic bacteria, and (2) characterize the hemocyte activity and diversity of cell types present in two forest caterpillars: Euclea delphinii and Lithacodes fasciola (Limacodidae). We found that although early and late instar (small and large size, respectively) naïve caterpillars had similar constitutive hemocyte densities in both species, late instar Lithacodes caterpillars injected with non-pathogenic E. coli produced more than a twofold greater density of hemocytes than those in early instars. We also found that both caterpillar species contained plasmatocytes, granulocytes and oenocytoids, all of which are found in other lepidopteran species, but lacked spherulocytes. Granulocytes and plasmatocytes were found to be strongly phagocytic in both species, but granulocytes exhibited a higher phagocytic activity than plasmatocytes. Our results strongly suggest that for at least one measure of immunological response, the production of hemocytes in response to infection, response magnitudes can increase over ontogeny. While the underlying raison d’ être for this improvement remains unclear, these findings may be useful in explaining natural patterns of stage-dependent parasitism and pathogen infection. PMID:23940679

Infection-Induced Interaction between the Mosquito Circulatory and Immune Systems

PubMed Central

King, Jonas G.; Hillyer, Julián F.

2012-01-01

Insects counter infection with innate immune responses that rely on cells called hemocytes. Hemocytes exist in association with the insect's open circulatory system and this mode of existence has likely influenced the organization and control of anti-pathogen immune responses. Previous studies reported that pathogens in the mosquito body cavity (hemocoel) accumulate on the surface of the heart. Using novel cell staining, microdissection and intravital imaging techniques, we investigated the mechanism of pathogen accumulation in the pericardium of the malaria mosquito, Anopheles gambiae, and discovered a novel insect immune tissue, herein named periostial hemocytes, that sequesters pathogens as they flow with the hemolymph. Specifically, we show that there are two types of endocytic cells that flank the heart: periostial hemocytes and pericardial cells. Resident periostial hemocytes engage in the rapid phagocytosis of pathogens, and during the course of a bacterial or Plasmodium infection, circulating hemocytes migrate to the periostial regions where they bind the cardiac musculature and each other, and continue the phagocytosis of invaders. Periostial hemocyte aggregation occurs in a time- and infection dose-dependent manner, and once this immune process is triggered, the number of periostial hemocytes remains elevated for the lifetime of the mosquito. Finally, the soluble immune elicitors peptidoglycan and β-1,3-glucan also induce periostial hemocyte aggregation, indicating that this is a generalized and basal immune response that is induced by diverse immune stimuli. These data describe a novel insect cellular immune response that fundamentally relies on the physiological interaction between the insect circulatory and immune systems. PMID:23209421

Effects of gibberellic acid on hemocytes of Galleria mellonella L. (Lepidoptera: Pyralidae).

PubMed

Altuntaş, H; Kılıç, A Y; Uçkan, F; Ergin, E

2012-06-01

The impacts of different doses of the plant growth regulator gibberellic acid (GA(3)) in diet on the number of total and differential hemocytes, frequency of apoptotic, and necrotic hemocytes, mitotic indices, encapsulation, and melanization responses were investigated using the greater wax moth Galleria mellonella L. (Lepidoptera: Pyralidae) larvae. Total hemocyte counts increased in G. mellonella larvae at all treatment doses whereas GA(3) application had no effect on the number of different hemocyte types. The occurrence of apoptosis, necrosis and mitotic indices in GA(3) treated and untreated last instars were detected by acridine orange or ethidium bromide double staining by fluorescence microscopy. While the ratio of necrotic hemocytes increased at all GA(3) treatments, that of late apoptotic cells was only higher at doses >200 ppm when compared with untreated larvae. The percentage of mitotic index also increased at 5,000 ppm. Positively charged DEAE Sephadex A-25 beads were used for analysis of the levels of encapsulation and melanization in GA(3) treated G. mellonella larvae. At four and 24 h posttreatments with Sephadex A-25 bead injection, insects were dissected under a stereomicroscope. Encapsulation rates of larval hemocytes were dependent on the extent of encapsulation and time but not treatment groups. While the extent of melanization of hemocytes showed differences related to time, in general, a decrease was observed at all doses of GA(3) treated larvae at 24 h. We suggest that GA(3) treatment negatively affects hemocyte physiology and cell immune responses inducing cells to die by necrosis and apoptosis in G. mellonella larvae.

The virus-like particles of a braconid endoparasitoid wasp, Meteorus pulchricornis, inhibit hemocyte spreading in its noctuid host, Pseudaletia separata.

PubMed

Suzuki, M; Miura, K; Tanaka, T

2008-06-01

We previously reported that the virus-like particles of Meteorus pulchricornis (MpVLPs) are capable of inducing apoptosis by around 6h in the hemocytes of the host, Pseudaletia separata [Suzuki, M., Tanaka, T., 2006. Virus-like particles in venom of Meteorus pulchricornis induce host hemocyte apoptosis. Journal of Insect Physiology 52, 602-611], thereby protecting the oviposited egg. In the present study, we focused on analyses of the earlier events caused by the MpVLPs upon the host immune response, namely their effects on hemocyte spreading. After recognition and attachment on foreign substance, the granulocytes and plasmatocytes assemble focal complexes and focal adhesions and spread by protruding filopodia/lamellipodia. The well-spread, cultured hemocytes were subjected to MpVLPs exposure, and the morphological changes were observed. The granulocytes lost the focal complexes/adhesions visualized as phosphotyrosine clusters and retracted the filopodia/lamellipodia within 30min after exposure, while the plasmatocytes exhibited similar but distinct responses. The two hemocyte species prepared from either parasitized or MpVLP-injected hosts lost the ability to form both filopodia/lamellipodia and phosphotyrosine clusters. A caspase inhibitor, Z-VAD-FMK, did not affect these MpVLP-induced morphological changes, indicating that these earlier changes found in the hemocytes precede apoptosis. The present study together with our previous data has established that the attenuation of host immune defense by the MpVLPs comprises at least two temporally distinguishable phases: immediate and early inhibition of hemocyte spreading and the eventual induction of hemocyte apoptosis.

Baculovirus induced transcripts in hemocytes from Heliothis virescens

USDA-ARS?s Scientific Manuscript database

Using RNA-sequencing digital difference expression profiling methods we have assessed the gene expression profiles of hemocytes harvested from Heliothis virescens that were challenged with Helicoverpa zea single nucleopolyhedrovirus (HzSNPV). A reference transcriptome of hemocyte-expressed transcri...

cDNA microarray analyses reveal candidate marker genes for the detection of ascidian disease in Korea.

PubMed

Azumi, Kaoru; Usami, Takeshi; Kamimura, Akiko; Sabau, Sorin V; Miki, Yasufumi; Fujie, Manabu; Jung, Sung-Ju; Kitamura, Shin-Ichi; Suzuki, Satoru; Yokosawa, Hideyoshi

2007-12-01

A serious disease of the ascidian Halocynthia roretzi has been spread extensively among Korean aquaculture sites. To reveal the cause of the disease and establish a monitoring system for it, we constructed a cDNA microarray spotted with 2,688 cDNAs derived from H. roretzi hemocyte cDNA libraries to detect genes differentially expressed in hemocytes between diseased and non-diseased ascidians. We detected 21 genes showing increased expression and 16 genes showing decreased expression in hemocytes from diseased ascidians compared with those from non-diseased ascidians. RT-PCR analyses confirmed that the expression levels of genes encoding astacin, lysozyme, ribosomal protein PO, and ubiquitin-ribosomal protein L40e fusion protein were increased in hemocytes from diseased ascidians, while those of genes encoding HSP40, HSP70, fibronectin, carboxypeptidase and lactate dehydrogenase were decreased. These genes were expressed not only in hemocytes but also in various other tissues in ascidians. Furthermore, the expression of glutathione-S transferase omega, which is known to be up-regulated in H. roretzi hemocytes during inflammatory responses, was strongly increased in hemocytes from diseased ascidians. These gene expression profiles suggest that immune and inflammatory reactions occur in the hemocytes of diseased ascidians. These genes will be good markers for detecting and monitoring this disease of ascidians in Korean aquaculture sites.

Comprehensive Study of the Influence of Altered Gravity on the Oxidative Burst of Mussel ( Mytilus edulis) Hemocytes

NASA Astrophysics Data System (ADS)

Unruh, E.; Brungs, S.; Langer, S.; Bornemann, G.; Frett, T.; Hansen, P.-D.

2016-06-01

Microgravity induces alterations in the functioning of immune cell; however, the underlying mechanisms have not yet been identified. In this study, hemocytes (blood cells) of the blue mussel Mytilus edulis were investigated under altered gravity conditions. The study was conducted on the ground in preparation for the BIOLAB TripleLux-B experiment, which will be performed on the International Space Station (ISS). On-line kinetic measurements of reactive oxygen species (ROS) production during the oxidative burst and thus cellular activity of isolated hemocytes were performed in a photomultiplier (PMT)-clinostat (simulated microgravity) and in the 1 g operation mode of the clinostat in hypergravity on the Short-Arm Human Centrifuge (SAHC) as well as during parabolic flights. In addition to studies with isolated hemocytes, the effect of altered gravity conditions on whole animals was investigated. For this purpose, whole mussels were exposed to hypergravity (1.8 g) on a multi-sample incubator centrifuge (MuSIC) or to simulated microgravity in a submersed clinostat. After exposure for 48 h, hemocytes were taken from the mussels and ROS production was measured under 1 g conditions. The results from the parabolic flights and clinostat studies indicate that mussel hemocytes respond to altered gravity in a fast and reversible manner. Hemocytes (after cryo-conservation) exposed to simulated microgravity ( μ g), as well as fresh hemocytes from clinorotated animals, showed a decrease in ROS production. Measurements during a permanent exposure of hemocytes to hypergravity (SAHC) show a decrease in ROS production. Hemocytes of mussels measured after the centrifugation of whole mussels did not show an influence to the ROS response at all. Hypergravity during parabolic flights led to a decrease but also to an increase in ROS production in isolated hemocytes, whereas the centrifugation of whole mussels did not influence the ROS response at all. This study is a good example how ground-based facility experiments can be used to prepare for an upcoming ISS experiment, in this case the TRIPLE LUX B experiment.

Morphological and functional characterization of the hemocytes from the pearl oyster Pteria hirundo and their immune responses against Vibrio infections.

PubMed

Vieira, Graziela Cleuza; da Silva, Patrícia Mirella; Barracco, Margherita Anna; Hering, Augusto Ferrari; Albuquerque, Marcos Caivano Pedroso de; Coelho, Jaqueline da Rosa; Schmidt, Éder Carlos; Bouzon, Zenilda Laurita; Rosa, Rafael Diego; Perazzolo, Luciane Maria

2017-11-01

Hemocyte populations of the pearl oyster Pteria hirundo were characterized at morphological, ultrastructural and functional levels. Three main hemocyte populations were identified: hyalinocytes, granulocytes and blast-like cells. Hyalinocytes were the most abundant population (88.2%) characterized by the presence of few or no granules in the cytoplasm and composed by two subpopulations, large and small hyalinocytes. Comparatively, granulocytes represented 2.2% of the hemocyte population and were characterized by the presence of numerous large electron-lucid granules in the cytoplasm. Finally, the blast-like cells (9.5%) were the smallest hemocytes, showing spherical shape and a high nucleus/cytoplasm ratio. Hemocytes exhibited a significant phagocytic capacity for inert particles (38.5%) and showed to be able to produce microbicidal molecules, such as reactive oxygen species (ROS) (ex vivo assays). The immune role of hemocytes was further investigated in the P. hirundo defense against the Gram-negative Vibrio alginolyticus. A significant decrease in the total number of hemocytes was observed at 24 h following injection of V. alginolyticus or sterile seawater (injury control) when compared to naïve (unchallenged) animals, indicating the migration of circulating hemocytes to the sites of infection and tissue damage. Bacterial agglutination was only observed against Gram-negative bacteria (Vibrio) but not against to marine Gram-positive-bacteria. Besides, an increase in the agglutination titer was observed against V. alginolyticus only in animals previously infected with this same bacterial strain. These results suggest that agglutinins or lectin-like molecules may have been produced in response to this particular microorganism promoting a specific recognition. The ultrastructural and functional characterization of P. hirundo hemocytes constitutes a new important piece of the molluscan immunity puzzle that can also contribute for the improvement of bivalve production sustainability. Copyright © 2017 Elsevier Ltd. All rights reserved.

Ultrastructural and functional characterization of circulating hemocytes from Galleria mellonella larva: Cell types and their role in the innate immunity.

PubMed

Wu, Gongqing; Liu, Yi; Ding, Ying; Yi, Yunhong

2016-08-01

Galleria mellonella larvae have been widely used as a model to study the virulence of various human pathogens. Hemocytes play important roles in the innate immune response of G. mellonella. In this study, the hemocytes of G. mellonella larvae were analyzed by transmission electron microscope, light microscope, and cytochemistry. The cytological and morphological analyses revealed four types of hemocytes; Plasmatocytes, granular cells, spherule cells and oenocytoids. Differential hemocyte counts showed that under our conditions plasmatocytes and granular cells were the most abundant circulating cell types in the hemolymph. We also investigated the role of different types of hemocytes in the cellular and humoral immune defenses. The in-vivo experiment showed that plasmatocytes, granular cells and oenocytoids phagocytized FITC-labelled Escherichia coli bacteria in larvae of G. mellonella, whereas the granular cells exhibited the strongest phagocytic ability against these microbial cells. After incubation with L-DOPA, plasmatocytes, granular cells and oenocytoids are stained brown, indicating the presence of phenoloxidase activity. These results shed new light on our understanding of the immune function of G. mellonella hemocytes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Serotonin modulates insect hemocyte phagocytosis via two different serotonin receptors

PubMed Central

Qi, Yi-xiang; Huang, Jia; Li, Meng-qi; Wu, Ya-su; Xia, Ren-ying; Ye, Gong-yin

2016-01-01

Serotonin (5-HT) modulates both neural and immune responses in vertebrates, but its role in insect immunity remains uncertain. We report that hemocytes in the caterpillar, Pieris rapae are able to synthesize 5-HT following activation by lipopolysaccharide. The inhibition of a serotonin-generating enzyme with either pharmacological blockade or RNAi knock-down impaired hemocyte phagocytosis. Biochemical and functional experiments showed that naive hemocytes primarily express 5-HT1B and 5-HT2B receptors. The blockade of 5-HT1B significantly reduced phagocytic ability; however, the blockade of 5-HT2B increased hemocyte phagocytosis. The 5-HT1B-null Drosophila melanogaster mutants showed higher mortality than controls when infected with bacteria, due to their decreased phagocytotic ability. Flies expressing 5-HT1B or 5-HT2B RNAi in hemocytes also showed similar sensitivity to infection. Combined, these data demonstrate that 5-HT mediates hemocyte phagocytosis through 5-HT1B and 5-HT2B receptors and serotonergic signaling performs critical modulatory functions in immune systems of animals separated by 500 million years of evolution. DOI: http://dx.doi.org/10.7554/eLife.12241.001 PMID:26974346

PERKINSUS-"CIDAL" ACTIVITY OF OYSTER HEMOCYTES USING A TETRAZOLIUM DYE REDUCTION ASSAY: OPTIMIZATION AND APPLICATIONS

EPA Science Inventory

A bactericidal assay developed to assess the ability of oyster (Crassostrea virginica) hemocytes to kill the human pathogen Vibrio parahaemolyticus was optimized to estimate killing of the oyster parasite Perkinsus marinus. Assay variables, temperature, hemocyte:parasite ratio, i...

Ultrastructures and classification of circulating hemocytes in 9 botryllid ascidians (chordata: ascidiacea).

PubMed

Hirose, Euichi; Shirae, Maki; Saito, Yasunori

2003-05-01

Ultrastructures of circulating hemocytes were studied in 9 botryllid ascidians. The hemocytes are classified into five types: hemoblasts, phagocytes, granulocytes, morula cells, and pigment cells. These five types are always found in the 9 species. They should represent the major hemocyte types of the circulating cells in the blood. Hemoblasts are small hemocytes having a high nucleus/cytoplasm ratio. There are few granular or vacuolar inclusions in the cytoplasm. Phagocytes have phagocytic activity and their shape is variable depending on the amount of engulfed materials. In granulocytes, shape and size of granules are different among the species. Morula cells are characterized by several vacuoles filled with electron dense materials. In pigment cells, the bulk of the cytoplasm is occupied by one or a few vacuoles containing pigment granules. We also described some other hemocyte types found in particular species. Furthermore, we encountered free oocytes circulating in the blood in two species, Botryllus primigenus and Botrylloides lentus.

The ascidian Styela plicata hemocytes as a potential biomarker of marine pollution: In vitro effects of seawater and organic mercury.

PubMed

Parrinello, D; Bellante, A; Parisi, M G; Sanfratello, M A; Indelicato, S; Piazzese, D; Cammarata, M

2017-02-01

Toxic metals, such as mercury, contribute substantially to anthropogenic pollution in many estuarine environments. Animals living in those environments, particularly invertebrate filter feeders like tunicates, can be used as bioindicators. In an attempt to identify cellular markers for revealing pollution, this study examined in vitro the effects of different concentrations of methyl mercury on Styela plicata hemocytes. The harvested hemocytes from S. plicata that were exposed to the metal had a significant mortality, cellular count and morphometric alterations. These findings provided evidence of MeHg immunotoxic effects on S. plicata, resulting in hemocyte death and morphological changes induced by cytoskeleton alterations. Thus, a morphometric cellular parameter, such as spreading ability, was used as a complementary method for differentiation between hemocytes treated with a marine solution (as a negative control) and hemocytes incubated with methylmercury and/or Sicilian seawater samples. Copyright © 2016 Elsevier Inc. All rights reserved.

Hemocytes of the palaemonids Macrobrachium rosenbergii and M. acanthurus, and of the penaeid Penaeus paulensis.

PubMed

Gargioni, R; Barracco, M A

1998-06-01

The hemocytes of two palaemonids and one penaeid were characterized using light and transmission electron microscopy (TEM). The blood cells in all three species were classified as hyaline hemocytes (HH), small granule hemocytes (SGH), and large granule hemocytes (LGH). The HH are unstable hemocytes with a characteristic high nucleo-cytoplasmic ratio. Their cytoplasm appears particularly dense and has from few to numerous granules that often exhibit a typical striated substructure. In both palaemonids, the great majority of the HH contain numerous granules, whereas in Penaeus paulensis, a small number of these cells have few or no granules. The cytoplasm of some HH of the penaeid exhibits typical electron-dense deposits. The granulocytes, LGH and SGH, contain abundant electron-dense granules that are usually smaller in the SGH. In both hemocyte types, the cytosol, but not the granules, is rich in carbohydrates (PAS positive) and numerous vesicles contain acid phosphatase (Gomori reactive). In all studied shrimps, the SGH and LGH were actively phagocytic when examined on blood cell monolayers incubated with the yeast Saccharomyces cerevisiae. A few mitotic figures (less than 1%) were observed in the granulocytes of P. paulensis, but not in the palaemonids. SGH is the main circulating blood cell type in both palaemonids, whereas HH is predominant in the penaeid. Based on morphological and functional features, it appears that the hyaline and the granular hemocytes of the three shrimp species represent different cell lineages.

FACTORS INFLUENCING IN VITRO KILLING OF BACTERIA BY HEMOCYTES OF THE EASTERN OYSTER (CRASSOSTREA VIRGINICA)

EPA Science Inventory

A tetrazolium dye reduction assay was used to study factors governing killing of bacteria by oyster hemocytes. In vitro tests were performed on bacterial strains by using hemocytes from oysters collected from the same location in winter and summer. Vibrio parahaemolyticus strains...

New perspectives on virus detection in shellfish: hemocytes as a source of concentrated virus

USDA-ARS?s Scientific Manuscript database

USDA ARS research indicates that circulating phagocytic cells (hemocytes) within oysters retain virus particles. We find that persistence of hepatitis A virus (HAV) within oyster hemocytes correlates with the presence of virus within whole oysters. Since bivalve shellfish have no self-nonself immun...

Identification and functional characterization of a novel locust peptide belonging to the family of insect growth blocking peptides.

PubMed

Duressa, Tewodros Firdissa; Boonen, Kurt; Hayakawa, Yoichi; Huybrechts, Roger

2015-12-01

Growth blocking peptides (GBPs) are recognized as insect cytokines that take part in multifaceted functions including immune system activation and growth retardation. The peptides induce hemocyte spreading in vitro, which is considered as the initial step in hemocyte activation against infection in many insect species. Therefore, in this study, we carried out a series of in vitro bioassay driven fractionations of Locusta migratoria hemolymph combined with mass spectrometry to identify locust hemocyte activation factors belonging to the family of insect GBPs. We identified the locust hemocyte spreading peptide (locust GBP) as a 28-mer peptide encoded at the C-terminus of a 64 amino acid long precursor polypeptide. As demonstrated by QRT-PCR, the gene encoding the locust GBP precursor (proGBP) was expressed in large quantities in diverse locust tissues including fat body, endocrine glands, central nervous system, reproductive tissues and flight muscles. In contrary, hemocytes, gut tissues and Malpighian tubules displayed little expression of the proGBP transcript. The bioactive peptide induces transient depletion of hemocytes in vivo and when injected in last instar nymphs it extends the larval growth phase and postpones adult molting. In addition, we identified a functional homologous hemocyte spreading peptide in Schistocerca gregaria. Copyright © 2015 Elsevier Inc. All rights reserved.

A systematic study on hemocyte identification and plasma prophenoloxidase from Culex pipiens quinquefasciatus at different developmental stages.

PubMed

Wang, Zhixiang; Lu, Anrui; Li, Xuquan; Shao, Qimiao; Beerntsen, Brenda T; Liu, Chaoliang; Ma, Yajun; Huang, Yamin; Zhu, Huaimin; Ling, Erjun

2011-01-01

Culexpipiens quinquefasciatus (C. quinquefasciatus) is an important vector that can transmit human diseases such as West Nile virus, lymphatic filariasis, Japanese encephalitis and St. Louis encephalitis. However, very limited research concerning the humoral and cellular immune defenses of C. quinquefasciatus has been done. Here we present the research on hemocyte identification and plasma including hemocyte prophenoloxidase from C. quinquefasciatus at all developmental stages in order to obtain a complete picture of C. quinquefasciatus innate immunity. We identified hemocytes into four types: prohemocytes, oenocytoids, plasmatocytes and granulocytes. Prophenoloxidase (PPO) is an essential enzyme to induce melanization after encapsulation. PPO-positive hemocytes and plasma PPO were observed at all developmental stages. As for specific hemocyte types, prophenoloxidase was found in the plasmatocytes at larval stage alone and in the smallest prohemocytes during almost all developmental stages. Moreover, the granulocytes were PPO-positive from blood-fed female mosquitoes and oenocytoids were observed PPO-positive in pupae and in adult females after blood-feeding. As for plasma, there were different patterns of PPO in C. quinquefasciatus at different developmental stages. These results are forming a basis for further studies on the function of C. quinquefasciatus hemocytes and prophenoloxidase as well as their involvement in fighting against mosquito-borne pathogens. Copyright © 2010 Elsevier Inc. All rights reserved.

Hemocytes and hemocytopoiesis in Silkworms.

PubMed

Beaulaton, J

1979-01-01

A brief review is presented of the current state of ultrastructure, cytochemistry, and physiology of the hemocytes and meso- and metathoracic peri-imaginal-wing organs in silkworms. According to the accepted morphological classification, five circulating types of hemocytes are recognized in Bombyx mori as well as in Antheraea pernyi. They are prophemocytes or stem cells, plasmatocytes or pre-differentiated cells and three specialized cells, granulocytes, spherule cells and oenocytoids. During post-embryonic development the last four types are the most common in the circulating hemolymph. Plasmatocytes are considered to be pluripotent cells from which granulocytes, spherule cells and oenocytoids are derived. Contrary to the situation in most insects the plasmatocytes are not phagocytic in Antheraea. The granulocytes are efficient phagocytes. Both plasmatocytes and granulocytes are involved in pinocytosis. Another possible function of the granulocytes is hemolymph coagulation. The function of the spherule cells which contain a paracrystalline material (muco- or glycoproteins) is by no means clear. The phenoloxidase activity found within the cytosol of oenocytoids appears effective against the natural monophenol and diphenol substrates. The involvement of oenocytoids in the complex metabolism of phenols and particularly in the production of plasma phenolases has been reported. The mitotic division of five circulating hemocyte types is well known and was long regarded as the only mechanism of postembryonic hemocyte production. We present for silkworms, experimental evidence of the hemocytopoietic function of the meso- and metathoracic organs surrounding the imaginal wing discs. Ablation experiments demonstrate that the mitotic activity of free hemocytes is unable to maintain the normal hemocytogram in the absence of the two paris of organs. These organs are typically divided into cell islets ensheathed by a connective tissue membrane. Two types of islets may be classified by the disposition of the cells : the compact islets or aggregations of stem cells and the reticulate islets which are mainly composed of hemocytes at different steps of differentiation. The relative number of prohemocytes in the total hemocyte population ranges from 84 to 97 p. cent in organs of Antheraea pernyi. This well-defined cell type appears to be the major hemocyte type in hemocytopoietic organs. In Antheraea, the mitotic index (the relative number of mitotic hemocytes in the total cell population) varies from 0.5 to about 3 p. cent. Finally, our data direct attention to cyclic functional changes such as mitotic divisions and hemocyte differentiation which run parallel to the molting cycle.

Endogenous growth factor stimulation of hemocyte proliferation induces resistance to Schistosoma mansoni challenge in the snail host.

PubMed

Pila, Emmanuel A; Gordy, Michelle A; Phillips, Valerie K; Kabore, Alethe L; Rudko, Sydney P; Hanington, Patrick C

2016-05-10

Digenean trematodes are a large, complex group of parasitic flatworms that infect an incredible diversity of organisms, including humans. Larval development of most digeneans takes place within a snail (Gastropoda). Compatibility between snails and digeneans is often very specific, such that suitable snail hosts define the geographical ranges of diseases caused by these worms. The immune cells (hemocytes) of a snail are sentinels that act as a crucial barrier to infection by larval digeneans. Hemocytes coordinate a robust and specific immunological response, participating directly in parasite killing by encapsulating and clearing the infection. Hemocyte proliferation and differentiation are influenced by unknown digenean-specific exogenous factors. However, we know nothing about the endogenous control of hemocyte development in any gastropod model. Here, we identify and functionally characterize a progranulin [Biomphalaria glabrata granulin (BgGRN)] from the snail B. glabrata, a natural host for the human blood fluke Schistosoma mansoni Granulins are growth factors that drive proliferation of immune cells in organisms, spanning the animal kingdom. We demonstrate that BgGRN induces proliferation of B. glabrata hemocytes, and specifically drives the production of an adherent hemocyte subset that participates centrally in the anti-digenean defense response. Additionally, we demonstrate that susceptible B. glabrata snails can be made resistant to infection with S. mansoni by first inducing hemocyte proliferation with BgGRN. This marks the functional characterization of an endogenous growth factor of a gastropod mollusc, and provides direct evidence of gain of resistance in a snail-digenean infection model using a defined factor to induce snail resistance to infection.

Hemocytes Are Sites of Enteric Virus Persistence within Oysters ▿

PubMed Central

Provost, Keleigh; Dancho, Brooke A.; Ozbay, Gulnihal; Anderson, Robert S.; Richards, Gary P.; Kingsley, David H.

2011-01-01

The goal of this study was to determine how enteric viruses persist within shellfish tissues. Several lines of novel evidence show that phagocytic blood cells (hemocytes) of Eastern oysters (Crassostrea virginica) play an important role in the retention of virus particles. Our results demonstrated an association of virus contamination with hemocytes but not with hemolymph. Live oysters contaminated overnight with hepatitis A virus (HAV) and murine norovirus (MNV) had 56% and 80% of extractable virus associated with hemocytes, respectively. Transfer of HAV-contaminated hemocytes to naïve (virus-free) oysters resulted in naïve oyster meat testing HAV positive for up to 3 weeks. Acid tolerance of HAV, MNV, poliovirus (PV), and feline calicivirus (FCV) correlated with the ability of each virus to persist within oysters. Using reverse transcription-PCR (RT-PCR) to evaluate persistence of these viruses in oysters, we showed that HAV persisted the longest (>21 days) and was most acid resistant, MNV and PV were less tolerant of acidic pH, persisting for up to 12 days and 1 day, respectively, and FCV did not persist (<1 day) within oysters and was not acid tolerant. This suggests that the ability of a virus to tolerate the acidic conditions typical of phagolysosomal vesicles within hemocytes plays a role in determining virus persistence in shellfish. Evaluating oyster and hemocyte homogenates and live contaminated oysters as a prelude to developing improved viral RNA extraction methods, we found that viruses were extracted more expediently from hemocytes than from whole shellfish tissues and gave similar RT-PCR detection sensitivities. PMID:21948840

Rhipicephalus microplus infected by Metarhizium: unveiling hemocyte quantification, GFP-fungi virulence, and ovary infection.

PubMed

de Paulo, Jéssica Fiorotti; Camargo, Mariana Guedes; Coutinho-Rodrigues, Caio Junior Balduino; Marciano, Allan Felipe; de Freitas, Maria Clemente; da Silva, Emily Mesquita; Gôlo, Patrícia Silva; Morena, Diva Denelle Spadacci; da Costa Angelo, Isabele; Bittencourt, Vânia Rita Elias Pinheiro

2018-06-01

Hemocytes, cells present in the hemocoel, are involved in the immune response of arthropods challenged with entomopathogens. The present study established the best methodology for harvesting hemocytes from Rhipicephalus microplus and evaluated the number of hemocytes in addition to histological analysis from ovaries of fungus-infected females and tested the virulence of GFP-fungi transformants. Different centrifugation protocols were tested, and the one in which presented fewer disrupted cells and higher cell recovery was applied for evaluating the effect of Metarhizium spp. on hemocytes against R. microplus. After processing, protocol number 1 (i.e., hemolymph samples were centrifuged at 500×g for 3 min at 4 °C) was considered more efficient, with two isolates used (Metarhizium robertsii ARSEF 2575 and Metarhizium anisopliae ARSEF 549), both wild types and GFP, to assess their virulence. In the biological assays, the GFP-fungi were as virulent as wild types, showing no significant differences. Subsequently, hemocyte quantifications were performed after inoculation, which exhibited notable changes in the number of hemocytes, reducing by approximately 80% in females previously treated with Metarhizium isolates in comparison to non-treated females. Complementarily, 48 h after inoculation, in which hemolymph could not be obtained, histological analysis showed the high competence of these fungi to colonize ovary from ticks. Here, for the first time, the best protocol (i.e., very low cell disruption and high cell recovery) for R. microplus hemocyte obtaining was established aiming to guide directions to other studies that involves cellular responses from ticks to fungi infection.

Molecular Profiling of Phagocytic Immune Cells in Anopheles gambiae Reveals Integral Roles for Hemocytes in Mosquito Innate Immunity*

PubMed Central

Smith, Ryan C.; King, Jonas G.; Tao, Dingyin; Zeleznik, Oana A.; Brando, Clara; Thallinger, Gerhard G.; Dinglasan, Rhoel R.

2016-01-01

The innate immune response is highly conserved across all eukaryotes and has been studied in great detail in several model organisms. Hemocytes, the primary immune cell population in mosquitoes, are important components of the mosquito innate immune response, yet critical aspects of their biology have remained uncharacterized. Using a novel method of enrichment, we isolated phagocytic granulocytes and quantified their proteomes by mass spectrometry. The data demonstrate that phagocytosis, blood-feeding, and Plasmodium falciparum infection promote dramatic shifts in the proteomic profiles of An. gambiae granulocyte populations. Of interest, large numbers of immune proteins were induced in response to blood feeding alone, suggesting that granulocytes have an integral role in priming the mosquito immune system for pathogen challenge. In addition, we identify several granulocyte proteins with putative roles as membrane receptors, cell signaling, or immune components that when silenced, have either positive or negative effects on malaria parasite survival. Integrating existing hemocyte transcriptional profiles, we also compare differences in hemocyte transcript and protein expression to provide new insight into hemocyte gene regulation and discuss the potential that post-transcriptional regulation may be an important component of hemocyte gene expression. These data represent a significant advancement in mosquito hemocyte biology, providing the first comprehensive proteomic profiling of mosquito phagocytic granulocytes during homeostasis blood-feeding, and pathogen challenge. Together, these findings extend current knowledge to further illustrate the importance of hemocytes in shaping mosquito innate immunity and their principal role in defining malaria parasite survival in the mosquito host. PMID:27624304

Physalin B inhibits Rhodnius prolixus hemocyte phagocytosis and microaggregation by the activation of endogenous PAF-acetyl hydrolase activities.

PubMed

Castro, D P; Figueiredo, M B; Genta, F A; Ribeiro, I M; Tomassini, T C B; Azambuja, P; Garcia, E S

2009-06-01

The effects of physalin B (a natural secosteroidal chemical from Physalis angulata, Solanaceae) on phagocytosis and microaggregation by hemocytes of 5th-instar larvae of Rhodnius prolixus were investigated. In this insect, hemocyte phagocytosis and microaggregation are known to be induced by the platelet-activating factor (PAF) or arachidonic acid (AA) and regulated by phospholipase A(2) (PLA(2)) and PAF-acetyl hydrolase (PAF-AH) activities. Phagocytic activity and formation of hemocyte microaggregates by Rhodnius hemocytes were strongly blocked by oral treatment of this insect with physalin B (1mug/mL of blood meal). The inhibition induced by physalin B was reversed for both phagocytosis and microaggregation by exogenous arachidonic acid (10microg/insect) or PAF (1microg/insect) applied by hemocelic injection. Following treatment with physalin B there were no significant alterations in PLA(2) activities, but a significant enhancement of PAF-AH was observed. These results show that physalin B inhibits hemocytic activity by depressing insect PAF analogous (iPAF) levels in hemolymph and confirm the role of PAF-AH in the cellular immune reactions in R. prolixus.

Varroa destructor parasitism reduces hemocyte concentrations and prophenol oxidase gene expression in bees from two populations.

PubMed

Koleoglu, Gun; Goodwin, Paul H; Reyes-Quintana, Mariana; Hamiduzzaman, Mollah Md; Guzman-Novoa, Ernesto

2018-04-01

Circulating hemocytes are responsible for defensive and healing mechanisms in the honey bee, Apis mellifera. Parasitism by the mite Varroa destructor and injection of V. destructor homogenate in buffer, but not buffer injection, showed similar reductions in total hemocyte concentrations in both Africanized and European adult honey bees. This indicated that compounds in V. destructor homogenate can have similar effects as V. destructor parasitism and that the response is not solely due to wounding. Samples from honey bees with different hemocyte concentrations were compared for the expression patterns of hemolectin (AmHml), prophenol oxidase (AmPpo), and class C scavenger receptor (AmSRC-C). Of the genes tested, only the expression of AmPpo correlated well with hemocyte counts for all the treatments, indicating that melanization is associated with those responses. Thus, the expression of AmPpo might be a suitable biomarker for hemocyte counts as part of cellular defenses against injection of buffer or mite compounds and V. destructor parasitism and perhaps other conditions involving healing and immunity.

The peripheral nervous system supports blood cell homing and survival in the Drosophila larva

PubMed Central

Makhijani, Kalpana; Alexander, Brandy; Tanaka, Tsubasa; Rulifson, Eric; Brückner, Katja

2011-01-01

Interactions of hematopoietic cells with their microenvironment control blood cell colonization, homing and hematopoiesis. Here, we introduce larval hematopoiesis as the first Drosophila model for hematopoietic colonization and the role of the peripheral nervous system (PNS) as a microenvironment in hematopoiesis. The Drosophila larval hematopoietic system is founded by differentiated hemocytes of the embryo, which colonize segmentally repeated epidermal-muscular pockets and proliferate in these locations. Importantly, we show that these resident hemocytes tightly colocalize with peripheral neurons and we demonstrate that larval hemocytes depend on the PNS as an attractive and trophic microenvironment. atonal (ato) mutant or genetically ablated larvae, which are deficient for subsets of peripheral neurons, show a progressive apoptotic decline in hemocytes and an incomplete resident hemocyte pattern, whereas supernumerary peripheral neurons induced by ectopic expression of the proneural gene scute (sc) misdirect hemocytes to these ectopic locations. This PNS-hematopoietic connection in Drosophila parallels the emerging role of the PNS in hematopoiesis and immune functions in vertebrates, and provides the basis for the systematic genetic dissection of the PNS-hematopoietic axis in the future. PMID:22071105

Colonization State Influences the Hemocyte Proteome in a Beneficial Squid–Vibrio Symbiosis*

PubMed Central

Schleicher, Tyler R.; VerBerkmoes, Nathan C.; Shah, Manesh; Nyholm, Spencer V.

2014-01-01

The squid Euprymna scolopes and the luminescent bacterium Vibrio fischeri form a highly specific beneficial light organ symbiosis. Not only does the host have to select V. fischeri from the environment, but it must also prevent subsequent colonization by non-symbiotic microorganisms. Host macrophage-like hemocytes are believed to play a role in mediating the symbiosis with V. fischeri. Previous studies have shown that the colonization state of the light organ influences the host's hemocyte response to the symbiont. To further understand the molecular mechanisms behind this process, we used two quantitative mass-spectrometry-based proteomic techniques, isobaric tags for relative and absolute quantification (iTRAQ) and label-free spectral counting, to compare and quantify the adult hemocyte proteomes from colonized (sym) and uncolonized (antibiotic-treated/cured) squid. Overall, iTRAQ allowed for the quantification of 1,024 proteins with two or more peptides. Thirty-seven unique proteins were determined to be significantly different between sym and cured hemocytes (p value < 0.05), with 20 more abundant proteins and 17 less abundant in sym hemocytes. The label-free approach resulted in 1,241 proteins that were identified in all replicates. Of 185 unique proteins present at significantly different amounts in sym hemocytes (as determined by spectral counting), 92 were more abundant and 93 were less abundant. Comparisons between iTRAQ and spectral counting revealed that 30 of the 37 proteins quantified via iTRAQ exhibited trends similar to those identified by the label-free method. Both proteomic techniques mutually identified 16 proteins that were significantly different between the two groups of hemocytes (p value < 0.05). The presence of V. fischeri in the host light organ influenced the abundance of proteins associated with the cytoskeleton, adhesion, lysosomes, proteolysis, and the innate immune response. These data provide evidence that colonization by V. fischeri alters the hemocyte proteome and reveals proteins that may be important for maintaining host–symbiont specificity. PMID:25038065

Effects of in vivo exposure of Mya arenaria to organic and inorganic mercury on phagocytic activity of hemocytes.

PubMed

Fournier, M; Pellerin, J; Clermont, Y; Morin, Y; Brousseau, P

2001-03-28

Marine bivalves are aquatic invertebrate organisms which can be used as bioindicators in environmental monitoring. In vivo effects of mercuric chloride (HgCl(2)) and methylmercury (CH(3)HgCl) on phagocytic function of Mya arenaria hemocytes were evaluated in this study. Clams were exposed to single metal in water for up to 28 days at concentrations ranging from 10(-9) to 10(-5) M. Phagocytic activity of hemocytes was determined by uptake of fluorescent microspheres and flow cytometry. All clams exposed to 10(-5) M HgCl(2) died by day 7 of exposure. The viability of hemocytes was decreased only in clams exposed to 10(-6) M HgCl(2) for 28 days. A significant decrease in phagocytic activity of hemocytes was observed in clams exposed to 10(-6) M of HgCl(2) for 28 days. A similar pattern was observed with CH(3)HgCl, but at an earlier time. Chemical analysis performed on the tissues of the animals clearly show a greater uptake of the organic form of mercury by clams. Furthermore, a clear correlation was established between body burden of mercury and effects on phagocytic activity of hemocytes. Overall, the results of this study show that both speciations of mercury inhibited phagocytic function of Mya arenaria hemocytes following in vivo exposures.

Transient expression of protein tyrosine phosphatases encoded in Cotesia plutellae bracovirus inhibits insect cellular immune responses

NASA Astrophysics Data System (ADS)

Ibrahim, Ahmed M. A.; Kim, Yonggyun

2008-01-01

Several immunosuppressive factors are associated with parasitism of an endoparasitoid wasp, Cotesia plutellae, on the diamondback moth, Plutella xylostella. C. plutellae bracovirus (CpBV) encodes a large number of putative protein tyrosine phosphatases (PTPs), which may play a role in inhibiting host cellular immunity. To address this inhibitory hypothesis of CpBV-PTPs, we performed transient expression of individual CpBV-PTPs in hemocytes of the beet armyworm, Spodoptera exigua, and analyzed their cellular immune responses. Two different forms of CpBV-PTPs were chosen and cloned into a eukaryotic expression vector under the control of the p10 promoter of baculovirus: one with the normal cysteine active site (CpBV-PTP1) and the other with a mutated active site (CpBV-PTP5). The hemocytes transfected with CpBV-PTP1 significantly increased in PTP activity compared to control hemocytes, but those with CpBV-PTP5 exhibited a significant decrease in the PTP activity. All transfected hemocytes exhibited a significant reduction in both cell spreading and encapsulation activities compared to control hemocytes. Co-transfection of CpBV-PTP1 together with its double-stranded RNA reduced the messenger RNA (mRNA) level of CpBV-PTP1 and resulted in recovery of both hemocyte behaviors. This is the first report demonstrating that the polydnaviral PTPs can manipulate PTP activity of the hemocytes to interrupt cellular immune responses.

Responses of Mytilus galloprovincialis hemocytes to environmental strains of Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio vulnificus.

PubMed

Ciacci, C; Manti, A; Canonico, B; Campana, R; Camisassi, G; Baffone, W; Canesi, L

2017-06-01

Marine bivalves are exposed to different types of bacteria in the surrounding waters, in particular of the Vibrio genus. In the hemocytes of the mussel Mytilus spp. immune responses to different vibrios have been largely characterized. However, little information is available on the hemocyte responses to human pathogenic vibrios commonly detected in coastal waters and bivalve tissues that are involved in seafood-borne diseases. In this work, functional parameters of the hemocytes from the Mediterranean mussel M. galloprovincialis were evaluated in response to in vitro challenge with different vibrios isolated from environmental samples of the Adriatic sea (Italy): V. parahaemolyticus Conero, V. alginolyticus 1513 and V. vulnificus 509. V. parahaemolyticus ATCC 43996 was used for comparison. At the 50:1 bacteria hemocyte ratio, only V. parahaemolyticus strains induced significant lysosomal membrane destabilisation. Stimulation of extracellular lysozyme release, total ROS, O 2 - and NO production were observed, although to different extents and with distinct time courses for different vibrios, V. vulnificus 509 in particular. Further comparisons between V. parahaemolyticus Conero and V. vulnificus 509 showed that only the latter induced dysregulation of the phosphorylation state of p38 MAP Kinase and apoptotic processes. The results indicate that mussel hemocytes can mount an efficient immune response towards V. parahaemolyticus and V. alginolyticus strains, whereas V. vulnificus 509 may affect the hemocyte function. This is the first report on immune responses of mussels to local environmental isolates of human pathogenic vibrios. These data reinforce the hypothesis that Mytilus hemocytes show specific responses to different vibrio species and strains. Copyright © 2017 Elsevier Ltd. All rights reserved.

Correlation of hemocyte counts with different developmental parameters during the last larval instar of the tobacco hornworm, Manduca sexta.

PubMed

Beetz, Susann; Holthusen, Traute K; Koolman, Jan; Trenczek, Tina

2008-02-01

We determined the changes in hemocyte titer and in the abundance of hemocyte types of the tobacco hornworm Manduca sexta during the fourth and fifth larval stadium and the beginning of the pupal stadium. As we analyzed the samples of individual insects at daily intervals, we were able to correlate phenotypical features, body weight, as well as total protein content and lysozyme activity in the hemolymph with the observations on hemocytes. In the course of the fifth larval stadium, the hemocyte titer decreased slightly and declined further after pupation. Using calculated values for total hemocyte numbers, females had about five times and males three times more hemocytes in the circulating population at the beginning of the wandering stage (in the middle of the fifth larval stadium) than immediately after the last larval--larval molt (from the fourth to the fifth larval stadium). This sexual difference was mainly due to an increase in the number of plasmatocytes, which was more prominent in females than in males. Granular cells were dominant in early fifth larval stadium while plasmatocytes were the most abundant cells in pupae. Oenocytoids and spherule cells disappeared during the wandering stage. Lysozyme activity in the hemolymph rose to a maximum during the wandering stage, with females having lysozyme values twice as high as those for males. These changes in lysozyme activity, however, did not correlate with the increase of total hemolymph protein titer which occurred already at the beginning of the wandering stage. We postulate that changes in hemocyte titers are under direct hormonal control, which has to be proven in future experiments. (c) 2007 Wiley-Liss, Inc.

Ocean Acidification Affects Hemocyte Physiology in the Tanner Crab (Chionoecetes bairdi)

PubMed Central

Meseck, Shannon L.; Alix, Jennifer H.; Swiney, Katherine M.; Long, W. Christopher; Wikfors, Gary H.; Foy, Robert J.

2016-01-01

We used flow cytometry to determine if there would be a difference in hematology, selected immune functions, and hemocyte pH (pHi), under two different, future ocean acidification scenarios (pH = 7.50, 7.80) compared to current conditions (pH = 8.09) for Chionoecetes bairdi, Tanner crab. Hemocytes were analyzed after adult Tanner crabs were held for two years under continuous exposure to acidified ocean water. Total counts of hemocytes did not vary among control and experimental treatments; however, there were significantly greater number of dead, circulating hemocytes in crabs held at the lowest pH treatment. Phagocytosis of fluorescent microbeads by hemocytes was greatest at the lowest pH treatment. These results suggest that hemocytes were dying, likely by apoptosis, at a rate faster than upregulated phagocytosis was able to remove moribund cells from circulation at the lowest pH. Crab hemolymph pH (pHe) averaged 8.09 and did not vary among pH treatments. There was no significant difference in internal pH (pHi) within hyalinocytes among pH treatments and the mean pHi (7.26) was lower than the mean pHe. In contrast, there were significant differences among treatments in pHi of the semi-granular+granular cells. Control crabs had the highest mean semi-granular+granular pHi compared to the lowest pH treatment. As physiological hemocyte functions changed from ambient conditions, interactions with the number of eggs in the second clutch, percentage of viable eggs, and calcium concentration in the adult crab shell was observed. This suggested that the energetic costs of responding to ocean acidification and maintaining defense mechanisms in Tanner crab may divert energy from other physiological processes, such as reproduction. PMID:26859148

Neuroglian-positive plasmatocytes of Manduca sexta and the initiation of hemocyte attachment to foreign surfaces.

PubMed

Nardi, James B; Pilas, Barbara; Bee, Charles Mark; Zhuang, Shufei; Garsha, Karl; Kanost, Michael R

2006-01-01

Observations of hemocyte aggregation on abiotic surfaces suggested that certain plasmatocytes from larvae of Manduca sexta act as foci for hemocyte aggregation. To establish how these particular plasmatocytes form initial attachments to foreign surfaces, they were cultured separately from other selected populations of hemocytes. While all circulating plasmatocytes immunolabel with anti-beta-integrin monoclonal antibody (MAb), only these larger plasmatocytes immunolabel with a MAb to the adhesion protein neuroglian. Neuroglian-negative plasmatocytes and granular cells that have been magnetically segregated from the majority of granular cells adhere to each other but fail to adhere to foreign substrata; by contrast, neuroglian-positive plasmatocytes that segregate with most granular cells adhere firmly to a substratum. Hemocytes form stable aggregates around the large, neuroglian-positive plasmatocytes. However, if neuroglian-positive plasmatocytes are separated from most granular cells, attachment of these plasmatocytes to foreign surfaces is suppressed.

Morphological and populational characteristics of hemocytes of Ornithodoros moubata nymphs during the ecdysial phase.

PubMed

Kadota, K; Walter, S; Claveria, F G; Igarashi, I; Taylor, D; Fujisaki, K

2003-11-01

The ultrastructure and characteristics of hemocytes of argasid tick species, Ornithodoros moubata, during the ecdysdial phase are herein presented. Hemocyte classes/populations characterized based on their affinity with Giemsa stain and ultrastructural differences comprised the prohemocytes, nongranular cells (Nc), eosinophilic granular cells (Ec), basophilic granular cells (Bc), and unidentified cells. Significant changes/shift in the ratio of hemocyte classes/population was apparent in ticks before and after the ecdysial phase. The granule-scant basophilic granular cells (sBc) constituted the most abundant hemocyte population in the ecdysial phase. Nymphs in ecdysis showed increases in Nc and sBc and decrease in Ec, a phenomenon that was reversed in unengorged nymphs and adults ticks. The significant increase in total Bc population in ecdysis relative to nonengorged ticks clearly point to blastogenesis of Bc taking place during the ecdysial phase and Bc's important role in the process of tissue remodeling.

The entomopathogenic fungus Nomuraea rileyi impairs cellular immunity of its host Helicoverpa armigera.

PubMed

Zhong, Ke; Liu, Zhan-Chi; Wang, Jia-Lin; Liu, Xu-Sheng

2017-09-01

In this study, we investigated the effect of the entomopathogenic fungus Nomuraea rileyi on Helicoverpa armigera cellular immune responses. Nomuraea rileyi infection had no effect on total hemocyte count (THC), but impaired hemocyte-mediated phagocytosis, nodulation, and encapsulation responses. Nomuraea rileyi infection led to a significant reduction in hemocyte spreading. An in vitro assay revealed that plasma from N. rileyi infected H. armigera larvae suppressed the spreading ability of hemocytes from naïve larvae. We infer that N. rileyi suppresses the cellular immune response of its host, possibly by secreting exogenous, cytotoxic compounds into the host's hemolymph. © 2017 Wiley Periodicals, Inc.

Neuroglian on hemocyte surfaces is involved in homophilic and heterophilic interactions of the innate immune system of Manduca sexta.

PubMed

Zhuang, Shufei; Kelo, Lisha; Nardi, James B; Kanost, Michael R

2007-01-01

Neuroglian, a member of the L1 family of cell adhesion molecules (L1-CAMs), is expressed on surfaces of granular cells and a subset of large plasmatocytes of Manduca sexta that act as foci for hemocyte aggregation during the innate immune response. Neuroglian expressed on surfaces of transfected Sf9 cells induced their homophilic aggregation, with the aggregation being abolished in the presence of recombinant immunoglobulin (Ig) domains of neuroglian. Neuroglian and its Ig domains also can interact with hemocyte-specific integrin (HS integrin) as demonstrated with an enzyme-linked immunoassay and a surface plasmon resonance (SPR) assay. Neuroglian double-stranded (ds) RNA not only depresses expression of neuroglian in hemocytes but also depresses the cell-mediated encapsulation response of these hemocytes to foreign surfaces. After injection of a monoclonal antibody (MAb 3B11) into M. sexta larvae that recognizes the Ig domains of neuroglian, the cell-mediated encapsulation response of hemocytes was likewise inhibited. The Ig domains of neuroglian are involved in both homophilic and heterophilic interactions, and subsets of these six different Ig domains may affect different functions of neuroglian.

Characterization of the Hemocytes in Larvae of Protaetia brevitarsis seulensis: Involvement of Granulocyte-Mediated Phagocytosis

PubMed Central

Cho, Saeyoull

2014-01-01

Hemocytes are key players in the immune response against pathogens in insects. However, the hemocyte types and their functions in the white-spotted flower chafers, Protaetia brevitarsis seulensis (Kolbe), are not known. In this study, we used various microscopes, molecular probes, and flow cytometric analyses to characterize the hemocytes in P. brevitarsis seulensis. The circulating hemocytes were classified based on their size, morphology, and dye-staining properties into six types, including granulocytes, plasmatocytes, oenocytoids, spherulocytes, prohemocytes, and adipohemocytes. The percentages of circulating hemocyte types were as follows: 13% granulocytes, 20% plasmatocytes, 1% oenocytoids, 5% spherulocytes, 17% prohemocytes, and 44% adipohemocytes. Next, we identified the professional phagocytes, granulocytes, which mediate encapsulation and phagocytosis of pathogens. The granulocytes were immunologically or morphologically activated and phagocytosed potentially hazardous substances in vivo. In addition, we showed that the phagocytosis by granulocytes is associated with autophagy, and that the activation of autophagy could be an efficient way to eliminate pathogens in this system. We also observed a high accumulation of autophagic vacuoles in activated granulocytes, which altered their shape and led to autophagic cell death. Finally, the granulocytes underwent mitotic division thus maintaining their number in vivo. PMID:25083702

Ultrastructural and functional characterization of circulating hemocytes from Plutella xylostella larva: cell types and their role in phagocytosis.

PubMed

Huang, Fang; Yang, Yan-yan; Shi, Min; Li, Jun-ying; Chen, Zong-qi; Chen, Fu-shou; Chen, Xue-xin

2010-12-01

The hemocytes of different types encountered in the diamondback moth Plutella xylostella larvae of each instar and the development of the differential hemocytes counts were herein presented. Hemocytes classes/populations characterized based on their affinity with fluorescent dye (acridine orange) and ultrastructural differences comprised the prohemcoytes (<10-16%), plasmatocytes (22-65%), granulocytes (25-72%), oenocytoids (<1-9%), and spherulocytes (<1%). Prohemcoytes were the smallest cells with a comparatively tremendous nucleus. Plasmatocytes and granulocytes occupied the main proportion of total cell numbers. Oenocytoids were in a most stable presence, i.e. rotund in a diameter of 10 μm and with a nucleus deviated from the central location; however, sometimes with two nuclei which were adjoining with each other. Spherulocytes were rare and only could be observed occasionally. Ultrastructural investigation revealed that hemocytes in the diamondback moth larvae were of the typical model as in the Lepidoptera insect larvae. It is interesting to find that the cell which could phagocytize bacteria in vitro was granulocyte, not the other types of hemocytes, although plasmatocyte was usually declared to participate in this reaction in various previous studies. Copyright © 2010 Elsevier Ltd. All rights reserved.

Sublethal toxicity of Roundup to immunological and molecular aspects of Biomphalaria alexandrina to Schistosoma mansoni infection.

PubMed

Mohamed, Azza H

2011-05-01

The present study was performed to elucidate the cellular mechanisms of Biomphalaria alexandrina snails hemocytes against sublethal concentration (10 mg/L) of herbicide Roundup (48% Glyphosate) and/or Schistosoma mansoni infection during 7 days of exposure. Obtained results indicated that herbicide treatment and/or infection led to significant increase (P<0.05) in total hemocytes count during exposure period. Examination of hemocytes monolayers resulted in observation of 3 morphologically different cell types, round small, hyalinocytes and spreading hemocytes. Spreading hemocytes are the dominant, more responsive and highly phagocytic cell type in all experimental groups. Moreover, the exposure to herbicide, infection or both together led to a significant increase (P<0.05) of in vitro phagocytic activity against yeast cells during 7 days of exposure. In addition, flow cytometric analysis of cell cycle and comet assay, resulted in DNA damage in B. alexandrina hemocytes exposed to herbicide and/or S. mansoni infection when compared to control group. The immunological responses as well as molecular aspects in B. alexandrina snails have been proposed as biomarkers of exposure to environmental pollutants. Copyright © 2010 Elsevier Inc. All rights reserved.

Differential cellular immune response of Galleria mellonella to Actinobacillus pleuropneumoniae.

PubMed

Arteaga Blanco, Luis Andrés; Crispim, Josicelli Souza; Fernandes, Kenner Morais; de Oliveira, Leandro Licursi; Pereira, Monalessa Fábia; Bazzolli, Denise Mara Soares; Martins, Gustavo Ferreira

2017-10-01

In the present work, we have investigate the cellular immune response of Galleria mellonella larvae against three strains of the gram-negative bacterium Actinobacillus pleuropneumoniae: low-virulence (780), high-virulence (1022) and the serotype 8 reference strain (R8). Prohemocytes, plasmatocytes, granulocytes, oenocytoids and spherulocytes were distinguished according to their size and morphology, their molecular markers and dye-staining properties and their role in the immune response. Total hemocyte count, differential hemocyte count, lysosome activity, autophagic response, cell viability and caspase-3 activation were determined in circulating hemocytes of naive and infected larvae. The presence of the autophagosome protein LC3 A/B within the circulating hemocytes of G. mellonella was dependent on and related to the infecting A. pleuropneumoniae strain and duration of infection. Hemocytes treated with the high-virulence strain expressed higher levels of LC3 A/B, whereas treatment with the low-virulence strain induced lower expression levels of this protein in the cells. Moreover, our results showed that apoptosis in circulating hemocytes of G. mellonella larvae after exposure to virulent bacterial strains occurred simultaneously with excessive cell death response induced by stress and subsequent caspase-3 activation.

Concholepas hemocyanin biosynthesis takes place in the hepatopancreas, with hemocytes being involved in its metabolism.

PubMed

Manubens, Augusto; Salazar, Fabián; Haussmann, Denise; Figueroa, Jaime; Del Campo, Miguel; Pinto, Jonathan Martínez; Huaquín, Laura; Venegas, Alejandro; Becker, María Inés

2010-12-01

Hemocyanins are copper-containing glycoproteins in some molluscs and arthropods, and their best-known function is O(2) transport. We studied the site of their biosynthesis in the gastropod Concholepas concholepas by using immunological and molecular genetic approaches. We performed immunohistochemical staining of various organs, including the mantle, branchia, and hepatopancreas, and detected C. concholepas hemocyanin (CCH) molecules in circulating and tissue-associated hemocytes by electron microscopy. To characterize the hemocytes, we purified them from hemolymph. We identified three types of granular cells. The most abundant type was a phagocyte-like cell with small cytoplasmic granules. The second type contained large electron-dense granules. The third type had vacuoles containing hemocyanin molecules suggesting that synthesis or catabolism occurred inside these cells. Our failure to detect cch-mRNA in hemocytes by reverse transcription with the polymerase chain reaction (RT-PCR) led us to propose that hemocytes instead played a role in CCH metabolism. This hypothesis was supported by colloidal gold staining showing hemocyanin molecules in electron-dense granules inside hemocytes. RT-PCR analysis, complemented by in situ hybridization analyses with single-stranded antisense RNAs as specific probes, demonstrated the presence of cch-mRNA in the hepatopancreas; this was consistent with the specific hybridization signal and confirmed the hepatopancreas as the site of CCH synthesis. Finally, we investigated the possibility that CCH catabolism in hemocytes was involved in the host immune response and in the generation of secondary metabolites such as antimicrobial peptides and phenoloxidase.

Immune Defenses of the Invasive Apple Snail Pomacea canaliculata (Caenogastropoda, Ampullariidae): Phagocytic Hemocytes in the Circulation and the Kidney

PubMed Central

Vega, Israel A.; Castro-Vazquez, Alfredo

2015-01-01

Hemocytes in the circulation and kidney islets, as well as their phagocytic responses to microorganisms and fluorescent beads, have been studied in Pomacea canaliculata, using flow cytometry, light microscopy (including confocal laser scanning microscopy) and transmission electron microscopy (TEM). Three circulating hemocyte types (hyalinocytes, agranulocytes and granulocytes) were distinguished by phase contrast microscopy of living cells and after light and electron microscopy of fixed material. Also, three different populations of circulating hemocytes were separated by flow cytometry, which corresponded to the three hemocyte types. Hyalinocytes showed a low nucleus/cytoplasm ratio, and no apparent granules in stained material, but showed granules of moderate electron density under TEM (L granules) and at least some L granules appear acidic when labeled with LysoTracker Red. Both phagocytic and non-phagocytic hyalinocytes lose most (if not all) L granules when exposed to microorganisms in vitro. The phagosomes formed differed whether hyalinocytes were exposed to yeasts or to Gram positive or Gram negative bacteria. Agranulocytes showed a large nucleus/cytoplasm ratio and few or no granules. Granulocytes showed a low nucleus/cytoplasm ratio and numerous eosinophilic granules after staining. These granules are electron dense and rod-shaped under TEM (R granules). Granulocytes may show merging of R granules into gigantic ones, particularly when exposed to microorganisms. Fluorescent bead exposure of sorted hemocytes showed phagocytic activity in hyalinocytes, agranulocytes and granulocytes, but the phagocytic index was significantly higher in hyalinocytes. Extensive hemocyte aggregates ('islets') occupy most renal hemocoelic spaces and hyalinocyte-like cells are the most frequent component in them. Presumptive glycogen deposits were observed in most hyalinocytes in renal islets (they also occur in the circulation but less frequently) and may mean that hyalinocytes participate in the storage and circulation of this compound. Injection of microorganisms in the foot results in phagocytosis by hemocytes in the islets, and the different phagosomes formed are similar to those in circulating hyalinocytes. Dispersed hemocytes were obtained after kidney collagenase digestion and cell sorting, and they were able to phagocytize fluorescent beads. A role for the kidney as an immune barrier is proposed for this snail. PMID:25893243

Immune Defenses of the Invasive Apple Snail Pomacea canaliculata (Caenogastropoda, Ampullariidae): Phagocytic Hemocytes in the Circulation and the Kidney.

PubMed

Cueto, Juan A; Rodriguez, Cristian; Vega, Israel A; Castro-Vazquez, Alfredo

2015-01-01

Hemocytes in the circulation and kidney islets, as well as their phagocytic responses to microorganisms and fluorescent beads, have been studied in Pomacea canaliculata, using flow cytometry, light microscopy (including confocal laser scanning microscopy) and transmission electron microscopy (TEM). Three circulating hemocyte types (hyalinocytes, agranulocytes and granulocytes) were distinguished by phase contrast microscopy of living cells and after light and electron microscopy of fixed material. Also, three different populations of circulating hemocytes were separated by flow cytometry, which corresponded to the three hemocyte types. Hyalinocytes showed a low nucleus/cytoplasm ratio, and no apparent granules in stained material, but showed granules of moderate electron density under TEM (L granules) and at least some L granules appear acidic when labeled with LysoTracker Red. Both phagocytic and non-phagocytic hyalinocytes lose most (if not all) L granules when exposed to microorganisms in vitro. The phagosomes formed differed whether hyalinocytes were exposed to yeasts or to Gram positive or Gram negative bacteria. Agranulocytes showed a large nucleus/cytoplasm ratio and few or no granules. Granulocytes showed a low nucleus/cytoplasm ratio and numerous eosinophilic granules after staining. These granules are electron dense and rod-shaped under TEM (R granules). Granulocytes may show merging of R granules into gigantic ones, particularly when exposed to microorganisms. Fluorescent bead exposure of sorted hemocytes showed phagocytic activity in hyalinocytes, agranulocytes and granulocytes, but the phagocytic index was significantly higher in hyalinocytes. Extensive hemocyte aggregates ('islets') occupy most renal hemocoelic spaces and hyalinocyte-like cells are the most frequent component in them. Presumptive glycogen deposits were observed in most hyalinocytes in renal islets (they also occur in the circulation but less frequently) and may mean that hyalinocytes participate in the storage and circulation of this compound. Injection of microorganisms in the foot results in phagocytosis by hemocytes in the islets, and the different phagosomes formed are similar to those in circulating hyalinocytes. Dispersed hemocytes were obtained after kidney collagenase digestion and cell sorting, and they were able to phagocytize fluorescent beads. A role for the kidney as an immune barrier is proposed for this snail.

Hemocyte responses of Manila clams, Ruditapes philippinarum, with varying parasite, Perkinsus olseni, severity to toxic-algal exposures.

PubMed

Hégaret, Hélène; da Silva, Patricia Mirella; Wikfors, Gary H; Lambert, Christophe; De Bettignies, Thibaut; Shumway, Sandra E; Soudant, Philippe

2007-10-30

This study assessed the possible combined effects of harmful algae and parasite infection on hemocyte and hemolymph parameters of a bivalve mollusc. Manila clams Ruditapes philippinarum, were exposed for 1 week, under controlled laboratory conditions, to bloom concentrations of two cultured dinoflagellates: Karenia selliformis, and Karenia mikimotoi, with demonstrated, sub-lethal, pathological effects upon these bivalves. Each dinoflagellate treatment was added to a basal diet of Chaetoceros neogracile; controls consisted of clams fed only C. neogracile. Hemocyte characteristics measured with flow-cytometric analyses, and agglutination titer, condition index, and prevalence and intensity of Perkinsus olseni, were assessed for individual clams before and after 3 and 6 days of microalgal exposure. Multifactor analysis of variance tests were conducted to determine possible effects of the harmful algae, time of exposure, and P. olseni intensity, as well as interactions between these three factors, upon each physiological variable measured. There was no relationship between P. olseni intensity and hemolymph measures. Both Karenia species, however, had a significant effect upon hemocyte profiles of the clams, and this effect was dependent upon duration of exposure; 3 days of exposure to the dinoflagellates generally was sufficient to resolve the effects on the clams. K. selliformis had a stronger effect than K. mikimotoi, which was intermediate between K. selliformis and clams fed the non-toxic control, C. neogracile. Total hemocyte counts increased in clams exposed to the harmful algae, while the percentage of dead hemocytes, as well as hemocyte size and complexity, decreased. Furthermore, these immunomodulating effects of K. selliformis were significantly more extreme in clams with a high parasite burden, compared with lightly infected clams. This report is, to our knowledge, the first study assessing the combined effects of harmful algae and parasite infection on a physiological function (hemocyte and hemolymph parameters) of a bivalve mollusc. These findings demonstrate that clams maintain hemocyte function when infected with P. olseni, that the clam immune system responds to harmful or toxic algal exposure, and that this response is modified by parasite infection.

Unique phagocytic properties of hemocytes of Pacific oyster Crassostrea gigas against yeast and yeast cell-wall derivatives.

PubMed

Takahashi, Keisuke G; Izumi-Nakajima, Nakako; Mori, Katsuyoshi

2017-11-01

For a marine bivalve mollusk such as Pacific oyster Crassostrea gigas, the elimination of foreign particles via hemocyte phagocytosis plays an important role in host defense mechanisms. The hemocytes of C. gigas have a high phagocytic ability for baker's yeast (Saccharomyces cerevisiae) and its cell-wall product zymosan. C. gigas hemocytes might phagocytose yeast cells after binding to polysaccharides on the cell-wall surface, but it is unknown how and what kinds of polysaccharide molecules are recognized. We conducted experiments to determine differences in the phagocytic ability of C. gigas hemocytes against heat-killed yeast (HK yeast), zymosan and zymocel, which are similarly sized and shaped but differ in the polysaccharide composition of their particle surface. We found that both the agranulocytes and granulocytes exerted strong phagocytic ability on all tested particles. The phagocytic index (PI) of granulocytes for zymosan was 9.4 ± 1.7, which significantly differed with that for HK yeast and zymocel (P < 0.05). To evaluate the PI for the three types of particles, and especially to understand the outcome of the much higher PI for zymosan, PI was gauged in increments of 5 (1-5, 6-10, 11-15, and ≥16), and the phagocytic frequencies were compared according to these increments. The results show that a markedly high PI of ≥16 was exhibited by 18.1% of granulocytes for zymosan, significantly higher than 1.7% and 3.9% shown for HK yeast and zymocel, respectively (P < 0.05). These findings indicate that the relatively high PI for zymosan could not be attributed to a situation wherein all phagocytic hemocytes shared a high mean PI, but rather to the ability of some hemocytes to phagocytose a larger portion of zymosan. To determine whether the phagocytosis of these respective particles depended on the recognition of specific polysaccharide receptors on the hemocyte surface, C. gigas hemocytes were pretreated with soluble α-mannan or β-laminarin and then allowed to phagocytose the three types of the particles. The percentage of phagocytic cells of β-laminarin-treated granulocytes decreased significantly for zymosan and zymocel, but not for yeast. These results suggest that C. gigas might possess at least two types of hemocytes, and that one type of the hemocytes (granulocytes) is more active for phagocytosis. The granulocytes were found to have multiple subtypes with different phagocytic abilities and multiple phagocytic receptors. Some of the granulocyte subtypes revealed a much stronger phagocytic ability, depending on the presence of β-glucan receptors for phagocytosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Drosophila hemocyte migration: an in vivo assay for directional cell migration.

PubMed

Moreira, Carolina G A; Regan, Jennifer C; Zaidman-Rémy, Anna; Jacinto, Antonio; Prag, Soren

2011-01-01

This protocol describes an in vivo assay for random and directed hemocyte migration in Drosophila. Drosophila is becoming an increasingly powerful model system for in vivo cell migration analysis, combining unique genetic tools with translucency of the embryo and pupa, which allows direct imaging and traceability of different cell types. In the assay we present here, we make use of the hemocyte response to epithelium wounding to experimentally induce a transition from random to directed migration. Time-lapse confocal microscopy of hemocyte migration in untreated conditions provides a random cell migration assay that allows identification of molecular mechanisms involved in this complex process. Upon laser-induced wounding of the thorax epithelium, a rapid chemotactic response changes hemocyte migratory behavior into a directed migration toward the wound site. This protocol provides a direct comparison of cells during both types of migration in vivo, and combined with recently developed resources such as transgenic RNAi, is ideal for forward genetic screens.

Immunolocalization of clavanins in Styela clava hemocytes.

PubMed

Menzel, Lorenzo P; Lee, In Hee; Sjostrand, Birgitta; Lehrer, Robert I

2002-07-01

Antimicrobial peptides play an important role in innate host defenses against infection. Clavanins are histidine-rich, amidated, 23-residue alpha-helical antimicrobial peptides that were isolated from a mixed population of Styela clava hemocytes. To learn which types of hemocytes contained clavanins, we raised a polyclonal antibody that recognized five different clavanins, and used it to localize these peptides by light and electron microscopy. Clavanins were present in the cytoplasmic granules and/or cytoplasm of five different types of granulocytes and they also occurred throughout the cytoplasm of macrophages. The orange G component of Mallory's trichrome stain had a high affinity for clavanins, and for the cytoplasmic granules of S. clava's hemocytes. Semiquantitative analysis of acid urea-PAGE gels suggested that clavanins and styelins comprised between 10 and 20% of the total cellular protein of eosinophilic granulocytes. Orange G and the century-old trichrome stain may provide simple screening tools for identifying cells that contain large amounts of antimicrobial peptides in mixed hemocyte populations.

Surface Interactions between Escherichia coli and Hemocytes of the Mediterranean Mussel Mytilus galloprovincialis Lam. Leading to Efficient Bacterial Clearance

PubMed Central

Canesi, Laura; Pruzzo, Carla; Tarsi, Renato; Gallo, Gabriella

2001-01-01

The role of type 1 fimbriae in the interactions between Escherichia coli and Mytilus galloprovincialis Lam. hemocytes was evaluated. The association of fimbriated strain MG155 with hemocyte monolayers at 18°C was 1.5- and 3- to 4-fold greater than the association of unfimbriated mutant AAEC072 in artificial seawater and in hemolymph serum, respectively. Such differences were apparently due to different adhesive properties since MG155 adhered more efficiently than AAEC072 when hemocytes were incubated at 4°C to inhibit the internalization process. Hemolymph serum increased both association and adherence of MG155 two- to threefold but did not affect association and adherence of AAEC072. MG155 was also 1.5- to 1.7-fold more sensitive to killing by hemocytes than AAEC072, as evaluated by the number of culturable bacteria after 60 and 120 min of incubation. The role of type 1 fimbriae in MG155 interactions with hemocytes was confirmed by the inhibitory effect of d-mannose. In in vivo experiments MG155 cells were cleared from circulating hemolymph more rapidly than AAEC072 cells were cleared. These results confirm that surface properties are crucial in influencing bacterial persistence and survival within mussel hemolymph. PMID:11133482

The granulocytes are the main immunocompetent hemocytes in Crassostrea gigas.

PubMed

Wang, Weilin; Li, Meijia; Wang, Lingling; Chen, Hao; Liu, Zhaoqun; Jia, Zhihao; Qiu, Limei; Song, Linsheng

2017-02-01

Hemocytes comprise diverse cell types with morphological and functional heterogeneity and play indispensable roles in immunological homeostasis of invertebrates. The morphological classification of different hemocytes in mollusk has been studied since the 1970's, yet the involvement of the different sub-populations in immune functions is far from clear. In the present study, three types of hemocytes were morphologically identified and separated as agranulocytes, semi-granulocytes and granulocytes by flow cytometry and Percoll ® density gradient centrifugation. The granulocytes were characterized functionally as the main phagocytic and encapsulating population, while semi-granulocytes and agranulocytes exhibited low or no such capacities, respectively. Meanwhile, the lysosome activity and the productions of ROS and NO were all mainly concentrated in granulocytes under both normal and immune-activated situations. Further, the mRNA transcripts of some immune related genes, including CgTLR, CgClathrin, CgATPeV, CgLysozyme, CgDefensin and CgIL-17, were mainly expressed in granulocytes, lower in semi-granulocytes and agranulocytes. These results collectively suggested that the granulocytes were the main immunocompetent hemocytes in oyster C. gigas, and a differentiation relationship among these three sub-population hemocytes was inferred based on the gradual changes in morphological, functional and molecular features. Copyright © 2016 Elsevier Ltd. All rights reserved.

Two hemocyte lineages exist in silkworm larval hematopoietic organ.

PubMed

Nakahara, Yuichi; Kanamori, Yasushi; Kiuchi, Makoto; Kamimura, Manabu

2010-07-28

Insects have multiple hemocyte morphotypes with different functions as do vertebrates, however, their hematopoietic lineages are largely unexplored with the exception of Drosophila melanogaster. To study the hematopoietic lineage of the silkworm, Bombyx mori, we investigated in vivo and in vitro differentiation of hemocyte precursors in the hematopoietic organ (HPO) into the four mature hemocyte subsets, namely, plasmatocytes, granulocytes, oenocytoids, and spherulocytes. Five days after implantation of enzymatically-dispersed HPO cells from a GFP-expressing transgenic line into the hemocoel of normal larvae, differentiation into plasmatocytes, granulocytes and oenocytoids, but not spherulocytes, was observed. When the HPO cells were cultured in vitro, plasmatocytes appeared rapidly, and oenocytoids possessing prophenol oxidase activity appeared several days later. HPO cells were also able to differentiate into a small number of granulocytes, but not into spherulocytes. When functionally mature plasmatocytes were cultured in vitro, oenocytoids were observed 10 days later. These results suggest that the hemocyte precursors in HPO first differentiate into plasmatocytes, which further change into oenocytoids. From these results, we propose that B. mori hemocytes can be divided into two major lineages, a granulocyte lineage and a plasmatocyte-oenocytoid lineage. The origins of the spherulocytes could not be determined in this study. We construct a model for the hematopoietic lineages at the larval stage of B. mori.

Differences in the number of hemocytes in the snail host Biomphalaria tenagophila, resistant and susceptible to Schistosoma mansoni infection.

PubMed

Oliveira, A L D; Levada, P M; Zanotti-Magalhaes, E M; Magalhães, L A; Ribeiro-Paes, J T

2010-12-21

The relationships between schistosomiasis and its intermediate host, mollusks of the genus Biomphalaria, have been a concern for decades. It is known that the vector mollusk shows different susceptibility against parasite infection, whose occurrence depends on the interaction between the forms of trematode larvae and the host defense cells. These cells are called amebocytes or hemocytes and are responsible for the recognition of foreign bodies and for phagocytosis and cytotoxic reactions. The defense cells mediate the modulation of the resistant and susceptible phenotypes of the mollusk. Two main types of hemocytes are found in the Biomphalaria hemolymph: the granulocytes and the hyalinocytes. We studied the variation in the number (kinetics) of hemocytes for 24 h after exposing the parasite to genetically selected and non-selected strains of Biomphalaria tenagophila, susceptible or not to infection by Schistosoma mansoni. The differences were analyzed referred to the variations in the number of hemocytes in mollusks susceptible or not to infection by S. mansoni. The hemolymph of the selected and non-selected snails was collected, and hemocytes were counted using a Neubauer chamber at six designated periods: 0 h (control, non-exposed individuals), 2 h, 6 h, 12 h, 18 h and, 24 h after parasite exposure. Samples of hemolymph of five selected mollusks and five non-selected mollusks were separately used at each counting time. There was a significant variation in the number of hemocytes between the strains, which indicates that defense cells have different behaviors in resistant and susceptible mollusks.

Morula-like cells in photo-symbiotic clams harboring zooxanthellae.

PubMed

Nakayama, K; Nishijima, M; Maruyama, T

1998-06-01

Symbiosis is observed between zooxanthellae, symbiotic dinoflagellates, and giant clams and related clams which belong to the families Tridacnidae and Cardiidae. We have previously shown that a photo-symbiotic clam Tridacna crocea has three types of hemocytes, the eosinophilic granular hemocyte with phagocytic activity, the agranular cell with electron lucent granules, and the morula-like cell with large (ca. 2 mum in diameter) colorless granules. The function of the morula-like cell is not clear, but it has not been reported in any other bivalves except photo-symbiotic clams T. crocea and Tridacna maxima. In order to clarify whether it is specific to photo-symbiotic clams or not, we studied hemocytes in the photo-symbiotic clams Tridacna derasa (Tridacnidae), Hippopus hippopus (Tridacnidae) and Corculum cardissa (Cardiidae), and a closely related non-symbiotic clam Fulvia mutica (Cardiidae). The eosinophilic granular hemocytes and the agranular cells were found in all of the clams examined. However, the morula-like cells which were packed with many large electron dense granules (ca. 2 mum in diameter), were observed only in the photo-symbiotic clams. In F. mutica, a closely related non-symbiotic clam, this type of hemocyte was not found. Instead a hemocyte with vacuoles and a few large granules containing peroxidase activity was observed. The large granules of F. mutica varied in size from ca. 1-9 mum in diameter. Present data suggests that the presence of morula-like cells is restricted to photo-symbiotic clams and that the hemocytes associated with the morula-like cells may have some functional relationship to symbiosis with zooxanthellae.

Differential sensitivity to cadmium of immunomarkers measured in hemocyte subpopulations of zebra mussel Dreissena polymorpha.

PubMed

Evariste, Lauris; Rioult, Damien; Brousseau, Pauline; Geffard, Alain; David, Elise; Auffret, Michel; Fournier, Michel; Betoulle, Stéphane

2017-03-01

Increasing discharge of industrial wastes into the environment results in pollution transfer towards hydrosystems. These activities release heavy metals such as cadmium, known as persistent pollutant that is accumulated by molluscs and exercise immunotoxicological effects. Among molluscs, the zebra mussel, Dreissena polymorpha constitutes a suitable support for freshwater ecotoxicological studies. In molluscs, homeostasis maintain is ensured in part by hemocytes that are composed of several cell populations involved in multiple physiological processes such as cell-mediated immune response or metal metabolism. Thus, hemocytes constitute a target of concern to study adverse effects of heavy metals. The objectives of this work were to determine whether immune-related endpoints assessed were of different sensitivity to cadmium and whether hemocyte functionalities were differentially affected depending on hemocyte subpopulation considered. Hemocytes were exposed ex vivo to concentrations of cadmium ranging from 10 -6 M to 10 -3 M for 21h prior flow cytometric analysis of cellular markers. Measured parameters (viability, phagocytosis, oxidative activity, lysosomal content) decreased in a dose-dependent manner with sensitivity differences depending on endpoint and cell type considered. Our results indicated that phagocytosis related endpoints were the most sensitive studied mechanisms to cadmium compared to other markers with EC 50 of 3.71±0.53×10 -4 M for phagocytic activity and 2.79±0.19×10 -4 M considering mean number of beads per phagocytic cell. Lysosomal content of granulocytes was less affected compared to other cell types, indicating lower sensitivity to cadmium. This suggests that granulocyte population is greatly involved in metal metabolism. Mitochondrial activity was reduced only in blast-like hemocytes that are considered to be cell precursors. Impairment of these cell functionalities may potentially compromise functions ensured by differentiated cells. We concluded that analysis of hemocyte activities should be performed at sub-population scale for more accurate results in ecotoxicological studies. Copyright © 2016 Elsevier Inc. All rights reserved.

Jack bean (Canavalia ensiformis) urease induces eicosanoid-modulated hemocyte aggregation in the Chagas' disease vector Rhodnius prolixus.

PubMed

Defferrari, M S; da Silva, R; Orchard, I; Carlini, C R

2014-05-01

Ureases are multifunctional proteins that display biological activities independently of their enzymatic function, such as induction of exocytosis and insecticidal effects. Rhodnius prolixus, a major vector of Chagas' disease, is a model for studies on the entomotoxicity of jack bean urease (JBU). We have previously shown that JBU induces the production of eicosanoids in isolated tissues of R. prolixus. In insects, the immune response comprises cellular and humoral reactions, and is centrally modulated by eicosanoids. Cyclooxygenase products signal immunity in insects, mainly cellular reactions, such as hemocyte aggregation. In searching for a link between JBU's toxic effects and immune reactions in insects, we have studied the effects of this toxin on R. prolixus hemocytes. JBU triggers aggregation of hemocytes after injection into the hemocoel and when applied to isolated cells. On in vitro assays, the eicosanoid synthesis inhibitors dexamethasone (phospholipase A2 indirect inhibitor) and indomethacin (cyclooxygenase inhibitor) counteracted JBU's effect, indicating that eicosanoids, more specifically cyclooxygenase products, are likely to mediate the aggregation response. Contrarily, the inhibitors esculetin and baicalein were inactive, suggesting that lipoxygenase products are not involved in JBU's effect. Extracellular calcium was also necessary for JBU's effect, in agreement to other cell models responsive to ureases. A progressive darkening of the medium of JBU-treated hemocytes was observed, suggestive of a humoral response. JBU was immunolocalized in the cultured cells upon treatment along with cytoskeleton damage. The highest concentration of JBU tested on cultured cells also led to nuclei aggregation of adherent hemocytes. This is the first time urease has been shown to affect insect hemocytes, contributing to our understanding of the entomotoxic mechanisms of action of this protein. Copyright © 2014 Elsevier Ltd. All rights reserved.

The Galectin CvGal1 from the Eastern Oyster (Crassostrea virginica) Binds to Blood Group A Oligosaccharides on the Hemocyte Surface*

PubMed Central

Feng, Chiguang; Ghosh, Anita; Amin, Mohammed N.; Giomarelli, Barbara; Shridhar, Surekha; Banerjee, Aditi; Fernández-Robledo, José A.; Bianchet, Mario A.; Wang, Lai-Xi; Wilson, Iain B. H.; Vasta, Gerardo R.

2013-01-01

The galectin CvGal1 from the eastern oyster (Crassostrea virginica), which possesses four tandemly arrayed carbohydrate recognition domains, was previously shown to display stronger binding to galactosamine and N-acetylgalactosamine relative to d-galactose. CvGal1 expressed by phagocytic cells is “hijacked” by the parasite Perkinsus marinus to enter the host, where it proliferates and causes systemic infection and death. In this study, a detailed glycan array analysis revealed that CvGal1 preferentially recognizes type 2 blood group A oligosaccharides. Homology modeling of the protein and its oligosaccharide ligands supported this preference over type 1 blood group A and B oligosaccharides. The CvGal ligand models were further validated by binding, inhibition, and competitive binding studies of CvGal1 and ABH-specific monoclonal antibodies with intact and deglycosylated glycoproteins, hemocyte extracts, and intact hemocytes and by surface plasmon resonance analysis. A parallel glycomic study carried out on oyster hemocytes (Kurz, S., Jin, C., Hykollari, A., Gregorich, D., Giomarelli, B., Vasta, G. R., Wilson, I. B. H., and Paschinger, K. (2013) J. Biol. Chem. 288,) determined the structures of oligosaccharides recognized by CvGal1. Proteomic analysis of the hemocyte glycoproteins identified β-integrin and dominin as CvGal1 “self”-ligands. Despite strong CvGal1 binding to P. marinus trophozoites, no binding of ABH blood group antibodies was observed. Thus, parasite glycans structurally distinct from the blood group A oligosaccharides on the hemocyte surface may function as potentially effective ligands for CvGal1. We hypothesize that carbohydrate-based mimicry resulting from the host/parasite co-evolution facilitates CvGal1-mediated cross-linking to β-integrin, located on the hemocyte surface, leading to cell activation, phagocytosis, and host infection. PMID:23824193

A cytokine-like factor astakine accelerates the hemocyte production in Pacific oyster Crassostrea gigas.

PubMed

Li, Yiqun; Jiang, Shuai; Li, Meijia; Xin, Lusheng; Wang, Lingling; Wang, Hao; Qiu, Limei; Song, Linsheng

2016-02-01

Astakine has been reported to be a hematopoietic growth factor of prokineticin homolog firstly found in arthropods freshwater crayfish Pacifastacus leniusculus. In the present study, an astakine homologous gene was identified from Pacific oyster Crassostrea gigas (designated CgAstakine). The full length cDNA of CgAstakine encoded a polypeptide of 103 amino acids containing a prokineticin (PK) domain homologous to that in astakine from freshwater crayfish P. leniusculus. The deduced amino acid sequence of CgAstakine shared higher similarity with those of other invertebrate astakines than prokineticins from vertebrates. The mRNA of CgAstakine was highly expressed in hepatopancreas and adductor muscle of oyster, while the CgAstakine protein was mainly distributed in hepatopancreas, gill and hemocytes. The mRNA expression of CgAstakine in hemocytes was significantly increased (p < 0.01) and maintained at a high level from 3 h to 9 h after Vibrio anguillarum challenge. After the oyster hemocytes were incubated with 5 μg/mL recombinant CgAstakine protein (rCgAstakine) for 24 h in vitro, the proliferation of hemocytes was significantly increased to 1.89 fold of that in control group (p < 0.05). Moreover, the total count of oyster hemocytes was significantly upregulated (2.45 fold of that in control group, p < 0.05) at 12 h after the oysters were received an injection of rCgAstakine (0.5 μg/g). These results collectively indicated that CgAstakine could modulate the hemocytes proliferation both in vitro and in vivo, and probably involved in the hematopoietic process fighting against the invasion of foreign pathogens. Copyright © 2015 Elsevier Ltd. All rights reserved.

Recognition between symbiotic Vibrio fischeri and the hemocytes of Euprymna scolopes

PubMed Central

Nyholm, Spencer V.; Stewart, Jennifer J.; Ruby, Edward G.; McFall-Ngai, Margaret J.

2008-01-01

Summary The light-organ crypts of the squid Euprymna scolopes permit colonization exclusively by the luminous bacterium Vibrio fischeri. Because the crypt interior remains in contact with seawater, the squid must not only foster the specific symbiosis but also continue to exclude other bacteria. Investigation of the role of the innate immune system in these processes revealed that macrophage-like hemocytes isolated from E. scolopes recognized and phagocytosed V. fischeri less than other closely related bacterial species common to the host’s environment. Interestingly, phagocytes isolated from hosts that had been cured of their symbionts bound five-times more V. fischeri cells than those from uncured hosts. No such change in the ability to bind other species of bacteria was observed, suggesting that the host adapts specifically to V. fischeri. Deletion of the gene encoding OmpU, the major outer membrane protein of V. fischeri, increased binding by hemocytes from uncured animals to the level observed for hemocytes from cured animals. Co-incubation with wild-type V. fischeri reduced this binding, suggesting they produce a factor that complements the mutant’s defect. Analyses of the phagocytosis of bound cells by fluorescence-activated cell sorting (FACS) indicated that, once binding to hemocytes had occurred, V. fischeri cells are phagocytosed as effectively as other bacteria. Thus, discrimination by this component of the squid immune system occurs at the level of hemocyte binding, and this response: (i) is modified by previous exposure to the symbiont and, (ii) relies on outer membrane and/or secreted components of the symbionts. These data suggest that regulation of host hemocyte binding by the symbiont may be one of many factors that contribute to specificity in this association. PMID:19196278

Genotoxic and Cytotoxic Effects on the Immune Cells of the Freshwater Bivalve Dreissena polymorpha Exposed to the Environmental Neurotoxin BMAA

PubMed Central

Milliote, Nadia; Bonnard, Marc; Palos-Ladeiro, Mélissa; Rioult, Damien; Bonnard, Isabelle; Bastien, Fanny; Geffard, Alain

2018-01-01

The environmental neurotoxin β-N-Methylamino-l-alanine (BMAA) has been pointed out to be involved in human neurodegenerative diseases. This molecule is known to be bioaccumulated by bivalves. However, little data about its toxic effects on freshwater mussels is available, particularly on the hemolymphatic compartment and its hemocyte cells involved in various physiological processes such as immune defenses, digestion and excretion, tissue repair, and shell production. Here we exposed Dreissena polymorpha to dissolved BMAA, at the environmental concentration of 7.5 µg of /mussel/3 days, during 21 days followed by 14 days of depuration in clear water, with the objective of assessing the BMAA presence in the hemolymphatic compartment, as well as the impact of the hemocyte cells in terms of potential cytotoxicity, immunotoxicity, and genotoxiciy. Data showed that hemocytes were in contact with BMAA. The presence of BMAA in hemolymph did not induce significant effect on hemocytes phagocytosis activity. However, significant DNA damage on hemocytes occurred during the first week (days 3 and 8) of BMAA exposure, followed by an increase of hemocyte mortality after 2 weeks of exposure. Those effects might be an indirect consequence of the BMAA-induced oxidative stress in cells. However, DNA strand breaks and mortality did not persist during the entire exposure, despite the BMAA persistence in the hemolymph, suggesting potential induction of some DNA-repair mechanisms. PMID:29494483

Hemocytes and Plasma of the Eastern Oyster (Crassostrea virginica) Display a Diverse Repertoire of Sulfated and Blood Group A-modified N-Glycans*

PubMed Central

Kurz, Simone; Jin, Chunsheng; Hykollari, Alba; Gregorich, Daniel; Giomarelli, Barbara; Vasta, Gerardo R.; Wilson, Iain B. H.; Paschinger, Katharina

2013-01-01

The eastern oyster (Crassostrea virginica) has become a useful model system for glycan-dependent host-parasite interactions due to the hijacking of the oyster galectin CvGal1 for host entry by the protozoan parasite Perkinsus marinus, the causative agent of Dermo disease. In this study, we examined the N-glycans of both the hemocytes, which via CvGal1 are the target of the parasite, and the plasma of the oyster. In combination with HPLC fractionation, exoglycosidase digestion, and fragmentation of the glycans, mass spectrometry revealed that the major N-glycans of plasma are simple hybrid structures, sometimes methylated and core α1,6-fucosylated, with terminal β1,3-linked galactose; a remarkable high degree of sulfation of such glycans was observed. Hemocytes express a larger range of glycans, including core-difucosylated paucimannosidic forms, whereas bi- and triantennary glycans were found in both sources, including structures carrying sulfated and methylated variants of the histo-blood group A epitope. The primary features of the oyster whole hemocyte N-glycome were also found in dominin, the major plasma glycoprotein, which had also been identified as a CvGal1 glycoprotein ligand associated with hemocytes. The occurrence of terminal blood group moieties on oyster dominin and on hemocyte surfaces can account in part for their affinity for the endogenous CvGal1. PMID:23824194

Two Hemocyte Lineages Exist in Silkworm Larval Hematopoietic Organ

PubMed Central

Nakahara, Yuichi; Kanamori, Yasushi; Kiuchi, Makoto; Kamimura, Manabu

2010-01-01

Background Insects have multiple hemocyte morphotypes with different functions as do vertebrates, however, their hematopoietic lineages are largely unexplored with the exception of Drosophila melanogaster. Methodology/Principal Findings To study the hematopoietic lineage of the silkworm, Bombyx mori, we investigated in vivo and in vitro differentiation of hemocyte precursors in the hematopoietic organ (HPO) into the four mature hemocyte subsets, namely, plasmatocytes, granulocytes, oenocytoids, and spherulocytes. Five days after implantation of enzymatically-dispersed HPO cells from a GFP-expressing transgenic line into the hemocoel of normal larvae, differentiation into plasmatocytes, granulocytes and oenocytoids, but not spherulocytes, was observed. When the HPO cells were cultured in vitro, plasmatocytes appeared rapidly, and oenocytoids possessing prophenol oxidase activity appeared several days later. HPO cells were also able to differentiate into a small number of granulocytes, but not into spherulocytes. When functionally mature plasmatocytes were cultured in vitro, oenocytoids were observed 10 days later. These results suggest that the hemocyte precursors in HPO first differentiate into plasmatocytes, which further change into oenocytoids. Conclusions/Significance From these results, we propose that B. mori hemocytes can be divided into two major lineages, a granulocyte lineage and a plasmatocyte-oenocytoid lineage. The origins of the spherulocytes could not be determined in this study. We construct a model for the hematopoietic lineages at the larval stage of B. mori. PMID:20676370

Exposure to the Neurotoxic Dinoflagellate, Alexandrium catenella, Induces Apoptosis of the Hemocytes of the Oyster, Crassostrea gigas

PubMed Central

Medhioub, Walid; Ramondenc, Simon; Vanhove, Audrey Sophie; Vergnes, Agnes; Masseret, Estelle; Savar, Veronique; Amzil, Zouher; Laabir, Mohamed; Rolland, Jean Luc

2013-01-01

This study assessed the apoptotic process occurring in the hemocytes of the Pacific oyster, Crassostrea gigas, exposed to Alexandrium catenella, a paralytic shellfish toxins (PSTs) producer. Oysters were experimentally exposed during 48 h to the toxic algae. PSTs accumulation, the expression of 12 key apoptotic-related genes, as well as the variation of the number of hemocytes in apoptosis was measured at time intervals during the experiment. Results show a significant increase of the number of hemocytes in apoptosis after 29 h of exposure. Two pro-apoptotic genes (Bax and Bax-like) implicated in the mitochondrial pathway were significantly upregulated at 21 h followed by the overexpression of two caspase executor genes (caspase-3 and caspase-7) at 29 h, suggesting that the intrinsic pathway was activated. No modulation of the expression of genes implicated in the cell signaling Fas-Associated protein with Death Domain (FADD) and initiation-phase (caspase-2) was observed, suggesting that only the extrinsic pathway was not activated. Moreover, the clear time-dependent upregulation of five (Bcl2, BI-1, IAP1, IAP7B and Hsp70) inhibitors of apoptosis-related genes associated with the return to the initial number of hemocytes in apoptosis at 48 h of exposure suggests the involvement of strong regulatory mechanisms of apoptosis occurring in the hemocytes of the Pacific oyster. PMID:24317471

Cellular and Transcriptional Responses of Crassostrea gigas Hemocytes Exposed in Vitro to Brevetoxin (PbTx-2)

PubMed Central

Mello, Danielle F.; de Oliveira, Eliza S.; Vieira, Renato C.; Simoes, Erik; Trevisan, Rafael; Dafre, Alcir Luiz; Barracco, Margherita Anna

2012-01-01

Hemocytes mediate a series of immune reactions essential for bivalve survival in the environment, however, the impact of harmful algal species and their associated phycotoxins upon bivalve immune system is under debate. To better understand the possible toxic effects of these toxins, Crassostrea gigas hemocytes were exposed to brevetoxin (PbTx-2). Hemocyte viability, monitored through the neutral red retention and MTT reduction assays, and apoptosis (Hoechst staining) remained unchanged during 12 h of exposure to PbTx-2 in concentrations up to 1000 µg/L. Despite cell viability and apoptosis remained stable, hemocytes incubated for 4 h with 1000 µg/L of PbTx-2 revealed higher expression levels of Hsp70 (p < 0.01) and CYP356A1 (p < 0.05) transcripts and a tendency to increase FABP expression, as evaluated by Real-Time quantitative PCR. The expression of other studied genes (BPI, IL-17, GSTO, EcSOD, Prx6, SOD and GPx) remained unchanged. The results suggest that the absence of cytotoxic effects of PbTx-2 in Crassostrea gigas hemocytes, even at high concentrations, allow early defense responses to be produced by activating protective mechanisms associated to detoxification (CYP356A1 and possibly FABP) and stress (Hsp70), but not to immune or to antioxidant (BPI, IL-17, EcSOD, Prx6, GPx and SOD) related genes. PMID:22611355

Intracellular pH Recovery Rates in Bivalve Hemocytes Following Exposure to Acidic Environmental Conditions

NASA Astrophysics Data System (ADS)

Croxton, A.; Wikfors, G. H.

2012-12-01

Predictions of ocean acidification effects upon carbonate shell-forming species have caused great concern for the future of shellfisheries. Nevertheless, bivalve species inhabiting an estuarine environment have evolved in these environments with fluctuating pH levels. Previous experimental studies conducted in our laboratory have demonstrated the ability of oyster hemocytes to maintain intracellular homeostasis under acidic external conditions. However, little information is known of this homeostatic mechanism in other molluscan shellfish species present in these same habitats. In the current study we propose to determine if other bivalve species of aquaculture interest also possess this intracellular regulation by applying an in vitro hemocyte pH-recovery assay, previously developed for oysters, on the northern quahog, Mercenaria mercenaria, the blue mussel, Mytilus edulis, and the softshell clam, Mya arenaria. Preliminary results from the determination of initial intracellular pH levels, the initial step in the rate recovery assay, indicated a pH range between 7.0-7.4. This range was comparable to initial values measured in oysters, and consistent with data reported in the current literature. The second step of the hemocyte pH-recovery assay involves exposing oyster hemocytes to acidic external conditions and measuring the ability of the hemocyte intracellular pH to maintain homeostasis (i.e. recovery rate). Results from the recovery rate process will be presented.

Gene expression of apoptosis-related genes, stress protein and antioxidant enzymes in hemocytes of white shrimp Litopenaeus vannamei under nitrite stress.

PubMed

Guo, Hui; Xian, Jian-An; Li, Bin; Ye, Chao-Xia; Wang, An-Li; Miao, Yu-Tao; Liao, Shao-An

2013-05-01

Apoptotic cell ratio and mRNA expression of caspase-3, cathepsin B (CTSB), heat shock protein 70 (HSP70), manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPx) and thioredoxin (TRx) in hemocytes of white shrimp Litopenaeus vannamei exposed to nitrite-N (20 mg/L) was investigated at different stress time (0, 4, 8, 12, 24, 48 and 72 h). The apoptotic cell ratio and mRNA expression level of CTSB were significantly increased in shrimp exposed to nitrite-N for 48 and 72 h. Caspase-3 mRNA expression level significantly increased by 766.50% and 1811.16% for 24 and 48 h exposure, respectively. HSP70 expression level significantly increased at 8 and 72 h exposure. MnSOD mRNA expression in hemocytes up-regulated at 8 and 48 h, while CAT mRNA expression level increased at 24 and 48 h. GPx expression showed a trend that increased first and then decreased. Significant increases of GPx expression were observed at 8 and 12 h exposure. Expression level of TRx reached its highest level after 48 h exposure. These results suggest that nitrite exposure induces expression of apoptosis-related genes in hemocytes, and subsequently caused hemocyte apoptosis. Meanwhile, expression levels of HSP70 and antioxidant enzymes up-regulated to protect the hemocyte against nitrite stress. Copyright © 2013 Elsevier Inc. All rights reserved.

Rab3 is involved in cellular immune responses of the cotton bollworm, Helicoverpa armigera.

PubMed

Li, Jie; Song, Cai-Xia; Li, Yu-Ping; Li, Li; Wei, Xiu-Hong; Wang, Jia-Lin; Liu, Xu-Sheng

2015-06-01

Rab3, a member of the Rab GTPase family, has been found to be involved in innate immunity. However, the precise function of this GTPase in innate immunity remains unknown. In this study, we identified a Rab3 gene (Ha-Rab3) from the cotton bollworm, Helicoverpa armigera and studied its roles in innate immune responses. Expression of Ha-Rab3 was upregulated in the hemocytes of H. armigera larvae after the injection of Escherichia coli or chromatography beads. The dsRNA-mediated knockdown of Ha-Rab3 gene in H. armigera larval hemocytes led to significant reduction in the phagocytosis and nodulation activities of hemocytes against E. coli, significant increase in the bacterial load in larval hemolymph, and significant reduction in the encapsulation activities of hemocytes toward invading chromatography beads. Furthermore, Ha-Rab3 knockdown significantly suppressed spreading of plasmatocytes. These results suggest that Ha-Rab3 plays important roles in H. armigera cellular immune responses, possibly by mediating spreading of hemocytes. Copyright © 2015 Elsevier Ltd. All rights reserved.

Hemocyte-mediated phagocytosis and melanization in the mosquito Armigeres subalbatus following immune challenge by bacteria.

PubMed

Hillyer, Julián F; Schmidt, Shelley L; Christensen, Bruce M

2003-07-01

Mosquitoes are important vectors of disease. These insects respond to invading organisms with strong cellular and humoral immune responses that share many similarities with vertebrate immune systems. The strength and specificity of these responses are directly correlated to a mosquito's ability to transmit disease. In the current study, we characterized the hemocytes (blood cells) of Armigeres subalbatus by morphology (ultrastructure), lectin binding, enzyme activity, immunocytochemistry, and function. We found four hemocyte types: granulocytes, oenocytoids, adipohemocytes, and thrombocytoids. Granulocytes contained acid phosphatase activity and bound the exogenous lectins Helix pomatia agglutinin, Galanthus nivalis lectin, and wheat germ agglutinin. Following bacteria inoculation, granulocytes mounted a strong phagocytic response as early as 5 min postexposure. Bacteria also elicited a hemocyte-mediated melanization response. Phenoloxidase, the rate-limiting enzyme in the melanization pathway, was present exclusively in oenocytoids and in many of the melanotic capsules enveloping bacteria. The immune responses mounted against different bacteria were not identical; gram(-) Escherichia coli were predominantly phagocytosed and gram(+) Micrococcus luteus were melanized. These studies implicate hemocytes as the primary line of defense against bacteria.

Phagocytic activities of hemocytes from the deep-sea symbiotic mussels Bathymodiolus japonicus, B. platifrons, and B. septemdierum.

PubMed

Tame, Akihiro; Yoshida, Takao; Ohishi, Kazue; Maruyama, Tadashi

2015-07-01

Deep-sea mytilid mussels harbor symbiotic bacteria in their gill epithelial cells that are horizontally or environmentally transmitted to the next generation of hosts. To understand the immune defense system in deep-sea symbiotic mussels, we examined the hemocyte populations of the symbiotic Bathymodiolus mussel species Bathymodiolus japonicus, Bathymodiolus platifrons, and Bathymodiolus septemdierum, and characterized three types of hemocytes: agranulocytes (AGs), basophilic granulocytes (BGs), and eosinophilic granulocytes (EGs). Of these, the EG cells were the largest (diameter, 8.4-10.0 μm) and had eosinophilic cytoplasm with numerous eosinophilic granules (diameter, 0.8-1.2 μm). Meanwhile, the BGs were of medium size (diameter, 6.7-8.0 μm) and contained small basophilic granules (diameter, 0.3-0.4 μm) in basophilic cytoplasm, and the AGs, the smallest of the hemocytes (diameter, 4.8-6.0 μm), had basophilic cytoplasm lacking granules. A lectin binding assay revealed that concanavalin A bound to all three hemocyte types, while wheat germ agglutinin bound exclusively to EGs and BGs. The total hemocyte population densities within the hemolymph of all three Bathymodiolus mussel species were similar (8.4-13.3 × 10(5) cells/mL), and the percentages of circulating AGs, BGs, and EGs in the hemolymph of these organisms were 44.7-48.5%, 14.3-17.6%, and 34.3-41.0%, respectively. To analyze the functional differences between these hemocytes, the phagocytic activity and post-phagocytic phagosome-lysosome fusion events were analyzed in each cell type using a fluorescent Alexa Fluor(®) 488-conjugated Escherichia coli bioparticle and a LysoTracker(®) lysosomal marker, respectively. While the AGs exhibited no phagocytic activity, both types of granulocytes were phagocytic. Of the three hemocyte types, the EGs exhibited the highest level of phagocytic activity as well as rapid phagosome-lysosome fusion, which occurred within 2 h of incubation. Meanwhile, the BGs showed lower phagocytic activity and lower rates of phagosome-lysosome fusion than the EGs. These findings indicate that the two types of granulocyte play distinct roles in the defense system. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

The Roles of Heat Shock Proteins 70 and 90 in Exopalaemon carinicauda After WSSV and Vibrio anguillarum Challenges

NASA Astrophysics Data System (ADS)

Li, Jitao; Li, Jian; Duan, Yafei; Chen, Ping; Liu, Ping

2018-04-01

Heat shock proteins (HSPs), such as HSP70 and HSP90, are a suite of highly conserved proteins produced in all cellular organisms when they are exposed to stresses. In aquatic animals, they have been proved to play important roles in response to environmental pollutants and particularly in the non-specific immune responses to pathogen infections. In the present study, the expression profiles of HSP70 and HSP90 genes in hemocytes and hepatopancreas from the ridgetail white prawn Exopalaemon carinicauda infected with WSSV and Vibrio anguillarum were detected using reverse transcription polymerase chain reaction (RT-PCR). After WSSV challenge, the expression level of HSP 70 gene transcripts in the hemocytes and hepatopancreas increased to peak level at 6 h and 48 h, respectively. HSP90 gene transcripts in hemocytes and hepatopancreas were up-regulated significantly at 12 h and 6 h, respectively. During V. anguillarum challenge, the mRNA content of HSP70 gene in hemocytes and hepatopancreas increased significantly at 12 h and 6 h post-infection, respectively. The expression level of HSP90 gene both in hemocytes and hepatopancreas were up-regulated in the first 3 h. The expression patterns of HSP70 and HSP90 genes in hemocytes and hepatopancreas showed temporal and spatial differences after challenged with WSSV and V. anguillarum. The results suggested that HSPs might be involved in immune responses to pathogens challenge in E. carinicauda.

The roles of heat shock proteins 70 and 90 in Exopalaemon carinicauda after WSSV and Vibrio anguillarum challenges

NASA Astrophysics Data System (ADS)

Li, Jitao; Li, Jian; Duan, Yafei; Chen, Ping; Liu, Ping

2017-12-01

Heat shock proteins (HSPs), such as HSP70 and HSP90, are a suite of highly conserved proteins produced in all cellular organisms when they are exposed to stresses. In aquatic animals, they have been proved to play important roles in response to environmental pollutants and particularly in the non-specific immune responses to pathogen infections. In the present study, the expression profiles of HSP70 and HSP90 genes in hemocytes and hepatopancreas from the ridgetail white prawn Exopalaemon carinicauda infected with WSSV and Vibrio anguillarum were detected using reverse transcription polymerase chain reaction (RT-PCR). After WSSV challenge, the expression level of HSP 70 gene transcripts in the hemocytes and hepatopancreas increased to peak level at 6 h and 48 h, respectively. HSP90 gene transcripts in hemocytes and hepatopancreas were up-regulated significantly at 12 h and 6 h, respectively. During V. anguillarum challenge, the mRNA content of HSP70 gene in hemocytes and hepatopancreas increased significantly at 12 h and 6 h post-infection, respectively. The expression level of HSP90 gene both in hemocytes and hepatopancreas were up-regulated in the first 3 h. The expression patterns of HSP70 and HSP90 genes in hemocytes and hepatopancreas showed temporal and spatial differences after challenged with WSSV and V. anguillarum. The results suggested that HSPs might be involved in immune responses to pathogens challenge in E. carinicauda.

Characterization of subpopulations and immune-related parameters of hemocytes in the green-lipped mussel Perna viridis.

PubMed

Wang, Youji; Hu, Menghong; Chiang, M W L; Shin, P K S; Cheung, S G

2012-03-01

The green-lipped mussel Perna viridis is distributed widely in the estuarine and coastal areas of the Indo-Pacific region and extensively cultured as an inexpensive protein source. Morphology and immunological activities of hemocytes of P. viridis were investigated using flow cytometry and light and electron microscopy. Three major types of hemocytes were identified in the hemolymph, including dense-granulocyte, semi-granulocyte (small and large size) and hyalinocyte. Other hemocytes, which occurred in low numbers, included granulocytes with different electron-dense/lucent granules and hemoblast-like cells. Based on flow cytometry, two subpopulations were identified. Granulocytes were larger cells, and the more abundant, containing numerous granules in the cytoplasm, and hyalinocytes were the smaller and less abundant with the fewest granules. Flow cytometry revealed that the granulocytes were more active in cell phagocytosis, contained the higher lysosomal content, and showed higher esterase activity and reactive oxygen species (ROS) generation compared with hyalinocytes. Immune functions assessed by the flow cytometry indicated that the granulocytes were the main hemocytes involved in the cellular defence in P. viridis. Copyright © 2011. Published by Elsevier Ltd.

Antimicrobial Histones and DNA Traps in Invertebrate Immunity

PubMed Central

Poirier, Aurore C.; Schmitt, Paulina; Rosa, Rafael D.; Vanhove, Audrey S.; Kieffer-Jaquinod, Sylvie; Rubio, Tristan P.; Charrière, Guillaume M.; Destoumieux-Garzón, Delphine

2014-01-01

Although antimicrobial histones have been isolated from multiple metazoan species, their role in host defense has long remained unanswered. We found here that the hemocytes of the oyster Crassostrea gigas release antimicrobial H1-like and H5-like histones in response to tissue damage and infection. These antimicrobial histones were shown to be associated with extracellular DNA networks released by hemocytes, the circulating immune cells of invertebrates, in response to immune challenge. The hemocyte-released DNA was found to surround and entangle vibrios. This defense mechanism is reminiscent of the neutrophil extracellular traps (ETs) recently described in vertebrates. Importantly, oyster ETs were evidenced in vivo in hemocyte-infiltrated interstitial tissues surrounding wounds, whereas they were absent from tissues of unchallenged oysters. Consistently, antimicrobial histones were found to accumulate in oyster tissues following injury or infection with vibrios. Finally, oyster ET formation was highly dependent on the production of reactive oxygen species by hemocytes. This shows that ET formation relies on common cellular and molecular mechanisms from vertebrates to invertebrates. Altogether, our data reveal that ET formation is a defense mechanism triggered by infection and tissue damage, which is shared by relatively distant species suggesting either evolutionary conservation or convergent evolution within Bilateria. PMID:25037219

Genetic Screen in Drosophila Larvae Links ird1 Function to Toll Signaling in the Fat Body and Hemocyte Motility

PubMed Central

Schmid, Martin R.; Anderl, Ines; Vo, Hoa T. M.; Valanne, Susanna; Yang, Hairu; Kronhamn, Jesper; Rämet, Mika; Rusten, Tor Erik

2016-01-01

To understand how Toll signaling controls the activation of a cellular immune response in Drosophila blood cells (hemocytes), we carried out a genetic modifier screen, looking for deletions that suppress or enhance the mobilization of sessile hemocytes by the gain-of-function mutation Toll10b (Tl10b). Here we describe the results from chromosome arm 3R, where five regions strongly suppressed this phenotype. We identified the specific genes immune response deficient 1 (ird1), headcase (hdc) and possibly Rab23 as suppressors, and we studied the role of ird1 in more detail. An ird1 null mutant and a mutant that truncates the N-terminal kinase domain of the encoded Ird1 protein affected the Tl10b phenotype, unlike mutations that affect the C-terminal part of the protein. The ird1 null mutant suppressed mobilization of sessile hemocytes, but enhanced other Tl10b hemocyte phenotypes, like the formation of melanotic nodules and the increased number of circulating hemocytes. ird1 mutants also had blood cell phenotypes on their own. They lacked crystal cells and showed aberrant formation of lamellocytes. ird1 mutant plasmatocytes had a reduced ability to spread on an artificial substrate by forming protrusions, which may explain why they did not go into circulation in response to Toll signaling. The effect of the ird1 mutation depended mainly on ird1 expression in hemocytes, but ird1-dependent effects in other tissues may contribute. Specifically, the Toll receptor was translocated from the cell membrane to intracellular vesicles in the fat body of the ird1 mutant, and Toll signaling was activated in that tissue, partially explaining the Tl10b-like phenotype. As ird1 is otherwise known to control vesicular transport, we conclude that the vesicular transport system may be of particular importance during an immune response. PMID:27467079

Changes in hemolymph characteristics of ark shell Scapharaca broughtonii dealt with Vibrio anguillarum challenge in vivo and various of anticoagulants in vitro.

PubMed

Zhou, Liqing; Yang, Aiguo; Liu, Zhihong; Wu, Biao; Sun, Xiujun; Lv, Zhenming; Tian, Ji-Teng; Du, Meirong

2017-02-01

The ark shell Scapharca broughtonii is a commercially important shellfish in China. Alserver's solution (AS), modified Alserver's solution (MAS) and Heparin sodium solution (HSS) are common anticoagulants used for shellfish blood. To observe the immune response mediated by its hemocytes, we challenged in vivo S. broughtonii hemolymph with Vibrio anguillarum and dealt with the following three anticoagulants in vitro: Alserver's solution (AS), modified Alserver's solution (MAS) and Heparin sodium solution (HSS). The methodologies we used were immunostimulation with V. anguillarum, Wright-Giemsa staining, micro-examination, and flow cytometric and hydrolyzing enzyme activity analysis. The results showed that all three types of anticoagulants effectively prevented blood clotting in ark shellfish. The morphology of hemocytes did not significantly change 30 h after anticoagulant treatment, except for the shrinking of hemocytes after administering HSS. The size and permeability of hemocytes changed when treated with the anticoagulants and when stimulated with V. anguillarum. Both alkaline phosphatase (AKP) and acid phosphatase (ACP) in hemocytes and Plasma were measured at different times after they were stimulated with V. anguillarum in HSS and MAS. The AKP enzymatic activity in HSS was somewhat higher than in the MAS anticoagulant, but changes in response to V. anguillarum challenge of enzymatic activity were almost the same in HSS and MAS groups. In conclusion, all three types of anticoagulants may be used for ark shell blood preservation. They all changed the cell-surface characteristics of hemocytes to inhibit clot formation. The AS anticoagulant was appropriate for maintaining white and red cell shapes, while MAS was ideal for retaining throbus cell function. Lastly, HSS was appropriate for maintaining enzymatic activity in hemolymph and function of hemocytes. Following this investigation, we gained insight into the changes in hemolymph characteristic during immune response. Copyright © 2016. Published by Elsevier Ltd.

Cellular Apoptosis of Hemocytes from Dendrolimus tabulaeformis Tsai et Liu Larvae Induced with the Secondary Metabolites of Beauveria brongniartii (Sacc.) Petch

PubMed Central

Fan, Jinhua; Xie, Yingping; Xue, Jiaoliang; Zhang, Yingling; Yang, Qian

2013-01-01

To investigate the effect of the secondary metabolites of entomopathogenic fungus on the hemocyte immunity of host insect, the secondary metabolite complex (SMC) of Beauveria brongniartii was used in three concentrations (5.5, 55, and 550 µg/mL), and the 4th instar larvae of the pine caterpillar Dendrolimus tabulaeformis were employed as host insects. The larvae were inoculated with the SMC solutions by injection in bioassays. Apoptosis of the larval hemocytes was observed using fluorescence microscopy (FM), transmission electron microscopy (TEM), and flow cytometry (FCM). The FM results showed that in the treated groups, larval hemocytes exhibited symptoms of early apoptosis at 6 h post-treatment by radiating a non-uniform kelly fluorescence and exhibited symptoms of late apoptosis at 12 h post-treatment by radiating a non-uniform orange fluorescence. Under TEM, the following ultra-structural changes associated with apoptosis of the larval hemocytes were observed in the treated groups: the nuclei were hypertrophied, slight folds were on the nuclear envelope, the chromatin became concentrated, the mitochondrial cristae disappeared or were disorderly, most cells developed blebs, and fibrillar aggregation appeared and accumulated in the cytoplasm. Apoptosis of the larval hemocytes was detected by FCM at 6 h post-treatment; the percentage of early apoptotic cells in the SMC 5.5, 55, and 550 µg/mL treatment groups were 11.93%, 13.10%, and 18.42%, respectively. Late apoptosis first occurred at 12 h post-treatment; the highest rate of apoptosis was 36.54 ± 4.37% at 24 h post-treatment in the SMC 55 µg/mL treatment group. In general, the cellular apoptosis rate was positively correlated with the SMC concentration and the time post-treatment. These results indicate that secondary metabolites of B . brongniartii are able to attack the hemocytes of D . tabulaeformis larvae and induce cellular apoptosis, thereby providing new evidence that secondary metabolites of mycopathogens can act on host immune systems. PMID:23940771

ACTIVATION OF OYSTER DEFENSES BY ENVIRONMENTAL CONTAMINANTS

EPA Science Inventory

Four field studies performed on eastern oysters Crassostrea virginica support a hypothesis that Cu, Zn, and perhaps butyltins and polycyclic aromatic hydrocarbons (PAH) can stimulate hemopoiesis, hemocyte locomotion and hemocyte bactericidal capacity. The first study found circul...

[Influence of trematode invasion and zinc ions on the histometric peculiarities of haemocytes and some hematological indices of Planorbarius purpura (Gastropoda: Pulmonata: Bulinidae)].

PubMed

Kirichuk, G E; Stadnichenko, A P

2010-01-01

Cellular components of the Planorbarius purpura hemolymph are represented by three phyla of haemocytes (prohemocytes, eosinophilis microgranulocytes, and basophilis granulocytes) and vesicular cells. As a result of the invasions of P. purpura with the trematode Echinoparyphium aconiatum, changes of the linear dimensions of granular hemocytes and their nuclei took place. Moreover, an increase of the hemocytes' number per l mm3 of hemolymph and change of the percentages of different hemocyte types were recorded. Under the influence of zinc ions, linear dimensions of prohemocytes and their nuclei (at 10 MPCns of the toxicant) were changed. In granular hemocytes and abnormalities of all histometrical and hematological parameters were observed. All cytometrical, karyometrical, and hematological alterations were expressed more clearly in infested mollusks than in non-infested ones, and occurred usually under lower concentrations of zinc ions.

Components of the cellular defense and detoxification system of the common cuttlefish Sepia officinalis (Mollusca, Cephalopoda).

PubMed

Beuerlein, Knut; Löhr, Sandra; Westermann, Bettina; Ruth, Peter; Schipp, Rudolf

2002-12-01

Endocytotic-active cells in the branchial heart complex of Sepia officinalis were studied by in situ injection of different types of xenobiotics and by in vitro perfusion of the organ complex with a bacterial suspension. The rhogocytes (ovoid cells) ingest particles of all tested sizes by endocytosis and phagocytosis. The hemocytes of the circulating blood and the adhesive hemocytes in the wall of the branchial heart incorporate all tested kinds of foreign materials, including bacterial cells due to phagocytosis achieved by the triangular mesenchymatic cells. The ultrastructural findings also give strong evidence that the triangular mesenchymatic cells are fixed hemocytes that have migrated into the branchial heart tissue. The ingestion and digestion of allogeneic substances and bacteria or their debris by rhogocytes and/or all (forms of) hemocytes suggests the involvement of these either fixed or mobile endocytotic-active cells in the defense and detoxification system of cephalopods.

Serratia marcescens Suppresses Host Cellular Immunity via the Production of an Adhesion-inhibitory Factor against Immunosurveillance Cells*

PubMed Central

Ishii, Kenichi; Adachi, Tatsuo; Hamamoto, Hiroshi; Sekimizu, Kazuhisa

2014-01-01

Injection of a culture supernatant of Serratia marcescens into the bloodstream of the silkworm Bombyx mori increased the number of freely circulating immunosurveillance cells (hemocytes). Using a bioassay with live silkworms, serralysin metalloprotease was purified from the culture supernatant and identified as the factor responsible for this activity. Serralysin inhibited the in vitro attachment of both silkworm hemocytes and murine peritoneal macrophages. Incubation of silkworm hemocytes or murine macrophages with serralysin resulted in degradation of the cellular immune factor BmSPH-1 or calreticulin, respectively. Furthermore, serralysin suppressed in vitro phagocytosis of bacteria by hemocytes and in vivo bacterial clearance in silkworms. Disruption of the ser gene in S. marcescens attenuated its host killing ability in silkworms and mice. These findings suggest that serralysin metalloprotease secreted by S. marcescens suppresses cellular immunity by decreasing the adhesive properties of immunosurveillance cells, thereby contributing to bacterial pathogenesis. PMID:24398686

Serratia marcescens suppresses host cellular immunity via the production of an adhesion-inhibitory factor against immunosurveillance cells.

PubMed

Ishii, Kenichi; Adachi, Tatsuo; Hamamoto, Hiroshi; Sekimizu, Kazuhisa

2014-02-28

Injection of a culture supernatant of Serratia marcescens into the bloodstream of the silkworm Bombyx mori increased the number of freely circulating immunosurveillance cells (hemocytes). Using a bioassay with live silkworms, serralysin metalloprotease was purified from the culture supernatant and identified as the factor responsible for this activity. Serralysin inhibited the in vitro attachment of both silkworm hemocytes and murine peritoneal macrophages. Incubation of silkworm hemocytes or murine macrophages with serralysin resulted in degradation of the cellular immune factor BmSPH-1 or calreticulin, respectively. Furthermore, serralysin suppressed in vitro phagocytosis of bacteria by hemocytes and in vivo bacterial clearance in silkworms. Disruption of the ser gene in S. marcescens attenuated its host killing ability in silkworms and mice. These findings suggest that serralysin metalloprotease secreted by S. marcescens suppresses cellular immunity by decreasing the adhesive properties of immunosurveillance cells, thereby contributing to bacterial pathogenesis.

Parasitism and venom of ectoparasitoid Scleroderma guani impairs host cellular immunity.

PubMed

Li, Li-Fang; Xu, Zhi-Wen; Liu, Nai-Yong; Wu, Guo-Xing; Ren, Xue-Min; Zhu, Jia-Ying

2018-06-01

Venom is a prominently maternal virulent factor utilized by parasitoids to overcome hosts immune defense. With respect to roles of this toxic mixture involved in manipulating hosts immunity, great interest has been mostly restricted to Ichneumonoidea parasitoids associated with polydnavirus (PDV), of which venom is usually considered as a helper component to enhance the role of PDV, and limited Chalcidoidea species. In contrast, little information is available in other parasitoids, especially ectoparasitic species not carrying PDV. The ectoparasitoid Scleroderma guani injects venom into its host, Tenebrio molitor, implying its venom was involved in suppression of hosts immune response for successful parasitism. Thus, we investigated the effects of parasitism and venom of this parasitoid on counteracting the cellular immunity of its host by examining changes of hemocyte counts, and hemocyte spreading and encapsulation ability. Total hemocyte counts were elevated in parasitized and venom-injected pupae. The spreading behavior of both granulocytes and plasmatocytes was impaired by parasitization and venom. High concentration of venom led to more severely increased hemocyte counts and suppression of hemocyte spreading. The ability of hemocyte encapsulation was inhibited by venom in vitro. In addition to immediate effects observed, venom showed persistent interference in hosts cellular immunity. These results indicate that venom alone from S. guani plays a pivotal role in blocking hosts cellular immune response, serving as a regulator that guarantees the successful development of its progenies. The findings provide a foundation for further investigation of the underlying mechanisms in immune inhibitory action of S. guani venom. © 2018 Wiley Periodicals, Inc.

Blood Feeding and Insulin-like Peptide 3 Stimulate Proliferation of Hemocytes in the Mosquito Aedes aegypti

PubMed Central

Castillo, Julio; Brown, Mark R.; Strand, Michael R.

2011-01-01

All vector mosquito species must feed on the blood of a vertebrate host to produce eggs. Multiple cycles of blood feeding also promote frequent contacts with hosts, which enhance the risk of exposure to infectious agents and disease transmission. Blood feeding triggers the release of insulin-like peptides (ILPs) from the brain of the mosquito Aedes aegypti, which regulate blood meal digestion and egg formation. In turn, hemocytes serve as the most important constitutive defense in mosquitoes against pathogens that enter the hemocoel. Prior studies indicated that blood feeding stimulates hemocytes to increase in abundance, but how this increase in abundance is regulated is unknown. Here, we determined that phagocytic granulocytes and oenocytoids express the A. aegypti insulin receptor (AaMIR). We then showed that: 1) decapitation of mosquitoes after blood feeding inhibited hemocyte proliferation, 2) a single dose of insulin-like peptide 3 (ILP3) sufficient to stimulate egg production rescued proliferation, and 3) knockdown of the AaMIR inhibited ILP3 rescue activity. Infection studies indicated that increased hemocyte abundance enhanced clearance of the bacterium Escherichia coli at lower levels of infection. Surprisingly, however, non-blood fed females better survived intermediate and high levels of E. coli infection than blood fed females. Taken together, our results reveal a previously unrecognized role for the insulin signaling pathway in regulating hemocyte proliferation. Our results also indicate that blood feeding enhances resistance to E. coli at lower levels of infection but reduces tolerance at higher levels of infection. PMID:21998579

Hemocytes of Rhipicephalus sanguineus (Acari: Ixodidae): Characterization, Population Abundance, and Ultrastructural Changes Following Challenge with Leishmania infantum.

PubMed

Feitosa, A P S; Alves, L C; Chaves, M M; Veras, D L; Silva, E M; Aliança, A S S; França, I R S; Gonçalves, G G A; Lima-Filho, J L; Brayner, F A

2015-11-01

Few studies have examined the cellular immune response of ticks, and further research on the characterization of the hemocytes of ticks is required, particularly on those of Rhipicephalus sanguineus (Latreille) because of the medical and veterinary importance of this tick. The aims of this study were to characterize the morphology and the ultrastructure of the different types of hemocytes of adult R. sanguineus and to determine the population abundance and the ultrastructural changes in the hemocytes of ticks infected with Leishmania infantum. The hemocytes were characterized through light and transmission electron microscopy. Within the variability of circulating cells in the hemolymph of adult R. sanguineus, five cell types were identified, which were the prohemocytes, plasmatocytes, granulocytes, spherulocytes, and adipohemocytes. The prohemocytes were the smallest cells found in the hemolymph. The plasmatocytes had polymorphic morphology with vesicles and cytoplasmic projections. The granulocytes had an elliptical shape with the cytoplasm filled with granules of different sizes and electrodensities. The spherulocytes were characterized by several spherules of uniform shapes and sizes that filled the entire cytoplasm, whereas the adipohemocytes had an irregular shape with multiple lipid inclusions that occupied almost the entire cytoplasmic space. The total counts of the hemocyte population increased in the group that was infected with L. infantum. Among the different cell types, the numbers increased and the ultrastructural changes occurred in the granulocytes and the plasmatocytes in the infected group of ticks. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

An immune-related gene expression atlas of the shrimp digestive system in response to two major pathogens brings insights into the involvement of hemocytes in gut immunity.

PubMed

Silveira, Amanda S; Matos, Gabriel M; Falchetti, Marcelo; Ribeiro, Fabio S; Bressan, Albert; Bachère, Evelyne; Perazzolo, Luciane M; Rosa, Rafael D

2018-02-01

Much of our current knowledge on shrimp immune system is restricted to the defense reactions mediated by the hemocytes and little is known about gut immunity. Here, we have investigated the transcriptional profile of immune-related genes in different organs of the digestive system of the shrimp Litopenaeus vannamei. First, the tissue distribution of 52 well-known immune-related genes has been assessed by semiquantitative analysis in the gastrointestinal tract (foregut, midgut and hindgut) and in the hepatopancreas and circulating hemocytes of shrimp stimulated or not with heat-killed bacteria. Then, the expression levels of 18 genes from key immune functional categories were quantified by fluorescence-based quantitative PCR in the midgut of animals experimentally infected with the Gram-negative Vibrio harveyi or the White spot syndrome virus (WSSV). Whereas the expression of some genes was induced at 48 h after the bacterial infection, any of the analyzed genes showed to be modulated in response to the virus. Whole-mount immunofluorescence assays confirmed the presence of infiltrating hemocytes in the intestines, indicating that the expression of some immune-related genes in gut is probably due to the migratory behavior of these circulating cells. This evidence suggests the participation of hemocytes in the delivery of antimicrobial molecules into different portions of the digestive system. Taken all together, our results revealed that gut is an important immune organ in L. vannamei with intimate association with hemocytes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Pacific oyster (Crassostrea gigas) hemocyte are not affected by a mixture of pesticides in short-term in vitro assays.

PubMed

Moreau, Pierrick; Burgeot, Thierry; Renault, Tristan

2014-04-01

Pesticides are frequently detected in estuaries among the pollutants found in estuarine and coastal areas and may have major ecological consequences. They could endanger organism growth, reproduction, or survival. In the context of high-mortality outbreaks affecting Pacific oysters, Crassostrea gigas, in France since 2008, it appears of importance to determine the putative effects of pesticides on oyster susceptibility to infectious agents. Massive mortality outbreaks reported in this species, mainly in spring and summer, may suggest an important role played by the seasonal use of pesticides and freshwater input in estuarine areas where oyster farms are frequently located. To understand the impact of some pesticides detected in French waters, their effects on Pacific oyster hemocytes were studied through short-term in vitro experiments. Bivalve immunity is mainly supported by hemocytes eliminating pathogens by phagocytosis and producing compounds including lysosomal enzymes and antimicrobial molecules. In this study, oyster hemocytes were incubated with a mixture of 14 pesticides and metaldehyde alone, a molecule used to eliminate land mollusks. Hemocyte parameters including dead/alive cells, nonspecific esterase activities, intracytoplasmic calcium, lysosome number and activity, and phagocytosis were monitored by flow cytometry. No significant effect of pesticides tested at different concentrations was reported on oyster hemocytes maintained in vitro for short-term periods in the present study. It could be assumed that these oyster cells were resistant to pesticide exposure in tested conditions and developing in vivo assays appears as necessary to better understand the effects of pollutants on immune system in mollusks.

The effect of Beauveria bassiana infection on cell mediated and humoral immune response in house fly, Musca domestica L.

PubMed

Mishra, Sapna; Kumar, Peeyush; Malik, Anushree

2015-10-01

Entomopathogenic fungi that manifest infections by overcoming insect's immune response could be a successful control agent for the house fly, Musca domestica L. which is a major domestic, medical, and veterinary pest. In this study, the immune response of house fly to Beauveria bassiana infection was investigated to reveal fundamental aspects of house fly hemocyte biology, such as hemocyte numbers and size, which is poorly understood. The total hemocyte counts (THCs) in B. bassiana-infected house fly showed an initial increase (from 6 to 9 h), followed by subsequent decrease (9 to 12 h) with increase in time of infection. The THCs was slightly greater in infected flies than the non-infected ones. Insight into relative hemocyte counts depicted a significant increase in prohemocyte (PR) and decrease in granulocyte (GR) in infected house flies compared to non-infected ones. The relative cell area of hemocyte cells showed a noticeable increase in PR and intermediate cells (ICs), while a considerable reduction was observed for plasmatocyte (PL) and GR. The considerable variation in relative cell number and cell area in the B. bassiana-infected house flies indicated stress development during infection. The present study highlights changes occurring during B. bassiana invasion to house fly leading to establishment of infection along with facilitation in understanding of basic hemocyte biology. The results of the study is expected to help in better understanding of house fly immune response during fungal infection, so as to assist production of more efficient mycoinsecticides for house fly control using B. bassiana.

The occurrence of chitin in the hemocytes of invertebrates

PubMed Central

Heath-Heckman, Elizabeth A.C.; McFall-Ngai, Margaret J.

2011-01-01

The light-organ symbiosis of Euprymna scolopes, the Hawaiian bobtail squid, is a useful model for the study of animal–microbe interactions. Recent analyses have demonstrated that chitin breakdown products play a role in communication between E. scolopes and its bacterial symbiont Vibrio fischeri. In this study, we sought to determine the source of chitin in the symbiotic organ. We used a commercially available chitin-binding protein (CBP) conjugated to fluorescein to label the polymeric chitin in host tissues. Confocal microscopy revealed that the only cells in contact with the symbionts that labeled with the probe were the macrophage-like hemocytes, which traffic into the light-organ crypts where the bacteria reside. Labeling of extracted hemocytes by CBP was markedly decreased following treatment with purified chitinase, providing further evidence that the labeled molecule is polymeric chitin. Further, CBP-positive areas co-localized with both a halide peroxidase antibody and Lysotracker, a lysosomal marker, suggesting that the chitin-like biomolecule occurs in the lysosome or acidic vacuoles. Reverse transcriptase polymerase chain reaction (PCR) of hemocytes revealed mRNA coding for a chitin synthase, suggesting that the hemocytes synthesize chitin de novo. Finally, upon surveying blood cells from other invertebrate species, we observed CBP-positive regions in all granular blood cells examined, suggesting that this feature is a shared character among the invertebrates; the vertebrate blood cells that we sampled did not label with CBP. Although the function of the chitin-like material remains undetermined, its presence and subcellular location in invertebrate hemocytes suggests a conserved role for this polysaccharide in the immune system of diverse animals. PMID:21723107

Wright-Giemsa staining to observe phagocytes in Locusta migratoria infected with Metarhizium acridum.

PubMed

Yu, Ying; Cao, Yueqing; Xia, Yuxian; Liu, Feihong

2016-09-01

Hemocytes are the first line of defense in the invertebrate immune system. Understanding their roles in cellular immunity is important for developing more efficient mycoinsecticides. However, the exact classification of hemocytes has been inconsistent and the various types of phagocytes in Locusta migratoria are poorly defined. Herein, the Wright-Giemsa staining method and microscopy were employed to characterize the hemocytes of L. migratoria following infection by Metarhizium acridum. Hemocytes were classified into four types, including granulocytes, plasmatocytes, prohemocytes, and oenocytoids, based on size, morphology, and dye-staining properties. Each type of hemocyte was classified into several subtypes according to different ultrastructural features. At least four subtypes of granulocytes or plasmatocytes, including small-nucleus plasmatocytes, basophil vacuolated plasmatocytes, homogeneous plasmatocytes, and eosinophilic granulocytes, carried out phagocytosis. The percentage of total phagocytes increased two days after infection by M. acridum, then gradually declined during the next two days, and then increased sharply again at the fifth day. Our data suggested that plasmatocytes and granulocytes may be the major phagocytes that protect against invasion by a fungal pathogen in L. migratoria. Total hemocytes in locusts significantly increased in the initial days after infection and decreased in the late period of infection compared to controls. In the hemocoel, hyphal bodies were recognized, enwrapped, and digested by the phagocytes. Then, the broken hyphal pieces were packaged as vesicles to be secreted from the cell. Moreover, locusts might have a sensitive and efficient cellular immune system that can regulate phagocyte differentiation and proliferation before fungi colonize the host hemolymph. Copyright © 2016 Elsevier Inc. All rights reserved.

Evaluation of the immune responses of the brown mussel Perna perna as indicators of fecal pollution.

PubMed

Silva Dos Santos, Fernanda; Neves, Raquel Almeida Ferrando; Carvalho, Wanderson Fernandes de; Krepsky, Natascha; Crapez, Mirian Araújo Carlos

2018-06-01

The mussel Perna perna is an intertidal bivalve that is widely distributed, cultivated and consumed in South Africa, Brazil and Venezuela. Among marine resources, bivalve mollusks are one of the most impacted by anthropogenic pollution, as they can accumulate pathogenic bacteria and water pollutants. Hemocytes are molluscan defense cells, and their abundance and functions can be affected in response to contaminants, such as bacterial load. However, no previous study has investigated the immune response of P. perna hemocytes. The aim of this study was to evaluate several immune parameters in P. perna as indicators of fecal pollution in mussel hemolymph and in seawater. We collected mussels and adjacent seawater from beaches with different levels of fecal contamination in Rio de Janeiro state (Brazil): Vermelha Beach (VB); Icaraí Beach (IB); Urca Beach (UB); and Jurujuba Beach (JB). Hemocyte parameters (density, morphology, phagocytic activity and production of Reactive Oxygen Species - ROS) were evaluated using flow cytometry. We quantified Fecal Indicator Bacteria (FIB) in seawater by the multiple tubes technique for each beach and for hemolymph by the spread-plate technique. In agreement with historical evaluation of fecal contamination levels, UB presented the highest FIB abundance in seawater (thermotolerant coliforms, TEC = 1600 NMP 100 mL -1 ), whereas VB exhibited the lowest (TEC = 17 NMP 100 mL -1 ). UB mussels had six and eight times higher hemocyte density and phagocytic activity, respectively, than mussels from VB. Mussels from VB and IB presented a significantly lower number of total coliforms in hemolymph and a significantly higher relative internal complexity of hemocytes than those from UB and JB (p ≤ 0.01, PERMANOVA). ROS production by hemocytes was significantly lower in mussels from VB compared to those from JB (p = 0.04, ANOVA). Our results indicate a significant relationship between the level of fecal contamination in aquatic environments and the immune response of mussel hemocytes. Immune-related parameters may therefore be useful as indicators of bivalve health and environmental quality. Our flow cytometric analysis of P. perna hemocytes represents a new approach for studying Perna perna biology and might represent a novel tool for measuring organic pollution and water quality. Copyright © 2018 Elsevier Ltd. All rights reserved.

RESPONSES OF OYSTER (CRASSOSTREA VIRGINICA) HEMOCYTES TO NONPATHOGENIC AND CLINICAL ISOLATES OF VIBRIO PARAHAEMOLYTICUS

EPA Science Inventory

Bacterial uptake by oysters (Crassostrea virginica) and bactericidal activity of oyster hemocytes were studied using four environmental isolates and three clinical isolates of Vibrio parahaemolyticus. Clinical isolates (2030, 2062, 2107) were obtained from gastroenteritis patien...

Association of reticular cells with CD34+/Sca-1+ apoptotic cells in the hemopoietic organ of grasshopper, Euprepocnemis shirakii.

PubMed

Lim, Jong Yeon; Lee, Bong Hee; Kang, Seok Woo; Wago, Haruhisa; Han, Sung Sik

2004-07-01

Hemopoiesis in orthopteran insects occurs in a hemopoietic organ that is located bilaterally along the aorta. This organ is also known as a reticulo-hemopoietic organ because of the rich presence of reticular cells. This study was performed to further elucidate hemopoiesis in the reticulo-hemopoietic organ of an orthopteran, Euprepocnemis shirakii. We focused on the question why reticular cells are so abundant (35% of cells in hemopoietic organ). Interestingly, 21% of these reticular cells surrounded hemocytes with their reticular cytoplasm. The surrounded hemocytes were distinguished by their different size and darkly stained nucleus. These cells were characterized by immunostaining using antibodies against several types of hemocytes: 45% of the surrounded hemocytes were CD34+, and these positive cells were double stained (over 85%) when immunostained by another hemopoietic pluripotent cell marker, Sca-1. Transmission electron microscopic analysis showed that reticular cells surrounded hemocytes containing large nuclei and poorly developed cytoplasmic organelles. This strongly suggests that the reticular cells surround hemopoietic stem cells. Additionally, surrounded hemopoietic progenitor cells are undergoing apoptosis as indicated by the TUNEL assay. The enclosed apoptotic cells are engulfed and then phagocytosed by reticular cells. Our results suggest that reticular cells are related to the differentiation and apoptosis of hemopoietic stem cells.

Cell lines, Md108 and Md66, from the hemocytes of Malacosoma disstria (Lepidoptera) display aspects of plasma-free innate non-self activities.

PubMed

Lapointe, Jason F; Dunphy, Gary B; Giannoulis, Paschalis; Mandato, Craig A; Nardi, James B; Gharib, Osama H; Niven, Donald F

2011-11-01

The innate non-self response systems of the deciduous tree pest, the forest tent caterpillar, Malacosoma disstria has been documented by us in terms of in vitro and in vivo reactions towards the Gram-positive nonpathogenic bacterium, Bacillus subtilis and Gram-negative pathogenic microbe, Xenorhabdus nematophila and their respective surface antigens, lipopoteichoic acids (LTA) and lipopolysaccharides (LPS). These studies, often conducted in whole and diluted hemolymph, preclude examination of plasma-free cellular (hemocyte) responses. Plasma-free hemocytes as primary cultures are difficult to obtain. The floating cell line Md66 and attached cell line Md108 from M. disstria hemocytes were examined as a model for plasma-free M. disstria hemocyte non-self responses. Herein, it was established that although both lines differed from each other and from the primary hemocyte cultures of M. disstria in growth parameters, cell composition and sizes both cell lines displayed granular cell-like (GL) cells and plasmatocyte-like (PL) cells according to morphological criteria and to some extent antigenic similarities based on labeling with anti-Chrysodeixis includens hemocyte monoclonal antibodies. Hemocyte-specific neuroglian-like protein was detected on cells of both cell lines and in the primary hemocyte cultures albeit with staining patterns differing according to culture and cell types, confluency levels and cell-cell adhesion. Both cell lines bound B. subtilis and X. nematophila, the reaction extent varying with the cell line and its cell types. LPS damaged both cell types in the two cell lines whereas LTA enhanced the adhesion of Md66 GL cells to flask surfaces followed by PL cell adhesion. PL cells of both lines, like the primary cultures, phagocytosed FITC-labeled B. subtilis; only Md108 GL cells phagocytosed B. subtilis. In either case phagocytosis was always less in frequency and intensity than the primary cultures. Proteins released from the cell lines differed in pattern and magnitude but contained bacterial binding proteins that enhanced differential bacterial adhesion to both cell types in both cell lines: the GL cells both cultures, and those of granular cells in primary cultures, were more involved than the primary plasmatocytes and PL cells. Only Md66 cells possessed lysozyme and both cell types of both lines contained phenoloxidase. Neither enzyme type was released during early phase reaction with the bacteria. LPS inhibited phenoloxidase activity. The similarities and differences between the lines and primary cultures make Md66 and Md108 useful for the systematic examination of plasma-free cellular non-self reactions. Copyright © 2011 Elsevier Inc. All rights reserved.

Effect of acclimatization on hemocyte functional characteristics of the Pacific oyster (Crassostrea gigas) and carpet shell clam (Ruditapes decussatus).

PubMed

Hurtado, Miguel Ángel; da Silva, Patricia Mirella; Le Goïc, Nelly; Palacios, Elena; Soudant, Philippe

2011-12-01

Most experimental procedures on molluscs are done after acclimatization of wild animals to lab conditions. Similarly, short-term acclimation is often unavoidable in a field survey when biological analysis cannot be done within the day of sample collection. However, acclimatization can affect the general physiological condition and particularly the immune cell responses of molluscs. Our aim was to study the changes in the hemocyte characteristics of the Pacific oyster Crassostrea gigas and the carpet shell clam Ruditapes decussatus acclimated 1 or 2 days under emersed conditions at 14 ± 1 °C and for 1, 2, 7, or 10 days to flowing seawater conditions (submerged) at 9 ± 1 °C, when compared to hemolymph withdrawn from organisms sampled in the field and immediately analyzed in the laboratory (unacclimated). The hemocyte characteristics assessed by flow cytometry were the total (THC) and differential hemocyte count, percentage of dead cells, phagocytosis, and reactive oxygen species (ROS) production. Dead hemocytes were lower in oysters acclimated both in emersed and submerged conditions (1%-5%) compared to those sampled in the field (7%). Compared to oysters, the percentage of dead hemocytes was lower in clams (0.4% vs. 1.1%) and showed a tendency to decrease during acclimatization in both emersed and submerged conditions. In comparison to organisms not acclimated, the phagocytosis of hemocytes decreased in both oysters and clams acclimated under submerged conditions, but was similar in those acclimated in emersed conditions. The ROS production remained stable in both oysters and clams acclimated in emersed conditions, whereas in submerged conditions ROS production did not change in both the hyalinocytes and granulocytes of oysters, but increased in clams. In oysters, the THC decreased when they were acclimated 1 and 2 days in submerged conditions and was mainly caused by a decrease in granulocytes, but the decrease in THC in oysters acclimated 2 days in emersed conditions was caused by a decrease in hyalinocytes and small agranular cells. In clams, the THC was significantly lower in comparison to those not acclimated, regardless of the conditions of the acclimatization. These findings demonstrate that hemocyte characteristics were differentially affected in both species by the tested conditions of acclimatization. The phagocytosis and ROS production in clams and phagocytosis in oysters were not different in those acclimated for 1 day under both conditions, i.e. emersed and submerged, and those sampled in the field (unacclimated). The THC was significantly affected by acclimatization conditions, so the differences between clams and oysters should be considered in studies where important concentrations of hemocytes are required. The difference in the immune response between both species could be related to their habitat (epifaunal vs. infaunal) and their ability of resilience to manipulation and adaptation to captivity. Our results suggest that functional characteristics of hemocytes should be analyzed in both oysters and clams during the first 1 or 2 days, preferably acclimated under emersed rather than submerged conditions. Copyright © 2011. Published by Elsevier Ltd.

INFLUENCE OF SEASONAL FACTORS ON OYSTER HEMOCYTE KILLING OF VIBRIO PARAHEMOLYTICUS

EPA Science Inventory

Seasonal variation of cellular defenses of oyster Crassostrea virginica against Vibrio parahaemolyticus were examined from June 1997 to December 1998 using a recently developed bactericidal assay that utilizes a tetrazolium dye. Mean hemocyte numbers, plasma lysozyme, and P. mari...

IN VITRO KILLING OF PERKINSUS MARINUS BY HEMOCYTES OF OYSTERS CRASSOSTREA VIRGINICA

EPA Science Inventory

A colorimetric microbicidal assay was adapted, optimized and applied in experiments to characterize the in vitro capacity of eastern oyster (Crassostrea virginica) hemocytes to kill cultured isolates of Perkinsus marinus, a protozoan parasite causing a highly destructive disease...

Immune responses of mussel hemocyte subpopulations are differentially regulated by enzymes of the PI 3-K, PKC, and ERK kinase families.

PubMed

García-García, Erick; Prado-Alvarez, Maria; Novoa, Beatriz; Figueras, Antonio; Rosales, Carlos

2008-01-01

Various hemocyte cell types have been described in invertebrates, but for most species a functional characterization of different hemocyte cell types is still lacking. In order to characterize some immunological properties of mussel (Mytilus galloprovincialis) hemocytes, cells were separated by flow cytometry and their capacity for phagocytosis, production of reactive oxygen species (ROS), and production of nitric oxide (NO), was examined. Phosphatidylinositol 3-kinase (PI 3-K), protein kinase C (PKC), and extracellular signal-regulated kinase (ERK) inhibitors were also used to biochemically characterize these cell responses. Four morphologically distinct subpopulations, designated R1-R4, were detected. R1, R2, and R3 cells presented different levels of phagocytosis towards zymosan, latex beads, and two bacteria species. Similarly, R1 to R3, but not R4, cells produced ROS, while all subpopulations produced NO, in response to zymosan. Internalization of all phagocytic targets was blocked by PI 3-K inhibition. In addition, internalization of latex particles, but not of bacteria, was partially blocked by PKC or ERK inhibition. Interestingly, phagocytosis of zymosan was impaired by PKC, or ERK inhibitors, only in R2 cells. Zymosan-induced ROS production was blocked by PI 3-K inhibition, but not by PKC, or ERK inhibition. In addition, zymosan-stimulated NO production was affected by PI 3-K inhibition in R1 and R2, but not in R3 or R4 cells. NO production in all cell types was unaffected by PKC inhibition, but ERK inhibition blocked it in R2 cells. These data reveal the existence of profound functional and biochemical differences in mussel hemocytes and indicate that M. galloprovincialis hemocytes are specialized cells fulfilling specific tasks in the context of host defense.

Immune responses and ultrastructural changes of hemocytes in freshwater crab Sinopotamon henanense exposed to elevated cadmium.

PubMed

Qin, Qin; Qin, Shengjuan; Wang, Lan; Lei, Wenwen

2012-01-15

Cadmium (Cd) is one of the most toxic heavy metals that can impact immunological parameters in aquatic animals. To investigate the immunotoxicity and ultrastructural changes of hemocytes, specimens of Sinopotamon henanense were exposed to different concentrations of cadmium and the differences in immunologic parameters between Cd exposure groups and control groups were investigated. Total hemocyte count (THC) in Cd-exposure groups were decreased significantly when compared with the control groups, especially in the groups treated with higher Cd concentrations and longer exposure time, while no significant differences were observed in the proportions of the three types of hemocytes. Phenoloxidase (PO) activities were significantly higher in Cd-exposure groups than the control groups. Superoxide dismutase (SOD) activities gradually increased in 7.25 and 14.5 mg L⁻¹ Cd groups, but in other higher Cd groups, they showed first increase and following decrease with the exposure time prolonged. Acid phosphatase (ACP) activities were induced at 48 h, and then decreased, while alkaline phosphatase (AKP) activities increased gradually until 96 h. Electron microscopic results showed that nucleus, mitochondria and rough endoplasm recutulum (rER) of three types of hemocytes were sensitive to acute Cd toxicity. In Cd-exposed groups, chromatin condensation, nucleus deformation and nucleus envelope rupture were noted. Additionally, mitochondrial dilation and rER degranulation were observed in Cd-treated crabs. These results suggested that immune response and organelles of hemocyte of S. henanense were impacted by Cd exposure, and the changes of these immunologic parameters reflect changes in crab immune response capability consequent to Cd exposure. Copyright © 2011 Elsevier B.V. All rights reserved.

IN VITRO KILLING OF PERKINSUS MARINUS BY HEMOCYTES OF OYSTERS CRASSOSTREA VIRGINICA

EPA Science Inventory

Presented at the 92nd Annual Meeting of the National Shellfisheries Association, 19-23 March 2000, Seattle, WA.

A colorimetric microbicidal assay was adapted, optimized and used in experiments to characterize the capacity of eastern oyster (Crassostrea virginica) hemocytes...

Hemocytes control stem cell activity in the Drosophila intestine

PubMed Central

Ayyaz, Arshad; Li, Hongjie; Jasper, Heinrich

2015-01-01

SUMMARY Coordination of stem cell activity with inflammatory responses is critical for regeneration and homeostasis of barrier epithelia. The temporal sequence of cell interactions during injury-induced regeneration is only beginning to be understood. Here we show that intestinal stem cells (ISCs) are regulated by macrophage-like hemocytes during the early phase of regenerative responses of the Drosophila intestinal epithelium. Upon tissue damage, hemocytes are recruited to the intestine and secrete the TGFβ/BMP homologue Dpp, inducing ISC proliferation by activating the Type I receptor Saxophone and the Smad homologue Smox. Activated ISCs then switch their response to Dpp by inducing expression of Thickveins, a second Type I receptor that has previously been shown to re-establish ISC quiescence by activating Mad. The interaction between hemocytes and ISCs promotes infection resistance, but also contributes to the development of intestinal dysplasia in aging flies. We propose that similar interactions influence pathologies like inflammatory bowel disease and colorectal cancer in humans. PMID:26005834

Morphologic, cytometric and functional characterization of the common octopus (Octopus vulgaris) hemocytes.

PubMed

Castellanos-Martínez, S; Prado-Alvarez, M; Lobo-da-Cunha, A; Azevedo, C; Gestal, C

2014-05-01

The hemocytes of Octopus vulgaris were morphologically and functionally characterized. Light and electron microscopy (TEM and SEM), and flow cytometry analyses revealed the existence of two hemocyte populations. Large granulocytes showed U-shaped nucleus, a mean of 11.6 μm±1.2 in diameter with basophilic granules, polysaccharide and lysosomic deposits in the cytoplasm. Small granulocytes measured a mean of 8.1 μm±0.7 in diameter, and have a round nucleus occupying almost the entire cell and few or not granules in the cytoplasm. Flow cytometry analysis showed that large granulocytes are the principal cells that develop phagocytosis of latex beads (rising up to 56%) and ROS after zymosan stimulation. Zymosan induced the highest production of both ROS and NO. This study is the first tread towards understanding the O. vulgaris immune system by applying new tools to provide a most comprehensive morpho-functional study of their hemocytes. Copyright © 2013 Elsevier Ltd. All rights reserved.

The immune response of hemocytes of the insect Oncopeltus fasciatus against the flagellate Phytomonas serpens.

PubMed

Alves e Silva, Thiago L; Vasconcellos, Luiz R C; Lopes, Angela H; Souto-Padrón, Thaïs

2013-01-01

The genus Phytomonas includes parasites that are etiological agents of important plant diseases, especially in Central and South America. These parasites are transmitted to plants via the bite of an infected phytophagous hemipteran. Despite the economic impact of these parasites, many basic questions regarding the genus Phytomonas remain unanswered, such as the mechanism by which the parasites cope with the immune response of the insect vector. In this report, using a model of systemic infection, we describe the function of Oncopeltus fasciatus hemocytes in the immune response towards the tomato parasite Phytomonas serpens. Hemocytes respond to infection by trapping parasites in nodular structures and phagocytizing the parasites. In electron microscopy of hemocytes, parasites were located inside vacuoles, which appear fused with lysosomes. The parasites reached the O. fasciatus salivary glands at least six hours post-infection. After 72 hours post-infection, many parasites were attached to the salivary gland outer surface. Thus, the cellular responses did not kill all the parasites.

The Immune Response of Hemocytes of the Insect Oncopeltus fasciatus against the Flagellate Phytomonas serpens

PubMed Central

Alves e Silva, Thiago L.; Vasconcellos, Luiz R. C.

2013-01-01

The genus Phytomonas includes parasites that are etiological agents of important plant diseases, especially in Central and South America. These parasites are transmitted to plants via the bite of an infected phytophagous hemipteran. Despite the economic impact of these parasites, many basic questions regarding the genus Phytomonas remain unanswered, such as the mechanism by which the parasites cope with the immune response of the insect vector. In this report, using a model of systemic infection, we describe the function of Oncopeltus fasciatus hemocytes in the immune response towards the tomato parasite Phytomonas serpens. Hemocytes respond to infection by trapping parasites in nodular structures and phagocytizing the parasites. In electron microscopy of hemocytes, parasites were located inside vacuoles, which appear fused with lysosomes. The parasites reached the O. fasciatus salivary glands at least six hours post-infection. After 72 hours post-infection, many parasites were attached to the salivary gland outer surface. Thus, the cellular responses did not kill all the parasites. PMID:24015207

Immunohistochemical Analysis of the Role of Hemocytin in Nodule Formation in the Larvae of the Silkworm, Bombyx mori

PubMed Central

Arai, Ikuyo; Ohta, Masayuki; Suzuki, Asahi; Tanaka, Shiho; Yoshizawa, Yasutaka; Sato, Ryoichi

2013-01-01

Hemocytin, a multidomain protein from Bombyx mori L. (Lepidoptera: Bombycidae), is an ortholog of von Willebrand factor and is expected to be a major mediator of hemocyte aggregation. Antiserum was generated against hemocytin, and immune staining of hemocytes, hemolymph, and nodules was performed. Hemocytin was observed in steady-state hemocytes but not in plasma, even after bacterial injection. When hemolymph was smeared on glass slides, hemocytin-containing fibrous structures formed a cellular network mainly consisting of granulocytes and oenocytoids. Hemocytin was stained only in the granules of the granulocytes. When nodule-like aggregates formed 30 sec after bacterial injection, both granulocytes and bacterial cells were observed binding to hemocytin-containing fibrous structures. When nodule sections were stained with antiserum, hemocytin was seen in the matrix of the nodules surrounding the hemocytes. These data suggest that hemocytin plays a major role in nodule formation as a component of the sticky fibrous structure exocytosed from granulocytes. PMID:24766322

Effects of high temperature and exposure to air on mussel (Mytilus galloprovincialis, Lmk 1819) hemocyte phagocytosis: modulation of spreading and oxidative response.

PubMed

Mosca, Francesco; Narcisi, Valeria; Calzetta, Angela; Gioia, Luisa; Finoia, Maria G; Latini, Mario; Tiscar, Pietro G

2013-06-01

Hemocytes are a critical component of the mussel defense system and the present study aims at investigating their spreading and oxidative properties during phagocytosis under in vivo experimental stress conditions. The spreading ability was measured by an automated cell analyzer on the basis of the circularity, a parameter corresponding to the hemocyte roundness. The oxidative activity was investigated by micromethod assay, measuring the respiratory burst as expression of the fluorescence generated by the oxidation of specific probe. Following the application of high temperature and exposure to air, there was evidence of negative modulation of spreading and oxidative response, as revealed by a cell roundness increase and fluorescence generation decrease. Therefore, the fall of respiratory burst appeared as matched with the inhibition of hemocyte morphological activation, suggesting a potential depression of the phagocytosis process and confirming the application of the circularity parameter as potential stress marker, both in experimental and field studies. Copyright © 2012 Elsevier Ltd. All rights reserved.

Serratia marcescens Induces Apoptotic Cell Death in Host Immune Cells via a Lipopolysaccharide- and Flagella-dependent Mechanism*

PubMed Central

Ishii, Kenichi; Adachi, Tatsuo; Imamura, Katsutoshi; Takano, Shinya; Usui, Kimihito; Suzuki, Kazushi; Hamamoto, Hiroshi; Watanabe, Takeshi; Sekimizu, Kazuhisa

2012-01-01

Injection of Serratia marcescens into the blood (hemolymph) of the silkworm, Bombyx mori, induced the activation of c-Jun NH2-terminal kinase (JNK), followed by caspase activation and apoptosis of blood cells (hemocytes). This process impaired the innate immune response in which pathogen cell wall components, such as glucan, stimulate hemocytes, leading to the activation of insect cytokine paralytic peptide. S. marcescens induced apoptotic cell death of silkworm hemocytes and mouse peritoneal macrophages in vitro. We searched for S. marcescens transposon mutants with attenuated ability to induce apoptosis of silkworm hemocytes. Among the genes identified, disruption mutants of wecA (a gene involved in lipopolysaccharide O-antigen synthesis), and flhD and fliR (essential genes in flagella synthesis) showed reduced motility and impaired induction of mouse macrophage cell death. These findings suggest that S. marcescens induces apoptosis of host immune cells via lipopolysaccharide- and flagella-dependent motility, leading to the suppression of host innate immunity. PMID:22859304

Rac1 mediates cytokine-stimulated hemocyte spreading via prostaglandin biosynthesis in the beet armyworm, Spodoptera exigua

USDA-ARS?s Scientific Manuscript database

Cell spreading is an integral component of insect hemocytic immune reactions to infections and invasions. Cell spreading is accomplished by cytoskeleton rearrangement, which is activated by three major immune mediators, biogenic monoamines, plasmatocyte-spreading peptide (PSP), and eicosanoids, part...

Oyster hemocyte mobilization and increased adhesion activity after beta glucan administration

USDA-ARS?s Scientific Manuscript database

In the eastern oyster (Crassostrea virginica) hemocytes are important effector cells for maintenance of defense against pathogenic microorganisms. Various forms of ß-glucans have been suggested for use in shrimp and fish aquaculture because of their potential to enhance disease resistance via hemoc...

RESPONSES OF OYSTERS AND THEIR HEMOCYTES TO CLINICAL AND ENVIRONMENTAL ISOLATES OF VIBRIO PARAHAEMOLYTICUS

EPA Science Inventory

Interactions of Vibrio parahaemolyticus with oysters and oyster hemocytes were studied using three environmental isolates (1094, 1163 and ATCC 17802) and three clinical isolates (2030, 2062, 2107). Clinical isolates were from patients who became ill during the June 1998 food pois...

Immunocompetence of bivalve hemocytes as evaluated by a miniaturized phagocytosis assay.

PubMed

Blaise, C; Trottier, S; Gagné, F; Lallement, C; Hansen, P-D

2002-01-01

Immune function in bivalves can be adversely affected by long-term exposure to environmental contaminants. Investigating alterations in immunity can therefore yield relevant information about the relationship between exposure to environmental contaminants and susceptibility to infectious diseases. We have developed a rapid, cost-effective, and miniaturized immunocompetence assay to evaluate the phagocytic activity, viability, and concentration of hemocytes in freshwater and marine bivalves. Preliminary experiments were performed to optimize various aspects of the assay including 1) the time required for adherence of hemocytes to polystyrene microplate wells, 2) the time required for internalization of fluorescent bacteria, 3) the ratio of hemocytes to fluorescent bacteria in relation to phagocytosis, 4) hemolymph plasma requirements, and 5) the elimination of fluorescence from (noninternalized) bacteria adhering to the external surface of hemocytes. The results of these experiments showed the optimal adherence time for hemocytes in microplate wells to be 1 h, that phagocytosis required at least 2 h of contact with fluorescently labeled E. coli cells, that the number of fluorescent E. coli cells had a positive effect on phagocytic activity, that at least 2.5 million cells/mL were required to measure a significant intake, and that a linear increase in uptake of bacteria (R = 0.91; p < 0.01) could be obtained with concentrations of up to 1.3 x 10(6) hemocytes/mL. Afterward, the assay was used in two field studies to identify sites having the potential to affect the immunocompetence of bivalves. The first study was conducted on Mya arenaria clams collected at selected contaminated sites in the Saguenay River (Quebec, Canada), and the second examined Elliptio complanata freshwater bivalves that had been exposed to a municipal effluent plume in the St. Lawrence River (Quebec, Canada). In the Saguenay River field study a significant increase in phagocytosis was observed at sites closest to polluted areas. Phagocytotic activity varied over time and was highest during the warmest months (June, July, and August), closely paralleling the spawning period of Mya arenaria clams. In contrast, a drop in phagocytic activity was observed in Elliptio complanata mussels exposed to surface water 4 km downstream of a major municipal effluent plume, with a concomitant increase in the number of hemocytes in the hemolymph. It appears that both immunosuppressive and immunostimulative effects are likely to occur in the field and that responses will be influenced by the type and intensity of contaminants at play. Copyright 2002 Wiley Periodicals, Inc.

Identification of expressed genes in cDNA library of hemocytes from the RLO-challenged oyster, Crassostrea ariakensis Gould with special functional implication of three complement-related fragments (CaC1q1, CaC1q2 and CaC3).

PubMed

Xu, Ting; Xie, Jiasong; Li, Jianming; Luo, Ming; Ye, Shigen; Wu, Xinzhong

2012-06-01

A SMARTer™ cDNA library of hemocyte from Rickettsia-like organism (RLO) challenged oyster, Crassostrea ariakensis Gould was constructed. Random clones (400) were selected and single-pass sequenced, resulted in 200 unique sequences containing 96 known genes and 104 unknown genes. The 96 known genes were categorized into 11 groups based on their biological process. Furthermore, we identified and characterized three complement-related fragments (CaC1q1, CaC1q2 and CaC3). Tissue distribution analysis revealed that all of three fragments were ubiquitously expressed in all tissues studied including hemocyte, gills, mantle, digestive glands, gonads and adductor muscle, while the highest level was seen in the hemocyte. Temporal expression profile in the hemocyte monolayers reveled that the mRNA expression levels of three fragments presented huge increase after the RLO incubation at 3 h and 6 h in post-challenge, respectively. And the maximal expression levels at 3 h in post-challenge are about 256, 104 and 64 times higher than the values detected in the control of CaC1q1, CaC1q2 and CaC3, respectively. Copyright © 2012 Elsevier Ltd. All rights reserved.

Effects of the endoparasitoid Cotesia chilonis (Hymenoptera: Braconidae) parasitism, venom, and calyx fluid on cellular and humoral immunity of its host Chilo suppressalis (Lepidoptera: Crambidae) larvae.

PubMed

Teng, Zi-Wen; Xu, Gang; Gan, Shi-Yu; Chen, Xuan; Fang, Qi; Ye, Gong-Yin

2016-02-01

The larval endoparasitoid Cotesia chilonis injects venom and bracoviruses into its host Chilo suppressalis during oviposition. Here we study the effects of the polydnavirus (PDV)-carrying endoparasitoid C. chilonis (Hymenoptera: Braconidae) parasitism, venom and calyx fluid on host cellular and humoral immunity, specifically hemocyte composition, cellular spreading, encapsulation and melanization. Total hemocyte counts (THCs) were higher in parasitized larvae than in unparasitized larvae in the late stages following parasitization. While both plasmatocyte and granulocyte fractions and hemocyte mortality did not differ between parasitized and unparasitized hosts, in vitro spreading behavior of hemocytes was inhibited significantly by parasitism throughout the course of parasitoid development. C. chilonis parasitism suppressed the encapsulation response and melanization in the early stages. Venom alone did not alter cellular immune responses, including effects on THCs, mortality, hemocyte composition, cell spreading and encapsulation, but venom did inhibit humoral immunity by reducing melanization within 6h after injection. In contrast to venom, calyx fluid had a significant effect on cell spreading, encapsulation and melanization from 6h after injection. Dose-response injection studies indicated the effects of venom and calyx fluid synergized, showing a stronger and more persistent reduction in immune system responses than the effect of either injected alone. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Imidacloprid intensifies its impact on honeybee and bumblebee cellular immune response when challenged with LPS (lippopolysacharide) of Escherichia coli.

PubMed

Walderdorff, Louise; Laval-Gilly, Philippe; Bonnefoy, Antoine; Falla-Angel, Jaïro

2018-07-01

Insect hemocytes play an important role in insects' defense against environmental stressors as they are entirely dependent on their innate immune system for pathogen defense. In recent years a dramatic decline of pollinators has been reported in many countries. The drivers of this declines appear to be associated with pathogen infections like viruses, bacteria or fungi in combination with pesticide exposure. The aim of this study was thus to investigate the impact of imidacloprid, a neonicotinoid insecticide, on the cellular immune response of two pollinators (Apis mellifera and Bombus terrestris) during simultaneous immune activation with LPS (lipopolysaccharide) of Escherichia coli. For this purpose the phagocytosis capacity as well as the production of H 2 O 2 and NO of larval hemocytes, exposed to five different imidacloprid concentrations in vitro, was measured. All used pesticide concentrations showed a weakening effect on phagocytosis with but also without LPS activation. Imidacloprid decreased H 2 O 2 and increased NO production in honeybees. Immune activation by LPS clearly reinforced the effect of imidacloprid on the immune response of hemocytes in all three immune parameters tested. Bumblebee hemocytes appeared more sensitive to imidacloprid during phagocytosis assays while imidacloprid showed a greater impact on honeybee hemocytes during H 2 O 2 and NO production. Copyright © 2018 Elsevier Ltd. All rights reserved.

Drosophila Embryos as Model Systems for Monitoring Bacterial Infection in Real Time

PubMed Central

Evans, Iwan R.; Waterfield, Nicholas; ffrench-Constant, Richard H.; Wood, Will

2009-01-01

Drosophila embryos are well studied developmental microcosms that have been used extensively as models for early development and more recently wound repair. Here we extend this work by looking at embryos as model systems for following bacterial infection in real time. We examine the behaviour of injected pathogenic (Photorhabdus asymbiotica) and non-pathogenic (Escherichia coli) bacteria and their interaction with embryonic hemocytes using time-lapse confocal microscopy. We find that embryonic hemocytes both recognise and phagocytose injected wild type, non-pathogenic E. coli in a Dscam independent manner, proving that embryonic hemocytes are phagocytically competent. In contrast, injection of bacterial cells of the insect pathogen Photorhabdus leads to a rapid ‘freezing’ phenotype of the hemocytes associated with significant rearrangement of the actin cytoskeleton. This freezing phenotype can be phenocopied by either injection of the purified insecticidal toxin Makes Caterpillars Floppy 1 (Mcf1) or by recombinant E. coli expressing the mcf1 gene. Mcf1 mediated hemocyte freezing is shibire dependent, suggesting that endocytosis is required for Mcf1 toxicity and can be modulated by dominant negative or constitutively active Rac expression, suggesting early and unexpected effects of Mcf1 on the actin cytoskeleton. Together these data show how Drosophila embryos can be used to track bacterial infection in real time and how mutant analysis can be used to genetically dissect the effects of specific bacterial virulence factors. PMID:19609447

RAPID TETRAZOLIUM DYE REDUCTION ASSAY TO ASSESS THE BACTERICIDAL ACTIVITY OF OYSTER (CRASSOSTREA VIRGINICA) HEMOCYTES AGAINST VIBRIO PARAHAEMOLYTICUS

EPA Science Inventory

An assay was developed to assess the ability of oyster, Crassostrea virginica, hemocytes to kill the human pathogenic bacterium, Vibrio parahaemolyticus (ATCC 17802). Bacterial killing was estimated colorimetrically by the enzymatic reduction of a tetrazolium dye, 3-(4,5-dimethyl...

[The effect in vitro of the toxins of the mushroom Metarrhizium anisopliae (Metsch.) Sorok. on the hemocytic reaction of Oryctes rhinoceros L. (Coleoptera)].

PubMed

Vey, A; Quiot, J M

1975-02-17

The filtrate of Metarhizium cultures contains toxic-substances inhibiting the process of formation of granuloma by Oryctes in vitro. This inhibition results from marked alterations of both nucleus and cytoplasm of hemocytes under the action of the fungal toxins.

Substantial changes in hemocyte parameters of Manila clam Ruditapes philippinarum two years after the Hebei Spirit oil spill off the west coast of Korea.

PubMed

Hong, Hyun-Ki; Donaghy, Ludovic; Kang, Chang-Keun; Kang, Hyun-Sil; Lee, Hee-Jung; Park, Heung-Sik; Choi, Kwang-Sik

2016-07-15

Two years after the Hebei Spirit oil spill occurred off the west coast of Korea, we determined sub-lethal effects of the spilled oil on hemocyte parameters of Ruditapes philippinarum in the damaged areas. Clams in the spilled sites displayed unusually high proportion of granulocytes, which may result in higher phagocytosis capacity and reactive oxygen species production. Hemocytes in clams from the polluted sites also displayed less DNA damage and mortality than in the control site, possibly due to a faster phagocytosis of the impaired cells. Glycogen, the major energetic reserve, was depleted in clams from the spilled sites, potentially due to energetic consumption for maintenance of a large pool of granulocytes, detoxification processes and oxidative stress. Modified hemocyte parameters in clams in the spilled area, may reflect sub-lethal physiological stresses caused by the residual oils in the sediment, in conjunction with environmental modifications such as food availability and pathogens pattern. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effect of dopamine injection on the hemocyte count and prophenoloxidase system of the white shrimp Litopenaeus vannamei

NASA Astrophysics Data System (ADS)

Pan, Luqing; Hu, Fawen; Zheng, Debin

2011-09-01

Effects of dopamine injection on the hemocyte count, phenoloxidase activity, serine proteinase activity, proteinase inhibitor activity and α2-macroglobulin-like activity in L. vannamei were studied. Results showed that dopamine injection resulted in a significant effect on the parameters measured ( P < 0.05), while no significant difference was observed in the control group (0.85% NaCl). In the experimental groups, the hemocyte count reached the minimum in 3 h; granular and semi-granular cells became stable after 12 h and hyaline cells and the total hemocyte count became stable after 18 h. Phenoloxidase activity reached the minimum in 6 h, and then became stable after 9 h. Serine protease activity and proteinase inhibitor activity reached the minimum in 3 h, and α2-macroglobulin-like activity reached the maximum in 3 h, and all the three parameters became stable after 12 h. The results suggest that the activating mechanisms of the proPO system triggered by dopamine are different from those triggered by invasive agents or spontaneously activated under a normal physical condition.

[Agglutination and phagocytosis of foreign abiotic particles by hemocytes of the blowely, Calliphora vicina in vivo. I. Dynamics of hemocyte activity during larval development].

PubMed

Kind, T V

2005-01-01

Three types of Calliphora larval hemocytes have been revealed to be involved in phagocytosis of abiotic foreign particles: thrombocytoids, larval plasmatocytes and plasmatocytes I. Thrombocytoids are the quickest to respond to the appearance of invaders. The onset of test particle entrapment by thrombocytoid cytoplasmic fragments was observed, depending on the larval age within 0.5-5.0 min after injection. Separated fragments were fused, forming strands or roundish agglutinates. Phagocytosis of carbon, carmine or Indian ink particles by larval plasmatocytes occurs far more lately, and no earlier than 20-30 min after injection. Plasmatocytes I are capable of foreign particles adhesion on their surface, with a subsequent morule formation, and of engulfing these particles. These two events start in different time periods: adhesion occurs in 5-10 min, while phagocytosis is observed in 1--3 h. The rate of test particle entrapment and stability of agglutinales clearly depends on the larval age. The most pronounced reaction of hemocytes to foreign particles may be observed by the end of feeding and crop emptying. The second, somewhat less expressed rise of activity occurs in mature larvae not long before the onset of pupariation. Diapause induction is accompanied by reducing activities of both plasmatocytes and thrompocytoids. The importance of different hemocyte types in cellular immune reaction of Calliphora vicina larvae, and co-ordination between plasmatocytes and thrombocytoids are discussed.

Hematopoiesis and hematopoietic organs in arthropods.

PubMed

Grigorian, Melina; Hartenstein, Volker

2013-03-01

Hemocytes (blood cells) are motile cells that move throughout the extracellular space and that exist in all clades of the animal kingdom. Hemocytes play an important role in shaping the extracellular environment and in the immune response. Developmentally, hemocytes are closely related to the epithelial cells lining the vascular system (endothelia) and the body cavity (mesothelia). In vertebrates and insects, common progenitors, called hemangioblasts, give rise to the endothelia and blood cells. In the adult animal, many differentiated hemocytes seem to retain the ability to proliferate; however, in most cases investigated closely, the bulk of hemocyte proliferation takes place in specialized hematopoietic organs. Hematopoietic organs provide an environment where undifferentiated blood stem cells are able to self-renew, and at the same time generate offspring that differentiate into different blood cell types. Hematopoiesis in vertebrates, taking place in the bone marrow, has been subject to intensive research by immunologists and stem cell biologists. Much less is known about blood cell formation in invertebrate animals. In this review, we will survey structural and functional properties of invertebrate hematopoietic organs, with a main focus on insects and other arthropod taxa. We will then discuss similarities, at the molecular and structural level, that are apparent when comparing the development of blood cells in hematopoietic organs of vertebrates and arthropods. Our comparative review is intended to elucidate aspects of the biology of blood stem cells that are more easily missed when focusing on one or a few model species.

TAT improves in vitro transportation of fortilin through midgut and into hemocytes of white shrimp Litopenaeus vannamei

NASA Astrophysics Data System (ADS)

Zhou, Yi; Zhang, Wenbing; Mai, Kangsen; Xu, Wei; Zhang, Yanjiao; Ai, Qinghui; Wang, Xiaojie

2012-06-01

Fortilin is a multifunctional protein implicated in many important cellular processes. Since injection of Pm-fortilin reduces shrimp mortality caused by white spot syndrome virus (WSSV), there is potential application of fortilin in shrimp culture. In the present study, in order to improve trans-membrane transportation efficiency, the protein transduction domain of the transactivator of transcription (TAT) peptide was fused to fortilin. The Pichia pastoris yeast expression system, which is widely accepted in animal feeds, was used for production of recombinant fusion protein. Green fluorescence protein (GFP) was selected as a reporter because of its intrinsic visible fluorescence. The fortilin, TAT and GFP fusion protein were constructed. Their trans-membrane transportation efficiency and effects on immune response of shrimp were analyzed in vitro. Results showed that TAT peptide improved in vitro uptake of fortilin into the hemocytes and midgut of Litopenaeus vannamei. The phenoloxidase (PO) activity of hemocytes incubated with GFP-Fortilin or GFP-Fortilin-TAT was significantly increased compared with that in the control without expressed fortilin. The PO activity of hemocytes incubated with 200 μg mL-1 GFP-Fortilin-TAT was significantly higher than that in the group with the same concentration of GFP-Fortilin. Hemocytes incubated with GFP-Fortilin-TAT at all concentrations showed significantly higher nitric oxide synthase (NOS) activity than those in the control or in the GFP-Fortilin treatment. The present in vitro study indicated that TAT fusion protein improved the immune effect of fortilin.

Studying longterm effects of micro gravity on basic immune functions - The development of an application based on the measuring of phagocytosis activity of Blue Mussel hemocytes

NASA Astrophysics Data System (ADS)

Unruh, Eckehardt

The immunsystem of astronauts exposed to microgravity is declining. Whether this effect is caused by microgravity or in combination with cosmic radiation is so far not clear. The immune system of vertebrates has several defence strategies but the basic immune response (Phagocytosis) is present as well in invertebrates. Phagocytotic cells are drawn by chemotaxis to the origin of an infection. By adhesion, ingestion and phagosome formation foreign particles, bacteria etc are transported inside of a cell were they are destroyed by native powerful biocides. Related to this biocide production is the formation of Reactive Oxygen Species (ROS). ROS can be measured by luminescence. The effects of microgravity will be simultaneously tested by exposure of phagocytotic hemocytes on orbit under microgravity, artificial gravity and, on ground under natural gravity. To address this purpose defined pools of Blue Mussel (Mytilus edulis) hemocytes will be launched frozen to the ISS. References for all batches will stay on ground. Shortly after arrival and then in three-month intervals batches of the same pool will be thawed and reconstituted. The phagocytosis related production of ROS will be stimulated with opsonized Zymosan. Luminescence will be measured and the data will be sent to ground. The experiment is scheduled for the Columbus Biolab early 2009. In preparation of this flight experiments the following procedures were investigated and the results will be presented: - a protocol for the cryoconservation and reconstituton of blue mussel hemocytes. - preliminary results of phagocytosis activity by reconstituted hemocytes after cryo-conservation and hemocytes without cryo-conservation treatment. The TRIPLELUX-B Experiment contributes to risk assessment concerning longterm immunotoxicity under space flight conditions. The immune system of invertebrates has not been studied so far in space. The choice of the phagocytes from invertebrates is justified by the claim to study the universal validity of innate immune responses.

Immunomodulation by different types of N-oxides in the hemocytes of the marine bivalve Mytilus galloprovincialis.

PubMed

Ciacci, Caterina; Canonico, Barbara; Bilaniĉovă, Dagmar; Fabbri, Rita; Cortese, Katia; Gallo, Gabriella; Marcomini, Antonio; Pojana, Giulio; Canesi, Laura

2012-01-01

The potential toxicity of engineered nanoparticles (NPs) for humans and the environment represents an emerging issue. Since the aquatic environment represents the ultimate sink for NP deposition, the development of suitable assays is needed to evaluate the potential impact of NPs on aquatic biota. The immune system is a sensitive target for NPs, and conservation of innate immunity represents an useful basis for studying common biological responses to NPs. Suspension-feeding invertebrates, such as bivalves, are particularly at risk to NP exposure, since they have extremely developed systems for uptake of nano and microscale particles integral to intracellular digestion and cellular immunity. Evaluation of the effects of NPs on functional parameters of bivalve immunocytes, the hemocytes, may help understanding the major toxic mechanisms and modes of actions that could be relevant for different NP types in aquatic organisms.In this work, a battery of assays was applied to the hemocytes of the marine bivalve Mytilus galloprovincialis to compare the in vitro effects of different n-oxides (n-TiO(2), n-SiO(2), n-ZnO, n-CeO(2)) chosen on the basis of their commercial and environmental relevance. Physico-chemical characterization of both primary particles and NP suspensions in artificial sea water-ASW was performed. Hemocyte lysosomal and mitochondrial parameters, oxyradical and nitric oxide production, phagocytic activity, as well as NP uptake, were evaluated. The results show that different n-oxides rapidly elicited differential responses hemocytes in relation to their chemical properties, concentration, behavior in sea water, and interactions with subcellular compartments. These represent the most extensive data so far available on the effects of NPs in the cells of aquatic organisms. The results indicate that Mytilus hemocytes can be utilized as a suitable model for screening the potential effects of NPs in the cells of aquatic invertebrates, and may provide a basis for future experimental work for designing environmentally safer nanomaterials.

Immunomodulation by Different Types of N-Oxides in the Hemocytes of the Marine Bivalve Mytilus galloprovincialis

PubMed Central

Ciacci, Caterina; Canonico, Barbara; Bilaniĉovă, Dagmar; Fabbri, Rita; Cortese, Katia; Gallo, Gabriella; Marcomini, Antonio; Pojana, Giulio; Canesi, Laura

2012-01-01

The potential toxicity of engineered nanoparticles (NPs) for humans and the environment represents an emerging issue. Since the aquatic environment represents the ultimate sink for NP deposition, the development of suitable assays is needed to evaluate the potential impact of NPs on aquatic biota. The immune system is a sensitive target for NPs, and conservation of innate immunity represents an useful basis for studying common biological responses to NPs. Suspension-feeding invertebrates, such as bivalves, are particularly at risk to NP exposure, since they have extremely developed systems for uptake of nano and microscale particles integral to intracellular digestion and cellular immunity. Evaluation of the effects of NPs on functional parameters of bivalve immunocytes, the hemocytes, may help understanding the major toxic mechanisms and modes of actions that could be relevant for different NP types in aquatic organisms.In this work, a battery of assays was applied to the hemocytes of the marine bivalve Mytilus galloprovincialis to compare the in vitro effects of different n-oxides (n-TiO2, n-SiO2, n-ZnO, n-CeO2) chosen on the basis of their commercial and environmental relevance. Physico-chemical characterization of both primary particles and NP suspensions in artificial sea water-ASW was performed. Hemocyte lysosomal and mitochondrial parameters, oxyradical and nitric oxide production, phagocytic activity, as well as NP uptake, were evaluated. The results show that different n-oxides rapidly elicited differential responses hemocytes in relation to their chemical properties, concentration, behavior in sea water, and interactions with subcellular compartments. These represent the most extensive data so far available on the effects of NPs in the cells of aquatic organisms. The results indicate that Mytilus hemocytes can be utilized as a suitable model for screening the potential effects of NPs in the cells of aquatic invertebrates, and may provide a basis for future experimental work for designing environmentally safer nanomaterials. PMID:22606310

Transcriptional analysis of disk abalone (Haliotis discus discus) antioxidant enzymes against marine bacteria and virus challenge.

PubMed

De Zoysa, Mahanama; Whang, Ilson; Nikapitiya, Chamilani; Oh, Chulhong; Choi, Cheol Young; Lee, Jehee

2011-07-01

Diverse antioxidant enzymes are essential for marine organisms to overcome oxidative stress as well as for the fine-tuning of immune reactions through activating different signal transduction pathways. This study describes the transcriptional analysis of antioxidant enzymes of disk abalone by challenging with bacteria (Vibrio alginolyticus, Vibrio parahemolyticus, and Listeria monocytogenes) and viral hemorrhagic septicemia virus (VHSV). Upon bacteria and VHSV challenge, Manganese superoxide dismutase (MnSOD), Copper, Zinc superoxide dismutase (CuZnSOD), catalase, thioredoxin peroxidase (TPx), Selenium-dependent glutathione peroxidase (SeGPx), and thioredoxin-2 (TRx-2) expression levels were altered in gills, and hemocytes at different magnitudes. In gills, only MnSOD, catalase, and SeGPx genes were completely upregulated by post-challenge of bacterial and VHSV. Among them, SeGPx demonstrated strong upregulation by 16-fold (bacteria) and 2-fold (VHSV) in gills, and 5-fold (bacteria) and 3.0-fold (VHSV) in hemocytes. None of the genes examined were downregulated (in gills and hemocytes) by bacteria challenge even though CuZnSOD and TPx showed downregulation (completely) in hemocytes by VHSV. In general, abalone hemocytes had lower potential to induce antioxidant enzyme transcripts upon bacteria and VHSV challenge than gills. Based upon these results, we suggest that abalones induce oxidative stress in tissues during the bacteria and VHSV challenge, and the identified response of antioxidant enzymes could be supported for maintaining a low-level of reactive oxygen species (ROS) that may serve as a signal for activating immune reactions against pathogenic conditions. Copyright © 2011 Elsevier Ltd. All rights reserved.

Hemocytic rickettsia-like organisms in ticks: serologic reactivity with antisera to Ehrlichiae and detection of DNA of agent of human granulocytic ehrlichiosis by PCR.

PubMed Central

Magnarelli, L A; Stafford, K C; Mather, T N; Yeh, M T; Horn, K D; Dumler, J S

1995-01-01

Ixodid ticks were collected from Connecticut, Massachusetts, Missouri, Pennsylvania, Rhode Island, and British Columbia (Canada) during 1991 to 1994 to determine the prevalence of infection with hemocytic (blood cell), rickettsia-like organisms. Hemolymph obtained from these ticks was analyzed by direct and indirect fluorescent antibody (FA) staining methods with dog, horse, or human sera containing antibodies to Ehrlichia canis, Ehrlichia equi, or Rickettsia rickettsii. Of the 693 nymphal and adult Amblyomma americanum, Dermacentor variabilis, Ixodes scapularis, and Ixodes pacificus ticks tested with dog anti-E. canis antiserum, 209 (32.5%) contained hemocytic bacteria. The prevalence of infected ticks varied greatly with species and locale. In parallel tests of duplicate hemolymph preparations from adult I. scapularis ticks, the hemocytic organisms reacted positively with E. canis and/or E. equi antisera, including sera from persons who had granulocytic ehrlichiosis. In separate PCR analyses, DNA of the agent of human granulocytic ehrlichiosis was detected in 59 (50.0%) of 118 adult and in 1 of 2 nymphal I. scapularis ticks tested from Connecticut. There was no evidence of Ehrlichia chaffeensis DNA in these ticks. In indirect FA tests of hemolymph for spotted fever group rickettsiae, the overall prevalence of infection was less than 4%. Specificity tests of antigens and antisera used in these studies revealed no cross-reactivity between E. canis and E. equi or between any of the ehrlichial reagents and those of R. rickettsii. The geographic distribution of hemocytic microorganisms with shared antigens to Ehrlichia species or spotted fever group rickettsiae is widespread. PMID:8567911

Honey bee hemocyte profiling by flow cytometry.

PubMed

Marringa, William J; Krueger, Michael J; Burritt, Nancy L; Burritt, James B

2014-01-01

Multiple stress factors in honey bees are causing loss of bee colonies worldwide. Several infectious agents of bees are believed to contribute to this problem. The mechanisms of honey bee immunity are not completely understood, in part due to limited information about the types and abundances of hemocytes that help bees resist disease. Our study utilized flow cytometry and microscopy to examine populations of hemolymph particulates in honey bees. We found bee hemolymph includes permeabilized cells, plasmatocytes, and acellular objects that resemble microparticles, listed in order of increasing abundance. The permeabilized cells and plasmatocytes showed unexpected differences with respect to properties of the plasma membrane and labeling with annexin V. Both permeabilized cells and plasmatocytes failed to show measurable mitochondrial membrane potential by flow cytometry using the JC-1 probe. Our results suggest hemolymph particulate populations are dynamic, revealing significant differences when comparing individual hive members, and when comparing colonies exposed to diverse conditions. Shifts in hemocyte populations in bees likely represent changing conditions or metabolic differences of colony members. A better understanding of hemocyte profiles may provide insight into physiological responses of honey bees to stress factors, some of which may be related to colony failure.

Honey Bee Hemocyte Profiling by Flow Cytometry

PubMed Central

Marringa, William J.; Krueger, Michael J.; Burritt, Nancy L.; Burritt, James B.

2014-01-01

Multiple stress factors in honey bees are causing loss of bee colonies worldwide. Several infectious agents of bees are believed to contribute to this problem. The mechanisms of honey bee immunity are not completely understood, in part due to limited information about the types and abundances of hemocytes that help bees resist disease. Our study utilized flow cytometry and microscopy to examine populations of hemolymph particulates in honey bees. We found bee hemolymph includes permeabilized cells, plasmatocytes, and acellular objects that resemble microparticles, listed in order of increasing abundance. The permeabilized cells and plasmatocytes showed unexpected differences with respect to properties of the plasma membrane and labeling with annexin V. Both permeabilized cells and plasmatocytes failed to show measurable mitochondrial membrane potential by flow cytometry using the JC-1 probe. Our results suggest hemolymph particulate populations are dynamic, revealing significant differences when comparing individual hive members, and when comparing colonies exposed to diverse conditions. Shifts in hemocyte populations in bees likely represent changing conditions or metabolic differences of colony members. A better understanding of hemocyte profiles may provide insight into physiological responses of honey bees to stress factors, some of which may be related to colony failure. PMID:25285798

Effects of the brominated flame retardant tetrabromobisphenol-A (TBBPA) on cell signaling and function of Mytilus hemocytes: involvement of MAP kinases and protein kinase C.

PubMed

Canesi, Laura; Lorusso, Lucia Cecilia; Ciacci, Caterina; Betti, Michele; Gallo, Gabriella

2005-11-10

Brominated flame retardants (BFRs) are a large group of compounds added to or applied as a treatment to polymeric materials to prevent fires. Tetrabisphenol A (TBBPA) is the most important individual BFR used in industry. Although TBBPA and its derivatives can be found in environmental samples, data are very limited on the presence of this compound in biota. Research on mammals indicates that TBBPA has low toxicity in vivo; however, in vitro TBBPA can act as a cytotoxicant, neurotoxicant, immunotoxicant, thyroid hormone agonist and has a weak estrogenic activity; in particular, the effects of TBBPA have been recently ascribed to its interactions with cellular signaling pathways, in particular with mitogen activated protein kinases (MAPKs). TBBPA has high acute toxicity to aquatic organisms, such as algae, molluscs, crustaceans and fish; however, little is known on the mechanisms of action of this compound in the cells of aquatic species. In this work, we investigated the possible effects and mechanisms of action of TBBPA on the immune cells, the hemocytes, of the marine mussel Mytilus galloprovincialis. The results demonstrate that TBBPA in the low micromolar range induces hemocyte lysosomal membrane destabilization. The effect was reduced or prevented by hemocyte pre-treatment by specific inhibitors of MAPKs and of protein kinase C (PKC). TBBPA stimulated phosphorylation of MAPK members and PKC, as evaluated by electrophoresis and Western blotting with anti-phospho-antibodies, although to a different extent and with distinct time-courses. A rapid (from 5 min) and transient increase in phosphoryation of the stress-activated JNK MAPKs and of PKC was observed, followed by a later increase (at 30-60 min) in phosphorylation of extracellularly regulated MAPKs (ERK2 MAPK) and of the stress-activated p38 MAPK. TBBPA significantly stimulated the hemocyte microbicidal activity towards E. coli, lysosomal enzyme release, phagocytic activity and extracellular superoxide (O2-) production. The results demonstrate that TBBPA in vitro activates the immune function of mussel hemocytes through kinase-mediated cell signaling and that common transduction pathways are involved in mediating the effects of this BFR in mammalian and aquatic invertebrate cells.

Investigation of microgravity effects on basic imune functions on the cellular level - The TRIPLELUX-B experiment

NASA Astrophysics Data System (ADS)

Unruh, Eckehardt; Hansen, Peter-Diedrich

Hemocytes are the primary defence of the Blue Mussel against invading microorganisms and foreign particles. The hemocytes of mussels as part of the immune system of invertebrates has not been studied so far in space. The choice of the phagocytes from invertebrates is justified by the claim to study the universal validity of innate immune responses. The hemocytes of mussels have a lot in common with macrophages of higher organisms. They are able to detect the presence of microorganisms and kill these microorganisms by phagocytosis. The phagocy-tosis related production of ROS will be stimulated with opsonised zymosan. The hemocytes will be stored frozen and reconstituted in-flight for the experiment. The signals of the im-muno cellular responses are translated into luminescence as a rapid optical reporter system. The primary aim of Triplelux B is to investigate under space flight conditions the effect of microgravity on the ability of isolated Blue Mussel hemocytes to perform phagocytosis. As a secondery objectiv, the results expected will allow to conclude whether the observed responses are caused by microgravity and/or radiation (change in permeability, endpoints in genotoxicity: DNA unwinding). The TRIPLELUX-B Experiment contributes to risk assessment concerning immunotoxicity under space flight conditions. The components of the fully automated AEC (Advanced Experimental Containment) will be demonstrated. The AEC of the TRIPLELUX-B experiment will contribute to a real time operational monitoring for immunotoxicity testing for earth. Blue mussels have been used repeatedly for monitoring imunotoxicity and genotoxicity in coastal waters. Based on the AEC an automatet measuring device will allow "real time monitoring" providing observations of immunotoxicity in coastal and inland waters.

The Hemolymph of the ascidian Styela plicata (Chordata-Tunicata) contains heparin inside basophil-like cells and a unique sulfated galactoglucan in the plasma.

PubMed

de Barros, Cintia M; Andrade, Leonardo R; Allodi, Silvana; Viskov, Christian; Mourier, Pierre A; Cavalcante, Moisés C M; Straus, Anita H; Takahashi, Helio K; Pomin, Vitor H; Carvalho, Vinicius F; Martins, Marco A; Pavão, Mauro S G

2007-01-19

The hemolymph of ascidians (Chordata-Tunicata) contains different types of hemocytes embedded in a liquid plasma. In the present study, heparin and a sulfated heteropolysaccharide were purified from the hemolymph of the ascidian Styela plicata. The heteropolysaccharide occurs free in the plasma, is composed of glucose ( approximately 60%) and galactose ( approximately 40%), and is highly sulfated. Heparin, on the other hand, occurs in the hemocytes, and high performance liquid chromatography of the products formed by degradation with specific lyases revealed that it is composed mainly by the disaccharides DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4)) (39.7%) and DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(6SO(4)) (38.2%). Small amounts of the 3-O-sulfated disaccharides DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(3SO(4)) (9.8%) and DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(3SO(4))(6SO(4)) (3.8%) were also detected. These 3-O-sulfated disaccharides were demonstrated to be essential for the binding of the hemocyte heparin to antithrombin III. Electron microscopy techniques were used to characterize the ultrastructure of the hemocytes and to localize heparin and histamine in these cells. At least five cell types were recognized and classified as univacuolated and multivacuolated cells, amebocytes, hemoblasts, and granulocytes. Immunocytochemistry showed that heparin and histamine co-localize in intracellular granules of only one type of hemocyte, the granulocyte. These results show for the first time that in ascidians, a sulfated galactoglucan circulates free in the plasma, and heparin occurs as an intracellular product of a circulating basophil-like cell.

The crustin-like peptide plays opposite role in shrimp immune response to Vibrio alginolyticus and white spot syndrome virus (WSSV) infection.

PubMed

Sun, Baozhen; Wang, Zhi; Zhu, Fei

2017-07-01

Crustin is an antimicrobial peptide (AMP) that plays a key role in innate immunity of crustaceans. In this study, we cloned the entire 660 bp crustin-like sequence with a 507 bp open reading frame encoding a 168 amino acid from Marsupenaeus japonicus. The crustin-like gene was primarily expressed in gills and over-expressed in shrimp hemocytes after challenge with WSSV or Vibrio alginolyticus. After knockdown crustin-like gene using specific double-stranded RNA (CRU-dsRNA), IMD, Rab7, L-lectin, mitogen-activated protein kinase, p53, prophenoloxidase and Rho were down-regulated and nitric oxide synthase, myosin and tumor necrosis factor-α were up-regulated in hemocytes at 24 h post dsRNA treatment. After WSSV challenge, The mortality, WSSV copy number and expressions of WSSV immediate early genes (IE1, IE2, DNA polymerase, VP28) were both decreased but the apoptosis rate was increased in CRU-dsRNA-treated shrimps, indicating that WSSV may take advantage of crustin-like to benefit its replication. After silenced the crustin-like, the results of phagocytosis showed that the phagocytic rate of shrimp hemocytes on WSSV decreased significantly. In contrast, the absence of crustin-like in shrimps increased the mortality following V. alginolyticus challenge, indicating that crustin-like may play a positive role in the antibacterial process. The phagocytosis experiment showed there was a higher phagocytosis rate of hemocytes after CRU-dsRNA treatment. The result indicated that V. alginolyticus may be able to use crustin-like to avoid phagocytosis of shrimp hemocytes. These results further added to our understanding of the function of crustin-like peptide and also provided its potential role in innate immunity in shrimp. Copyright © 2017 Elsevier Ltd. All rights reserved.

Endogenous Molecules Induced by a Pathogen-Associated Molecular Pattern (PAMP) Elicit Innate Immunity in Shrimp

PubMed Central

Chen, Yu-Yuan; Chen, Jiann-Chu; Lin, Yong-Chin; Kitikiew, Suwaree; Li, Hui-Fang; Bai, Jia-Chin; Tseng, Kuei-Chi; Lin, Bo-Wei; Liu, Po-Chun; Shi, Yin-Ze; Kuo, Yi-Hsuan; Chang, Yu-Hsuan

2014-01-01

Invertebrates rely on an innate immune system to combat invading pathogens. The system is initiated in the presence of cell wall components from microbes like lipopolysaccharide (LPS), β-1,3-glucan (βG) and peptidoglycan (PG), altogether known as pathogen-associated molecular patterns (PAMPs), via a recognition of pattern recognition protein (PRP) or receptor (PRR) through complicated reactions. We show herein that shrimp hemocytes incubated with LPS, βG, and PG caused necrosis and released endogenous molecules (EMs), namely EM-L, EM-β, and EM-P, and found that shrimp hemocytes incubated with EM-L, EM-β, and EM-P caused changes in cell viability, degranulation and necrosis of hemocytes, and increased phenoloxidase (PO) activity and respiratory burst (RB) indicating activation of immunity in vitro. We found that shrimp receiving EM-L, EM-β, and EM-P had increases in hemocyte count and other immune parameters as well as higher phagocytic activity toward a Vibrio pathogen, and found that shrimp receiving EM-L had increases in proliferation cell ratio and mitotic index of hematopoietic tissues (HPTs). We identified proteins of EMs deduced from SDS-PAGE and LC-ESI-MS/MS analyses. EM-L and EM-P contained damage-associated molecular patterns (DAMPs) including HMGBa, HMGBb, histone 2A (H2A), H2B, and H4, and other proteins including proPO, Rab 7 GPTase, and Rab 11 GPTase, which were not observed in controls (EM-C, hemocytes incubated in shrimp salt solution). We concluded that EMs induced by PAMPs contain DAMPs and other immune molecules, and they could elicit innate immunity in shrimp. Further research is needed to identify which individual molecule or combined molecules of EMs cause the results, and determine the mechanism of action in innate immunity. PMID:25517999

In vivo effects of metaldehyde on Pacific oyster, Crassostrea gigas: comparing hemocyte parameters in two oyster families.

PubMed

Moreau, Pierrick; Burgeot, Thierry; Renault, Tristan

2015-06-01

Pollutants via run-off into the ocean represent a potential threat to marine organisms, especially bivalves such as oysters living in coastal environments. These organisms filter large volumes of seawater and may accumulate contaminants within their tissues. Pesticide contamination in water could have a direct or indirect toxic action on tissues or cells and could induce alteration of immune system. Bivalve immunity is mainly supported by hemocytes and participates directly by phagocytosis to eliminate pathogens. Some studies have shown that pesticides can reduce immune defences and/or modify genomes in vertebrates and invertebrates. Metaldehyde is used to kill slugs, snails and other terrestrial gastropods. Although metaldehyde has been detected in surface waters, its effects on marine bivalves including the Pacific oyster, Crassostrea gigas, have never been studied. Given the mode of action of this molecule and its targets (molluscs), it could be potentially more toxic to oysters than other pesticides (herbicides, fungicides, insecticides, etc.). Effects of metaldehyde on oyster hemocyte parameters were thus monitored through in vivo experiments based on a short-term exposure. In this work, metaldehyde at 0.1 μg/L, which corresponds to an average concentration detected in the environment, modulated hemocyte activities of Pacific oysters after an in vivo short-term contact. Individuals belonging to two families showed different behaviours for some hemocyte activities after contamination by metaldehyde. These results suggested that effects of pollutants on oysters may differ from an individual to another in relation to genetic diversity. Finally, it appears essential to take an interest in the effects of metaldehyde on a wide variety of aquatic invertebrates including those that have a significant economic impact.

A shell-formation related carbonic anhydrase in Crassostrea gigas modulates intracellular calcium against CO2 exposure: Implication for impacts of ocean acidification on mollusk calcification.

PubMed

Wang, Xiudan; Wang, Mengqiang; Jia, Zhihao; Song, Xiaorui; Wang, Lingling; Song, Linsheng

2017-08-01

Ocean acidification (OA) could decrease the shells and skeletons formation of mollusk by reducing the availability of carbonate ions at calcification sites. Carbonic anhydrases (CAs) convert CO 2 to HCO 3 - and play important roles in biomineralization process from invertebrate to vertebrate. In the present study, a CA (designated as CgCA) was identified and characterized in Pacific oyster C. gigas. The cDNA of CgCA was of 927bp encoding a predicted polypeptide of 308 amino acids with a signal peptide and a CA catalytic function domain. The mRNA transcripts of CgCA were constitutively expressed in all tested tissues with the highest levels in mantle and hemocytes. During the early development period, the mRNA transcripts of CgCA could be detected in all the stages with the highest level in D-veliger larvae. Elevated CO 2 increased the mRNA transcripts of CgCA in muscle, mantle, hepatopancreas, gill and hemocytes significantly (p<0.05) and induced the translocation of CgCA in hemocytes and mantle. Moreover, elevated CO 2 also caused the decrease of intracellular Ca 2+ in hemocytes (p<0.05). The inhibition of CA by acetazolamide and suppression of CgCA gene via RNA interference could increase the intracellular Ca 2+ in hemocytes (p<0.05). Besides, the decrease of intracellular Ca 2+ content caused by Ca 2+ reagent ionomycin could affect localization of CgCA in mantle tissue. The results indicated CgCA played essential roles in calcification and elevated CO 2 accelerated the mutual modulation between calcium and CgCA, implying reduced calcification rate and dissolved shells under OA. Copyright © 2017 Elsevier B.V. All rights reserved.

Differential Protein Expression in the Hemolymph of Bithynia siamensis goniomphalos Infected with Opisthorchis viverrini

PubMed Central

Suwannatrai, Kulwadee; Suwannatrai, Apiporn; Tabsripair, Pairat; Welbat, Jariya Umka; Tangkawattana, Sirikachorn; Cantacessi, Cinzia; Mulvenna, Jason; Tesana, Smarn; Loukas, Alex

2016-01-01

Bithynia siamensis goniomphalos is a freshwater snail that serves as the first intermediate host of the human liver fluke Opisthorchis viverrini. This parasite is a major public health problem in different countries throughout the Greater Mekong sub-region (Thailand, southern Vietnam, Lao PDR and Cambodia). Chronic O. viverrini infection also results in a gradual increase of fibrotic tissues in the biliary tract that are associated with hepatobiliary diseases and contribute to cholangiocarcinoma (a fatal type of bile duct cancer). Infectivity of the parasite in the snail host is strongly correlated with destruction of helminths by the snail’s innate immune system, composed of cellular (hemocyte) and humoral (plasma) defense factors. To better understand this important host-parasite interface we applied sequential window acquisition of all theoretical spectra mass spectrometry (SWATH-MS) to identify and quantify the proteins from the hemolymph of B. siamensis goniomphalos experimentally infected with O. viverrini and compare them to non-infected snails (control group). A total of 362 and 242 proteins were identified in the hemocytes and plasma, respectively. Of these, 145 and 117 proteins exhibited significant differences in expression upon fluke infection in hemocytes and plasma, respectively. Among the proteins with significantly different expression patterns, we found proteins related to immune response (up-regulated in both hemocyte and plasma of infected snails) and proteins belonging to the structural and motor group (mostly down-regulated in hemocytes but up-regulated in plasma of infected snails). The proteins identified and quantified in this work will provide important information for the understanding of the factors involved in snail defense against O. viverrini and might facilitate the development of new strategies to control O. viverrini infection in endemic areas. PMID:27893749

Immunotoxicity of surface waters contaminated by municipal effluents to the snail Lymnaea stagnalis.

PubMed

Gust, M; Fortier, M; Garric, J; Fournier, M; Gagné, F

2013-01-15

The immunotoxic effects of surface waters contaminated by a municipal effluent dispersion plume were examined in the snail Lymnaea stagnalis. Snails were exposed to surface waters where changes in hemocyte counts, viability, levels of reactive oxygen species (ROS), reduced thiols and phagocytic activity were tracked following exposure periods of 3h and 3 and 7d. Changes in mRNA expression of some genes in the hemocytes were also assessed after 7d of exposure, as follows: genes coding for catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GSR), selenium-dependent glutathione peroxidase (SeGPX), two isoforms of the nitric oxide synthetase (NOS1 and NOS2), molluscan defensive molecule (MDM), toll-like receptor 4 (TLR4), allograft inflammatory factor-1 (AIF), and heat-shock protein 70 (HSP70). At the sites closest to the discharge point, exposure led to impaired hemocyte viability and intracellular thiol levels and also an increase of hemocyte count, ROS levels and phagocytosis. Phagocytosis and ROS levels in hemocytes were correlated with heterotrophic bacterial counts in snails. We found four genes with increased mRNA expression as a response to exposure of municipal wastewaters: TLR4 (6-fold), HSP70 (2-fold), SeGPx (4-fold) and CAT (2-fold). Immunocompetence responses were analyzed by canonical analysis to seek out relationships with mRNA expression of the genes involved in stress, pattern recognition, cellular and humoral responses. The data revealed that genes involved in oxidative stress were strongly involved with immunocompetence and that the resulting immune responses were influenced both by the bacterial and pollutant loadings of the effluent. Copyright © 2012 Elsevier B.V. All rights reserved.

Oxidative stress and cytotoxicity elicited lipid peroxidation in hemocytes of Bombyx mori larva infested with dipteran parasitoid, Exorista bombycis.

PubMed

Pooja, Makwana; Pradeep, Appukuttan Nair R; Hungund, Shambhavi P; Sagar, Chandrashekhar; Ponnuvel, Kangayam M; Awasthi, Arvind K; Trivedy, Kanika

2017-12-20

Parasitization of silkworm, Bombyx mori by invasive larva of dipteran parasitoid Exorista bombycis caused upto 20% revenue loss in sericulture. The parasitism was successful by suppressing host immune system however mechanism of immune suppression induced by E. bombycis is unknown which is unravelled here. The infestation induced cytotoxic symptoms in host hemocytes, such as vacuolated cytoplasm, porous plasma membrane, indented nuclei with condensed chromatin and dilated RER. One of the markers of necrosis is cell permeabilization, which can be measured as released lactate dehydrogenase (LDH). LDH level showed significantly (P<0.01) high release into extracellular medium in vitro after exposure of hemocytes to parasitoid larval tissue protein compared with control revealing membrane permeability and loss of cell integrity. At five minutes after exposure, cytotoxicity was 43% and was increased to 99% at 3h. The cytotoxicity is signalled by increased content of hydrogen peroxide (H2O2) causing lipid peroxidation followed by porosity in plasma membrane. A test for lipid peroxidation by measurement of lipid peroxidation breakdown product, malondialdehyde (MDA) revealed significant increase in peroxidation from one to 24 h post-invasion, with maximum at 12 h (P<0.008). Level of reactive oxygen species measured as H2O2 production increased from 6 to 12 h post-invasion and continued to increase significantly (P<0.03) reaching maximum at 48 h. These observations reveal that dipteran endoparasitoid invasion induced H2O2 production in the hemocytes causing cytotoxicity, lipid peroxidation and membrane porosity that suppressed both humoral- and cell-mediated immune responses of hemocytes in B. mori.

Over-Expression of Superoxide Dismutase Ameliorates Cr(VI) Induced Adverse Effects via Modulating Cellular Immune System of Drosophila melanogaster

PubMed Central

Pragya, Prakash; Shukla, Arvind Kumar; Murthy, Ramesh Chandra; Abdin, Malik Zainul; Kar Chowdhuri, Debapratim

2014-01-01

The evolutionarily conserved innate immune system plays critical role for maintaining the health of an organism. However, a number of environmental chemicals including metals are known to exert adverse effects on immune system. The present study assessed the in vivo effect of a major environmental chemical, Cr(VI), on cellular immune response using Drosophila melanogaster and subsequently the protective role of superoxide dismutase (SOD) based on the comparable performance of the tested anti-oxidant enzymes. The immuno-modulatory potential of Cr(VI) was demonstrated by observing a significant reduction in the total hemocyte count along with impaired phagocytic activity in exposed organism. Concurrently, a significant increase in the percentage of Annexin V-FITC positive cells, activation of DEVDase activity, generation of free radical species along with inhibition of anti-oxidant enzyme activities was observed in the hemocytes of exposed organism. In addition, we have shown that ONOO− is primarily responsible for Cr(VI) induced adverse effects on Drosophila hemocytes along with O2 −. While generation of O2 −/ONOO− in Cr(VI) exposed Drosophila hemocytes was found to be responsible for the suppression of Drosophila cellular immune response, Cr(VI) induced alteration was significantly reduced by the over-expression of sod in Drosophila hemocytes. Overall, our results suggest that manipulation of one of the anti-oxidant genes, sod, benefits the organism from Cr(VI) induced alteration in cellular immunity. Further, this study demonstrates the applicability of D. melanogaster to examine the possible effects of environmental chemicals on innate immunity which can be extrapolated to higher organisms due to evolutionary conservation of innate immune system between Drosophila and mammals. PMID:24505420

Comparative venom toxicity between Pteromalus puparum and Nasonia vitripennis (Hymenoptera: Pteromalidae) toward the hemocytes of their natural hosts, non-target insects and cultured insect cells.

PubMed

Zhang, Zhong; Ye, Gong-Yin; Cai, Jun; Hu, Cui

2005-09-01

Crude venoms from two parasitoid species, Pteromalus puparum and Nasonia vitripennis (Hymenoptera: Pteromalidae) were assayed for biological activities toward hemocytes from two species of their natural hosts and eight species of their non-natural hosts as well as two lines of cultured Lepidoptera cells, respectively. By inhibiting the spreading and viability of insect hemocytes, the venom from P. puparum displayed significantly higher activities toward plasmatocytes and granular cells from both larvae and pupae of two natural hosts, Pieris rapae and Papilio xuthus, and lower activity toward those from Spodoptera litura, Musca domestica and Sarcophaga peregrina. However, no effect was found towards any type of hemocytes from other five insects tested, namely, Ectropis oblique, Galleria mellonella, Sesamia inferens, Bombyx mori and Parnara guttata. In contrast, the venom from N. vitripennis showed a narrower range of targeted insects. It appeared to have highly adverse effects on the spreading and viability of plasmatocytes and granular cells only from the natural hosts, M. domestica and S. peregrina, little toxicity to cells from P. rapae and P. xuthus, and no effect on any of the other insects tested. Pteromalus puparum venom also apparently presented a high ability to block the spreading of Tn-5B1-4 cells derived from Trichoplusia ni, and high cytotoxicity to the cells and Ha cells derived from Helicoverpa armigera. Nasonia vitripennis venom, however, only had a marked lethal effect to Ha cells. In addition, the possibility that the host range of a defined parasitoid could be assessed using our method of treating hemocytes from candidate insects with venom in vitro, and the potential of our venoms tested in the development of bio-insecticides, insect-resistant transgenic plants, are discussed.

Characterization of a Monoclonal Antibody Directed against Mytilus spp Larvae Reveals an Antigen Involved in Shell Biomineralization

PubMed Central

Calvo-Iglesias, Juan; Pérez-Estévez, Daniel; Lorenzo-Abalde, Silvia; Sánchez-Correa, Beatriz; Quiroga, María Isabel; Fuentes, José M.; González-Fernández, África

2016-01-01

The M22.8 monoclonal antibody (mAb) developed against an antigen expressed at the mussel larval and postlarval stages of Mytilus galloprovincialis was studied on adult samples. Antigenic characterization by Western blot showed that the antigen MSP22.8 has a restricted distribution that includes mantle edge tissue, extrapallial fluid, extrapallial fluid hemocytes, and the shell organic matrix of adult samples. Other tissues such as central mantle, gonadal tissue, digestive gland, labial palps, foot, and byssal retractor muscle did not express the antigen. Immunohistochemistry assays identified MSP22.8 in cells located in the outer fold epithelium of the mantle edge up to the pallial line. Flow cytometry analysis showed that hemocytes from the extrapallial fluid also contain the antigen intracellularly. Furthermore, hemocytes from hemolymph have the ability to internalize the antigen when exposed to a cell-free extrapallial fluid solution. Our findings indicate that hemocytes could play an important role in the biomineralization process and, as a consequence, they have been included in a model of shell formation. This is the first report concerning a protein secreted by the mantle edge into the extrapallial space and how it becomes part of the shell matrix framework in M. galloprovincialis mussels. PMID:27008638

Multixenobiotic resistance in Mytilus edulis: Molecular and functional characterization of an ABCG2- type transporter in hemocytes and gills.

PubMed

Ben Cheikh, Yosra; Xuereb, Benoit; Boulangé-Lecomte, Céline; Le Foll, Frank

2018-02-01

Among the cellular protection arsenal, ABC transporters play an important role in xenobiotic efflux in marine organisms. Two pumps belonging to B and C subfamily has been identified in Mytilus edulis. In this study, we investigated the presence of the third major subtype ABCG2/BCRP protein in mussel tissues. Transcript was expressed in hemocytes and with higher level in gills. Molecular characterization revealed that mussel ABCG2 transporter shares the sequence and organizational structure with mammalian and molluscan orthologs. Overall identity of the predicted amino acid sequence with corresponding homologs from other organisms was between 49% and 98%. Moreover, protein efflux activity was demonstrated using a combination of fluorescent allocrites and specific inhibitors. The accumulation of bodipy prazosin and pheophorbide A was heterogeneous in gills and hemocytes. Most of the used blockers enhanced probe accumulation at different levels, most significantly for bodipy prazosin. Moreover, Mrp classical blocker MK571 showed a polyspecificity. In conclusion, our data demonstrate that several ABC transporters contribute to MXR phenotype in the blue mussel including ABCG2 that forms an active pump in hemocytes and gills. Efforts are needed to distinguish between the different members and to explore their single function and specificity towards allocrites and chemosensitizers. Copyright © 2017 Elsevier B.V. All rights reserved.

Comprehensive Genetic Dissection of the Hemocyte Immune Response in the Malaria Mosquito Anopheles gambiae

PubMed Central

Lombardo, Fabrizio; Ghani, Yasmeen; Kafatos, Fotis C.; Christophides, George K.

2013-01-01

Reverse genetics in the mosquito Anopheles gambiae by RNAi mediated gene silencing has led in recent years to an advanced understanding of the mosquito immune response against infections with bacteria and malaria parasites. We developed RNAi screens in An. gambiae hemocyte-like cells using a library of double-stranded RNAs targeting 109 genes expressed highly or specifically in mosquito hemocytes to identify novel regulators of the hemocyte immune response. Assays included phagocytosis of bacterial bioparticles, expression of the antimicrobial peptide CEC1, and basal and induced expression of the mosquito complement factor LRIM1. A cell viability screen was also carried out to assess dsRNA cytotoxicity and to identify genes involved in cell growth and survival. Our results identify 22 novel immune regulators, including proteins putatively involved in phagosome assembly and maturation (Ca2+ channel, v-ATPase and cyclin-dependent protein kinase), pattern recognition (fibrinogen-domain lectins and Nimrod), immune modulation (peptidase and serine protease homolog), immune signaling (Eiger and LPS-induced factor), cell adhesion and communication (Laminin B1 and Ninjurin) and immune homeostasis (Lipophorin receptor). The development of robust functional cell-based assays paves the way for genome-wide functional screens to study the mosquito immune response to infections with human pathogens. PMID:23382679

Molecular cloning of crustins from the hemocytes of Brazilian penaeid shrimps.

PubMed

Rosa, Rafael Diego; Bandeira, Paula Terra; Barracco, Margherita Anna

2007-09-01

Crustins are antimicrobial peptides initially identified in the hemocytes of the crab Carcinus maenas (11.5-kDa peptide or carcinin) and recently also recognized in penaeid shrimps and other crustacean species. The aim of this study was to identify sequences encoding for crustins from the hemocytes of four Brazilian penaeid species: Farfantepenaeus paulensis, Farfantepenaeus subtilis, Farfantepenaeus brasiliensis and Litopenaeus schmitti. Using primers based on consensus nucleotide alignment of crustins from different crustaceans, cDNA sequences coding for crustins in all indigenous penaeid species were amplified. The obtained four crustin sequences encoded for peptides containing a hydrophobic N-terminal region rich in glycine repeats and a C-terminal part with 12 cysteine residues and a conserved whey acidic protein domain. All obtained crustin sequences showed high amino acidic similarity among each other and with crustins from litopenaeid shrimps (76-98%). This is the first report of crustins in native Brazilian penaeid shrimps.

Relevant principal factors affecting the reproducibility of insect primary culture.

PubMed

Ogata, Norichika; Iwabuchi, Kikuo

2017-06-01

The primary culture of insect cells often suffers from problems with poor reproducibility in the quality of the final cell preparations. The cellular composition of the explants (cell number and cell types), surgical methods (surgical duration and surgical isolation), and physiological and genetic differences between donors may be critical factors affecting the reproducibility of culture. However, little is known about where biological variation (interindividual differences between donors) ends and technical variation (variance in replication of culture conditions) begins. In this study, we cultured larval fat bodies from the Japanese rhinoceros beetle, Allomyrina dichotoma, and evaluated, using linear mixed models, the effect of interindividual variation between donors on the reproducibility of the culture. We also performed transcriptome analysis of the hemocyte-like cells mainly seen in the cultures using RNA sequencing and ultrastructural analyses of hemocytes using a transmission electron microscope, revealing that the cultured cells have many characteristics of insect hemocytes.

Regulation of Drosophila hematopoietic sites by Activin-β from active sensory neurons

PubMed Central

Makhijani, Kalpana; Alexander, Brandy; Rao, Deepti; Petraki, Sophia; Herboso, Leire; Kukar, Katelyn; Batool, Itrat; Wachner, Stephanie; Gold, Katrina S.; Wong, Corinna; O’Connor, Michael B.; Brückner, Katja

2017-01-01

An outstanding question in animal development, tissue homeostasis and disease is how cell populations adapt to sensory inputs. During Drosophila larval development, hematopoietic sites are in direct contact with sensory neuron clusters of the peripheral nervous system (PNS), and blood cells (hemocytes) require the PNS for their survival and recruitment to these microenvironments, known as Hematopoietic Pockets. Here we report that Activin-β, a TGF-β family ligand, is expressed by sensory neurons of the PNS and regulates the proliferation and adhesion of hemocytes. These hemocyte responses depend on PNS activity, as shown by agonist treatment and transient silencing of sensory neurons. Activin-β has a key role in this regulation, which is apparent from reporter expression and mutant analyses. This mechanism of local sensory neurons controlling blood cell adaptation invites evolutionary parallels with vertebrate hematopoietic progenitors and the independent myeloid system of tissue macrophages, whose regulation by local microenvironments remain undefined. PMID:28748922

Analysis of the shapes of hemocytes of Callista brevisiphonata in vitro (Bivalvia, Veneridae).

PubMed

Karetin, Yu A; Pushchin, I I

2015-08-01

Fractal formalism in conjunction with linear methods of image analysis is suitable for the comparative analysis of such "irregular" shapes (from the point of view of classical Euclidean geometry) as flattened amoeboid cells of invertebrates in vitro. Cell morphology of in vitro spreading hemocytes from the bivalve mollusc Callista brevisiphonata was analyzed using correlation, factor and cluster analysis. Four significantly different cell types were identified on the basis of 36 linear and nonlinear parameters. The analysis confirmed the adequacy of the selected methodology for numerical description of the shape and the adequacy of classification of nonlinear shapes of spread hemocytes belonging to the same species. Investigation has practical significance for the description of the morphology of cultured cells, since cell shape is a result of summation of a number of extracellular and intracellular factors. © 2015 International Society for Advancement of Cytometry.

Inter-Cellular Forces Orchestrate Contact Inhibition of Locomotion

PubMed Central

Davis, John R.; Luchici, Andrei; Mosis, Fuad; Thackery, James; Salazar, Jesus A.; Mao, Yanlan; Dunn, Graham A.; Betz, Timo; Miodownik, Mark; Stramer, Brian M.

2015-01-01

Summary Contact inhibition of locomotion (CIL) is a multifaceted process that causes many cell types to repel each other upon collision. During development, this seemingly uncoordinated reaction is a critical driver of cellular dispersion within embryonic tissues. Here, we show that Drosophila hemocytes require a precisely orchestrated CIL response for their developmental dispersal. Hemocyte collision and subsequent repulsion involves a stereotyped sequence of kinematic stages that are modulated by global changes in cytoskeletal dynamics. Tracking actin retrograde flow within hemocytes in vivo reveals synchronous reorganization of colliding actin networks through engagement of an inter-cellular adhesion. This inter-cellular actin-clutch leads to a subsequent build-up in lamellar tension, triggering the development of a transient stress fiber, which orchestrates cellular repulsion. Our findings reveal that the physical coupling of the flowing actin networks during CIL acts as a mechanotransducer, allowing cells to haptically sense each other and coordinate their behaviors. PMID:25799385

Wash functions downstream of Rho1 GTPase in a subset of Drosophila immune cell developmental migrations

PubMed Central

Verboon, Jeffrey M.; Rahe, Travis K.; Rodriguez-Mesa, Evelyn; Parkhurst, Susan M.

2015-01-01

Drosophila immune cells, the hemocytes, undergo four stereotypical developmental migrations to populate the embryo, where they provide immune reconnoitering, as well as a number of non–immune-related functions necessary for proper embryogenesis. Here, we describe a role for Rho1 in one of these developmental migrations in which posteriorly located hemocytes migrate toward the head. This migration requires the interaction of Rho1 with its downstream effector Wash, a Wiskott–Aldrich syndrome family protein. Both Wash knockdown and a Rho1 transgene harboring a mutation that prevents Wash binding exhibit the same developmental migratory defect as Rho1 knockdown. Wash activates the Arp2/3 complex, whose activity is needed for this migration, whereas members of the WASH regulatory complex (SWIP, Strumpellin, and CCDC53) are not. Our results suggest a WASH complex–independent signaling pathway to regulate the cytoskeleton during a subset of hemocyte developmental migrations. PMID:25739458

Effects of Copper on Hemocyte Apoptosis, ROS Production, and Gene Expression in White Shrimp Litopenaeus vannamei.

PubMed

Guo, Hui; Li, Kexu; Wang, Wei; Wang, Chenggui; Shen, Yuchun

2017-10-01

Copper, a common chemical contaminant in aquatic environment, is known to be toxic to aquatic life at high concentrations. In the present study, we evaluated the apoptotic cell ratio and ROS production in hemocytes of the white shrimp Litopenaeus vannamei exposed to 1 or 5 mg L -1 Cu for 0, 3, 6, 12, 24, and 48 h. The expression changes of antioxidant biomarker genes, i.e., copper-zinc superoxide dismutase (Cu-Zn SOD) and catalase (CAT), apoptosis-related genes, i.e., caspase-3 and inhibitor of apoptosis protein (IAP), and a specific biomarker gene of heavy metal pollution, i.e., metallothionein (MT), were also determined in hemocytes. Significant increases in ROS production were observed in both treatment groups at each time points. The apoptotic cell ratios were significantly increased at 6-48 h among shrimp exposed to 1 mg L -1 Cu and at each time points in 5 mg L -1 Cu group. These results indicated that Cu would induce oxidative stress and apoptosis in the hemocyte of L. vannamei. Quantitative real-time PCR analysis revealed that the relative expression levels of Cu-Zn SOD, CAT, caspase-3, IAP, and MT were upregulated in a dose-dependent and time-dependent manner, suggesting the involvement of these genes in stress response against Cu exposure.

Impact of fluorescent silicon nanoparticles on circulating hemolymph and hematopoiesis in an invertebrate model organism.

PubMed

Xing, Rui; Li, Kai-Le; Zhou, Yan-Feng; Su, Yuan-Yuan; Yan, Si-Qi; Zhang, Kai-Long; Wu, Si-Cong; Sima, Yang-Hu; Zhang, Ke-Qin; He, Yao; Xu, Shi-Qing

2016-09-01

Silicon nanoparticles (SiNPs) have attractive potential applications in biological and medical fields, and yet their impact on animals is still controversial, and there have been no reports of their effects on hematopoiesis. In this study, the effects of SiNPs on hemocytes and hematopoiesis were investigated by administering SiNPs via a vascular injection into an invertebrate model, the silkworm. Our results show that the ability of SiNPs to enter different types of circulating hemocytes and their impact on those hemocytes differed significantly. Rapid accumulation of SiNPs was observed in granulocytes, oenocytoids, and spherulocytes, which have immune functions in the circulating hemolymph, whereas SiNPs did not easily enter prohemocytes, which can differentiate into granulocytes, oenocytoids, and spherulocytes and replenish them. The SiNPs that entered the hemocytes initiated autophagy and apoptosis via the lysosomal/mitochondrial pathway. High-dose SiNPs weakly stimulated lysosomal activity in hematopoietic organs, but did not lead to a significant increase in reactive oxygen species or severe autophagy or apoptosis in the organ tissues. We suggest that the damage caused by high-dose SiNPs to hematopoiesis is self-healing, because few SiNPs entered the hematopoietic stem cells in the circulating hemolymph, so the damage to the hematopoietic tissues was limited. Copyright © 2016 Elsevier Ltd. All rights reserved.

Nitric oxide production by hemocytes of the ascidian Styela plicata.

PubMed

de Barros, Cintia Monteiro; de Carvalho, Danielle Ronald; Andrade, Leonardo R; Pavão, Mauro Sérgio G; Allodi, Silvana

2009-10-01

Ascidian hemolymph contains various types of blood cells (hemocytes), which are believed to be involved in defense mechanisms. We have studied nitric-oxide (NO) synthase activity in hemocytes of the ascidian Styela plicata after exposure to lipopolysaccharide (LPS). To investigate which cell types are involved in NO production, we first identified, by electron microscopy, the types of hemocytes previously described, mainly by light microscopy, by others. Five types of blood cells could be recognized in the hemolymph: granulocytes, hemoblasts, lymphocyte-like cells, morula cells, and pigment cells. The lymphocyte-like cells produced the most NO. In agreement with studies of other invertebrates, nitrite generation did not change after LPS stimulation in assays in vitro, under either different concentrations of LPS or different time periods. Therefore, we performed an in vivo assay by injecting a known quantity of Escherichia coli into the tunic of the ascidians in order to investigate possible differences in NO levels. No increase of NO occurred accompanying the inflammatory reaction suggesting that another molecule in the pathway was involved. We found that nuclear factor kappaB (NFkappaB) was activated. Since NFkappaB is involved in the production of many substances related to immune responses, additional molecules might also be generated in response to E. coli infection. These observations may improve our understanding of the reaction of animals to eutrophic conditions.

In Situ Subcellular Imaging of Copper and Zinc in Contaminated Oysters Revealed by Nanoscale Secondary Ion Mass Spectrometry.

PubMed

Weng, Nanyan; Jiang, Haibo; Wang, Wen-Xiong

2017-12-19

Determining the in situ localization of trace elements at high lateral resolution levels in the biological system is very challenging, but critical for our understanding of metal sequestration and detoxification. Here, the cellular and subcellular distributions of Cu and Zn in contaminated oysters of Crassostrea hongkongensis were for the first time mapped using nanoscale secondary ion mass spectrometry (nanoSIMS). Three types of metal-containing cells were revealed in the gill and mantle of oysters, including Cu-specific hemocytes, Cu and Zn-containing granular hemocytes, and Cu and Zn-containing calcium cells. Obvious intercellular distribution of Cu was found in the gill tissue, indicating the potential role of hemolymph in the transportation of Cu in oysters. The distribution of Cu showed a strong colocalization with sulfur and nitrogen in Cu-specific hemocyte and intercellular hemolymph. In the Cu and Zn-containing granular hemocytes and calcium cells, the co-occurrence of Cu and Zn with phosphorus and calcium was also found. Different relationships of distributions between Cu/Zn and macronutrient elements (nitrogen, sulfur and phosphorus) implied the differential metal complexation in oysters. Interestingly, quantitative analysis of the ratios of 32 S - / 12 C 14 N - and 31 P - / 12 C 14 N - of metal-deposited sites suggested the dynamic process of transfer of Cu and Zn from the metabolized protein pool to a more thermodynamically stable and detoxified form.

Comparative studies on the internal defense system of schistosome-resistant and -susceptible amphibious snail Oncomelania nosophora 1. Comparative morphological and functional studies on hemocytes from both snails.

PubMed

Sasaki, Yuri; Furuta, Emiko; Kirinoki, Masashi; Seo, Naomi; Matsuda, Hajime

2003-10-01

Two morphologically distinct blood cell types (hemocytes), Type I and Type II were found coexisting in hemolymph from two kinds of snails, Oncomelania nosophora strain, viz. from the Nirasaki strain (schistosome-resistant snail) and the Kisarazu strain (schistosome-susceptible snail). Ten min after inoculation of SRBC, the majority of Type I cells from Nirasaki strain flattened and spread over the surface of the glass plate by extending pseudopodia. In the Kisarazu strain, Type I cells adhered to the surface of substrate with spike-like filopodia, but did not form spreading lamellipodia. Type I cell from the Nirasaki strain phagocytosed SRBC but that from the Kisarazu strain did not. The starting time of recognition of foreign materials was slightly different in the Type I hemocytes from the two strains. Type II cells from both strains were round and lymphocyte-like. Ten or sixty min after incubation, Type II cells from neither strain adhered to the surface of substrate or SRBC, and did not phagocytose SRBC. Type II cells from the Nirasaki strain were quite similar to those from the Kisarazu strain. We concluded that Type I cells from the schistosome-resistant snail, Nirasaki strain, possessed higher phagocytic activity than those from the susceptible snail, Kisarazu strain, despite the morphological similarities of the hemocytes from both strains.

Insight into the messenger role of reactive oxygen intermediates in immunostimulated hemocytes from the scallop Argopecten purpuratus.

PubMed

Oyanedel, Daniel; Gonzalez, Roxana; Brokordt, Katherina; Schmitt, Paulina; Mercado, Luis

2016-12-01

Reactive oxygen intermediates (ROI) are metabolites produced by aerobic cells which have been linked to oxidative stress. Evidence reported in vertebrates indicates that ROI can also act as messengers in a variety of cellular signaling pathways, including those involved in innate immunity. In a recent study, an inhibitor of NF-kB transcription factors was identified in the scallop Argopecten purpuratus, and its functional characterization suggested that it may regulate the expression of the big defensin antimicrobial peptide ApBD1. In order to give new insights into the messenger role of ROI in the immune response of bivalve mollusks, the effect of ROI production on gene transcription of ApBD1 was assessed in A. purpuratus. The results showed that 48 h-cultured hemocytes were able to display phagocytic activity and ROI production in response to the β-glucan zymosan. The immune stimulation also induced the transcription of ApBD1, which was upregulated in cultured hemocytes. After neutralizing the ROI produced by the stimulated hemocytes with the antioxidant trolox, the transcription of ApBD1 was reduced near to base levels. The results suggest a potential messenger role of intracellular ROI on the regulation of ApBD1 transcription during the immune response of scallops. Copyright © 2016 Elsevier Ltd. All rights reserved.

The impact of expired commercial drugs on non-target marine species: A case study with the use of a battery of biomarkers in hemocytes of mussels.

PubMed

Politakis, Nektarios; Belavgeni, Alexia; Efthimiou, Ioanna; Charalampous, Nikolina; Kourkouta, Chara; Dailianis, Stefanos

2018-02-01

The present study investigated the effects of two expired commercial medicines, like Buscopan Plus and Mesulid, commonly classified as household medical wastes, on hemocytes of mussel Mytilus galloprovincialis. Mussel hemocytes' lysosomal membrane stability (in terms of neutral red retention assay), superoxide anions (O 2 · - ) and nitric oxides (NO, in terms of nitrites) production, lipid peroxidation (in terms of malondialdehyde/MDA content) and the formation of nuclear abnormalities (using the micronucleus/MN assay) were assessed in hemocytes of mussels treated for 7 days with appropriate amounts of each drug (the concentrations of active substances were considered in each case, due to the absence of data related with the excipients) as well as in hemocytes of post-treated/recovered mussels (7 days post-treatment/recovery period). According to the results, treated mussels showed significantly decreased NRRT values, enhanced O 2 · - , NO and MDA levels, as well as high frequencies of nuclear abnormalities in both cases. Thοse effects showed a drastic reduction in almost all cases, after the post-treatment/recovery period. Moreover, the "stress on stress" method, commonly performed for estimating mussels' ability to survive in air, showed significantly reduced LT 50 values in challenged mussels, compared to values observed in control mussels. The current findings revealed for the first time that both expired commercial drugs could affect mussels, probably via the formation of active substances bioactivated metabolites, as well as excipients, such as TiO 2 and SiO 2 , at least in case of Buscopan plus. Although further research is needed, the current findings indicate the environmental impact of expired commercial drugs, thus revealing the need for the proper disposal of household medical wastes. Copyright © 2017 Elsevier Inc. All rights reserved.

Steroid Hormone Signaling Is Essential to Regulate Innate Immune Cells and Fight Bacterial Infection in Drosophila

PubMed Central

Regan, Jennifer C.; Brandão, Ana S.; Leitão, Alexandre B.; Mantas Dias, Ângela Raquel; Sucena, Élio; Jacinto, António; Zaidman-Rémy, Anna

2013-01-01

Coupling immunity and development is essential to ensure survival despite changing internal conditions in the organism. Drosophila metamorphosis represents a striking example of drastic and systemic physiological changes that need to be integrated with the innate immune system. However, nothing is known about the mechanisms that coordinate development and immune cell activity in the transition from larva to adult. Here, we reveal that regulation of macrophage-like cells (hemocytes) by the steroid hormone ecdysone is essential for an effective innate immune response over metamorphosis. Although it is generally accepted that steroid hormones impact immunity in mammals, their action on monocytes (e.g. macrophages and neutrophils) is still not well understood. Here in a simpler model system, we used an approach that allows in vivo, cell autonomous analysis of hormonal regulation of innate immune cells, by combining genetic manipulation with flow cytometry, high-resolution time-lapse imaging and tissue-specific transcriptomic analysis. We show that in response to ecdysone, hemocytes rapidly upregulate actin dynamics, motility and phagocytosis of apoptotic corpses, and acquire the ability to chemotax to damaged epithelia. Most importantly, individuals lacking ecdysone-activated hemocytes are defective in bacterial phagocytosis and are fatally susceptible to infection by bacteria ingested at larval stages, despite the normal systemic and local production of antimicrobial peptides. This decrease in survival is comparable to the one observed in pupae lacking immune cells altogether, indicating that ecdysone-regulation is essential for hemocyte immune functions and survival after infection. Microarray analysis of hemocytes revealed a large set of genes regulated at metamorphosis by EcR signaling, among which many are known to function in cell motility, cell shape or phagocytosis. This study demonstrates an important role for steroid hormone regulation of immunity in vivo in Drosophila, and paves the way for genetic dissection of the mechanisms at work behind steroid regulation of innate immune cells. PMID:24204269

The role of phosphatidylinositol-3-OH-kinase (PI3-kinase) and respiratory burst enzymes in the [omim][BF4]-mediated toxic mode of action in mussel hemocytes.

PubMed

Belavgeni, Alexia; Dailianis, Stefanos

2017-09-01

The present study investigates the role of phosphatidylinositol-3-OH-kinase (PI3-kinase) and respiratory burst enzymes, NADPH oxidase and NO synthase, in the 1-methyl-3-octylimidazolium tetrafluoroborate ([omim][BF 4 ])-mediated toxic mode of action in mussel hemocytes. Specifically, cell viability (using the neutral red uptake assay) was primarily tested in hemocytes treated with different concentrations of [omim][BF 4 ] (0.1-10 mg L -1 ) and thereafter [omim][BF 4 ]-mediated oxidative (in terms of superoxide anions/O 2 - and nitric oxide/NO generation, as well as the enhancement of lipid peroxidation by-products, in terms of malondialdehyde/MDA) and genotoxic (in terms of DNA damage) effects were determined in hemocytes treated with 1 mg L -1 [omim][BF 4 ]. Moreover, in order to investigate, even indirectly and non-entirely specific, the role of PI3-kinase, NADPH oxidase and NO synthase, the [omim][BF 4 ]-mediated effects were also investigated in hemocytes pre-incubated with wortmannin (50 nM), diphenyleneiodonium chloride (DPI 10 μM) and N G -nitro- l -arginine methyl ester (l-NAME 10 μM), respectively. The results showed that [omim][BF 4 ] ability to enhance O 2 - , NO, MDA and DNA damage, via its interaction with cellular membranes, was significantly attenuated in the presence of each inhibitor in almost all cases. The current findings revealed for the first time that certain signaling molecules, such as PI3-kinase, as well as respiratory burst enzymes activation, such as NADPH oxidase and NO synthase, could merely attribute to the [omim][BF 4 ]-mediated mode of action, thus enriching our knowledge for the molecular mechanisms of ILs toxicity. Copyright © 2017 Elsevier Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Warren, J.E.; Klaine, S.J.

The field cricket, Acheta domesticus, was used as a test organism to determine the effects of heavy metal exposure on cellular immunity. Insects were separated by sex and exposed to cadmium chloride or mercuric chloride at concentrations of 0, 2.5, and 5.0 ug/g. Exposures consisted of injecting the chemicals into the hemocoel of each insect on days 0, 2, and 4. Hemolymph was collected on day 7 of the study to determine total hemocyte counts, protein levels, and phenoloxidase activity in individual insects. Cadmium chloride decreased the total number of hemocytes in male crickets at 2.5 and 5.0 ug/g andmore » in female crickets at 5.0 ug/g. Protein levels increased in a dose dependent manner in the males but only slightly increased in the females. Mercuric chloride caused a dose-dependent increase in total hemocytes in both male and female crickets. In addition, mercuric chloride caused a dose-dependent increase in protein levels in males but not females.« less

dOCRL maintains immune cell quiescence by regulating endosomal traffic

PubMed Central

Del Signore, Steven J.; Biber, Sarah A.; Lehmann, Katherine S.; Heimler, Stephanie R.; Rosenfeld, Benjamin H.; Eskin, Tania L.

2017-01-01

Lowe Syndrome is a developmental disorder characterized by eye, kidney, and neurological pathologies, and is caused by mutations in the phosphatidylinositol-5-phosphatase OCRL. OCRL plays diverse roles in endocytic and endolysosomal trafficking, cytokinesis, and ciliogenesis, but it is unclear which of these cellular functions underlie specific patient symptoms. Here, we show that mutation of Drosophila OCRL causes cell-autonomous activation of hemocytes, which are macrophage-like cells of the innate immune system. Among many cell biological defects that we identified in docrl mutant hemocytes, we pinpointed the cause of innate immune cell activation to reduced Rab11-dependent recycling traffic and concomitantly increased Rab7-dependent late endosome traffic. Loss of docrl amplifies multiple immune-relevant signals, including Toll, Jun kinase, and STAT, and leads to Rab11-sensitive mis-sorting and excessive secretion of the Toll ligand Spåtzle. Thus, docrl regulation of endosomal traffic maintains hemocytes in a poised, but quiescent state, suggesting mechanisms by which endosomal misregulation of signaling may contribute to symptoms of Lowe syndrome. PMID:29028801

Hemocytes of the cochineal insect: ultrastructure.

PubMed

Caselín-Castro, Sandra; Llanderal-Cázares, Celina; Méndez-Gallegos, Santiago de Jesús; Ramírez-Cruz, Arturo; Hernández-Hernández, Fidel de la Cruz

2010-03-01

Using transmission electron microscopy, light microscopy (Giemsa May-Grumwald), and the Periodic Acid-Schif (PAS) and Sudan Black B staining techniques, hemocytes in the hemolymph of adult female Dactylopius coccus were characterized. The following, in order of abundance, were found: granulocytes, plasmatocytes, prohemocytes, and oenocytoids. Granulocytes varied in size with granulations in the cytoplasm, a large quantity of mitochondria, rugose endoplasmatic reticulum, ribosomes and vesicles, central or exocentric, spherical and occasionally lobulate nucleus. Plasmatocytes were polymorphic with irregularities in the plasma membrane; cytoplasm contained mitochondria, rugose endoplasmatic reticulum and vesicles, and exocentric, spherical, or irregular nucleus. In both types of hemocytes, scant polysaccharides and lipids were found. Prohemocytes were small and spherical with homogeneous cytoplasm and large exocentric nuclei. Oenocytoids were oval or irregular with dense homogeneous cytoplasm and elongated exocentric nuclei. The percentages of granulocytes on different days (d 1 and 10) during the life of the adult female were significantly different, as were those of plasmatocytes on d 30 and 50 and prohemocytes on d 1 and 50. (c) 2010 Wiley Periodicals, Inc.

A novel immune-related gene HDD1 of silkworm Bombyx mori is involved in bacterial response.

PubMed

Zhang, Kui; Pan, Guangzhao; Zhao, Yuzu; Hao, Xiangwei; Li, Chongyang; Shen, Li; Zhang, Rui; Su, Jingjing; Cui, Hongjuan

2017-08-01

Insects have evolved an effective immune system to respond to various challenges. In this study, a novel immune-related gene, called BmHDD1, was first charactered in silkworm, Bombyx mori. BmHDD1 contained an ORF of 837bp and encoding a deduced protein of 278 amino acids. BmHDD1 was specifically expressed in hemocytes, and highly expressed at the molting and metamorphosis stages under normal physiological conditions. Our results suggested that BmHDD1 was mainly generated by hemocytes and secreted into hemolymph. Our results also showed that the expression level of BmHDD1 was significantly increased after 20E injection, which indicated that BmHDD1 might be regulated by ecdysone. More importantly, BmHDD1 was dramatically induced after injected with different types of PAMPs or bacteria, either in hemocytes or fat body. Those results suggested that BmHDD1 plays a role in developing and immunity system in silkworm, Bombyx mori. Copyright © 2017. Published by Elsevier Ltd.

Edin Expression in the Fat Body Is Required in the Defense Against Parasitic Wasps in Drosophila melanogaster.

PubMed

Vanha-Aho, Leena-Maija; Anderl, Ines; Vesala, Laura; Hultmark, Dan; Valanne, Susanna; Rämet, Mika

2015-05-01

The cellular immune response against parasitoid wasps in Drosophila involves the activation, mobilization, proliferation and differentiation of different blood cell types. Here, we have assessed the role of Edin (elevated during infection) in the immune response against the parasitoid wasp Leptopilina boulardi in Drosophila melanogaster larvae. The expression of edin was induced within hours after a wasp infection in larval fat bodies. Using tissue-specific RNAi, we show that Edin is an important determinant of the encapsulation response. Although edin expression in the fat body was required for the larvae to mount a normal encapsulation response, it was dispensable in hemocytes. Edin expression in the fat body was not required for lamellocyte differentiation, but it was needed for the increase in plasmatocyte numbers and for the release of sessile hemocytes into the hemolymph. We conclude that edin expression in the fat body affects the outcome of a wasp infection by regulating the increase of plasmatocyte numbers and the mobilization of sessile hemocytes in Drosophila larvae.

Hemocitical responses to environmental stress in invertebrates: a review.

PubMed

Perez, Danielli Giuliano; Fontanetti, Carmem Silvia

2011-06-01

Although invertebrates are recognized by the great facility to accumulate pollutants present in their environment and many of them are used as sentinel species in biomonitoring studies, little is known about the impact of toxicants on the immune system of these animals. In this regard, hemocytes play a fundamental role: these cells circulate freely through the hemolymph of invertebrates and act on the recognition of foreign material to the organism, mediating and effecting the cellular defense, such as phagocytosis, nodulation, and encapsulation. Different morphological types can be recognized but still there is controversy among the researchers about the exact classification of the hemocytes due to the diversity of techniques for the preservation and observation of these cells. In the present study, a review on the main hemocyte responses to environmental stress in different invertebrate organisms is presented, emphasizing the contamination by heavy metals. It is discussed parameters such as: alteration in the number of cells involved in the defense reaction, phagocytic activity, lysosomal responses, and production of reactive oxygen species.

A continuous cell line, SYSU-OfHe-C, from hemocytes of Ostrinia furnacalis possesses immune ability depending on the presence of larval plasma.

PubMed

Hu, Jian; Feng, Xiangping; Yang, Zhongguo; Chen, Zhuoxin; Zhang, Wenqing

2014-07-01

A continuous cell line, SYSU-OfHe-C, from larval hemocytes of corn borer, Ostrinia furnacalis was established. With increasing passages, the cells grew increasingly faster, and approximately 45% of the cells were in division at passage 55. The culture was mainly composed of two types of cells, granulocytes and plasmatocytes, which showed different division and proliferation behaviors, but possessed similar phagocytic ability. Its spreading ability was significantly weaker than that of hemocytes from naïve larva; however, it could be promoted by larval plasma. Furthermore, its encapsulation ability was also promoted by larval plasma to form multilayer capsules on Sephadex A-25 beads. Finally, the expression of several immune-related genes was verified after provocation by microbes or Sephadex beads. These results indicated that the cell line possessed immune ability depending on the presence of plasma of naïve larvae and are beneficial to studies of insect cellular systems. Copyright © 2014 Elsevier Ltd. All rights reserved.

Allatotropin: A pleiotropic neuropeptide that elicits mosquito immune responses.

PubMed

Hernández-Martínez, Salvador; Sánchez-Zavaleta, Minerva; Brito, Kevin; Herrera-Ortiz, Antonia; Ons, Sheila; Noriega, Fernando G

2017-01-01

Allatotropins (AT) are neuropeptides with pleotropic functions on a variety of insect tissues. They affect processes such as juvenile hormone biosynthesis, cardiac rhythm, oviduct and hindgut contractions, nutrient absorption and circadian cycle. The present work provides experimental evidence that AT elicits immune responses in two important mosquito disease vectors, Anopheles albimanus and Aedes aegypti. Hemocytes and an immune-competent mosquito cell line responded to AT by showing strong morphological changes and increasing bacterial phagocytic activity. Phenoloxidase activity in hemolymph was also increased in Ae. aegypti mosquitoes treated with AT but not in An. albimanus, suggesting differences in the AT-dependent immune activation in the two species. In addition, two important insect immune markers, nitric oxide levels and expression of antimicrobial peptide genes, were increased in An. albimanus guts after AT treatment. AT conjugated to quantum dot nanocrystals (QDots) specifically labeled hemocytes in vivo in both mosquito species, implying molecular interactions between AT and hemocytes. The results of our studies suggest a new role for AT in the modulation of the immune response in mosquitoes.

Inter-cellular forces orchestrate contact inhibition of locomotion.

PubMed

Davis, John R; Luchici, Andrei; Mosis, Fuad; Thackery, James; Salazar, Jesus A; Mao, Yanlan; Dunn, Graham A; Betz, Timo; Miodownik, Mark; Stramer, Brian M

2015-04-09

Contact inhibition of locomotion (CIL) is a multifaceted process that causes many cell types to repel each other upon collision. During development, this seemingly uncoordinated reaction is a critical driver of cellular dispersion within embryonic tissues. Here, we show that Drosophila hemocytes require a precisely orchestrated CIL response for their developmental dispersal. Hemocyte collision and subsequent repulsion involves a stereotyped sequence of kinematic stages that are modulated by global changes in cytoskeletal dynamics. Tracking actin retrograde flow within hemocytes in vivo reveals synchronous reorganization of colliding actin networks through engagement of an inter-cellular adhesion. This inter-cellular actin-clutch leads to a subsequent build-up in lamellar tension, triggering the development of a transient stress fiber, which orchestrates cellular repulsion. Our findings reveal that the physical coupling of the flowing actin networks during CIL acts as a mechanotransducer, allowing cells to haptically sense each other and coordinate their behaviors. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

MicroRNA-100 is involved in shrimp immune response to white spot syndrome virus (WSSV) and Vibrio alginolyticus infection

PubMed Central

Wang, Zhi; Zhu, Fei

2017-01-01

In this study, we discovered that shrimp miR-100 was up-regulated at 24 h after WSSV or Vibrio alginolyticus infection, confirming its participation in the innate immune system of shrimp. The anti-miRNA oligonucleotide (AMO-miR-100) was applied to inhibit the expression of miR-100. After AMO-miR-100 treatment, the shrimp was challenged with WSSV or V. alginolyticus. The knockdown of miR-100 expression decreased the mortality of WSSV-infected shrimp from 24 h to 72 h post-infection and enhanced the mortality of V. alginolyticus-infected shrimp significantly. The knockdown of miR-100 affected phenoloxidase (PO) activity, superoxide dismutase (SOD) activity and total hemocyte count (THC) after the infection with WSSV or V. alginolyticus, indicating a regulative role of miR-100 in the immune potential of shrimp in the response to WSSV or V. alginolyticus infection. The knockdown of miR-100 induced the apoptosis of shrimp hemocytes, and V. alginolyticus + AMO-miR-100 treatment caused more hemocyte apoptosis than V. alginolyticus treatment. The miR-100 influenced also the morphology of shrimp hemocytes and regulated the phagocytosis of WSSV or V. alginolyticus. Thus, we concluded that miR-100 may promote the anti-Vibrio immune response of shrimp through regulating apoptosis, phagocytosis and PO activity and affects the progression of WSSV infection at a certain level. PMID:28181552

Effects of destruxins on free calcium and hydrogen ions in insect hemocytes.

PubMed

Chen, Xiu-Run; Hu, Qiong-Bo; Yu, Xiao-Qiang; Ren, Shun-Xiang

2014-02-01

Destruxins, cyclohexadepsipeptidic mycotoxins isolated from the entomopathogenic fungus Metarhizium anisopliae, inhibit innate insect immunity. However, their mechanism of action remains unclear. In this study, the effects of destruxins on changes in free calcium and hydrogen ions in the hemocytes of Exolontha serrulata, Bombyx mori and the Spodoptera litura SL-1 cell line were detected using laser scanning confocal microscopy (LSCM). An instant Ca(2+) influx of hemocytes induced by destruxins A and B (DA and DB) was recorded. The DA/DB-dependent Ca(2+) influx was not influenced by the Ca(2+) channel inhibitors 2-aminoethoxydiphenyl borane (2-APB) and U73122. It also had an apparently different LSCM profile from that of the ionomycin-dependent Ca(2+) influx. However, the instant Ca(2+) influx was not seen in the SL-1 cells; on the contrary, a slow, moderate enhancement of intracellular Ca(2+) was observed. Meanwhile, an instant intracellular free H(+) decrease aroused by DA and DB was found. DB at 20 μmol/L and DA at 690 μmol/L significantly reduced intracellular free H(+) levels. Furthermore, the vacuolar H(+)-ATPase (V-ATPase) inhibitor bafilomycin A1 had obvious effects on the decreases of intracellular free H(+) in hemocytes. These results suggest that the mechanism of DA/DB-dependent Ca(2+) influx is perhaps not related to Ca(2+) channels and ionophores; rather, the intracellular free H(+) decrease might be due to V-ATPase inhibition. © 2013 Institute of Zoology, Chinese Academy of Sciences.

Apoptosis in Hemocytes Induces a Shift in Effector Mechanisms in the Drosophila Immune System and Leads to a Pro-Inflammatory State

PubMed Central

Arefin, Badrul; Kucerova, Lucie; Krautz, Robert; Kranenburg, Holger; Parvin, Farjana; Theopold, Ulrich

2015-01-01

Apart from their role in cellular immunity via phagocytosis and encapsulation, Drosophila hemocytes release soluble factors such as antimicrobial peptides, and cytokines to induce humoral responses. In addition, they participate in coagulation and wounding, and in development. To assess their role during infection with entomopathogenic nematodes, we depleted plasmatocytes and crystal cells, the two classes of hemocytes present in naïve larvae by expressing proapoptotic proteins in order to produce hemocyte-free (Hml-apo, originally called Hemoless) larvae. Surprisingly, we found that Hml-apo larvae are still resistant to nematode infections. When further elucidating the immune status of Hml-apo larvae, we observe a shift in immune effector pathways including massive lamellocyte differentiation and induction of Toll- as well as repression of imd signaling. This leads to a pro-inflammatory state, characterized by the appearance of melanotic nodules in the hemolymph and to strong developmental defects including pupal lethality and leg defects in escapers. Further analysis suggests that most of the phenotypes we observe in Hml-apo larvae are alleviated by administration of antibiotics and by changing the food source indicating that they are mediated through the microbiota. Biochemical evidence identifies nitric oxide as a key phylogenetically conserved regulator in this process. Finally we show that the nitric oxide donor L-arginine similarly modifies the response against an early stage of tumor development in fly larvae. PMID:26322507

MicroRNA-100 is involved in shrimp immune response to white spot syndrome virus (WSSV) and Vibrio alginolyticus infection.

PubMed

Wang, Zhi; Zhu, Fei

2017-02-09

In this study, we discovered that shrimp miR-100 was up-regulated at 24 h after WSSV or Vibrio alginolyticus infection, confirming its participation in the innate immune system of shrimp. The anti-miRNA oligonucleotide (AMO-miR-100) was applied to inhibit the expression of miR-100. After AMO-miR-100 treatment, the shrimp was challenged with WSSV or V. alginolyticus. The knockdown of miR-100 expression decreased the mortality of WSSV-infected shrimp from 24 h to 72 h post-infection and enhanced the mortality of V. alginolyticus-infected shrimp significantly. The knockdown of miR-100 affected phenoloxidase (PO) activity, superoxide dismutase (SOD) activity and total hemocyte count (THC) after the infection with WSSV or V. alginolyticus, indicating a regulative role of miR-100 in the immune potential of shrimp in the response to WSSV or V. alginolyticus infection. The knockdown of miR-100 induced the apoptosis of shrimp hemocytes, and V. alginolyticus + AMO-miR-100 treatment caused more hemocyte apoptosis than V. alginolyticus treatment. The miR-100 influenced also the morphology of shrimp hemocytes and regulated the phagocytosis of WSSV or V. alginolyticus. Thus, we concluded that miR-100 may promote the anti-Vibrio immune response of shrimp through regulating apoptosis, phagocytosis and PO activity and affects the progression of WSSV infection at a certain level.

Intracellular pH Recovery Rates of Hemocytes from Estuarine and Open Ocean Bivalve Species Following In vitro Acid Challenge

NASA Astrophysics Data System (ADS)

Croxton, A.; Wikfors, G.

2013-12-01

Decreasing pH in estuarine systems is a growing concern for researchers studying mollusk species. Debates continue on whether estuarine bivalve species are more or less vulnerable to ocean acidification than marine species because estuaries can present multiple environmental stressors. The aim of this study is to understand the homeostatic mechanisms of bivalve hemocytes following exposure to extracellular acid treatment. Previous measurements using fluorescent SNARF probes and flow-cytometry have determined the intracellular pH of hemocytes from several bivalve species (eastern oyster, bay scallop, northern quahog, soft-shell clam, and blue mussel) to range between 7.0-7.4. In the present study of four bivalve species, recovery rate profiles were determined for intracellular hemocyte pH following addition of acid to hemolymph in vitro. These profiles indicate that soft-shell clams and bay scallops maintained homeostasis with very little change in intracellular pH. In contrast, an initial drop in intracellular pH in northern quahogs was followed by a steady recovery of intracellular pH. Contrasting results between species appear to be unrelated to mineral shell composition (aragonite vs. calcite) or habitat location (infaunal vs. epifaunal). The next phase of this study will be to determine if offshore species (surfclams and sea scallops) will have similar responses. Results from these studies will provide a better understanding of the physiological responses of estuarine and marine species exposed to acidified environments.

Immunotoxicity in green mussels under perfluoroalkyl substance (PFAS) exposure: Reversible response and response model development.

PubMed

Liu, Changhui; Gin, Karina Yew-Hoong

2018-04-01

The immunotoxicity of 4 commonly detected perfluoroalkyl substances (PFASs), namely, perfluorooctanesulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and perfluorodecanoic acid (PFDA) was investigated by measuring biomarkers of the immune profile of green mussels, Perna viridis. The biomarkers included neutral red retention, phagocytosis, and spontaneous cytotoxicity, all of which were tested on mussel hemocytes. Hemocytes are an important component of the invertebrate immune system. We found that exposure to PFASs could lead to reduced hemocyte cell viability and suppress immune function by up to 50% of normal performance within the experimental exposure range. The results indicate that PFASs have an immunotoxic potential and thus could pose severe health risks to aquatic organisms. The reported immunotoxicity is likely to result from the compounds' direct and indirect interactions with the hemocyte membrane, and therefore likely to affect the functionality of these cells. The immunotoxic response was found to be related to the organism's burden of PFASs, and was reversible when the compounds were removed from the test organisms. Based on this relationship, models using an organism's PFAS concentration and bioaccumulation factor (BAF) as the independent variables were established to quantify PFAS-induced immunotoxicity. The models help us to gain a better understanding of the toxic mechanism of PFASs, and provide a tool to evaluate adverse effects for the whole group of compounds with one mathematical equation. Environ Toxicol Chem 2018;37:1138-1145. © 2018 SETAC. © 2018 SETAC.

The Silencing of a 14-3-3ɛ Homolog in Tenebrio molitor Leads to Increased Antimicrobial Activity in Hemocyte and Reduces Larval Survivability.

PubMed

Seo, Gi Won; Jo, Yong Hun; Seong, Jeong Hwan; Park, Ki Beom; Patnaik, Bharat Bhusan; Tindwa, Hamisi; Kim, Sun-Am; Lee, Yong Seok; Kim, Yu Jung; Han, Yeon Soo

2016-08-20

The 14-3-3 family of phosphorylated serine-binding proteins acts as signaling molecules in biological processes such as metabolism, division, differentiation, autophagy, and apoptosis. Herein, we report the requirement of 14-3-3ɛ isoform from Tenebrio molitor (Tm14-3-3ɛ) in the hemocyte antimicrobial activity. The Tm14-3-3ɛ transcript is 771 nucleotides in length and encodes a polypeptide of 256 amino acid residues. The protein has the typical 14-3-3 domain, the nuclear export signal (NES) sequence, and the peptide binding residues. The Tm14-3-3ɛ transcript shows a significant three-fold expression in the hemocyte of T. molitor larvae when infected with Escherichia coli Tm14-3-3ɛ silenced larvae show significantly lower survival rates when infected with E. coli. Under Tm14-3-3ɛ silenced condition, a strong antimicrobial activity is elicited in the hemocyte of the host inoculated with E. coli. This suggests impaired secretion of antimicrobial peptides (AMP) into the hemolymph. Furthermore, a reduction in AMP secretion under Tm14-3-3ɛ silenced condition would be responsible for loss in the capacity to kill bacteria and might explain the reduced survivability of the larvae upon E. coli challenge. This shows that Tm14-3-3ɛ is required to maintain innate immunity in T. molitor by enabling antimicrobial secretion into the hemolymph and explains the functional specialization of the isoform.

The Silencing of a 14-3-3ɛ Homolog in Tenebrio molitor Leads to Increased Antimicrobial Activity in Hemocyte and Reduces Larval Survivability

PubMed Central

Seo, Gi Won; Jo, Yong Hun; Seong, Jeong Hwan; Park, Ki Beom; Patnaik, Bharat Bhusan; Tindwa, Hamisi; Kim, Sun-Am; Lee, Yong Seok; Kim, Yu Jung; Han, Yeon Soo

2016-01-01

The 14-3-3 family of phosphorylated serine-binding proteins acts as signaling molecules in biological processes such as metabolism, division, differentiation, autophagy, and apoptosis. Herein, we report the requirement of 14-3-3ɛ isoform from Tenebrio molitor (Tm14-3-3ɛ) in the hemocyte antimicrobial activity. The Tm14-3-3ɛ transcript is 771 nucleotides in length and encodes a polypeptide of 256 amino acid residues. The protein has the typical 14-3-3 domain, the nuclear export signal (NES) sequence, and the peptide binding residues. The Tm14-3-3ɛ transcript shows a significant three-fold expression in the hemocyte of T. molitor larvae when infected with Escherichia coli Tm14-3-3ɛ silenced larvae show significantly lower survival rates when infected with E. coli. Under Tm14-3-3ɛ silenced condition, a strong antimicrobial activity is elicited in the hemocyte of the host inoculated with E. coli. This suggests impaired secretion of antimicrobial peptides (AMP) into the hemolymph. Furthermore, a reduction in AMP secretion under Tm14-3-3ɛ silenced condition would be responsible for loss in the capacity to kill bacteria and might explain the reduced survivability of the larvae upon E. coli challenge. This shows that Tm14-3-3ɛ is required to maintain innate immunity in T. molitor by enabling antimicrobial secretion into the hemolymph and explains the functional specialization of the isoform. PMID:27556493

De novo transcriptome sequencing of the Octopus vulgaris hemocytes using Illumina RNA-Seq technology: response to the infection by the gastrointestinal parasite Aggregata octopiana.

PubMed

Castellanos-Martínez, Sheila; Arteta, David; Catarino, Susana; Gestal, Camino

2014-01-01

Octopus vulgaris is a highly valuable species of great commercial interest and excellent candidate for aquaculture diversification; however, the octopus' well-being is impaired by pathogens, of which the gastrointestinal coccidian parasite Aggregata octopiana is one of the most important. The knowledge of the molecular mechanisms of the immune response in cephalopods, especially in octopus is scarce. The transcriptome of the hemocytes of O. vulgaris was de novo sequenced using the high-throughput paired-end Illumina technology to identify genes involved in immune defense and to understand the molecular basis of octopus tolerance/resistance to coccidiosis. A bi-directional mRNA library was constructed from hemocytes of two groups of octopus according to the infection by A. octopiana, sick octopus, suffering coccidiosis, and healthy octopus, and reads were de novo assembled together. The differential expression of transcripts was analysed using the general assembly as a reference for mapping the reads from each condition. After sequencing, a total of 75,571,280 high quality reads were obtained from the sick octopus group and 74,731,646 from the healthy group. The general transcriptome of the O. vulgaris hemocytes was assembled in 254,506 contigs. A total of 48,225 contigs were successfully identified, and 538 transcripts exhibited differential expression between groups of infection. The general transcriptome revealed genes involved in pathways like NF-kB, TLR and Complement. Differential expression of TLR-2, PGRP, C1q and PRDX genes due to infection was validated using RT-qPCR. In sick octopuses, only TLR-2 was up-regulated in hemocytes, but all of them were up-regulated in caecum and gills. The transcriptome reported here de novo establishes the first molecular clues to understand how the octopus immune system works and interacts with a highly pathogenic coccidian. The data provided here will contribute to identification of biomarkers for octopus resistance against pathogens, which could improve octopus farming in the near future.

De Novo Transcriptome Sequencing of the Octopus vulgaris Hemocytes Using Illumina RNA-Seq Technology: Response to the Infection by the Gastrointestinal Parasite Aggregata octopiana

PubMed Central

Castellanos-Martínez, Sheila; Arteta, David; Catarino, Susana; Gestal, Camino

2014-01-01

Background Octopus vulgaris is a highly valuable species of great commercial interest and excellent candidate for aquaculture diversification; however, the octopus’ well-being is impaired by pathogens, of which the gastrointestinal coccidian parasite Aggregata octopiana is one of the most important. The knowledge of the molecular mechanisms of the immune response in cephalopods, especially in octopus is scarce. The transcriptome of the hemocytes of O. vulgaris was de novo sequenced using the high-throughput paired-end Illumina technology to identify genes involved in immune defense and to understand the molecular basis of octopus tolerance/resistance to coccidiosis. Results A bi-directional mRNA library was constructed from hemocytes of two groups of octopus according to the infection by A. octopiana, sick octopus, suffering coccidiosis, and healthy octopus, and reads were de novo assembled together. The differential expression of transcripts was analysed using the general assembly as a reference for mapping the reads from each condition. After sequencing, a total of 75,571,280 high quality reads were obtained from the sick octopus group and 74,731,646 from the healthy group. The general transcriptome of the O. vulgaris hemocytes was assembled in 254,506 contigs. A total of 48,225 contigs were successfully identified, and 538 transcripts exhibited differential expression between groups of infection. The general transcriptome revealed genes involved in pathways like NF-kB, TLR and Complement. Differential expression of TLR-2, PGRP, C1q and PRDX genes due to infection was validated using RT-qPCR. In sick octopuses, only TLR-2 was up-regulated in hemocytes, but all of them were up-regulated in caecum and gills. Conclusion The transcriptome reported here de novo establishes the first molecular clues to understand how the octopus immune system works and interacts with a highly pathogenic coccidian. The data provided here will contribute to identification of biomarkers for octopus resistance against pathogens, which could improve octopus farming in the near future. PMID:25329466

Analysis of the Contribution of Hemocytes and Autophagy to Drosophila Antiviral Immunity.

PubMed

Lamiable, Olivier; Arnold, Johan; de Faria, Isaque Joao da Silva; Olmo, Roenick Proveti; Bergami, Francesco; Meignin, Carine; Hoffmann, Jules A; Marques, Joao Trindade; Imler, Jean-Luc

2016-06-01

Antiviral immunity in the model organism Drosophila melanogaster involves the broadly active intrinsic mechanism of RNA interference (RNAi) and virus-specific inducible responses. Here, using a panel of six viruses, we investigated the role of hemocytes and autophagy in the control of viral infections. Injection of latex beads to saturate phagocytosis, or genetic depletion of hemocytes, resulted in decreased survival and increased viral titers following infection with Cricket paralysis virus (CrPV), Flock House virus (FHV), and vesicular stomatitis virus (VSV) but had no impact on Drosophila C virus (DCV), Sindbis virus (SINV), and Invertebrate iridescent virus 6 (IIV6) infection. In the cases of CrPV and FHV, apoptosis was induced in infected cells, which were phagocytosed by hemocytes. In contrast, VSV did not trigger any significant apoptosis but we confirmed that the autophagy gene Atg7 was required for full virus resistance, suggesting that hemocytes use autophagy to recognize the virus. However, this recognition does not depend on the Toll-7 receptor. Autophagy had no impact on DCV, CrPV, SINV, or IIV6 infection and was required for replication of the sixth virus, FHV. Even in the case of VSV, the increases in titers were modest in Atg7 mutant flies, suggesting that autophagy does not play a major role in antiviral immunity in Drosophila Altogether, our results indicate that, while autophagy plays a minor role, phagocytosis contributes to virus-specific immune responses in insects. Phagocytosis and autophagy are two cellular processes that involve lysosomal degradation and participate in Drosophila immunity. Using a panel of RNA and DNA viruses, we have addressed the contribution of phagocytosis and autophagy in the control of viral infections in this model organism. We show that, while autophagy plays a minor role, phagocytosis contributes to virus-specific immune responses in Drosophila This work brings to the front a novel facet of antiviral host defense in insects, which may have relevance in the control of virus transmission by vector insects or in the resistance of beneficial insects to viral pathogens. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Analysis of the Contribution of Hemocytes and Autophagy to Drosophila Antiviral Immunity

PubMed Central

Lamiable, Olivier; Arnold, Johan; de Faria, Isaque Joao da Silva; Olmo, Roenick Proveti; Bergami, Francesco; Meignin, Carine; Hoffmann, Jules A.; Marques, Joao Trindade

2016-01-01

ABSTRACT Antiviral immunity in the model organism Drosophila melanogaster involves the broadly active intrinsic mechanism of RNA interference (RNAi) and virus-specific inducible responses. Here, using a panel of six viruses, we investigated the role of hemocytes and autophagy in the control of viral infections. Injection of latex beads to saturate phagocytosis, or genetic depletion of hemocytes, resulted in decreased survival and increased viral titers following infection with Cricket paralysis virus (CrPV), Flock House virus (FHV), and vesicular stomatitis virus (VSV) but had no impact on Drosophila C virus (DCV), Sindbis virus (SINV), and Invertebrate iridescent virus 6 (IIV6) infection. In the cases of CrPV and FHV, apoptosis was induced in infected cells, which were phagocytosed by hemocytes. In contrast, VSV did not trigger any significant apoptosis but we confirmed that the autophagy gene Atg7 was required for full virus resistance, suggesting that hemocytes use autophagy to recognize the virus. However, this recognition does not depend on the Toll-7 receptor. Autophagy had no impact on DCV, CrPV, SINV, or IIV6 infection and was required for replication of the sixth virus, FHV. Even in the case of VSV, the increases in titers were modest in Atg7 mutant flies, suggesting that autophagy does not play a major role in antiviral immunity in Drosophila. Altogether, our results indicate that, while autophagy plays a minor role, phagocytosis contributes to virus-specific immune responses in insects. IMPORTANCE Phagocytosis and autophagy are two cellular processes that involve lysosomal degradation and participate in Drosophila immunity. Using a panel of RNA and DNA viruses, we have addressed the contribution of phagocytosis and autophagy in the control of viral infections in this model organism. We show that, while autophagy plays a minor role, phagocytosis contributes to virus-specific immune responses in Drosophila. This work brings to the front a novel facet of antiviral host defense in insects, which may have relevance in the control of virus transmission by vector insects or in the resistance of beneficial insects to viral pathogens. PMID:27009948

Bombyx mori and Aedes aegypti form multi-functional immune complexes that integrate pattern recognition, melanization, coagulants, and hemocyte recruitment

PubMed Central

Phillips, Dennis R.

2017-01-01

The innate immune system of insects responds to wounding and pathogens by mobilizing multiple pathways that provide both systemic and localized protection. Key localized responses in hemolymph include melanization, coagulation, and hemocyte encapsulation, which synergistically seal wounds and envelop and destroy pathogens. To be effective, these pathways require a targeted deposition of their components to provide protection without compromising the host. Extensive research has identified a large number of the effectors that comprise these responses, but questions remain regarding their post-translational processing, function, and targeting. Here, we used mass spectrometry to demonstrate the integration of pathogen recognition proteins, coagulants, and melanization components into stable, high-mass, multi-functional Immune Complexes (ICs) in Bombyx mori and Aedes aegypti. Essential proteins common to both include phenoloxidases, apolipophorins, serine protease homologs, and a serine protease that promotes hemocyte recruitment through cytokine activation. Pattern recognition proteins included C-type Lectins in B. mori, while A. aegypti contained a protein homologous to Plasmodium-resistant LRIM1 from Anopheles gambiae. We also found that the B. mori IC is stabilized by extensive transglutaminase-catalyzed cross-linking of multiple components. The melanization inhibitor Egf1.0, from the parasitoid wasp Microplitis demolitor, blocked inclusion of specific components into the IC and also inhibited transglutaminase activity. Our results show how coagulants, melanization components, and hemocytes can be recruited to a wound surface or pathogen, provide insight into the mechanism by which a parasitoid evades this immune response, and suggest that insects as diverse as Lepidoptera and Diptera utilize similar defensive mechanisms. PMID:28199361

Hematological Analysis of the Ascidian Botrylloides leachii (Savigny, 1816) During Whole-Body Regeneration.

PubMed

Blanchoud, Simon; Zondag, Lisa; Lamare, Miles D; Wilson, Megan J

2017-06-01

Whole-body regeneration (WBR)-the formation of an entire adult from only a small fragment of its own tissue-is extremely rare among chordates. Exceptionally, in the colonial ascidian Botrylloides leachii (Savigny, 1816) a fully functional adult is formed from their common vascular system after ablation of all adults from the colony in just 10 d, thanks to their high blastogenetic potential. While previous studies have identified key genetic markers and morphological changes, no study has yet focused on the hematological aspects of regeneration despite the major involvement of the remaining vascular system and the contained hemocytes in this process. To dissect this process, we analyzed colony blood flow patterns using time-lapse microscopy to obtain a quantitative description of the velocity, reversal pattern, and average distance traveled by hemocytes. We also observed that flows present during regeneration are powered by temporally and spatially synchronized contractions of the terminal ampullae. In addition, we revised previous studies of B. leachii hematology as well as asexual development using histological sectioning and compared the role played by hemocytes during WBR. We found that regeneration starts with a rapid healing response characterized by hemocyte aggregation and infiltration of immunocytes, followed by increased activity of hemoblasts, recruitment of macrophage-like cells for clearing the tissues of debris, and their subsequent disappearance from the circulation concomitant with the maturation of a single regenerated adult. Overall, we provide a detailed account of the hematological properties of regenerating B. leachii colonies, providing novel lines of inquiry toward the decipherment of regeneration in chordates.

In Vitro Analysis of Early Genotoxic and Cytotoxic Effects of Okadaic Acid in Different Cell Types of the Mussel Mytilus galloprovincialis.

PubMed

Prego-Faraldo, María Verónica; Valdiglesias, Vanessa; Laffon, Blanca; Eirín-López, José M; Méndez, Josefina

2015-01-01

Okadaic acid (OA) is the predominant biotoxin responsible for diarrhetic shellfish poisoning (DSP) syndrome in humans. While its harmful effects have been extensively studied in mammalian cell lines, the impact on marine organisms routinely exposed to OA is still not fully known. Few investigations available on bivalve molluscs suggest less genotoxic and cytotoxic effects of OA at high concentrations during long exposure times. In contrast, no apparent information is available on how sublethal concentrations of OA affect these organisms over short exposure times. In order to fill this gap, this study addressed for the first time in vitro analysis of early genotoxic and cytotoxic effects attributed to OA in two cell types of the mussel Mytilus galloprovincialis. Accordingly, hemocytes and gill cells were exposed to low OA concentrations (10, 50, 100, 200, or 500 nM) for short periods of time (1 or 2 h). The resulting DNA damage, as apoptosis and necrosis, was subsequently quantified using the comet assay and flow cytometry, respectively. Data demonstrated that (1) mussel hemocytes seem to display a resistance mechanism against early genotoxic and cytotoxic OA-induced effects, (2) mussel gill cells display higher sensitivity to early OA-mediated genotoxicity than hemocytes, and (3) mussel gill cells constitute more suitable systems to evaluate the genotoxic effect of low OA concentrations in short exposure studies. Taken together, this investigation provides evidence supporting the more reliable suitability of mussel gill cells compared to hemocytes to evaluate the genotoxic effect of low short-duration exposure to OA.

Ultrastructural characterization of the hemocytes of Lasiodora sp. (Koch, 1850) (Araneae: Theraphosidae).

PubMed

Soares, Tatiana; Cavalcanti, Marília Gabriela Dos Santos; Ferreira, Felipe Roberto Borba; Cavalcanti, Maria do Socorro de Mendonça; Alves, Luiz Carlos; Brayner, Fábio André; Paiva, Patrícia Maria Guedes

2013-05-01

This paper is the first descriptive review of hemolymph cell types in the circulation of the tarantula spider Lasiodora sp. These animals are more long-lived than other arthropods, and may live for approximately twenty years. Such remarkable longevity may result from a highly successful immune system, which in turn is directly correlated with hemocyte function. Since the literature on the genus Lasiodora sp. is limited, the main goal of the present study was to identify the different cell types by optical and transmission microscope. Six hemocyte types were characterized and called prohemocyte, granulocyte type I, granulocyte type II, spherulocyte, oenocytoid and plasmatocyte. Prohemocytes presented a large nucleus, elongated granulocytes type I showed the nucleus with the same cell format, elliptical granulocytes type II showed the central nucleus of identical shape, spherulocytes exhibited the nucleus filling almost the whole cell, oval oenocytoids showed eccentric nucleus and less dense cytoplasm, and irregular plasmatocytes showed a nucleus and no granules in cytoplasm. These polymorphic granulocytes presented a round, elongated, elliptical, oval or irregular profile with large and varied numbers of granules, except for plasmatocytes, that were agranular. Different densities and different concentrations of these granules were found at the periphery of the cell. The possible reasons and implications of differences and similarities between arthropods hemocytes are discussed. It can be concluded that there are six cell types in Lasiodora sp. This study is of the first step in the elucidation of the role these cells play in the circulatory and immune system in spiders. Copyright © 2013 Elsevier Ltd. All rights reserved.

Virus-like particles in venom of Meteorus pulchricornis induce host hemocyte apoptosis.

PubMed

Suzuki, M; Tanaka, T

2006-06-01

Ultrastructural studies on the reproductive tract and venom apparatus of a female braconid, Meteorus pulchricornis, revealed that the parasitoid lacks the calyx region in its oviduct, but possesses a venom gland with two venom gland filaments and a venom reservoir filled with white and cloudy fluid. Its venom gland cell is concaved and has a lumen filled with numerous granules. Transmisson electron microscopic (TEM) observation revealed that virus-like particles (VLPs) were produced in venom gland cells. The virus-like particle observed in M. pulchricornis (MpVLP) is composed of membranous envelopes with two different parts: a high-density core and a whitish low-density part. The VLPs of M. pulchricornis is also found assembling ultimately in the lumen of venom gland cell. Microvilli were found thrusting into the lumen of the venom gland cell and seem to aid in driving the matured MpVLPs to the common duct of the venom gland filament. Injection of MpVLPs into non-parasitized Pseudaletia separata hosts induced apoptosis in hemocytes, particularly granulocytes (GRs). Rate of apoptosis induced in GRs peaked 48h after VLP injection. While a large part of the GR population collapsed due to apoptosis caused by MpVLPs, the plasmatocyte population was minimally affected. The capacity of MpVLPs to cause apoptosis in host's hemocytes was further demonstrated by a decrease ( approximately 10-fold) in ability of host hemocytes to encapsulate fluorescent latex beads when MpVLPs were present. Apparently, the reduced encapsulation ability was due to a decrease in the GR population resulting from MpVLP-induced apoptosis.

Bombyx mori and Aedes aegypti form multi-functional immune complexes that integrate pattern recognition, melanization, coagulants, and hemocyte recruitment.

PubMed

Phillips, Dennis R; Clark, Kevin D

2017-01-01

The innate immune system of insects responds to wounding and pathogens by mobilizing multiple pathways that provide both systemic and localized protection. Key localized responses in hemolymph include melanization, coagulation, and hemocyte encapsulation, which synergistically seal wounds and envelop and destroy pathogens. To be effective, these pathways require a targeted deposition of their components to provide protection without compromising the host. Extensive research has identified a large number of the effectors that comprise these responses, but questions remain regarding their post-translational processing, function, and targeting. Here, we used mass spectrometry to demonstrate the integration of pathogen recognition proteins, coagulants, and melanization components into stable, high-mass, multi-functional Immune Complexes (ICs) in Bombyx mori and Aedes aegypti. Essential proteins common to both include phenoloxidases, apolipophorins, serine protease homologs, and a serine protease that promotes hemocyte recruitment through cytokine activation. Pattern recognition proteins included C-type Lectins in B. mori, while A. aegypti contained a protein homologous to Plasmodium-resistant LRIM1 from Anopheles gambiae. We also found that the B. mori IC is stabilized by extensive transglutaminase-catalyzed cross-linking of multiple components. The melanization inhibitor Egf1.0, from the parasitoid wasp Microplitis demolitor, blocked inclusion of specific components into the IC and also inhibited transglutaminase activity. Our results show how coagulants, melanization components, and hemocytes can be recruited to a wound surface or pathogen, provide insight into the mechanism by which a parasitoid evades this immune response, and suggest that insects as diverse as Lepidoptera and Diptera utilize similar defensive mechanisms.

Jaburetox-induced toxic effects on the hemocytes of Rhodnius prolixus (Hemiptera: Reduviidae).

PubMed

Moyetta, Natalia R; Broll, Valquiria; Perin, Ana Paula A; Uberti, Augusto F; Coste Grahl, Matheus V; Staniscuaski, Fernanda; Carlini, Celia R; Fruttero, Leonardo L

2017-10-01

Jaburetox is a recombinant peptide derived from a Canavalia ensiformis urease that presents toxic effects upon several species of insects, phytopathogenic fungi and yeasts of medical importance. So far, no toxicity of Jaburetox to mammals has been shown. Previous reports have identified biochemical targets of this toxic peptide in insect models, although its mechanism of action is not completely understood. In this work, we aimed to characterize the effects of Jaburetox in hemolymphatic insect cells. For this purpose, the model insect and Chagas' disease vector Rhodnius prolixus was used. In vivo and in vitro experiments indicated that Jaburetox interacts with a subset of hemocytes and it can be found in various subcellular compartments. In insects injected with Jaburetox there was an increase in the gene expression of the enzymes UDP-N-acetylglucosamine pyrophosphorylase (UAP), chitin synthase and nitric oxide synthase (NOS). Nevertheless, the expression of NOS protein, the enzyme activities of UAP and acid phosphatase (a possible link between UAP and NOS) as well as the phosphorylation state of proteins remained unchanged upon the in vivo Jaburetox treatment. Nitric oxide (NO) imaging using fluorescent probes showed that Jaburetox augmented NO production in the hemocyte aggregates when compared to controls. Even though Jaburetox activated the hemocytes, as demonstrated by wheat germ agglutinin binding assays, the peptide did not lead to an increase of their phagocytic behavior. Taken together, these findings contribute to our understanding of toxic effects of Jaburetox, a peptide with biotechnological applications and a prospective tool for rational insect control. Copyright © 2017 Elsevier Inc. All rights reserved.

Clustering of adhesion receptors following exposure of insect blood cells to foreign surfaces.

PubMed

Nardi, James B; Zhuang, Shufei; Pilas, Barbara; Bee, Charles Mark; Kanost, Michael R

2005-05-01

Cell-mediated immune responses of insects involve interactions of two main classes of blood cells (hemocytes) known as granular cells and plasmatocytes. In response to a foreign surface, these hemocytes suddenly transform from circulating, non-adherent cells to cells that interact and adhere to each other and the foreign surface. This report presents evidence that during this adhesive transformation the extracellular matrix (ECM) proteins lacunin and a ligand for peanut agglutinin (PNA) lectin are released by granular cells and bind to surfaces of both granular cells and plasmatocytes. ECM protein co-localizes on cell surfaces with the adhesive receptors integrin and neuroglian, a member of the immunoglobulin superfamily. The ECM protein(s) secreted by granular cells are hypothesized to interact with adhesion receptors such as neuroglian and integrin by cross linking and clustering them on hemocyte surfaces. This clustering of receptors is known to enhance the adhesiveness (avidity) of interacting mammalian immune cells. The formation of ring-shaped clusters of these adhesion receptors on surfaces of insect immune cells represents an evolutionary antecedent of the mammalian immunological synapse.

Allatotropin: A pleiotropic neuropeptide that elicits mosquito immune responses

PubMed Central

Sánchez-Zavaleta, Minerva; Brito, Kevin; Herrera-Ortiz, Antonia; Ons, Sheila; Noriega, Fernando G.

2017-01-01

Allatotropins (AT) are neuropeptides with pleotropic functions on a variety of insect tissues. They affect processes such as juvenile hormone biosynthesis, cardiac rhythm, oviduct and hindgut contractions, nutrient absorption and circadian cycle. The present work provides experimental evidence that AT elicits immune responses in two important mosquito disease vectors, Anopheles albimanus and Aedes aegypti. Hemocytes and an immune-competent mosquito cell line responded to AT by showing strong morphological changes and increasing bacterial phagocytic activity. Phenoloxidase activity in hemolymph was also increased in Ae. aegypti mosquitoes treated with AT but not in An. albimanus, suggesting differences in the AT-dependent immune activation in the two species. In addition, two important insect immune markers, nitric oxide levels and expression of antimicrobial peptide genes, were increased in An. albimanus guts after AT treatment. AT conjugated to quantum dot nanocrystals (QDots) specifically labeled hemocytes in vivo in both mosquito species, implying molecular interactions between AT and hemocytes. The results of our studies suggest a new role for AT in the modulation of the immune response in mosquitoes. PMID:28426765

Transdifferentiation and Proliferation in Two Distinct Hemocyte Lineages in Drosophila melanogaster Larvae after Wasp Infection

PubMed Central

Ihalainen, Teemu O.; Vanha-aho, Leena-Maija; Andó, István; Rämet, Mika

2016-01-01

Cellular immune responses require the generation and recruitment of diverse blood cell types that recognize and kill pathogens. In Drosophila melanogaster larvae, immune-inducible lamellocytes participate in recognizing and killing parasitoid wasp eggs. However, the sequence of events required for lamellocyte generation remains controversial. To study the cellular immune system, we developed a flow cytometry approach using in vivo reporters for lamellocytes as well as for plasmatocytes, the main hemocyte type in healthy larvae. We found that two different blood cell lineages, the plasmatocyte and lamellocyte lineages, contribute to the generation of lamellocytes in a demand-adapted hematopoietic process. Plasmatocytes transdifferentiate into lamellocyte-like cells in situ directly on the wasp egg. In parallel, a novel population of infection-induced cells, which we named lamelloblasts, appears in the circulation. Lamelloblasts proliferate vigorously and develop into the major class of circulating lamellocytes. Our data indicate that lamellocyte differentiation upon wasp parasitism is a plastic and dynamic process. Flow cytometry with in vivo hemocyte reporters can be used to study this phenomenon in detail. PMID:27414410

Transdifferentiation and Proliferation in Two Distinct Hemocyte Lineages in Drosophila melanogaster Larvae after Wasp Infection.

PubMed

Anderl, Ines; Vesala, Laura; Ihalainen, Teemu O; Vanha-Aho, Leena-Maija; Andó, István; Rämet, Mika; Hultmark, Dan

2016-07-01

Cellular immune responses require the generation and recruitment of diverse blood cell types that recognize and kill pathogens. In Drosophila melanogaster larvae, immune-inducible lamellocytes participate in recognizing and killing parasitoid wasp eggs. However, the sequence of events required for lamellocyte generation remains controversial. To study the cellular immune system, we developed a flow cytometry approach using in vivo reporters for lamellocytes as well as for plasmatocytes, the main hemocyte type in healthy larvae. We found that two different blood cell lineages, the plasmatocyte and lamellocyte lineages, contribute to the generation of lamellocytes in a demand-adapted hematopoietic process. Plasmatocytes transdifferentiate into lamellocyte-like cells in situ directly on the wasp egg. In parallel, a novel population of infection-induced cells, which we named lamelloblasts, appears in the circulation. Lamelloblasts proliferate vigorously and develop into the major class of circulating lamellocytes. Our data indicate that lamellocyte differentiation upon wasp parasitism is a plastic and dynamic process. Flow cytometry with in vivo hemocyte reporters can be used to study this phenomenon in detail.

Circulating Hemocytes from Larvae of the Japanese Rhinoceros Beetle Allomyrina dichotoma (Linnaeus) (Coleoptera: Scarabaeidae) and the Cellular Immune Response to Microorganisms.

PubMed

Hwang, Sejung; Bang, Kyeongrin; Lee, Jiae; Cho, Saeyoull

2015-01-01

Hemocytes of the last larva of the Japanese rhinoceros beetle A. dichotoma (Linnaeus) (Coleoptera: Scarabaeidae) were classified as granulocytes, plasmatocytes, oenocytoids, spherulocytes, prohemocytes, and adipohemocytes. Among these cell types, only the granulocytes became immunologically activated with obvious morphological changes, displaying large amoeba-like, lobopodia-like, and fan-like structures. In addition, their cytoplasmic granules became larger and greatly increased in number. To explore whether these granules could be immunologically generated as phagosomes, total hemocytes were stained with LysoTracker. Greater than 90% of the granulocytes retained the LysoTracker dye at 4 h post-bacterial infection. In flow cytometry analysis, the red fluorescent signal was highly increased at 4 h post-bacterial infection (60.36%) compared to controls (5.08%), as was confirmed by fluorescent microscopy. After 12 h post-infection, these signals returned to basal levels. The uptake of pathogens by granulocytes rapidly triggered the translocation of the microtubule-associated protein 1 light chain 3 alpha (LC3) to the phagosome, which may result in enhanced pathogen killing.

Circulating Hemocytes from Larvae of the Japanese Rhinoceros Beetle Allomyrina dichotoma (Linnaeus) (Coleoptera: Scarabaeidae) and the Cellular Immune Response to Microorganisms

PubMed Central

Hwang, Sejung; Bang, Kyeongrin; Lee, Jiae; Cho, Saeyoull

2015-01-01

Hemocytes of the last larva of the Japanese rhinoceros beetle A. dichotoma (Linnaeus) (Coleoptera: Scarabaeidae) were classified as granulocytes, plasmatocytes, oenocytoids, spherulocytes, prohemocytes, and adipohemocytes. Among these cell types, only the granulocytes became immunologically activated with obvious morphological changes, displaying large amoeba-like, lobopodia-like, and fan-like structures. In addition, their cytoplasmic granules became larger and greatly increased in number. To explore whether these granules could be immunologically generated as phagosomes, total hemocytes were stained with LysoTracker. Greater than 90% of the granulocytes retained the LysoTracker dye at 4 h post-bacterial infection. In flow cytometry analysis, the red fluorescent signal was highly increased at 4 h post-bacterial infection (60.36%) compared to controls (5.08%), as was confirmed by fluorescent microscopy. After 12 h post-infection, these signals returned to basal levels. The uptake of pathogens by granulocytes rapidly triggered the translocation of the microtubule-associated protein 1 light chain 3 alpha (LC3) to the phagosome, which may result in enhanced pathogen killing. PMID:26030396

[The age-related changes in hemolymph cellular composition and in the spectrum of cytomorphological traits of hemocyte genetic damages in snail Lymnaea stagnalis].

PubMed

Koneva, O Iu; Afonin, V Iu; Dromashko, S E

2006-01-01

The age-related changes in hemolymph cellular composition of snail Lymnaea stagnalis (Gastropoda, Pulmonata) obtained from individuals of a natural population (the river Pripayt, Gomel region, Belarus) as well as in the spectrum of cytomorphological traits of hemocyte genetic damages have been studied. The percentage of the distinguished hemolymph cell types during the chosen age period was not revealed to change. The percentage of cells with different morphological attributes of cell death varied during ageing. The tendency to increase in the total level of dying cells was observed.

Savicalin, a lipocalin from hemocytes of the soft tick, Ornithodoros savignyi.

PubMed

Cheng, Paul H; Mans, Ben J; Neitz, Albert W H; Gaspar, Anabella R M

2010-11-01

Savicalin, is a lipocalin found in the hemocytes of the soft tick, Ornithodoros savignyi. It could be assigned to the tick lipocalin family based on BLAST analysis. Savicalin is the first non-salivary gland lipocalin described in ticks. The mature sequence is composed of 188 amino acids with a molecular mass of 21481.9 Da. A homolog for savicalin was found in a whole body EST-library from a related soft tick O. porcinus, while other tick salivary gland derived lipocalins retrieved from the non-redundant sequence database are more distantly related. Homology modeling supports the inclusion of savicalin into the lipocalin family. The model as well as multiple alignments suggests the presence of five disulphide bonds. Two conserved disulphide bonds are found in hard and soft tick lipocalins. A third disulphide bond is shared with the TSGP4-clade of leukotriene C4 binding soft tick lipocalins and a fourth is shared with a lipocalin from the hard tick Ixodes scapularis. The fifth disulphide bond is unique and links strands D-E. Phylogenetic analysis showed that savicalin is a distant relative of salivary gland derived lipocalins, but groups within a clade that is possibly non-salivary gland derived. It lacks the biogenic amine-binding motif associated with tick histamine and serotonin binding proteins. Expression profiles indicate that savicalin is found in hemocytes, midgut and ovaries, but not in the salivary glands. Up-regulation occurs in hemocytes after bacterial challenge and in midguts and ovaries after feeding. Given its tissue distribution and up-regulation of expression, it is possible that this lipocalin functions in tick development after feeding or in an anti-microbial capacity.

Impact of exposure time, particle size and uptake pathway on silver nanoparticle effects on circulating immune cells in mytilus galloprovincialis.

PubMed

Bouallegui, Younes; Ben Younes, Ridha; Turki, Faten; Oueslati, Ridha

2017-12-01

Nanomaterials have increasingly emerged as potential pollutants to aquatic organisms. Nanomaterials are known to be taken up by hemocytes of marine invertebrates including Mytilus galloprovincialis. Indeed, assessments of hemocyte-related parameters are a valuable tool in the determination of potentials for nanoparticle (NP) toxicity. The present study assessed the effects from two size types of silver nanoparticles (AgNP: <50 nm and <100 nm) on the frequency of hemocytes subpopulations as immunomodulation biomarkers exposed in a mollusk host. Studies were performed using exposures prior to and after inhibition of potential NP uptake pathways (i.e. clathrin- and caveolae-mediated endocytosis) and over different durations of exposure (3, 6 and 12 h). Differential hemocyte counts (DHC) revealed significant variations in frequency of different immune cells in mussels exposed for 3 hr to either AgNP size. However, as exposure duration progressed cell levels were subsequently differentially altered depending on particle size (i.e. no significant effects after 3 h with larger AgNP). AgNP effects were also delayed/varied after blockade of either clathrin- or caveolae-mediated endocytosis. The results also noted significant negative correlations between changes in levels hyalinocytes and acidophils or in levels basophils and acidophils as a result of AgNP exposure. From these results, we concluded AgNP effects on mussels were size and duration of exposure dependent. This study highlighted how not only was NP size important, but that differing internalization mechanisms could be key factors impacting on the potential for NP in the environment to induce immunomodulation in a model/test sentinel host like M. galloprovincialis.

Scavenger Receptor C Mediates Phagocytosis of White Spot Syndrome Virus and Restricts Virus Proliferation in Shrimp

PubMed Central

Yang, Ming-Chong; Shi, Xiu-Zhen; Yang, Hui-Ting; Sun, Jie-Jie; Xu, Ling; Wang, Xian-Wei; Zhao, Xiao-Fan

2016-01-01

Scavenger receptors are an important class of pattern recognition receptors that play several important roles in host defense against pathogens. The class C scavenger receptors (SRCs) have only been identified in a few invertebrates, and their role in the immune response against viruses is seldom studied. In this study, we firstly identified an SRC from kuruma shrimp, Marsupenaeus japonicus, designated MjSRC, which was significantly upregulated after white spot syndrome virus (WSSV) challenge at the mRNA and protein levels in hemocytes. The quantity of WSSV increased in shrimp after knockdown of MjSRC, compared with the controls. Furthermore, overexpression of MjSRC led to enhanced WSSV elimination via phagocytosis by hemocytes. Pull-down and co-immunoprecipitation assays demonstrated the interaction between MjSRC and the WSSV envelope protein. Electron microscopy observation indicated that the colloidal gold-labeled extracellular domain of MjSRC was located on the outer surface of WSSV. MjSRC formed a trimer and was internalized into the cytoplasm after WSSV challenge, and the internalization was strongly inhibited after knockdown of Mjβ-arrestin2. Further studies found that Mjβ-arrestin2 interacted with the intracellular domain of MjSRC and induced the internalization of WSSV in a clathrin-dependent manner. WSSV were co-localized with lysosomes in hemocytes and the WSSV quantity in shrimp increased after injection of lysosome inhibitor, chloroquine. Collectively, this study demonstrated that MjSRC recognized WSSV via its extracellular domain and invoked hemocyte phagocytosis to restrict WSSV systemic infection. This is the first study to report an SRC as a pattern recognition receptor promoting phagocytosis of a virus. PMID:28027319

Effects of Sublethal Concentrations of the Chitin Synthesis Inhibitor, Hexaflumuron, on the Development and Hemolymph Physiology of the Cutworm, Spodoptera litura

PubMed Central

Zhu, Qiqi; He, Yuan; Yao, Jing; Liu, Yinzhao; Tao, Liming; Huang, Qingchun

2012-01-01

The effects of sublethal concentrations 0.1, 0.5, and 1.2 µg mL-1of the chitin synthesis inhibitor, hexaflumuron, on larval growth and development, the count and proportion of hemocytes, and carbohydrate content (trehalose and glyceride) in hemolymph were investigated in the cutworm, Spodoptera litura (Fabricious) (Lepidoptera: Noctuidae). When 3rdinstar larvae were subjected to the sublethal concentrations, there were dose-dependent effects on larval weight and length of each instar larvae, percent pupation and the duration of development. Most of the larvae died during the molting process at all concentrations. Few individuals from 0.5 and 1.2 µg mL -1concentrations could develop to the 6thinstar, while the pupae emerging from the 0.1 µg mL -1concentrations did not exceed 16% of the number of the initial larvae. In 5thinstar S. litura, the total number of hemocytes was significantly increased at 24 hours post—treatment, whereas the proliferation of hemocytes was inhibited, plasmatocyte pseudopodia contracted, and granulocyte expanded at 96 hours post—treatment. The increases of plasmatocyte count and the decreases of granulocyte count were dose—dependent. The longer treatment time of the sublethal concentrations increased the content of total carbohydrate and trehalose in hematoplasma, and was dose—dependent in hemocytes. The content of glyceride in hemolymph was significantly higher at 24 hours post—treatment, but gradually returned to normal levels at 96 hours post—treatment as compared with the control. The results suggested that sublethal concentrations of hexaflumuron reduced S. litura larval survival and interfered with hemolymph physiological balances. PMID:22958164

The B-cell translocation gene 1 (CgBTG1) identified in oyster Crassostrea gigas exhibit multiple functions in immune response.

PubMed

Liu, Rui; Cheng, Qi; Wang, Xiudan; Chen, Hao; Wang, Weilin; Zhang, Huan; Wang, Lingling; Song, Linsheng

2017-02-01

B-cell translocation gene 1 (BTG1) is a member of the anti-proliferative gene family, which plays important roles in regulation of cell cycle. In the present study, a B-cell translocation gene 1 molecule homologue (designed CgBTG1) are identified and characterized in oyster Crassostrea gigas. CgBTG1 contains a conserved BTG domain with Box A and Box B motifs, and it shares high similarities with both BTG1 and BTG2 proteins in vertebrates. CgBTG1 mRNA is predominantly expressed in hemocytes, and its expression level in hemocytes is significantly up-regulated at 6 h (5.40-fold, p < 0.01) post Vibrio splendidus stimulation. The apoptosis rate of oyster hemocytes is significantly decreased (p < 0.05) after CgBTG1 interfered by dsRNA (dsCgBTG1). This is indicated that CgBTG1 participated in the regulation of oyster hemocytes apoptosis. Furthermore, CgBTG1 could also induce the apoptosis of cancer cells (HeLa, A549 and BEL7402) in vitro. Compared with normal oysters, both vessel-like structures and muscle fibers in CgBTG1 interfered oysters are severely damaged after V. splendidus challenge in paraffin section, considering that CgBTG1 possessed an analogous feature of angiogenesis for maintenance of vessel-like structures in adductor muscle of oyster. The results suggests that CgBTG1 is a multi-functional molecule involved in the immune response of C. gigas against pathogen infection, which provides more clues for intensive studies of BTG family proteins in invertebrates. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Antimicrobial Defense of the Pacific Oyster, Crassostrea gigas. How Diversity may Compensate for Scarcity in the Regulation of Resident/Pathogenic Microflora

PubMed Central

Schmitt, Paulina; Rosa, Rafael Diego; Duperthuy, Marylise; de Lorgeril, Julien; Bachère, Evelyne; Destoumieux-Garzón, Delphine

2012-01-01

Healthy oysters are inhabited by abundant microbial communities that vary with environmental conditions and coexist with immunocompetent cells in the circulatory system. In Crassostrea gigas oysters, the antimicrobial response, which is believed to control pathogens and commensals, relies on potent oxygen-dependent reactions and on antimicrobial peptides/proteins (AMPs) produced at low concentrations by epithelial cells and/or circulating hemocytes. In non-diseased oysters, hemocytes express basal levels of defensins (Cg-Defs) and proline-rich peptides (Cg-Prps). When the bacterial load dramatically increases in oyster tissues, both AMP families are driven to sites of infection by major hemocyte movements, together with bactericidal permeability/increasing proteins (Cg-BPIs) and given forms of big defensins (Cg-BigDef), whose expression in hemocytes is induced by infection. Co-localization of AMPs at sites of infection could be determinant in limiting invasion as synergies take place between peptide families, a phenomenon which is potentiated by the considerable diversity of AMP sequences. Besides, diversity occurs at the level of oyster AMP mechanisms of action, which range from membrane lysis for Cg-BPI to inhibition of metabolic pathways for Cg-Defs. The combination of such different mechanisms of action may account for the synergistic activities observed and compensate for the low peptide concentrations in C. gigas cells and tissues. To overcome the oyster antimicrobial response, oyster pathogens have developed subtle mechanisms of resistance and evasion. Thus, some Vibrio strains pathogenic for oysters are equipped with AMP-sensing systems that trigger resistance. More generally, the known oyster pathogenic vibrios have evolved strategies to evade intracellular killing through phagocytosis and the associated oxidative burst. PMID:22783227

Co-expression of heat shock protein (HSP) 40 and HSP70 in Pinctada martensii response to thermal, low salinity and bacterial challenges.

PubMed

Li, Jun; Zhang, Yuehuan; Liu, Ying; Zhang, Yang; Xiao, Shu; Yu, Ziniu

2016-01-01

Heat shock protein (HSP) 40 proteins are a family of molecular chaperones that bind to HSP70 through their J-domain and regulate the function of HSP70 by stimulating its adenosine triphosphatase activity. In the present study, a HSP40 homolog named PmHSP40 was cloned from the hemocytes of pearl oyster Pinctada martensii using EST and rapid amplification of cDNA ends (RACE) techniques. The full-length cDNA of PmHSP40 was 1251 bp in length, which included a 5' untranslated region (UTR) of 75 bp, an open reading frame (ORF) of a 663 bp, and a 3' UTR of 513 bp. The deduced amino acid sequence of PmHSP40 contains a J domain in the N-terminus. In response to thermal and low salinity stress challenges, the expression of PmHSP40 in hemocytes and the gill were inducible in a time-dependent manner. After bacterial challenge, PmHSP40 transcripts in hemocytes increased and peaked at 6 h post injection. In the gill, PmHSP40 expression increased, similar to expression in hemocytes; however, transcript expression of PmHSP40 was significantly up-regulated at 12 h post injection. Furthermore, the transcripts of PmHSP70 showed similar kinetics as that of PmHSP40, with highest induction during thermal, low salinity stress and bacterial challenges. Altogether these results demonstrate that PmHSP40 is an inducible protein under thermal, low salinity and bacterial challenges, suggesting its involvement in both environmental and biological stresses, and in the innate immunity of the pearl oyster. Copyright © 2015 Elsevier Ltd. All rights reserved.

Consequences of Food Restriction for Immune Defense, Parasite Infection, and Fitness in Monarch Butterflies.

PubMed

McKay, Alexa Fritzsche; Ezenwa, Vanessa O; Altizer, Sonia

2016-01-01

Organisms have a finite pool of resources to allocate toward multiple competing needs, such as development, reproduction, and enemy defense. Abundant resources can support investment in multiple traits simultaneously, but limited resources might promote trade-offs between fitness-related traits and immune defenses. We asked how food restriction at both larval and adult life stages of the monarch butterfly (Danaus plexippus) affected measures of immunity, fitness, and immune-fitness interactions. We experimentally infected a subset of monarchs with a specialist protozoan parasite to determine whether parasitism further affected these relationships and whether food restriction influenced the outcome of infection. Larval food restriction reduced monarch fitness measures both within the same life stage (e.g., pupal mass) as well as later in life (e.g., adult lifespan); adult food restriction further reduced adult lifespan. Larval food restriction lowered both hemocyte concentration and phenoloxidase activity at the larval stage, and the effects of larval food restriction on phenoloxidase activity persisted when immunity was sampled at the adult stage. Adult food restriction reduced only adult phenoloxidase activity but not hemocyte concentration. Parasite spore load decreased with one measure of larval immunity, but food restriction did not increase the probability of parasite infection. Across monarchs, we found a negative relationship between larval hemocyte concentration and pupal mass, and a trade-off between adult hemocyte concentration and adult life span was evident in parasitized female monarchs. Adult life span increased with phenoloxidase activity in some subsets of monarchs. Our results emphasize that food restriction can alter fitness and immunity across multiple life stages. Understanding the consequences of resource limitation for immune defense is therefore important for predicting how increasing constraints on wildlife resources will affect fitness and resistance to natural enemies.

Cloning and characterization of a shrimp clip domain serine protease homolog (c-SPH) as a cell adhesion molecule.

PubMed

Lin, Chun-Yu; Hu, Kuang-Yu; Ho, Shih-Hu; Song, Yen-Ling

2006-01-01

Clip domain serine protease homologs (c-SPHs) are involved in various innate immune functions in arthropods such as antimicrobial activity, cell adhesion, pattern recognition, opsonization, and regulation of the prophenoloxidase system. In the present study, we cloned a c-SPH cDNA from tiger shrimp (Penaeus monodon) hemocytes. It is 1337 bp in length with a coding region of 1068 bp consisting a protein of 355 amino acid residues. The deduced protein includes one clip domain and one catalytically inactive serine protease-like (SP-like) domain. Its molecular weight is estimated to be 38 kDa with an isoelectric point of 7.9. The predicted cutting site of the signal peptide is located between Gly(21) and Gln(22). We aligned 15 single clip domain SPH protein sequences from 12 arthropod species; the identity of these clip domains is low and that of SP-like domains is from 34% to 46%. The conserved regions are located near the amino acid residues which served as substrate interaction sites in catalytically active serine protease. Phylogenetically, the tiger shrimp c-SPH is most similar to a low molecular mass masquerade-like protein of crayfish, but less similar to c-SPHs in Chelicerata and Insecta. Nested reverse transcription polymerase chain reaction (RT-PCR) revealed that c-SPH mRNA is expressed most in tissues with the highest hemocyte abundance. Antimicrobial and opsonization activities of the molecule were not detected. The expression of c-SPH mRNA in hemocytes was up-regulated at the 12-day post beta-glucan immersion. Recombinant c-SPH could significantly enhance hemocyte adhesion. The result suggests that the shrimp c-SPH protein plays a role in innate immunity.

Transcriptomic and Quantitative Proteomic Analyses Provide Insights Into the Phagocytic Killing of Hemocytes in the Oyster Crassostrea gigas

PubMed Central

Jiang, Shuai; Qiu, Limei; Wang, Lingling; Jia, Zhihao; Lv, Zhao; Wang, Mengqiang; Liu, Conghui; Xu, Jiachao; Song, Linsheng

2018-01-01

As invertebrates lack an adaptive immune system, they depend to a large extent on their innate immune system to recognize and clear invading pathogens. Although phagocytes play pivotal roles in invertebrate innate immunity, the molecular mechanisms underlying this killing remain unclear. Cells of this type from the Pacific oyster Crassostrea gigas were classified efficiently in this study via fluorescence-activated cell sorting (FACS) based on their phagocytosis of FITC-labeled latex beads. Transcriptomic and quantitative proteomic analyses revealed a series of differentially expressed genes (DEGs) and proteins present in phagocytes; of the 352 significantly high expressed proteins identified here within the phagocyte proteome, 262 corresponding genes were similarly high expressed in the transcriptome, while 140 of 205 significantly low expressed proteins within the proteome were transcriptionally low expressed. A pathway crosstalk network analysis of these significantly high expressed proteins revealed that phagocytes were highly activated in a number of antimicrobial-related biological processes, including oxidation–reduction and lysosomal proteolysis processes. A number of DEGs, including oxidase, lysosomal protease, and immune receptors, were also validated in this study using quantitative PCR, while seven lysosomal cysteine proteases, referred to as cathepsin Ls, were significantly high expressed in phagocytes. Results show that the expression level of cathepsin L protein in phagocytes [mean fluorescence intensity (MFI): 327 ± 51] was significantly higher (p < 0.01) than that in non-phagocytic hemocytes (MFI: 83 ± 26), while the cathepsin L protein was colocalized with the phagocytosed Vibrio splendidus in oyster hemocytes during this process. The results of this study collectively suggest that oyster phagocytes possess both potent oxidative killing and microbial disintegration capacities; these findings provide important insights into hemocyte phagocytic killing as a component of C. gigas innate immunity. PMID:29942306

No Detectable Trade-Offs Among Immune Function, Fecundity, and Survival via a Juvenile Hormone Analog in the House Cricket.

PubMed

Nava-Sánchez, A; Munguía-Steyer, R; Córdoba-Aguilar, A

2014-08-01

Hormones are key regulators of resource allocation among functions and thus play an important role in resource-based trade-offs. The juvenile hormone (JH) is an insect hormone that mediates resource allocation between immunity and life history components. Here, we have tested whether this is the case using the house cricket. We investigated whether increased levels of JH (using methoprene, a JH analog) enable an enhanced survival and fecundity (via egg number) at the cost of reduced hemocyte number (a trait that is associated with immune response in insects) in the house cricket, Acheta domesticus L. We had three groups of adult crickets of both sexes: experimental (methoprene and acetone), positive control (methoprene), and negative control (no manipulation). Prior to and after experimental treatments, we counted the number of hemocytes (for the case of both sexes) and recorded the number of eggs laid and survival of females after the manipulation. There was no difference in hemocyte number, egg number, and survival. These results do not support a JH-mediated trade-off among immune ability, survival, and fecundity. We provide arguments to explain the lack of JH-mediated trade-offs in the house cricket.

Mediated effect of ultrasound treated Diclofenac on mussel hemocytes: First evidence for the involvement of respiratory burst enzymes in the induction of DCF-mediated unspecific mode of action.

PubMed

Toufexi, Eirini; Dailianis, Stefanos; Vlastos, Dimitris; Manariotis, Ioannis D

2016-06-01

The present study investigates the toxic behavior of diclofenac (DCF) before and after its ultrasound (US) treatment, as well as the involvement of intracellular target molecules, such as NADPH oxidase and NO synthase, in the DCF-induced adverse effects on hemocytes of mussel Mytilus galloprovincialis. In this context, appropriate volumes (350 and 500mL) of DCF solutions (at concentrations of 2, 2.5, 5 and 10mgL(-1)) were treated under different ultrasound operating conditions (frequency at 582 and 862kHz, electric power density at 133 and 167W) for assessing US method efficiency. In parallel, DCF and US DCF-mediated cytotoxic (in terms of cell viability measured with the use of neutral red uptake/NRU method), oxidative (in terms of superoxide anions/(.)O2(-), nitric oxides such as NO2(-) and lipid peroxidation products, such as malondialdehyde/MDA content) and genotoxic (DNA damage measured by the use of Comet assay method) effects were investigated in hemocytes exposed for 1h to 5, 10 and 100ngL(-1) and 1, 10 and 20μgL(-1) of DCF. The involvement of NADPH oxidase and NO synthase to the DCF-induced toxicity was further investigated by the use of 10μΜ L-NAME, a NO synthase inhibitor and 10μΜ DPI, a NADPH oxidase inhibitor. According to the results, 350mL of 2mgL(-1) DCF showed higher degradation (>50%) under 167W electric power density and frequency at 862kHz for 120min, compared to degradation in all other cases, followed by a significant elimination of its toxicity. Specifically, US DCF-treated hemocytes showed a significant attenuation of DCF-mediated cytotoxic, oxidative and genotoxic effects, which appeared to be caused by NADPH oxidase and NO synthase activation, since their inhibition was followed by a significant elimination of (.)O2(-) and NO2(-) generation and the concomitant oxidative damage within cells. The results of the present study showed for the first time that unspecific mode of action of DCF, associated with the induction of NADPH oxidase and NO synthase in mussel hemocytes, could be significantly diminished after partial US degradation of DCF, at least under optimized operating conditions currently tested. Copyright © 2016 Elsevier B.V. All rights reserved.

Immunological responses of the mangrove oysters Crassostrea gasar naturally infected by Perkinsus sp. in the Mamanguape Estuary, Paraíba state (Northeastern, Brazil).

PubMed

Queiroga, Fernando Ramos; Marques-Santos, Luis Fernando; Hégaret, Hélène; Soudant, Philippe; Farias, Natanael Dantas; Schlindwein, Aline Daiane; Mirella da Silva, Patricia

2013-08-01

Perkinsus genus includes protozoan parasites of marine mollusks, especially bivalves. In the last four years, this parasite has been detected in mangrove oysters Crassostrea rhizophorae and Crassostrea gasar from the Northeastern region of Brazil. Hemocytes are the key cells of the oyster immune system, being responsible for a variety of cellular and humoral reactions, such as phagocytosis, encapsulation and the release of several effector molecules that control the invasion and proliferation of microorganisms. In Brazil, there is little information on perkinsosis and none on the immune responses of native oysters' species against Perkinsus spp. The objective of this study was to determine the effects of natural infection by Perkinsus sp. on the immunological parameters of mangrove oysters C. gasar cultured in the Mamanguape River Estuary (Paraíba, Brazil). Adults oysters (N = 40/month) were sampled in December 2011, March, May, August and October 2012. Gills were removed and used to determine the presence and intensity of the Perkinsus sp. infection, according to a scale of four levels (1-4), using the Ray's fluid thioglycollate medium assay. Immunological parameters were measured in hemolymph samples by flow cytometry, including: total hemocyte count (THC), differential hemocyte count (DHC), cell mortality, phagocytic capacity, and production of Reactive Oxygen Species (ROS). The plasma was used to determine the hemagglutination activity. The results showed the occurrence of Perkinsus sp. with the highest mean prevalence (93.3%) seen so far in oyster populations in Brazil. Despite that, no oyster mortality was associated. In contrast, we observed an increase in hemocyte mortality and a suppression of two of the main defense mechanisms, phagocytosis and ROS production in infected oysters. The increase in the percentage of blast-like cells on the hemolymph, and the increase in THC in oysters heavily infected (at the maximum intensity, 4) suggest an induction of hemocytes proliferation. The immunological parameters varied over the studied months, which may be attributed to the dynamics of infection by Perkinsus sp. The results of the present study demonstrate that Perkinsus sp. has a deleterious effect on C. gasar immune system, mainly in high intensities, which likely renders oysters more susceptible to other pathogens and diseases. Copyright © 2013 Elsevier Ltd. All rights reserved.

Condition-Dependent Trade-Off Between Weapon Size and Immunity in Males of the European Earwig.

PubMed

Körner, Maximilian; Vogelweith, Fanny; Foitzik, Susanne; Meunier, Joël

2017-08-11

Investigating the expression of trade-offs between key life-history functions is central to our understanding of how these functions evolved and are maintained. However, detecting trade-offs can be challenging due to variation in resource availability, which masks trade-offs at the population level. Here, we investigated in the European earwig Forficula auricularia whether (1) weapon size trades off with three key immune parameters - hemocyte concentration, phenoloxidase and prophenoloxidase activity - and whether (2) expression and strength of these trade-offs depend on male body condition (body size) and/or change after an immune challenge. Our results partially confirmed condition dependent trade-offs between weapon size and immunity in male earwigs. Specifically, we found that after an immune challenge, weapon size trades off with hemocyte concentrations in low-condition, but not in good-condition males. Contrastingly, weapon size was independent of pre-challenge hemocyte concentration. We also found no trade-off between weapon size and phenoloxidase activity, independent of body condition and immune challenge. Overall, our study reveals that trade-offs with sexual traits may weaken or disappear in good-condition individuals. Given the importance of weapon size for male reproductive success, our results highlight how low-condition individuals may employ alternative life-history investment strategies to cope with resource limitation.

Hematopoietic organs of Manduca sexta and hemocyte lineages.

PubMed

Nardi, James B; Pilas, Barbara; Ujhelyi, Elizabeth; Garsha, Karl; Kanost, Michael R

2003-10-01

Cells of the moth immune system are derived from organs that loosely envelop the four wing imaginal discs. The immune response in these insects is believed to depend on the activities of two main classes of hemocytes: plasmatocytes and granular cells. The fates of cells that arise from these hematopoietic organs have been followed by immunolabeling with plasmatocyte-specific and granular-cell-specific antibodies. Cells within each hematopoietic organ differ in their coherence and in their expression of two plasmatocyte-specific surface proteins, integrin and neuroglian. Within an organ there is no overlap in the expression of these two surface proteins; neuroglian is found on the surfaces of the coherent cells while integrin is expressed on cells that are losing coherence, rounding up, and dispersing. A granular-cell-specific marker for the protein lacunin labels the basal lamina that delimits each organ but only a small number of granular cells that lie on or near the periphery of the hematopoietic organ. When organs are cultured in the absence of hemolymph, all cells derived from hematopoietic organs turn out to immunolabel with the plasmatocyte-specific antibody MS13. The circulating plasmatocytes derived from hematopoietic organs have higher ploidy levels than the granular cells and represent a separate lineage of hemocytes.

Isolation of prawn ( Exopalaemon carinicauda) lipopolysaccharide and β-1, 3-glucan binding protein gene and its expression in responding to bacterial and viral infections

NASA Astrophysics Data System (ADS)

Ge, Qianqian; Li, Jian; Duan, Yafei; Li, Jitao; Sun, Ming; Zhao, Fazhen

2016-04-01

The pattern recognition proteins (PRPs) play a major role in immune response of crustacean to resist pathogens. In the present study, as one of PRPs, lipopolysaccharide and β-1, 3-glucan binding protein (LGBP) gene in the ridge tail white prawn ( Exopalaemon carinicauda) ( EcLGBP) was isolated. The full-length cDNA of EcLGBP was 1338 bp, encoding a polypeptide of 366 amino acid residules. The deduced amino acid sequence of EcLGBP shared high similarities with LGBP and BGBP from other crustaceans. Some conservative domains were predicted in EcLGBP sequence. EcLGBP constitutively expressed in most tissues at different levels, and the highest expression was observed in hepatopancreas. With infection time, the cumulative mortality increased gradually followed by the proliferation of Vibrio parahaemolyticus and white spot syndrome virus (WSSV). The expression of EcLGBP in response to V. parahaemolyticus infection was up-regulated in hemocytes and hepatopancreas, and the up-regulation in hepatopancreas was earlier than that in hemocytes. EcLGBP expression after WSSV infection increased at 3 h, then significantly decreased in both hemocytes and hepatopancreas. The results indicated that EcLGBP was involved in the immune defense against bacterial and viral infections.

[Response of Calliphora vicina larval hemocytes to abiotic and biotic foreign particles injection].

PubMed

Kind, T V

2012-01-01

Human erythrocytes injection into the body cavity of Calliphora vicina postfeeding larvae results to their fast binding by thrombocytoidal fragments with agglutinates formation. There were almost none sites of lysis and degradation of erythrocytes in agglutinates even after shape modification and strands generation. Exceptions are zones of agglutinates with juvenile hemocytes, where destruction of erythrocytes is seen. The sequential injection of erythrocytes and charcoal particles leads to charcoal adhesion at first to agglutinates periphery and later to more deep stratum of cytoplasm between the erythrocytes. Under such conditions agglutinate formation period is accompanied with morphology variations which do not influence the intensity of agglutinating reaction. Juvenile plasmatocytes phagocytized the charcoal particles regardless of their concentration and duration of previous contact with erythrocytes. When mixture of abiotic and biotic particles was injected into post feeding larvae, crythrocytes and charcoal generate independent aggregations in the range of separate agglutinates. At the same time plasmatocytes form nodules consisting of temporary cell aggregations covered with cores of non phagocytized charcoal particles. These data testified that presumably lectin receptors responsible for foreign biotic and abiotic particles recognition are very near but not identical for different types of hemocytes. They may be specifical (for plasmatocytes) or integrated to different parts of cellular membrane (in thrombocytoids).

Hemocyte-lineage marker proteins in a crustacean, the freshwater crayfish, Pacifastacus leniusculus.

PubMed

Wu, Chenglin; Söderhäll, Irene; Kim, Young-A; Liu, Haipeng; Söderhäll, Kenneth

2008-10-01

To identify proteins associated with development of different hemocyte types in the freshwater crayfish Pacifastacus leniusculus, 2-DE followed by MS analysis was carried out with hematopoietic tissue (Hpt) cells, semigranular cells (SGC) and granular cells (GC). Within the hemocyte lineages one two-domain Kazal proteinase inhibitor (KPI) was found to be specific for SGC, while a superoxide dismutase (SOD) was specific for GC at protein as well as at mRNA level. The proliferation cell nuclear antigen (PCNA) was detected at the mRNA level in Hpt cells only. We also provide evidence that SGC and GC most likely differentiate to maturation as separate lineages. We found that after laminarin or lipopolysaccharide (LPS) injection into crayfish, the transcript levels of PCNA and SOD increased in the Hpt cells, whereas the KPI transcript never was present in Hpt regardless of any challenge. RNA interference of PCNA in the Hpt cells led to that most of the cells did not spread or attach to the tissue culture dish. These results suggest that PCNA, KPI and SOD can be used as markers for Hpt cells, SGC and GC, respectively, and in conjunction with these results, a model is proposed how the Hpt responds to a microbial challenge by proliferation and release of Hpt cells.

Immunological Profile of the Yellow Clam Mesodesma mactroides (Mesodesmatidae) from the Southern Coast of Rio Grande do Sul, Brazil.

PubMed

Silva Santos, Juan Jethro; Carvalho, Yuri Bovi; de Alcantara Lopes, Diogo Luiz; Romano, Luis Alberto

2016-03-01

The yellow clam Mesodesma mactroides (Mesodesmatidae) is a sandy beach bivalve that is distributed from Rio de Janeiro, Brazil, to the south of Buenos Aires province, Argentina. The yellow clam population has been declining in recent decades. To increase our understanding of this species, we evaluated the immunological status of yellow clams collected during different seasons from various areas in the state of Rio Grande do Sul, Brazil. We characterized the hemocytes, determined the differential hemocyte counts (DHCs), calculated the apoptotic index, and evaluated the incidence of parasites in yellow clams through histological analysis. We identified two types of hemocyte (hyaline and granular) that showed significant variation in DHCs among sampling areas during the summer and winter. The apoptotic index only exhibited significant variation during the summer. Histopathological analysis results did not significantly differ among sampling areas. This work demonstrated that environmental variation (e.g., temperature and salinity) associated with anthropogenic actions may be affecting the immune system of yellow clams. However, more studies are needed to determine the full influence of these factors on the yellow clam's immune system and thus contribute to future management and aquaculture of the species. Received May 10, 2015; accepted October 28, 2015.

TWO INNEXINS OF Spodoptera litura INFLUENCES HEMICHANNEL AND GAP JUNCTION FUNCTIONS IN CELLULAR IMMUNE RESPONSES.

PubMed

Pang, Zunyu; Li, Ming; Yu, Dongshuai; Yan, Zhang; Liu, Xinyi; Ji, Xinglai; Yang, Yang; Hu, Jiansheng; Luo, Kaijun

2015-09-01

Insect cellular immune responses include encapsulation, nodule formation, and phagocytosis. Hemichannels and gap junctions are involved in these cellular actions. Innexins (Inxs: analogous to the vertebrate connexins) form hemichannels and gap junctions, but the molecular mechanisms underlying their biology is still unclear. In this article, we reported a steady-state level of Inxs (SpliInxs) in hemocytes of Spodoptera litura, which formed nonfunctional hemichannels on the cell surface to maintain normal metabolism. We also reported that two innnexins (SpliInx2 and SpliInx3) were expressed significantly higher in hemocytes compared to other tissues, suggesting that they play important roles in hemocytes. Amino acid analysis found that two cysteine residues in two extracellular loops provided the capability for SpliInx2 and SpliInx3 hemichannels to dock into gap junctions. Western blotting demonstrated that both extracellular and intracellular loops of SpliInx3 and the extracellular loops of SpliInx2 might undergo posttranslational modification during the formation of a steady-state hemichannel. During hemichannel formation, SpliInx2 presented as one isoform, while SpliInx3 presented as three isoforms. These results provide fundamental knowledge for further study of how steady-state levels of SpliInxs are dynamically adjusted to perform cellular immune responses under immune challenge. © 2015 Wiley Periodicals, Inc.

Activation of PmRelish from Penaeus monodon by yellow head virus.

PubMed

Visetnan, Suwattana; Supungul, Premruethai; Hirono, Ikuo; Tassanakajon, Anchalee; Rimphanitchayakit, Vichien

2015-02-01

Humoral innate immune response against pathogenic infection is partly responsible by the Imd pathway in which a transcription factor Relish relays the infection signals to the nuclei for the expression of antimicrobial proteins. A PmRelish gene which encoded a protein of 1195 amino acids was cloned. The PmRelish was constitutively expressed in all tissues tested and mostly up-regulated upon YHV infection. In hemocytes, the PmRelish expression was up-regulated upon Vibrio harveyi, yellow head virus (YHV) and white spot syndrome virus (WSSV) challenges. Using dsRNA silencing of PmRelish gene, it was shown that the expression of penaeidin5 but not anti-lipopolysaccharide factor ALFPm3, crustinPm1 and penaeidin3 was under the regulation of Imd pathway. Under PmRelish silencing, the shrimp were more susceptible to infection by YHV with the 50% survival rate reduced from about 72 h to 42 h. The PmRelish was detected in the cytoplasm of all the hemocytes from both uninfected and YHV-infected shrimp. The accumulation of activated PmRelish in the nuclei was not clearly observed but the activated PmRelish was detected in the YHV-infected hemocytes by Western blot analysis. Thus, the PmRelish and, hence, the Imd pathway respond to the YHV infection. Copyright © 2014 Elsevier Ltd. All rights reserved.

The tick plasma lectin, Dorin M, is a fibrinogen-related molecule.

PubMed

Rego, Ryan O M; Kovár, Vojtĕch; Kopácek, Petr; Weise, Christoph; Man, Petr; Sauman, Ivo; Grubhoffer, Libor

2006-04-01

A lectin, named Dorin M, previously isolated and characterized from the hemolymph plasma of the soft tick, Ornithodoros moubata, was cloned and sequenced. The immunofluorescence using confocal microscopy revealed that Dorin M is produced in the tick hemocytes. A tryptic cleavage of Dorin M was performed and the resulting peptide fragments were sequenced by Edman degradation and/or mass spectrometry. Two of three internal peptide sequences displayed a significant similarity to the family of fibrinogen-related molecules. Degenerate primers were designed and used for PCR with hemocyte cDNA as a template. The sequence of the whole Dorin M cDNA was completed by the method of RACE. The tissue-specific expression investigated by RT-PCR revealed that Dorin M, in addition to hemocytes, is significantly expressed in salivary glands. The derived amino-acid sequence clearly shows that Dorin M has a fibrinogen-like domain, and exhibited the most significant similarity with tachylectins 5A and 5B from a horseshoe crab, Tachypleus tridentatus. In addition, other protein and binding characteristics suggest that Dorin M is closely related to tachylectins-5. Since these lectins have been reported to function as non-self recognizing molecules, we believe that Dorin M may play a similar role in an innate immunity of the tick and, possibly, also in pathogen transmission by this vector.

Differential immune response in the hard clam (mercenaria mercenaria) against bacteria and the protistan pathogen QPX (quahog parasite unknown).

PubMed

Perrigault, Mickael; Allam, Bassem

2012-06-01

The immune response of the hard clam (quahog) Mercenaria mercenaria following challenge with live bacteria (Vibrio alginolyticus) and the protist QPX (Quahog Parasite Unknown) was investigated. The study also compared immune responses following QPX challenge in two different hard clam broodstocks exhibiting different degrees of susceptibility toward this parasite. Different immune and stress-related cellular and humoral factors were assessed including general hemocyte parameters (total and differential hemocyte counts, percentage of dead cells, reactive oxygen production, phagocytosis), parameters geared toward QPX (anti-QPX activity in plasma and hemocyte resistance to the cytotoxicity of QPX extracellular products). Two genes (ferritin and metallothionein) previously shown to be modulated following QPX exposure were molecularly characterized by rapid amplification of cDNA ends (RACE) and their transcription levels were determined in resistant and susceptible clams in response to QPX and bacterial challenge. Results indicated that both V. alginolyticus and QPX challenge triggered significant immune responses in clams with similar trends for most measured parameters. However, specific responses were observed for anti-QPX activity in plasma and hemocyte resistance to QPX products as well as ferritin and metallothionein expression according to each inoculum. Similarly, different response patterns were detected following QPX challenge in susceptible and resistant clam stocks. Resistant clams were able to elicit effective response against the parasite leading to the elimination of QPX and the restoration of constitutive immune status whereas QPX-susceptible clams triggered a strong immune modulation characterized by an acute phase response and associated acute phase protein but appeared to be less active in eliminating the parasite. These results suggest that different signaling pathways are triggered during V. alginolyticus and QPX challenge. Moreover, differences in the immune response toward QPX might be linked to the susceptibility or resistance of different clam stocks to the infection by this parasite. Copyright © 2012 Elsevier Ltd. All rights reserved.

Nematocytes: Discovery and characterization of a novel anculeate hemocyte in Drosophila falleni and Drosophila phalerata.

PubMed

Bozler, Julianna; Kacsoh, Balint Z; Bosco, Giovanni

2017-01-01

Immune challenges, such as parasitism, can be so pervasive and deleterious that they constitute an existential threat to a species' survival. In response to these ecological pressures, organisms have developed a wide array of novel behavioral, cellular, and molecular adaptations. Research into these immune defenses in model systems has resulted in a revolutionary understanding of evolution and functional biology. As the field has expanded beyond the limited number of model organisms our appreciation of evolutionary innovation and unique biology has widened as well. With this in mind, we have surveyed the hemolymph of several non-model species of Drosophila. Here we identify and describe a novel hemocyte, type-II nematocytes, found in larval stages of numerous Drosophila species. Examined in detail in Drosophila falleni and Drosophila phalerata, we find that these remarkable cells are distinct from previously described hemocytes due to their anucleate state (lacking a nucleus) and unusual morphology. Type-II nematocytes are long, narrow cells with spindle-like projections extending from a cell body with high densities of mitochondria and microtubules, and exhibit the ability to synthesize proteins. These properties are unexpected for enucleated cells, and together with our additional characterization, we demonstrate that these type-II nematocytes represent a biological novelty. Surprisingly, despite the absence of a nucleus, we observe through live cell imaging that these cells remain motile with a highly dynamic cellular shape. Furthermore, these cells demonstrate the ability to form multicellular structures, which we suggest may be a component of the innate immune response to macro-parasites. In addition, live cell imaging points to a large nucleated hemocyte, type-I nematocyte, as the progenitor cell, leading to enucleation through a budding or asymmetrical division process rather than nuclear ejection: This study is the first to report such a process of enucleation. Here we describe these cells in detail for the first time and examine their evolutionary history in Drosophila.

[Cellular defense system of some synanthropic dipterans inhabitant of bacterially aggressive environment].

PubMed

Kind, T V

2014-01-01

The hemocytic count and defense reaction within 4 families of higher Diptera: Tabanidae, Syrphidae, Muscidae and Sarcophagidae, whose larvae inhabit bacterially aggressive environment, were investigated. The least hemocytes types (3) were revealed in Tabanidae and Syrphidae larvae--prohemocytes, plasmatocytes and prophenoloxydase-containing unstable hyaline cells (oenocytoids). In Sarcophaga crassipalpis and Musca domestica stable hyaline cells and thrombocytoids or podocytoid-like cells can be added to this set. At the time of pupariation in Sarcophaga, new generation of prohemocytes is segregated into the hemolymph, which form small round or spindle-shaped hyaline cells. So, the number of plasmatocyte types in Sarcophaga increase to six. Typical to Calliphoridae juvenile plasmatocytes in the members of investigated families are absent. Among the one hemocyte type morphology also can vary, especially in unstable prophenoloxydase hyaline cells. In Drosophila there are crystal cells containing in the cytoplasm paracrystalloidal inclusions. In Calliphoridae there are big hyaline cells with homogenous cytoplasm producing circumferential bubbles. Both in Sarcophaga and Tabanidae they contain in their cytoplasm big globules. However in Sarcophaga they rapidly disintegrate, while in Tabanidae are maintained unchanged during hours. In Muscidae and Syrphidae prophenoloxydase extrusion occurs very early and these cells obtain pycnotic nuclei and very liquid cytoplasm with strings of granules. Thrombocytoids in Musca larvae are represented by big flattened anucleated irregular cytoplasm and "naked" nuclei and cytoplasmic fragments often with fan-like projections. Plasmatocytes in all species studied are the cells with pronounced phylopodies. In larvae they contain cytoplasmic catabolic inclusions and in pupa--ragments of apoptotic tissues. Clearance of hemolymph from alien particles in Sarcophagidae and Muscidae occur by thrombocytoides, while in Tabanidae by plasmatocyte nodulation. A differing case is Syrphidae whe-e charcoal injection produce depletion of hemolymph both from particles and all types of hemocytes. So the specimen of different higher Diptera families can use different schemes of cellular defense reaction.

Molecular cloning of Kuruma shrimp Marsupenaeus japonicus endonuclease-reverse transcriptase and its positive role in white spot syndrome virus and Vibrio alginolyticus infection.

PubMed

Ma, Xiongchao; Sun, Baozhen; Zhu, Fei

2018-02-01

This study investigated the function of endonuclease-reverse transcriptase (mjERT) in Marsupenaeus japonicus. The 1129 bp cDNA sequence of mjERT was cloned from M. japonicus using rapid amplification of cDNA ends (RACE) PCR, and RT-qPCR analysis indicated that mjERT was highly expressed in the gills and hepatopancreas of M. japonicus. We also found that white spot syndrome virus (WSSV) or Vibrio alginolyticus challenge could enhance the expression of mjERT. When mjERT was inhibited, immune genes such as toll, p53, hemocyanin and tumor necrosis factor-α (TNF-α) were significantly down-regulated (P < .01) in the hemocytes of shrimp, while myosin was significantly up-regulated (P < .01). We demonstrated that mjERT is very important for the progression of WSSV infection and that the cumulative mortality of WSSV-infected and V. alginolyticus-infected shrimps was significantly increased following mjERT RNA interfere (RNAi). Apoptosis data provided information to suggest that mjERT-dsRNA challenge caused less apoptosis in hemocytes in both the disease-free and viral group. We also revealed that mjERT-dsRNA treatment resulted in a lower phagocytosis rate in the hemocytes of V. alginolyticus-challenged shrimp. Finally, we found that the absence of mjERT had an significantly negative impact upon shrimp phenoloxidase (PO) activity, superoxide dismutase (SOD) activity and total hemocyte count (THC) following WSSV or V. alginolyticus infection, indicating a regulative role for mjERT in the innate immunity of shrimp in response to pathogenic infection. In summary, we concluded that mjERT might promote the anti-WSSV immune response of shrimp by regulating apoptosis, PO activity, THC and SOD activity, and also exert a positive role in the immune response against V. alginolyticus by regulating phagocytosis, SOD activity, PO activity and THC. Copyright © 2017 Elsevier Ltd. All rights reserved.

Ocean Acidification Affects the Cytoskeleton, Lysozymes, and Nitric Oxide of Hemocytes: A Possible Explanation for the Hampered Phagocytosis in Blood Clams, Tegillarca granosa.

PubMed

Su, Wenhao; Rong, Jiahuan; Zha, Shanjie; Yan, Maocang; Fang, Jun; Liu, Guangxu

2018-01-01

An enormous amount of anthropogenic carbon dioxide (CO 2 ) has been dissolved into the ocean, leading to a lower pH and changes in the chemical properties of seawater, which has been termed ocean acidification (OA). The impacts of p CO 2 -driven acidification on immunity have been revealed recently in various marine organisms. However, the mechanism causing the reduction in phagocytosis still remains unclear. Therefore, the impacts of p CO 2 -driven OA at present and near-future levels (pH values of 8.1, 7.8, and 7.4) on the rate of phagocytosis, the abundance of cytoskeleton components, the levels of nitric oxide (NO), and the concentration and activity of lysozymes (LZM) of hemocytes were investigated in a commercial bivalve species, the blood clam ( Tegillarca granosa ). In addition, the effects of OA on the expression of genes regulating actin skeleton and nitric oxide synthesis 2 ( NOS2 ) were also analyzed. The results obtained showed that the phagocytic rate, cytoskeleton component abundance, concentration and activity of LZM of hemocytes were all significantly reduced after a 2-week exposure to the future OA scenario of a pH of 7.4. On the contrary, a remarkable increase in the concentration of NO compared to that of the control was detected in clams exposed to OA. Furthermore, the expression of genes regulating the actin cytoskeleton and NOS were significantly up-regulated after OA exposure. Though the mechanism causing phagocytosis seemed to be complicated based on the results obtained in the present study and those reported previously, our results suggested that OA may reduce the phagocytosis of hemocytes by (1) decreasing the abundance of cytoskeleton components and therefore hampering the cytoskeleton-mediated process of engulfment, (2) reducing the concentration and activity of LZM and therefore constraining the degradation of the engulfed pathogen through an oxygen-independent pathway, and (3) inducing the production of NO, which may negatively regulate immune responses.

Integrated Immune and Cardiovascular Function in Pancrustacea: Lessons from the Insects.

PubMed

Hillyer, Julián F

2015-11-01

When pathogens invade the insect hemocoel (body cavity) they immediately confront two major forces: immune-responses and circulatory currents. The immune response is mediated by circulating and sessile hemocytes, the fat body, the midgut, and the salivary glands. These tissues drive cellular and humoral immune processes that kill pathogens via phagocytosis, melanization, lysis, encapsulation, and nodulation. Moreover, immune-responses take place within a three-dimensional and dynamic space that is governed by the forces of the circulatory system. The circulation of hemolymph (insect blood) is primarily controlled by the wave-like contraction of a dorsal vessel, which is a muscular tube that extends the length of the insect and is divided into a thoracic aorta and an abdominal heart. Distributed along the heart are valves, called ostia, that allow hemolymph to enter the vessel. Once inside the heart, hemolymph is sequentially propelled to the anterior and to the posterior of the body. During an infection, circulatory currents sweep small pathogens to all regions of the body. As they circulate, pathogens encounter immune factors of the insect that range from soluble cytotoxic peptides to phagocytic hemocytes. A prominent location for these encounters is the surface of the heart. Specifically, periostial hemocytes aggregate in the extracardiac regions that flank the heart's ostia (the periostial regions) and phagocytoze pathogens in areas of high flow of hemolymph. This review summarizes the biology of the immune and circulatory systems of insects, including how these two systems have co-adapted to fight infection. This review also compares the immune and circulatory systems of insects to that of crustaceans, and details how attachment of hemocytes to cardiac tissues and the biology of the lymphoid organ demonstrate that dynamic interactions between the immune and circulatory systems also occur in lineages of crustaceans. © The Author 2015. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.

C-type lectin B (SpCTL-B) regulates the expression of antimicrobial peptides and promotes phagocytosis in mud crab Scylla paramamosain.

PubMed

Wei, Xiaoyuan; Wang, Limin; Sun, Wanwei; Zhang, Ming; Ma, Hongyu; Zhang, Yueling; Zhang, Xinxu; Li, Shengkang

2018-07-01

As pattern recognition receptors, C-type lectins (CTLs) play important roles in immune system of crustaceans through identifying and binding to the conservative pathogen-associated molecular patterns (PAMPs) on pathogen surfaces. In this study, a new CTL, SpCTL-B, was identified from the hemocytes of mud crab Scylla paramamosain. The full-length of SpCTL-B cDNA was 1278 bp with an open reading frame (ORF) of 348 bp. The predicted SpCTL-B protein contains a single carbohydrate-recognition domain (CRD). SpCTL-B transcripts were distributed in all examined tissues with the highest levels in hepatopancreas. After challenged with Vibrio parahaemolyticus, LPS, polyI:C and white spot syndrome virus (WSSV), the mRNA levels of SpCTL-B in hemocytes and hepatopancreas were up-regulated. The recombinant SpCTL-B (rSpCTL-B) purified by Ni-affinity chromatography showed stronger binding activities with Staphylococcus aureus, β-hemolytic Streptococcus, Escherichia coli, Aeromonas hydrophila, Vibrio alginolyticus than those with V. parahaemolyticus and Saccharomyces cerevisiae. rSpCTL-B exhibited a broad spectrum of microorganism-agglutination activities against Gram-positive bacteria (S. aureus, β-hemolytic Streptococcus) and Gram-negative bacteria (E. coli, V. parahaemolyticus, A. hydrophila, V. alginolyticus) in a Ca 2+ -dependent manner. The agglutination activities of rSpCTL-B could be inhibited by D-mannose and LPS, but not by d-fructose and galactose. The antimicrobial assay showed that rSpCTL-B exhibited the growth inhibition against all examined gram-positive bacteria and gram-negative bacteria. When SpCTL-B was silenced by RNAi, the bacterial clearance ability in mud crab was decreased and the transcript levels of five antimicrobial peptides (AMPs) (SpCrustin, SpHistin, SpALF4 (anti-lipopolysaccharide factor), SpALF5 and SpALF6) were significantly decreased in hemocytes. In our study, knockdown of SpCTL-B could down-regulate the expression of SpSTAT at mRNA transcriptional level and protein translational level in mud crab. Meantime, the phagocytosis rate and the expression of three phagocytosis related genes were declined after RNAi of SpCTL-B in hemocytes in mud crab. Collectively, our results suggest that SpCTL-B might play its roles as a pattern recognition receptor (PRR) in immune response towards pathogens infection through influencing the expression of AMPs and the phagocytosis of hemocytes in mud crab S. paramamosain. Copyright © 2018 Elsevier Ltd. All rights reserved.

A clip domain serine protease regulates the expression of proPO and hemolymph clotting in mud crab, Scylla paramamosain.

PubMed

Zhang, Daimeng; Wan, Weisong; Kong, Tongtong; Zhang, Ming; Aweya, Jude Juventus; Gong, Yi; Li, Shengkang

2018-05-07

The clip domain serine proteinases (clip-SPs) play vital roles in embryonic development and in various innate immune functions in invertebrates such as antimicrobial activity, cell adhesion, hemolymph clotting, pattern recognition and regulation of the prophenoloxidase system. However, little is known about the role of the clip domain serine proteinase in Scylla paramamosain (designated SpcSP) immunity. In the present study, we cloned a clip-SP from S. paramamosain hemocytes using rapid amplification of cDNA end (RACE) approach. The full-length cDNA of SpcSP was 1823 bp, containing a 5' untranslated region (UTR) of 334 bp, an open reading frame of 1122 bp, and a 3' UTR of 367 bp. The open reading frame encoded a polypeptide of 373 amino acids with a calculated molecular weight of 39.7 kDa and an isoelectric point of 6.64. Structurally, SpcSP has a predicted 21-residue signal peptide and possessed the characteristic features of the clip domain family of serine proteases, namely one clip domain in the amino-terminal with six highly conserved cysteine residues and one enzyme active serine proteinase domain in the carboxyl-terminal with a highly conserved catalytic triad (His 156 , Asp 226 , Ser 321 ). Phylogenetic analysis showed that SpcSP was clustered together with PtcSP (clip domain serine proteinase from Portunus trituberculatus). Quantitative real-time PCR (qPCR) analysis showed that the mRNA of SpcSP was constitutively expressed at different levels in all tested tissues in untreated S. paramamosain, with hemocytes and skin expressing the most. The transcriptional level of SpcSP in hemocytes was significantly up-regulated upon challenge with V. parahaemolyticus and LPS, indicating its involvement in antibacterial immune response. Indirect immunofluorescence analysis showed that SpcSP was expressed in the cytoplasm of all three hemocyte cell types (hyaline, semigranular and granular cells). Further, recombinant SpcSP protein exhibited strong binding ability and has antimicrobial activity against both Gram-positive and Gram-negative bacteria as well as fungi. Moreover, knockdown of SpcSP resulted in increased hemolymph clotting time and decreased the mRNA expression of SpproPO mRNA in hemocytes. These findings therefore suggest that SpcSP plays an important role in the antimicrobial defense mechanism of S. paramamosain by regulating the expression of SpproPO and hemolymph clotting in S. paramamosain. Copyright © 2018. Published by Elsevier Ltd.

L-3,4-Dihydroxyphenylalanine (l-DOPA) induces neuroendocrinological, physiological, and immunological regulation in white shrimp, Litopenaeus vannamei.

PubMed

Mapanao, Ratchaneegorn; Kuo, Hsin-Wei; Chang, Chin-Chuan; Liu, Kuan-Fu; Cheng, Winton

2018-03-01

L-3,4-Dihydroxyphenylalanine (l-DOPA) is a precursor for dopamine (DA) synthesis. Assessments were conducted to analyze the effects of l-DOPA on mediating regulation of neuroendocrinological, immunological, and physiological parameters in the shrimp, Litopenaeus vannamei when they were individually injected with 0.01 N HCl or l-DOPA at 0.5 or 1.0 μmol shrimp -1 for 60, 120, and 240 min. For catecholamine synthesis evaluation, tyrosine hydroxylase (TH) and DA beta hydroxylase (DBH) activities, l-DOPA, DA, and norepinephrine (NE) levels in hemolymph were determined. The total hemocyte count (THC), differential hemocyte count (DHC), phenoloxidase (PO) activity, respiratory bursts (RBs), superoxide dismutase (SOD) activity, phagocytic activity, and clearance efficiency in response to the pathogen, Vibrio alginolyticus were assessed for immune responses, and plasma glucose and lactate levels were for physiological response. Results showed that the TH activity, THC, hyaline cells (HCs), and semigranular cells (SGCs) at 120 min, DA levels at 60-240 min, PO activity in hemocytes per 50 μL of hemolymph at 60-120 min, and PO activity per granulocyte (granular cells (GCs) + SGCs) at 60 min significantly increased, but TH activity, l-DOPA levels, GCs, SGCs, and respiratory bursts in hemocytes per 10 μL of hemolymph at 60 min, respiratory bursts per hemocyte and SOD activity at 120 min, phagocytic activity at 60-240 min, and the clearance efficiency at 60-120 min significantly decreased in shrimp injected with l-DOPA at 1.0 μmol shrimp -1 . In another experiment, 60 min after shrimp had received l-DOPA at 0.5 or 1.0 μmol shrimp -1 , they were challenged with an injection of V. alginolyticus at 2 × 10 5  colony-forming units (cfu) shrimp -1 . The injection of l-DOPA at 1.0 μmol shrimp -1 also significantly increased the cumulative mortality of shrimp by 16.7%, compared to the HCl-challenged control after 120 h. These results suggest that l-DOPA administration at 1.0 μmol shrimp -1 can mediate the transient regulation of neuroendocrinological, immunological, and physiologic responses resulting in immunosuppression, which in turn promoted the susceptibility of L. vannamei to V. alginolyticus. Copyright © 2018 Elsevier Ltd. All rights reserved.

Characterization of myosin light chain in shrimp hemocytic phagocytosis.

PubMed

Han, Fang; Wang, Zhiyong; Wang, Xiaoqing

2010-11-01

Myosin light chain, a well-known cytoskeleton gene, regulates multiple processes that are involved in material transport, muscle shrink and cell division. However, its function in phagocytosis against invading pathogens in crustacean remains unknown. In this investigation, a myosin light chain gene was obtained from Marsupenaeus japonicus shrimp. The full-length cDNA of this gene was of 766 bp and an open reading frame (ORF) of 462 bp encoding a polypeptide of 153 amino acids. The myosin light chain protein was expressed in Escherichia coli and purified. Subsequently the specific antibody was raised using the purified GST fusion protein. As revealed by immuno-electron microscopy, the myosin light chain protein was only expressed in the dark bands of muscle. In the present study, the myosin light chain gene was up-regulated in the WSSV-resistant shrimp as revealed by real-time PCR and western blot. And the phagocytic percentage and phagocytic index using FITC-labeled Vibrio parahemolyticus were remarkably increased in the WSSV-resistant shrimp, suggesting that the myosin light chain protein was essential in hemocytic phagocytosis. On the other hand, RNAi assays indicated that the phagocytic percentage and phagocytic index were significantly decreased when the myosin light chain gene was silenced by sequence-specific siRNA. These findings suggested that myosin light chain protein was involved in the regulation of hemocytic phagocytosis of shrimp. Copyright 2010 Elsevier Ltd. All rights reserved.

PmVRP15, a novel viral responsive protein from the black tiger shrimp, Penaeus monodon, promoted white spot syndrome virus replication.

PubMed

Vatanavicharn, Tipachai; Prapavorarat, Adisak; Jaree, Phattarunda; Somboonwiwat, Kunlaya; Tassanakajon, Anchalee

2014-01-01

Suppression subtractive hybridization of Penaeus monodon hemocytes challenged with white spot syndrome virus (WSSV) has identified the viral responsive gene, PmVRP15, as the highest up-regulated gene ever reported in shrimps. Expression analysis by quantitative real time RT-PCR revealed 9410-fold up-regulated level at 48 h post WSSV injection. Tissue distribution analysis showed that PmVRP15 transcript was mainly expressed in the hemocytes of shrimp. The full-length cDNA of PmVRP15 transcript was obtained and showed no significant similarity to any known gene in the GenBank database. The predicted open reading frame of PmVRP15 encodes for a deduced 137 amino acid protein containing a putative transmembrane helix. Immunofluorescent localization of the PmVRP15 protein revealed it accumulated around the nuclear membrane in all three types of shrimp hemocytes and that the protein was highly up-regulated in WSSV-infected shrimps. Double-stranded RNA interference-mediated gene silencing of PmVRP15 in P. monodon significantly decreased WSSV propagation compared to the control shrimps (injected with GFP dsRNA). The significant decrease in cumulative mortality rate of WSSV-infected shrimp following PmVRP15 knockdown was observed. These results suggest that PmVRP15 is likely to be a nuclear membrane protein and that it acts as a part of WSSV propagation pathway.

Experimental infection of octopus vulgaris (Cuvier, 1797) with Photobacterium damsela subsp. piscicida. Immunohistochemical tracking of antigen and tissue responses.

PubMed

Bakopoulos, Vasileios; White, Daniella; Valsamidis, Michail-Aggelos; Vasilaki, Feli

2017-03-01

Adult common octopus individuals were intramuscularly infected with Photobacterium damsela subsp. piscicida in order to investigate if this species is sensitive to this common and important fish pathogen. The fate of the bacterial antigens and the tissue responses of Octopus vulgaris were studied employing immunohistochemical techniques. Strong reaction at the site of injection was evident from day 2 post-infection that continued until day 14. Great numbers of hemocytes that were attracted at the site of infection were involved in phagocytosis of bacteria. Very early in the infection, a transition of cells to fibroblasts and an effort to isolate the infection was observed. During the course of the study, very large necrotic cells were seen at the site of infection, whereas during the later stages hemocytes with phagocytosed bacteria were observed in well-defined pockets inside the muscle tissue. None of the internal organs tested for the presence of the bacterium were positive with the exception of the digestive gland where antigen staining was observed which was not associated with hemocyte infiltration. The high doses of bacterial cells used in this experimental infection and the lack of disease signs from Octopus vulgaris suggest that, under normal conditions, octopus is resistant to Photobacterium damsela subsp. piscicida. Copyright © 2017 Elsevier Inc. All rights reserved.

Monosaccharide transport into hemocytes of a sipunculan worm Themiste dyscrita

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ingermann, R.L.; Hall, R.E.; Bissonnette, J.M.

1985-07-01

The hemerythrin-containing blood cells, or hemocytes, of the sipunculan worm Themiste dyscrita were found to have a stereospecific and nonconcentrative monosaccharide transport system. The transport system transferred both D-glucose and 3-O-methyl-D-glucose (3-OMG), and transport into cells by this system was rapid, reaching 50% equilibrium in approximately 20 s at 10 degrees C with an initial concentration gradient of 0.1 mM; the contribution to total uptake by simple diffusion was very small. 3-OMG uptake showed saturation kinetics with a low half-saturation constant (Km less than or equal to 0.1 mM). The uptake of labeled 3-OMG by the hemocytes was strongly inhibitedmore » by unlabeled 3-OMG, 2-deoxy-D-glucose, alpha- and beta-D-glucose, D-galactose, and D-mannose. It was moderately inhibited by D-xylose, only slightly by alpha-methyl-D-glucoside and D-fructose, and uninhibited by sucrose, L-glucose, or D-sorbitol. Phloretin was more potent than phloridzin in blocking entry of 3-OMG. Cytochalasin B did not bind tightly to the T. dyscrita transporter and was not a potent inhibitor of transport; it half-maximally inhibited 3-OMG transport at 0.1 mM. Therefore, despite some differences the data suggest functional similarities in the mechanism of monosaccharide transport into blood cells of mammals and this invertebrate.« less

Coronatin-2 from the entomopathogenic fungus Conidiobolus coronatus kills Galleria mellonella larvae and incapacitates hemocytes.

PubMed

Boguś, M I; Wieloch, W; Ligęza-Żuber, M

2017-02-01

Coronatin-2, a 14.5 kDa protein, was isolated from culture filtrates of the entomopathogenic fungus Conidiobolus coronatus (Costantin) Batko (Entomophthoramycota: Entomophthorales). After LC-MS/MS (liquid chromatography tandem mass spectrometry) analysis of the tryptic peptide digest of coronatin-2 and a mass spectra database search no orthologs of this protein could be found in fungi. The highest homology was observed to the partial translation elongation factor 1a from Sphaerosporium equinum (protein sequence coverage, 21%), with only one peptide sequence, suggesting that coronatin-2 is a novel fungal protein that has not yet been described. In contrast to coronatin-1, an insecticidal 36 kDa protein, which shows both elastolytic and chitinolytic activity, coronatin-2 showed no enzymatic activity. Addition of coronatin-2 into cultures of hemocytes taken from larvae of Galleria mellonella Linnaeus (Lepidoptera: Pyralidae), resulted in progressive disintegration of nets formed by granulocytes and plasmatocytes due to rapid degranulation of granulocytes, extensive vacuolization of plasmatocytes accompanied by cytoplasm expulsion, and cell disintegration. Spherulocytes remained intact, while oenocytes rapidly disintegrated. Coronatin-2 produced 80% mortality when injected into G. mellonella at 5 µg larva-1. Further study is warranted to determine the relevance of the acute toxicity of coronatin-2 and its effects on hemocytes in vitro to virulence of C. coronatus against its hosts.

Molecular cloning and tissue expression of the fatty acid-binding protein (Es-FABP) gene in female Chinese mitten crab (Eriocheir sinensis).

PubMed

Gong, Ya-Nan; Li, Wei-Wei; Sun, Jiang-Ling; Ren, Fei; He, Lin; Jiang, Hui; Wang, Qun

2010-09-16

Fatty acid-binding proteins (FABPs), small cytosolic proteins that function in the uptake and utilization of fatty acids, have been extensively studied in higher vertebrates while invertebrates have received little attention despite similar nutritional requirements during periods of reproductive activity. Therefore, a cDNA encoding Eriocheir sinensis FABP (Es-FABP) was cloned based upon EST analysis of a hepatopancreas cDNA library. The full length cDNA was 750 bp and encoded a 131 aa polypeptide that was highly homologous to related genes reported in shrimp. The 9108 bp Es-FABP gene contained four exons that were interrupted by three introns, a genomic organization common among FABP multigene family members in vertebrates. Gene expression analysis, as determined by RT-PCR, revealed the presence of Es-FABP transcripts in hepatopancreas, hemocytes, ovary, gills, muscle, thoracic ganglia, heart, and intestine, but not stomach or eyestalk. Real-time quantitative RT-PCR analysis revealed that Es-FABP expression in ovary, hemocytes, and hepatopancreas was dependent on the status of ovarian development, with peak expression observed in January. Evidence provided in the present report supports a role of Es-FABP in lipid transport during the period of rapid ovarian growth in E. sinensis, and indirectly confirms the participation of the hepatopancreas, ovary, and hemocytes in lipid nutrient absorption and utilization processes.

Selectively enhanced expression of prophenoloxidase activating enzyme 1 (PPAE1) at a bacteria clearance site in the white shrimp, Litopenaeus vannamei

PubMed Central

2011-01-01

Background The prophenoloxidase-activating (PO activating) system plays an important role in the crustacean innate immunity, particularly in wound healing and pathogen defense. A key member of this system is prophenoloxidase-activating enzyme (PPAE), which is the direct activator of prophenoloxidase (proPO). Despite their importance in crustacean PO activating system, the studies on them remain limited. Results Here we report on a PPAE of white shrimp, Litopenaeus vannamei (lvPPAE1), which showed 94% similarity to PPAE1 of Penaeus monodon. We found that lvPPAE1 in fluid hemocytes was down regulated after challenge by Vibrio harveyi but was enhanced when shrimps were exposed to a bacteria-rich environment for long-term. In vivo gene silence of lvPPAE1 by RNAi can significantly reduce the phenoloxidase activity (PO) and increase the susceptibility of shrimps to V. harveyi. Although lvPPAE1 was down-regulated in fluid hemocytes by Vibrio challenge, its expression increased significantly in gill after bacteria injection, which is the primary bacteria-clearance tissue. Conclusion Suppressed expression in fluid hemocytes and enhanced expression in gill indicates selectively enhanced expression at the bacterial clearance site. This is a novel feature for PPAE expression. The results will contribute to our understanding of the PO activating system in crustaceans. PMID:22208405

Cellular Immune Reactions of the Sunn Pest, Eurygaster integriceps, to the Entomopathogenic Fungus, Beauveria bassiana and Its Secondary Metabolites

PubMed Central

Zibaee, Arash; Bandani, Ali Reza; Talaei-Hassanlouei, Reza; Malagoli, Davide

2011-01-01

In this study, five morphological types of circulating hemocytes were recognized in the hemolymph of the adult sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae), namely prohemocytes, plasmatocytes, granulocytes, adipohemocytes, and oenocytoids. The effects of the secondary metabolites of the entomopathogenic fungus Beauveria bassiana on cellular immune defenses of Eurygaster integriceps were investigated. The results showed that the fungal secondary metabolites inhibited phagocytic activity of E. integriceps hemocytes and hampered nodule formation. A reduction of phenoloxidase activity was also observed. The data suggest that B. bassiana produce secondary metabolites that disable several immune mechanisms allowing the fungus to overcome and then kill its host. This characteristic makes B. bassiana a promising model for biological control of insect pests such as E. integriceps. PMID:22233481

Silvernanotherapy on the viral borne disease of silkworm Bombyx mori L.

NASA Astrophysics Data System (ADS)

Govindaraju, K.; Tamilselvan, S.; Kiruthiga, V.; Singaravelu, G.

2011-12-01

Antiviral assays of chemically and biologically synthesized silver nanoparticles were made against BmNPV ( Bombyx mori Nuclear Polyhedrosis Virus). Reduction of silver ions by sodium citrate and Spirulina platensis led to the formation of spherical silver nanoparticles of 40-60 and 7-16 nm size. Single cell protein ( Spirulina platensis)-synthesized silver nanoparticles showed the strongest antiviral activity. Immunological studies made on the silkworm Bombyx mori disclosed that a significant increase in the total hemocyte count and differential hemocyte count due to S. platensis-synthesized silver nanoparticles supplementation. Improvement in the defense mechanism was noticed from the strengthened peritrophic membrane of the digestive tract and the increased total protein. Overall, the results presented illustrate that single cell protein-synthesized silver nanoparticles supplementation is effective in controlling viral-borne diseases of the silkworm.

Immunocompetence analysis of the aquatic snail Lymnaea stagnalis exposed to urban wastewaters.

PubMed

Boisseaux, Paul; Noury, Patrice; Delorme, Nicolas; Perrier, Lucile; Thomas-Guyon, Helene; Garric, Jeanne

2018-04-02

Wastewater treatment plant effluents from urban area are a well-known source of chronic multiple micropollution to the downstream living organisms. In this study, ecologically relevant laboratory-bred freshwater gastropods, Lymnaea stagnalis, were exposed for 29 days to raw effluents of a wastewater treatment plant in Lyon area (France). A time-course analysis of individual markers of immunocompetence (hemocyte density and viability, hemocyte NADPH activity, phenol oxidase activity, and capacity of phagocytosis) has shown slight trends of inflammatory-like responses induced by the 100% effluents. So far, no short-term hazard for L. stagnalis can be revealed. However, over the long term, such environmental stress-stimulating immune responses could provoke deleterious life history trade-offs because the immune system is known to be highly energy-consuming.

Identification of cDNAs encoding HSP70 and HSP90 in the abalone Haliotis tuberculata: Transcriptional induction in response to thermal stress in hemocyte primary culture.

PubMed

Farcy, Emilie; Serpentini, Antoine; Fiévet, Bruno; Lebel, Jean-Marc

2007-04-01

Heat-shock proteins are a multigene family of proteins whose expression is induced by a variety of stress factors. This work reports the cloning and sequencing of HSP70 and HSP90 cDNAs in the gastropod Haliotis tuberculata. The deduced amino acid sequences of both HSP70 and HSP90 from H. tuberculata shared a high degree of homology with their homologues in other species, including typical eukaryotic HSP70 and HSP90 signature sequences. We examined their transcription expression pattern in abalone hemocytes exposed to thermal stress. Real-time PCR analysis indicated that both HSP70 and HSP90 mRNA were expressed in control animals but rapidly increased after heat-shock.

The effects of defoliation-induced delayed changes in silver birch foliar chemistry on gypsy moth fitness, immune response, and resistance to baculovirus infection.

PubMed

Martemyanov, Vyacheslav V; Dubovskiy, Ivan M; Rantala, Markus J; Salminen, Juha-Pekka; Belousova, Irina A; Pavlushin, Sergey V; Bakhvalov, Stanislav A; Glupov, Victor V

2012-03-01

We tested the effects of defoliation-induced changes in silver birch, Betula pendula, foliar chemistry (delayed induced resistance, DIR) on the fitness and immune defense of the gypsy moth, Lymantria dispar. We measured larval developmental time, pupal weight, rate of survival to the adult stage, and five characteristics of larval immune defense: (1) encapsulation response; (2) phenoloxidase activity; (3) hemocyte concentration and (4) lysozyme-like activity in the hemolymph; and (5) resistance to infection by L. dispar nucleopolyhedrovirus (LdMNPV). The latter is an entomopathogenic baculovirus that often causes epizootics during outbreaks of L. dispar. We also measured the involvement of foliage non-tannin phenolic compounds in resistance of B. pendula to herbivory as well as the relationship between the compounds we identified and L. dispar development, growth, and survival. Leaves of B. pendula with previous defoliation history contained increased levels of myricetin glycoside, two flavonoid aglycones (acacetin and tetrahydroxy-flavone dimethyl ether), as well as one unidentified simple phenolic. The concentrations of two glycosides of quercetin, as well as the content of one unidentified flavonoid glycoside were significantly decreased under defoliation treatment. DIR of B. pendula retarded larval growth rate and increased lysozyme-like activity in the hemolymph, but did not affect encapsulation response, phenoloxidase activity, or hemocyte count. We did not find any DIR-mediated tritrophic interactions among birch, gypsy moth, and LdMNPV. After viral inoculation, the mean hemocyte counts in larvae reared on an individual tree correlated significantly with the survival of larvae reared on that same tree, indicating that hemocyte density in hemolymph might be associated with resistance to viral infection. We found a strong positive correlation between the concentration of 1-(4″-hydroxyphenyl)-3'-oxopropyl-β-D-glucopyranose and L. dispar survival rate, which may indicate an unlikely role of this dominant non-tannin phenolic in B. pendula defense against L. dispar. Our study also shows that several immune characteristics of insects that function as barriers against different groups of parasites are differently affected by plant induced defenses. This underscores the importance of considering multiple factors when characterizing barriers to insect immunity.

Molecular characterization of shrimp harbinger transposase derived 1 (HARBI1)-like and its role in white spot syndrome virus and Vibrio alginolyticus infection.

PubMed

Sun, Baozhen; Qian, Xiyi; Zhu, Fei

2018-07-01

The role of the nuclease, HARBI1-like protein (mjHARBI1-like) in the innate immunity of Marsupenaeus japonicus was explored in this study. The 1361 bp cDNA sequence of mjHARBI1-like was cloned from M. japonicus using RACE. RT-qPCR analysis results showed that the gills and hepatopancreas of M. japonicus were the main tissues where mjHARBI1-like is expressed. In addition, it was also found that white spot syndrome virus (WSSV) or Vibrio alginolyticus challenge could stimulate mjHARBI1-like expression. After mjHARBI1-likewas inhibited, expression of immune genes such as toll, p53, myosin, and proPO were significantly downregulated (P < 0.01). However, in shrimp hemocytes, hemocyanin and tumor necrosis factor-α (TNF-α) were up-regulated significantly (P < 0.01). This study demonstrated that mjHARBI1-like plays a key role in the progression of WSSV and V. alginolyticus infection. Specifically, the cumulative mortality of WSSV-infected and V. alginolyticus-infected shrimp was significantly advanced by double-strand RNA interference (dsRNAi) of mjHARBI1-like. Apoptosis studies indicated that mjHARBI1-dsRNA treatment caused a reduction in hemocyte apoptosis in bacterial and viral groups. In addition, phagocytosis experiments illustrated that mjHARBI1-dsRNA treatment led to a lower phagocytosis rate in hemocytes of V. alginolyticus-challenged shrimp. It was also found that knockdown of mjHARBI1-like inhibited shrimp phenoloxidase (PO) activity, superoxide dismutase (SOD) activity, and total hemocyte count (THC) after WSSV or V. alginolyticus infection. These data indicate a regulative role of mjHARBI1-likein the immunity of shrimp in response to pathogen infection. Resultantly, it was concluded that mjHARBI1-like might have a positive effect on the anti-WSSV immune response of shrimp by regulating apoptosis, THC, PO activity, and SOD activity. Additionally, mjHARBI1-like might promote anti-V. alginolyticus infection by participating in regulating phagocytosis, apoptosis, SOD activity, PO activity, and THC. Copyright © 2018 Elsevier Ltd. All rights reserved.

Molecular cloning, characterization and expression analysis of a putative C-type lectin (Fclectin) gene in Chinese shrimp Fenneropenaeus chinensis.

PubMed

Liu, Yi-Chen; Li, Fu-Hua; Dong, Bo; Wang, Bing; Luan, Wei; Zhang, Xiao-Jun; Zhang, Liu-Suo; Xiang, Jian-Hai

2007-01-01

Lectin is regarded as a potential molecule involved in immune recognition and phagocytosis through opsonization in crustacean. Knowledge on lectin at molecular level would help us to understand its regulation mechanism in crustacean immune system. A novel C-type lectin gene (Fclectin) was cloned from hemocytes of Chinese shrimp Fenneropenaeus chinensis by 3' and 5' rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA consists of 1482 bp with an 861 bp open reading frame, encoding 287 amino acids. The deduced amino acid sequence contains a putative signal peptide of 19 amino acids. It also contains two carbohydrate recognition domains/C-type lectin-like domains (CRD1 and CRD2), which share 78% identity with each other. CRD1 and CRD2 showed 34% and 30% identity with that of mannose-binding lectin from Japanese lamprey (Lethenteron japonicum), respectively. Both CRD1 and CRD2 of Fclectin have 11 amino acids residues, which are relatively invariant in animals' C-type lectin CRDs. Five residues at Ca2+ binding site 1 are conserved in Fclectin. The potential Ca2+/carbohydrate-binding (site 2) motif QPD, E, NP (Gln-Pro-Asp, Glu, Asn-Pro) presented in the two CRDs of Fclectin may support its ability to bind galactose-type sugars. It could be deduced that Fclectin is a member of C-type lectin superfamily. Transcripts of Fclectin were found only in hemocytes by Northern blotting and RNA in situ hybridization. The variation of mRNA transcription level in hemocytes during artificial infection with bacteria and white spot syndrome virus (WSSV) was quantitated by capillary electrophoresis after RT-PCR. An exploration of mRNA expression variation after LPS stimulation was carried out in primarily cultured hemocytes in vitro. Expression profiles of Fclectin gene were greatly modified after bacteria, LPS or WSSV challenge. The above-stated data can provide us clues to understand the probable role of C-type lectin in innate immunity of shrimp and would be helpful to shrimp disease control.

Granulocytes of the red claw crayfish Cherax quadricarinatus can endocytose beads, E. coli and WSSV, but in different ways.

PubMed

Duan, Hu; Jin, Songjun; Zhang, Yan; Li, Fuhua; Xiang, Jianhai

2014-10-01

The hemocytes of the red claw crayfish Cherax quadricarinatus are classified by morphologic observation into the following types: hyalinocytes (H), semi-granulocytes (SG) and granulocytes (G). Density gradient centrifugation with Percoll was developed to separate these three subpopulations of hemocytes. Beads, Escherichia coli, and FITC labeling WSSV were used to investigate the characteristics of granulocytes by using scanning electron microscope, transmission electron microscope, and laser scan confocal microscope. Results showed that granulocytes could phagocytose beads and E. coli by endocytic pathways. WSSV could rely on caveolae-mediated endocytosis to mainly enter into granulocytes. These results could elucidate the mechanism of the innate immunity function of granulocytes, and it also showed the mechanism by which WSSV invaded granulocytes in the red claw crayfish. Copyright © 2014 Elsevier Ltd. All rights reserved.

Synthesis of prolyl 4-hydroxylase alpha subunit and type IV collagen in hemocytic granular cells of silkworm, Bombyx mori: Involvement of type IV collagen in self-defense reaction and metamorphosis.

PubMed

Adachi, Takahiro; Tomita, Masahiro; Yoshizato, Katsutoshi

2005-04-01

The present study shows that hemocytic granular cells synthesize and secrete type IV collagen (ColIV) in the silkworm Bombyx mori (B. mori) and suggests that these cells play roles in the formation of basement membrane, the encapsulation of foreign bodies, and the metamorphic remodeling of the gut. The full- and partial-length cDNA of B. mori prolyl 4-hydroxylase alpha subunit (BmP4Halpha) and B. mori ColIV (BmColIV) were cloned, respectively. In situ hybridization and immunocytochemistry on larval tissues and cells identified hemocytic granular cells as the cells that express mRNAs and proteins of both BmP4Halpha and BmColIV. Immunohistochemistry and immunocytochemistry demonstrated that BmColIV was present in the basement membrane and in the secretory granules of granular cells, respectively. Granular cells in culture secreted BmColIV without accompanying the degranulation and discharged it from the granules when the cells were degranulated. Nylon threads were inserted into the hemocoel of larvae. Granular cells concentrated around the nylon threads and encapsulated them as a self-defense reaction. BmColIV was found to be a component of the capsules. Furthermore, the present study showed that actively BmColIV-expressing granular cells accumulated around the midgut epithelium and formed BmColIV-rich thick basal lamina-like structures there in larval to pupal metamorphosis.

PmVRP15, a Novel Viral Responsive Protein from the Black Tiger Shrimp, Penaeus monodon, Promoted White Spot Syndrome Virus Replication

PubMed Central

Vatanavicharn, Tipachai; Prapavorarat, Adisak; Jaree, Phattarunda; Somboonwiwat, Kunlaya; Tassanakajon, Anchalee

2014-01-01

Suppression subtractive hybridization of Penaeus monodon hemocytes challenged with white spot syndrome virus (WSSV) has identified the viral responsive gene, PmVRP15, as the highest up-regulated gene ever reported in shrimps. Expression analysis by quantitative real time RT-PCR revealed 9410–fold up-regulated level at 48 h post WSSV injection. Tissue distribution analysis showed that PmVRP15 transcript was mainly expressed in the hemocytes of shrimp. The full-length cDNA of PmVRP15 transcript was obtained and showed no significant similarity to any known gene in the GenBank database. The predicted open reading frame of PmVRP15 encodes for a deduced 137 amino acid protein containing a putative transmembrane helix. Immunofluorescent localization of the PmVRP15 protein revealed it accumulated around the nuclear membrane in all three types of shrimp hemocytes and that the protein was highly up-regulated in WSSV-infected shrimps. Double-stranded RNA interference-mediated gene silencing of PmVRP15 in P. monodon significantly decreased WSSV propagation compared to the control shrimps (injected with GFP dsRNA). The significant decrease in cumulative mortality rate of WSSV-infected shrimp following PmVRP15 knockdown was observed. These results suggest that PmVRP15 is likely to be a nuclear membrane protein and that it acts as a part of WSSV propagation pathway. PMID:24637711

Immune-physiological aspects of synergy between avermectins and the entomopathogenic fungus Metarhizium robertsii in Colorado potato beetle larvae.

PubMed

Tomilova, Oksana G; Kryukov, Vadim Yu; Duisembekov, Bahytzhan A; Yaroslavtseva, Olga N; Tyurin, Maksim V; Kryukova, Natalia A; Skorokhod, Valery; Dubovskiy, Ivan M; Glupov, Viktor V

2016-10-01

The interaction between the entomopathogenic fungus Metarhizium robertsii and natural avermectin metabolites of the actinomycete Streptomyces avermitilis were investigated on Colorado potato beetle larvae. A synergy in the mortality of larvae was detected after simultaneous treatment with half-lethal doses of avermectins (commercial name actarophit) 0.005% and fungus (5×10 5 conidia/ml). The treatment with avermectins led to rapid fungal colonization of the hemolymph. The defense strategies of insects infected by fungus and treated with avermectins and untreated insects were compared to investigate the mechanisms of this synergy. We have shown an increase in hemocytes, especially immunocompetent cells - plasmatocytes and granular cells in the initial stages of mycosis (third day post inoculation). In contrast, avermectins suppressed cellular immunity in hemolymph. Specifically, avermectins dramatically decreased the count of granular cells in larvae infected and uninfected with fungus. Apoptosis inducement and hemocyte necrosis under the influence of avermectins has been shown in vitro as one of the possible reasons for hemocyte mortality. In addition, avermectins enhanced the activity of phenoloxidases in integuments and hemolymph and increased the activity of glutathione-S-transferases activity in the fat body and hemolymph of infected and uninfected larvae, thereby intensifying the development of fungal infection by M. robertsii in Colorado potato beetle larvae. The combination of fungal infection and avermectins constitutes a new perspective for developing multicomponent bioinsecticides. Copyright © 2016 Elsevier Inc. All rights reserved.

Noradrenaline and alpha-adrenergic signaling induce the hsp70 gene promoter in mollusc immune cells.

PubMed

Lacoste, A; De Cian, M C; Cueff, A; Poulet, S A

2001-10-01

Expression of heat shock proteins (hsp) is a homeostatic mechanism induced in both prokaryotic and eukaryotic cells in response to metabolic and environmental insults. A growing body of evidence suggests that in mammals, the hsp response is integrated with physiological responses through neuroendocrine signaling. In the present study, we have examined the effect of noradrenaline (NA) on the hsp70 response in mollusc immune cells. Oyster and abalone hemocytes transfected with a gene construct containing a gastropod hsp70 gene promoter linked to the luciferase reporter-gene were exposed to physiological concentrations of NA, or to various alpha- and beta-adrenoceptor agonists and antagonists. Results show that NA and alpha-adrenergic stimulations induced the expression of luciferase in transfected mollusc immunocytes. Furthermore, exposure of hemocytes to NA or to the alpha-adrenoceptor agonist phenylephrine (PE) resulted in the expression of the inducible isoform of the hsp70 protein. Pertussis toxin (PTX), the phospholipase C (PLC) inhibitor U73122, the protein kinase C (PKC) inhibitor calphostin C, the Ca(2+)-dependent PKC inhibitor Gö 6976 and the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY294002 blocked the PE-mediated induction of the hsp70 gene promoter. These results suggest that alpha-adrenergic signaling induces the transcriptionnal upregulation of hsp70 in mollusc hemocytes through a PTX-sensitive G-protein, PLC, Ca(2+)-dependent PKC and PI 3-kinase. Thus, a functional link exists between neuroendocrine signaling and the hsp70 response in mollusc immune cells.

New activity of yamamarin, an insect pentapeptide, on immune system of mealworm, Tenebrio molitor.

PubMed

Walkowiak-Nowicka, K; Nowicki, G; Kuczer, M; Rosiński, G

2017-09-12

In insects, two types of the immune responses, cellular and humoral, constitute a defensive barrier against various parasites and pathogens. In response to pathogens, insects produce a wide range of immune agents that act on pathogens directly, such as cecropins or lysozyme, or indirectly by the stimulation of hemocyte migration or by increasing phenoloxidase (PO) activity. Recently, many new immunologically active substances from insects, such as peptides and polypeptides, have been identified. Nevertheless, in the most cases, their physiological functions are not fully known. One such substance is yamamarin - a pentapeptide isolated from the silk moth Antheraea yamamai. This yamamarin possesses strong antiproliferative properties and is probably involved in diapause regulation. Here, we examined the immunotropic activity of yamamarin by testing its impact on selected functions of the immune system in heterologous bioassays with the beetle Tenebrio molitor, commonly known as a stored grains pest. Our results indicate that the pentapeptide affects the activity of immune processes in the beetle. We show that yamamarin induces changes in both humoral and cellular responses. The yamamarin increases the activity of PO, as well as causes changes in the hemocyte cytoskeleton and stimulates phagocytic activity. We detected an increased number of apoptotic hemocytes, however after the yamamarin injection, no significant variations in the antibacterial activity in the hemolymph were observed. The obtained data suggest that yamamarin could be an important controller of the immune system in T. molitor.

IS COPPER REQUIRED FOR EASTERN OYSTER SETTING AND METAMORPHOSIS?

EPA Science Inventory

Recent field research with eastern oysters demonstrated higher defense activities, including hemocyte numbers, locomotion and bactericidal ability, associated with locations exhibiting relatively high contamination. Copper and zinc, found in high concentrations in tissues of oyst...

Molecular cloning of Kazal-type proteinase inhibitor of the shrimp Fenneropenaeus chinensis.

PubMed

Kong, Hee Jeong; Cho, Hyun Kook; Park, Eun-Mi; Hong, Gyeong-Eun; Kim, Young-Ok; Nam, Bo-Hye; Kim, Woo-Jin; Lee, Sang-Jun; Han, Hyon Sob; Jang, In-Kwon; Lee, Chang Hoon; Cheong, Jaehun; Choi, Tae-Jin

2009-01-01

Proteinase inhibitors play important roles in host defence systems involving blood coagulation and pathogen digestion. We isolated and characterized a cDNA clone for a Kazal-type proteinase inhibitor (KPI) from a hemocyte cDNA library of the oriental white shrimp Fenneropenaeus chinensis. The KPI gene consists of three exons and two introns. KPI cDNA contains an open reading frame of 396 bp, a polyadenylation signal sequence AATAAA, and a poly (A) tail. KPI cDNA encodes a polypeptide of 131 amino acids with a putative signal peptide of 21 amino acids. The deduced amino acid sequence of KPI contains two homologous Kazal domains, each with six conserved cysteine residues. The mRNA of KPI is expressed in the hemocytes of healthy shrimp, and the higher expression of KPI transcript is observed in shrimp infected with the white spot syndrome virus (WSSV), suggesting a potential role for KPI in host defence mechanisms.

Digital gene expression analysis in hemocytes of the white shrimp Litopenaeus vannamei in response to low salinity stress.

PubMed

Zhao, Qun; Pan, Luqing; Ren, Qin; Hu, Dongxu

2015-02-01

The white shrimp Litopenaeus vannamei has been greatly impacted by low salinity stress. To gain knowledge on the immune response in L. vannamei under such stress, we investigated digital gene expression (DEG) in L. vannamei hemocytes using the deep-sequencing platform Illumina HiSeq 2000. In total, 38,155 high quality unigenes with average length 770 bp were generated; 145 and 79 genes were identified up- or down-regulated, respectively. Functional categorization and pathways of the differentially expressed genes revealed that immune signaling pathways, cellular immunity, humoral immunity, apoptosis, cellular protein synthesis, lipid transport and energy metabolism were the differentially regulated processes occurring during low salinity stress. These results will provide a resource for subsequent gene expression studies regarding environmental stress and a valuable gene information for a better understanding of immune mechanisms of L. vannamei under low salinity stress. Copyright © 2014 Elsevier Ltd. All rights reserved.

Gene discovery in Boophilus microplus, the cattle tick: the transcriptomes of ovaries, salivary glands, and hemocytes.

PubMed

Santos, Isabel K F de Miranda; Valenzuela, Jesus G; Ribeiro, José Marcos C; de Castro, Marilia; Costa, Juliana Nardelli; Costa, Ana Maria; da Silva, Edson Ramiro; Neto, Olavo Bilac Rego; Rocha, Clarisse; Daffre, Sirlei; Ferreira, Beatriz R; da Silva, João Santana; Szabó, Matias Pablo; Bechara, Gervasio Henrique

2004-10-01

The quest for new control strategies for ticks can profit from high throughput genomics. In order to identify genes that are involved in oogenesis and development, in defense, and in hematophagy, the transcriptomes of ovaries, hemocytes, and salivary glands from rapidly ingurgitating females, and of salivary glands from males of Boophilus microplus were PCR amplified, and the expressed sequence tags (EST) of random clones were mass sequenced. So far, more than 1,344 EST have been generated for these tissues, with approximately 30% novelty, depending on the the tissue studied. To date approximately 760 nucleotide sequences from B. microplus are deposited in the NCBI database. Mass sequencing of partial cDNAs of parasite genes can build up this scant database and rapidly generate a large quantity of useful information about potential targets for immunobiological or chemical control.

Dual role of wingless signaling in stem-like hematopoietic precursor maintenance in Drosophila.

PubMed

Sinenko, Sergey A; Mandal, Lolitika; Martinez-Agosto, Julian A; Banerjee, Utpal

2009-05-01

In Drosophila, blood development occurs in a specialized larval hematopoietic organ, the lymph gland (LG), within which stem-like hemocyte precursors or prohemocytes differentiate to multiple blood cell types. Here we show that components of the Wingless (Wg) signaling pathway are expressed in prohemocytes. Loss- and gain-of-function analysis indicates that canonical Wg signaling is required for maintenance of prohemocytes and negatively regulates their differentiation. Wg signals locally in a short-range fashion within different compartments of the LG. In addition, Wg signaling positively regulates the proliferation and maintenance of cells that function as a hematopoietic niche in Drosophila, the posterior signaling center (PSC), and in the proliferation of crystal cells. Our studies reveal a conserved function of Wg signaling in the maintenance of stem-like blood progenitors and reveal an involvement of this pathway in the regulation of hemocyte differentiation through its action in the hematopoietic niche.

The dipteran parasitoid Exorista bombycis induces pro- and anti-oxidative reactions in the silkworm Bombyx mori: Enzymatic and genetic analysis.

PubMed

Makwana, Pooja; Pradeep, Appukuttan Nair R; Hungund, Shambhavi P; Ponnuvel, Kangayam M; Trivedy, Kanika

2017-02-01

Hymenopteran parasitoids inject various factors including polydnaviruses along with their eggs into their host insects that suppress host immunity reactions to the eggs and larvae. Less is known about the mechanisms evolved in dipteran parasitoids that suppress host immunity. Here we report that the dipteran, Exorista bombycis, parasitization leads to pro-oxidative reactions and activation of anti-oxidative enzymes in the silkworm Bombyx mori larva. We recorded increased activity of oxidase, superoxide dismutase, thioredoxin peroxidase, catalase, glutathione-S-transferase (GST), and peroxidases in the hemolymph plasma, hemocytes, and fat body collected from B. mori after E. bombycis parasitization. Microarray and qPCR showed differential expression of genes encoding pro- and anti-oxidant enzymes in the hemocytes. The significance of this work lies in increased understanding of dipteran parasitoid biology. © 2017 Wiley Periodicals, Inc.

Structural and functional characterization of the contractile aorta and associated hemocytes of the mosquito Anopheles gambiae.

PubMed

Sigle, Leah T; Hillyer, Julián F

2018-06-22

The primary pump of the circulatory system of insects is a dorsal vessel that traverses the length of the insect. The anterior portion, located in the head, neck and thorax, is the aorta, and the posterior portion, located in the abdomen, is the heart. Here, we characterize the structure and function of the aorta and conical chamber of the mosquito, Anopheles gambiae The aorta begins in the head with an excurrent opening located above the dorsal pharyngeal plate and ends at the thoraco-abdominal junction where it joins the conical chamber of the heart. The aorta lacks ostia, and based on the diameter of the vessel as well as the density and helical orientation of muscle, consists of three regions: the anterior aorta, the bulbous chamber, and the posterior aorta. The aorta contracts in the anterograde direction, but these contractions are independent of heart contractions and do not play a major role in hemolymph propulsion. Intravital imaging of the venous channels, the first abdominal segment and the neck revealed that hemolymph only travels through the aorta in the anterograde direction, and does so only during periods of anterograde heart flow. Furthermore, hemolymph only enters the thoraco-abdominal ostia of the conical chamber when the heart contracts in the retrograde direction, propelling this hemolymph to the posterior of the body. Finally, very few hemocytes associate with the aorta, and unlike what is seen in the periostial regions of the heart, infection does not induce the aggregation of hemocytes on the aorta. © 2018. Published by The Company of Biologists Ltd.

In vitro effects of suspensions of selected nanoparticles (C60 fullerene, TiO2, SiO2) on Mytilus hemocytes.

PubMed

Canesi, Laura; Ciacci, Caterina; Vallotto, Davide; Gallo, Gabriella; Marcomini, Antonio; Pojana, Giulio

2010-01-31

As the nanotechnology industries increase production, nanoscale products will enter the aquatic environment, posing a possible threat to aquatic organisms. Suspension-feeding invertebrates may represent a unique target group for nanoparticle (NP) ecotoxicity, since they have highly developed processes for the cellular internalisation of nano- and microscale particles (endocytosis and phagocytosis), which are integral to key physiological functions such as intracellular digestion and cellular immunity. In the marine bivalve Mytilus, short-term exposure to nanosized carbon black (NCB) was shown to significantly affect immune parameters of immune cells, the hemocytes, in vitro. In this work, we further investigated the effects of other types of commercial NPs (C60 fullerene, TiO(2) and SiO(2) at 1, 5, 10 microg/ml) on Mytilus hemocytes. Characterization of NP suspensions in artificial sea water (ASW) was performed, indicating the formation of agglomerates of different sizes for different types of NPs. None of the NP tested significantly affected lysosomal membrane stability, indicating the lack of a major toxic effect. However, all NP suspensions induced a concentration-dependent lysozyme release, extracellular oxyradical and nitric oxide (NO) production, to a different extent and with different time courses depending on the concentration and the NP type. The inflammatory effects of NPs were mediated by rapid activation of the stress-activated p38 MAPK. The results further support the hypothesis that in bivalves the immune system represents a significant target for NPs. Copyright (c) 2009 Elsevier B.V. All rights reserved.

Epstein-Barr Virus DNA Enhances Diptericin Expression and Increases Hemocyte Numbers in Drosophila melanogaster via the Immune Deficiency Pathway.

PubMed

Sherri, Nour; Salloum, Noor; Mouawad, Carine; Haidar-Ahmad, Nathaline; Shirinian, Margret; Rahal, Elias A

2018-01-01

Infection with the Epstein-Barr virus (EBV) is associated with several malignancies and autoimmune diseases in humans. The following EBV infection and establishment of latency, recurrences frequently occur resulting in potential viral DNA shedding, which may then trigger the activation of immune pathways. We have previously demonstrated that levels of the pro-inflammatory cytokine IL-17, which is associated with several autoimmune diseases, are increased in response to EBV DNA injection in mice. Whether other pro-inflammatory pathways are induced in EBV DNA pathobiology remains to be investigated. The complexity of mammalian immune systems presents a challenge to studying differential activities of their intricate immune pathways in response to a particular immune stimulus. In this study, we used Drosophila melanogaster to identify innate humoral and cellular immune pathways that are activated in response to EBV DNA. Injection of wild-type adult flies with EBV DNA induced the immune deficiency (IMD) pathway resulting in enhanced expression of the antimicrobial peptide diptericin. Furthermore, EBV DNA increased the number of hemocytes in flies. Conditional silencing of the IMD pathway decreased diptericin expression in addition to curbing of hemocyte proliferation in response to challenge with EBV DNA. Comparatively, upon injecting mice with EBV DNA, we detected enhanced expression of tumor necrosis factor-α (TNFα); this enhancement is rather comparable to IMD pathway activation in flies. This study hence indicates that D. melanogaster could possibly be utilized to identify immune mediators that may also play a role in the response to EBV DNA in higher systems.

Shrimp hemocyte homeostasis-associated protein (PmHHAP) interacts with WSSV134 to control apoptosis in white spot syndrome virus infection.

PubMed

Apitanyasai, Kantamas; Amparyup, Piti; Charoensapsri, Walaiporn; Sangsuriya, Pakkakul; Tassanakajon, Anchalee

2018-05-01

Hemocyte homeostasis-associated protein (PmHHAP) was first identified as a viral-responsive gene, due to a high upregulation in transcription following white spot syndrome virus (WSSV) infection. Functional studies using RNA interference have suggested that PmHHAP is involved in hemocyte homeostasis by controlling apoptosis during WSSV infection. In this study, the role of PmHHAP in host-viral interactions was further investigated. Yeast two-hybrid assay and co-immunoprecipitation revealed that PmHHAP binds to an anti-apoptosis protein, WSSV134. The viral protein WSSV134 is a late protein of WSSV, expressed 24 h post infection (hpi). Gene silencing of WSSV134 in WSSV-infected shrimp resulted in a reduction of the expression level of the viral replication marker genes VP28, wsv477, and ie-1, which suggests that WSSV134 is likely involved in viral propagation. However, co-silencing of PmHHAP and WSSV134 counteracted the effects on WSSV infection, which implies the importance of the host-pathogen interaction between PmHHAP and WSSV134 in WSSV infection. In addition, caspase 3/7 activity was noticeably induced in the PmHHAP and WSSV134 co-silenced shrimp upon WSSV infection. Moreover, PmHHAP and WSSV134 inhibited caspase-induced activation of PmCasp in vitro in a non-competitive manner. Taken together, these results suggest that PmHHAP and WSSV134 play a role in the host-pathogen interaction and work concordantly to control apoptosis in WSSV infection. Copyright © 2018 Elsevier Ltd. All rights reserved.

The Immune Phenotype of Three Drosophila Leukemia Models.

PubMed

Arefin, Badrul; Kunc, Martin; Krautz, Robert; Theopold, Ulrich

2017-07-05

Many leukemia patients suffer from dysregulation of their immune system, making them more susceptible to infections and leading to general weakening (cachexia). Both adaptive and innate immunity are affected. The fruit fly Drosophila melanogaster has an innate immune system, including cells of the myeloid lineage (hemocytes). To study Drosophila immunity and physiology during leukemia, we established three models by driving expression of a dominant-active version of the Ras oncogene ( Ras V12 ) alone or combined with knockdowns of tumor suppressors in Drosophila hemocytes. Our results show that phagocytosis, hemocyte migration to wound sites, wound sealing, and survival upon bacterial infection of leukemic lines are similar to wild type. We find that in all leukemic models the two major immune pathways (Toll and Imd) are dysregulated. Toll-dependent signaling is activated to comparable extents as after wounding wild-type larvae, leading to a proinflammatory status. In contrast, Imd signaling is suppressed. Finally, we notice that adult tissue formation is blocked and degradation of cell masses during metamorphosis of leukemic lines, which is akin to the state of cancer-dependent cachexia. To further analyze the immune competence of leukemic lines, we used a natural infection model that involves insect-pathogenic nematodes. We identified two leukemic lines that were sensitive to nematode infections. Further characterization demonstrates that despite the absence of behavioral abnormalities at the larval stage, leukemic larvae show reduced locomotion in the presence of nematodes. Taken together, this work establishes new Drosophila models to study the physiological, immunological, and behavioral consequences of various forms of leukemia. Copyright © 2017 Arefin et al.

Gypsy moth larval defense mechanisms against pathogenic microorganisms

Treesearch

Kathleen S. Shields; Tariq M. Butt

1991-01-01

We investigated the response of gypsy moth, Lymantria dispar, larval hemocytes to L. dispar nuclear polyhedrosis virus (LdMNPV) administered per os and by injection, and to injected hyphal bodies and natural protoplasts of some entomopathogenic, entomophthoralean fungi.

FACTORS INFLUENCING IN VITRO KILLING OF BACTERIA BY HEMOCYTES OF THE EASTERN OYSTER (CRASSOSTREA VIRGINICA)

EPA Science Inventory

Vibrio parahaemolyticus strains altered in motility or colonial morphology (opaque versus translucent), Listeria monocytogenes mutants lacking catalase, superoxide dismutase, hemolysin, or phospholipase activities, and Vibrio vulnificus strains, possessing and lacking capsules we...

REACTIVE NITROGEN SPECIES IN OYSTER HEMOCYTES. (R827100)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Nitric oxide mediates insect cellular immunity via phospholipase A2 activation

USDA-ARS?s Scientific Manuscript database

After infection or invasion is recognized, biochemical mediators act in signaling insect immune functions. These include biogenic amines, insect cytokines, eicosanoids and nitric oxide (NO). Treating insects or isolated hemocyte populations with different mediators often leads to similar results. Se...

Rapid extraction of virus-contaminated hemocytes from oysters

USDA-ARS?s Scientific Manuscript database

Rapid viral detection methods are necessary to employ diagnostic testing for viral contamination in shellfish to prevent and control foodborne illness. Current shellfish viral RNA extraction methods, which are time-consuming and not applicable for routine monitoring, require the testing of whole or ...

Hemocytes are sites of persistence for virus-contaminated oysters

USDA-ARS?s Scientific Manuscript database

Like fecal bacteria, waterborne enteric viruses are readily bioconcentrated by bivalve shellfish. However while many bacteria decline rapidly when bivalves are placed in uncontaminated water, viruses tend to be retained within shellfish. In this study, we offer evidence that phagocytic blood cells...

Comparative transcriptomic analysis of shrimp hemocytes in response to acute hepatopancreas necrosis disease (AHPND) causing Vibrio parahemolyticus infection.

PubMed

Zheng, Zhihong; Wang, Fan; Aweya, Jude Juventus; Li, Ruiwei; Yao, Defu; Zhong, Mingqi; Li, Shengkang; Zhang, Yueling

2018-03-01

The recent emergence of acute hepatopancreas necrosis disease (AHPND) in shrimps has posed a major challenge in the shrimp aquaculture industry. The Pir toxin proteins carried by some strains of Vibrio parahaemolyticus are believed to play essential roles in the pathogenesis of AHPND. However, few studies have so far explored how the host immune system responds to these bacteria. In this study, AHPND V. parahaemolyticus (with Pir) and non-AHPND V. parahaemolyticus (without Pir) were injected into two groups of shrimps, and the hemocytes collected for comparative transcriptomic analyses. A total of 1064 differentially expressed genes (DEGs) were identified, of which 910 were up-regulated and 154 were down-regulated. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that many DEGs were involved in a number of biological processes such as cellular process, metabolic process and single-organism process in the AHPND V. parahaemolyticus injected group than the non-AHPND V. parahaemolyticus injected group. Among these, major metabolic processes such as carbohydrate metabolism, lipid metabolism and amino acid metabolism were further identified as the major responsive gene groups. We observed that genes involved in cell growth and anti-apoptosis including src, iap2, cas2, cytochrome P450, gst and cytochromecoxidase were strongly activated in the AHPND V. parahaemolyticus group than in the non-AHPND V. parahaemolyticus group. Collectively, our results unveiled that shrimp hemocytes respond to AHPND related strain of Vibrio parahaemolyticus infection at the transcriptional level, which is useful in furthering our understanding of AHPND. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cloning and characterisation of the SpToll gene from green mud crab, Scylla paramamosain.

PubMed

Lin, Zhongyang; Qiao, Jie; Zhang, Yueling; Guo, Lingling; Huang, He; Yan, Fang; Li, Yuanyou; Wang, Xiuying

2012-05-01

Toll/Toll-like receptors (TLRs), one of the most important pattern recognition receptors (PRRs), play a crucial role in innate immune responses in both invertebrates and vertebrates. In this study, we cloned and characterised a Toll gene from Scylla paramamosain (SpToll). Bioinformatic analysis predicted that SpToll contained one open reading frame of 3018bp and encoded a single-pass transmembrane domain protein of 1005 amino acids. Further, SpToll could be clustered into one branch along with other arthropod Tolls in a phylogenetic tree. SpToll transcripts could be detected by RT-PCR from all tissues examined including the heart, gill, hepatopancreas, stomach, intestine, muscle, eyestalk and hemocytes. Infection by Vibrio parahemolyticus up-regulated SpToll mRNA expression in hemocytes after 48h. The profile of single nucleotide polymorphisms (SNPs) in the leucine-rich repeats (LRRs) domain of SpToll in three healthy crabs was then evaluated. Two hundred and twenty SNPs with a frequency of about 1.0-4.0% were identified in hemocyte DNA/cDNA. Surprisingly, the adenine to guanine transition at position 1372 (c.1372A>G) had a frequency of about 50%. Finally, the results showed that challenge with V. parahemolyticus stimulated the appearance of two sets of SNPs in crabs. More importantly, the c.1372A>G mutation could contribute to a low mortality after V. parahemolyticus infection and introduce variation of charge and secondary structure into the SpToll polypeptide. In summary, these studies suggested a novel Toll homologue in crab and identified a SNP with potential pathogen-resistant activities. The result will be important for the investigation of crab immune defense mechanisms. Copyright © 2011 Elsevier Ltd. All rights reserved.

Kazal-type proteinase inhibitor from disk abalone (Haliotis discus discus): molecular characterization and transcriptional response upon immune stimulation.

PubMed

Wickramaarachchi, W D Niroshana; De Zoysa, Mahanama; Whang, Ilson; Wan, Qiang; Lee, Jehee

2013-09-01

Proteinases and proteinase inhibitors are involved in several biological and physiological processes in all multicellular organisms. Proteinase inhibitors play a key role in regulating the activity of the respective proteinases. Among serine proteinase inhibitors, kazal-type proteinase inhibitors (KPIs) are widely found in mammals, avians, and a variety of invertebrates. In this study, we describe the identification of a kazal-type serine proteinase inhibitor (Ab-KPI) from the disk abalone, Haliotis discus discus, which is presumably involved in innate immunity. The full-length cDNA of Ab-KPI includes 600 bp nucleotides with an open reading frame (ORF) encoding a polypeptide of 143 amino acids. The deduced amino acid sequence of Ab-KPI contains a putative 17-amino acid signal peptide and two tandem kazal domains with high similarity to other kazal-type SPIs. Each kazal domain consists of reactive site (P1) residue containing a leucine (L), and a threonine (T) located in the second amino acid position after the second conserved cysteine of each domain. Temporal expression of Ab-KPI was assessed by real time quantitative PCR in hemocytes and mantle tissue following bacterial and viral hemorrhagic septicemia virus (VHSV) challenge, and tissue injury. At 6 h post-bacterial and -VHSV challenge, Ab-KPI expression in hemocytes was increased 14-fold and 4-fold, respectively, compared to control samples. The highest up-regulations upon tissue injury were shown at 9 h and 12 h in hemocytes and mantle, respectively. The transcriptional modulation of Ab-KPI following bacterial and viral challenges and tissue injury indicates that it might be involved in immune defense as well as wound healing process in abalone. Copyright © 2013 Elsevier Ltd. All rights reserved.

Bombyx mori cecropin A has a high antifungal activity to entomopathogenic fungus Beauveria bassiana.

PubMed

Lu, Dingding; Geng, Tao; Hou, Chengxiang; Huang, Yuxia; Qin, Guangxing; Guo, Xijie

2016-05-25

A cDNA encoding cecropin A (CecA) was cloned from the larvae of silkworm, Bombyx mori, using RT-PCR. It encodes a protein of 63 amino acids, containing a 22 amino acid signal peptide and a 37 amino acid mat peptide of functional domain. The CecA secondary structure contains two typical amphiphilic α-helices. Real-time qPCR analysis revealed that CecA was expressed in all the tissues tested, including cuticle, fat body, hemocytes, Malpighian tubule, midgut and silk gland in the silkworm larvae with the highest expression in the fat body and hemocytes. The gene expression of B. mori CecA was rapidly induced by Beauveria bassiana challenge and reached maximum levels at 36h after inoculation in third instar larvae. In the fifth instar larvae infected with B. bassiana, the relative expression level of CecA was upregulated in fat body and hemocytes, but not in cuticle, Malpighian tubule, midgut and silk gland. The cDNA segment of the CecA was inserted into the expression plasmid pET-30a(+) to construct a recombinant expression plasmid. Western blot results revealed that his-tagged fusion protein was successfully expressed and purified. Then the mat peptide of CecA was chemically synthesized with C-terminus amidation for in vivo antifungal assay and purity achieved 93.7%. Mass spectrometry and SDS-PAGE showed its molecular weight to be 4046.95Da. Antifungal assays indicated that the B. mori CecA had a high antifungal activity to entomopathogenic fungus B. bassiana both in vitro and in vivo in the silkworm larvae. This is the first report that the CecA is effective to inhibit B. bassiana inside the body of silkworm. Copyright © 2016 Elsevier B.V. All rights reserved.

Lactobacillus paracasei modulates the immune system of Galleria mellonella and protects against Candida albicans infection

PubMed Central

Rossoni, Rodnei Dennis; Fuchs, Beth Burgwyn; de Barros, Patrícia Pimentel; Velloso, Marisol dos Santos; Jorge, Antonio Olavo Cardoso; Junqueira, Juliana Campos; Mylonakis, Eleftherios

2017-01-01

Probiotics have been described as a potential strategy to control opportunistic infections due to their ability to stimulate the immune system. Using the non-vertebrate model host Galleria mellonella, we evaluated whether clinical isolates of Lactobacillus spp. are able to provide protection against Candida albicans infection. Among different strains of Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus fermentum, we verified that L. paracasei 28.4 strain had the greatest ability to prolong the survival of larvae infected with a lethal dose of C. albicans. We found that the injection of 107 cells/larvae of L. paracasei into G. mellonella larvae infected by C. albicans increased the survival of these insects compared to the control group (P = 0.0001). After that, we investigated the immune mechanisms involved in the protection against C. albicans infection, evaluating the number of hemocytes and the gene expression of antifungal peptides. We found that L. paracasei increased the hemocyte quantity (2.38 x 106 cells/mL) in relation to the control group (1.29 x 106 cells/mL), indicating that this strain is capable of raising the number of circulating hemocytes into the G. mellonella hemolymph. Further, we found that L. paracasei 28.4 upregulated genes that encode the antifungal peptides galiomicin and gallerymicin. In relation to the control group, L. paracasei 28.4 increased gene expression of galiomicin by 6.67-fold and 17.29-fold for gallerymicin. Finally, we verified that the prophylactic provision of probiotic led to a significant reduction of the number of fungal cells in G. mellonella hemolymph. In conclusion, L. paracasei 28.4 can modulate the immune system of G. mellonella and protect against candidiasis. PMID:28267809

The hot-water extract of leaves of noni, Morinda citrifolia, promotes the immunocompetence of giant freshwater prawn, Macrobrachium rosenbergii.

PubMed

Marisa Halim, Atika; Lee, Pai-Po; Chang, Zhong-Wen; Chang, Chin-Chyuan

2017-05-01

The hot-water Morinda citrifolia leaf extract (HMLE) was prepared for in vitro assessment on phenoloxidase (PO) activity, respiratory bursts (RBs), and phagocytic activity (PA). Furthermore, the HMLE was administrated in the diet at 0.6, 3, and 6 g (kg diet) -1 for Macrobrachium rosenbergii, and the potential effects on the immunocompetence of prawns were evaluated. PO activity, RBs, and PA in hemocytes incubated with the HMLE at 140, 20, 20, and 140 mg l -1 significantly increased. The immune parameters of the total hemocyte count (THC), differential hemocyte count (DHC), RBs, PO activity, superoxide dismutase (SOD) activity, PA, transglutaminase (TG) activity and hemolymph clotting time were evaluated before and after 1, 3, 5, 7, and 9 weeks of the feeding trial. During 9 weeks of the feeding trial, higher THCs, DHCs, RBs, PO, and TG as well as accelerated clotting times were observed in prawns fed HMLE-containing diets at 0.6 g kg -1 . The mRNA expressions of prophenoloxidase, TG, crustin, and lysozyme of prawns fed HMLE-containing diets at 0.6 g kg -1 for 9 weeks of the feeding trial significantly increased. The susceptibility of prawns fed the HMLE at 0.6 g kg -1 to Lactococcus garvieae infection significantly decreased, and the relative survival percentage was 23.1%. We therefore found that HMLE administrated through the diet at 0.6 g kg -1 was capable of enhancing the immunity and resistance against L. garvieae in M. rosenbergii. Copyright © 2017 Elsevier Ltd. All rights reserved.

Acetylcholine Protects against Candida albicans Infection by Inhibiting Biofilm Formation and Promoting Hemocyte Function in a Galleria mellonella Infection Model.

PubMed

Rajendran, Ranjith; Borghi, Elisa; Falleni, Monica; Perdoni, Federica; Tosi, Delfina; Lappin, David F; O'Donnell, Lindsay; Greetham, Darren; Ramage, Gordon; Nile, Christopher

2015-08-01

Both neuronal acetylcholine and nonneuronal acetylcholine have been demonstrated to modulate inflammatory responses. Studies investigating the role of acetylcholine in the pathogenesis of bacterial infections have revealed contradictory findings with regard to disease outcome. At present, the role of acetylcholine in the pathogenesis of fungal infections is unknown. Therefore, the aim of this study was to determine whether acetylcholine plays a role in fungal biofilm formation and the pathogenesis of Candida albicans infection. The effect of acetylcholine on C. albicans biofilm formation and metabolism in vitro was assessed using a crystal violet assay and phenotypic microarray analysis. Its effect on the outcome of a C. albicans infection, fungal burden, and biofilm formation were investigated in vivo using a Galleria mellonella infection model. In addition, its effect on modulation of host immunity to C. albicans infection was also determined in vivo using hemocyte counts, cytospin analysis, larval histology, lysozyme assays, hemolytic assays, and real-time PCR. Acetylcholine was shown to have the ability to inhibit C. albicans biofilm formation in vitro and in vivo. In addition, acetylcholine protected G. mellonella larvae from C. albicans infection mortality. The in vivo protection occurred through acetylcholine enhancing the function of hemocytes while at the same time inhibiting C. albicans biofilm formation. Furthermore, acetylcholine also inhibited inflammation-induced damage to internal organs. This is the first demonstration of a role for acetylcholine in protection against fungal infections, in addition to being the first report that this molecule can inhibit C. albicans biofilm formation. Therefore, acetylcholine has the capacity to modulate complex host-fungal interactions and plays a role in dictating the pathogenesis of fungal infections. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Acetylcholine Protects against Candida albicans Infection by Inhibiting Biofilm Formation and Promoting Hemocyte Function in a Galleria mellonella Infection Model

PubMed Central

Rajendran, Ranjith; Borghi, Elisa; Falleni, Monica; Perdoni, Federica; Tosi, Delfina; Lappin, David F.; O'Donnell, Lindsay; Greetham, Darren; Ramage, Gordon

2015-01-01

Both neuronal acetylcholine and nonneuronal acetylcholine have been demonstrated to modulate inflammatory responses. Studies investigating the role of acetylcholine in the pathogenesis of bacterial infections have revealed contradictory findings with regard to disease outcome. At present, the role of acetylcholine in the pathogenesis of fungal infections is unknown. Therefore, the aim of this study was to determine whether acetylcholine plays a role in fungal biofilm formation and the pathogenesis of Candida albicans infection. The effect of acetylcholine on C. albicans biofilm formation and metabolism in vitro was assessed using a crystal violet assay and phenotypic microarray analysis. Its effect on the outcome of a C. albicans infection, fungal burden, and biofilm formation were investigated in vivo using a Galleria mellonella infection model. In addition, its effect on modulation of host immunity to C. albicans infection was also determined in vivo using hemocyte counts, cytospin analysis, larval histology, lysozyme assays, hemolytic assays, and real-time PCR. Acetylcholine was shown to have the ability to inhibit C. albicans biofilm formation in vitro and in vivo. In addition, acetylcholine protected G. mellonella larvae from C. albicans infection mortality. The in vivo protection occurred through acetylcholine enhancing the function of hemocytes while at the same time inhibiting C. albicans biofilm formation. Furthermore, acetylcholine also inhibited inflammation-induced damage to internal organs. This is the first demonstration of a role for acetylcholine in protection against fungal infections, in addition to being the first report that this molecule can inhibit C. albicans biofilm formation. Therefore, acetylcholine has the capacity to modulate complex host-fungal interactions and plays a role in dictating the pathogenesis of fungal infections. PMID:26092919

Simulated climate change causes immune suppression and protein damage in the crustacean Nephrops norvegicus.

PubMed

Hernroth, Bodil; Sköld, Helen Nilsson; Wiklander, Kerstin; Jutfelt, Fredrik; Baden, Susanne

2012-11-01

Rising atmospheric carbon dioxide concentration is causing global warming, which affects oceans by elevating water temperature and reducing pH. Crustaceans have been considered tolerant to ocean acidification because of their retained capacity to calcify during subnormal pH. However, we report here that significant immune suppression of the Norway lobster, Nephrops norvegicus, occurs after a 4-month exposure to ocean acidification (OA) at a level predicted for the year 2100 (hypercapnic seawater with a pH lowered by 0.4 units). Experiments carried out at different temperatures (5, 10, 12, 14, 16, and 18°C) demonstrated that the temperature within this range alone did not affect lobster immune responses. In the OA-treatment, hemocyte numbers were reduced by almost 50% and the phagocytic capacity of the remaining hemocytes was inhibited by 60%. The reduction in hemocyte numbers was not due to increased apoptosis in hematopoetic tissue. Cellular responses to stress were investigated through evaluating advanced glycation end products (AGE) and lipid oxidation in lobster hepatopancreata, and OA-treatment was shown to significantly increase AGEs', indicating stress-induced protein alterations. Furthermore, the extracellular pH of lobster hemolymph was reduced by approximately 0.2 units in the OA-treatment group, indicating either limited pH compensation or buffering capacity. The negative effects of OA-treatment on the nephropidae immune response and tissue homeostasis were more pronounced at higher temperatures (12-18°C versus 5°C), which may potentially affect disease severity and spread. Our results signify that ocean acidification may have adverse effects on the physiology of lobsters, which previously had been overlooked in studies of basic parameters such as lobster growth or calcification. Copyright © 2012 Elsevier Ltd. All rights reserved.

A Carbonic Anhydrase Serves as an Important Acid-Base Regulator in Pacific Oyster Crassostrea gigas Exposed to Elevated CO2: Implication for Physiological Responses of Mollusk to Ocean Acidification.

PubMed

Wang, Xiudan; Wang, Mengqiang; Jia, Zhihao; Qiu, Limei; Wang, Lingling; Zhang, Anguo; Song, Linsheng

2017-02-01

Carbonic anhydrases (CAs) have been demonstrated to play an important role in acid-base regulation in vertebrates. However, the classification and modulatory function of CAs in marine invertebrates, especially their responses to ocean acidification remain largely unknown. Here, a cytosolic α-CA (designated as CgCAII-1) was characterized from Pacific oyster Crassostrea gigas and its molecular activities against CO 2 exposure were investigated. CgCAII-1 possessed a conserved CA catalytic domain, with high similarity to invertebrate cytoplasmic or mitochondrial α-CAs. Recombinant CgCAII-1 could convert CO 2 to HCO 3 - with calculated activity as 0.54 × 10 3  U/mg, which could be inhibited by acetazolamide (AZ). The mRNA transcripts of CgCAII-1 in muscle, mantle, hepatopancreas, gill, and hemocytes increased significantly after exposure to elevated CO 2 . CgCAII-1 could interact with the hemocyte membrane proteins and the distribution of CgCAII-1 protein became more concentrated and dense in gill and mantle under CO 2 exposure. The intracellular pH (pHi) of hemocytes under CO 2 exposure increased significantly (p < 0.05) and CA inhibition reduced the pHi value. Besides, there was no increase in CA activity in gill and mantle after CO 2 exposure. The impact of CO 2 exposure on CA activity coupled with the mRNA expression level and protein translocation of CgCAII-1 provided evidences that CgCAII-1 could respond to ocean acidification and participate in acid-base regulation. Such cytoplasmic CA-based physiological regulation mechanism might explain other physiological responses of marine organisms to OA.

[Functional morphology of blowfly Calliphora vicina hemocytes].

PubMed

Kind, T V

2012-01-01

In the hemolymph of Calliphora seven types of hemocytes were revealed. These are prohemocytes, which are the stem cells, stable and unstable hyaline cells, thrombocytoids, spindle cells, juvenile plasmatocytes and plasmatocytes I-IV, which represent sequential stages of one cell line differentiation were registered. The margin between them is completion of the crop emptying and beginning of wandering stage. In the feeding and crop emptying larvae take place rising of hyaline cells, thrombocytoids and hyaline cells amount with parallel growth of their defense function. The second wave of hemogenesis occur in the end of crop emptying period. It is accompanied by burst of plasmatocyte I production with their subsequent differentiation to plasmatocytes II-IV. Production of stable hyaline cells and respectively prothrombocytoids may be regulated not only by hormonal background but also by inorganic or organic particles invaded into the hemocel. Three types of hemocytes are involved in loosing of hemolymph from alien particles, notably thrombocytoids, juvenile plasmatocytes and plasmatocytes I and II. Thrombocytoids are responsible for parasitic eggs encapsulation. In addition they can phagocytize tiny organic and inorganic particles. Juvenile plasmatocytes respond to alien invasion almost as quickly as thrombocytoids at the onset of invasion. Plasmatocytes I and II start phagocytosis more slowly, hours post invasion, frequently accumulating the particles previously catched by thrombocytoids. Plasmatocytes I can absorb foreign particles and group in morules and can also surround filled thrombocytoids forming distinctive capsules. Both morules and capsules are temporary structures and disintegrate some hours lately. It is supposed the existence of three levels of immune defence: the fast response reaction of thrombocytoids and juvenile plasmatocytes and slow cellular reactions of plasmatocytes I. They are prerequisites for more extensive humoral response.

Protection from Oxidative Stress in Immunocytes of the Colonial Ascidian Botryllus schlosseri: Transcript Characterization and Expression Studies.

PubMed

Franchi, Nicola; Ballin, Francesca; Ballarin, Loriano

2017-02-01

Botryllus schlosseri is a cosmopolitan colonial ascidian that undergoes cyclical generation changes, or take-overs, during which adult zooids are resorbed and replaced by their buds. At take-over, adult tissues undergo diffuse apoptosis and effete cells are massively ingested by circulating phagocytes, with a consequent increase in oxygen consumption and in production of reactive oxygen species (ROS). The latter are responsible for the death of phagocytes involved in the clearance of apoptotic cells and corpses by phagocytosis-induced apoptosis. However, the majority of phagocytes and hemocytes do not die, even if they experience oxidative stress. This fact suggests the presence of detoxification mechanisms assuring their protection. To test this assumption, we searched for transcripts of genes involved in detoxification in the transcriptome of B. schlosseri. We identified and characterized transcripts for Cu/Zn superoxide dismutase (SOD), γ-glutamyl-cysteine ligase modulatory subunit (GCLM), glutathione synthase (GS), and two glutathione peroxidases (i.e., GPx3 and GPx5), all involved in protection from ROS. We also carried out a phylogenetic analysis of the putative amino acid sequences, confirming their similarity to their vertebrate counterparts, and studied the location of their mRNAs by in situ hybridization on hemocyte monolayers. We also analyzed gene transcription during the colonial blastogenetic cycle, which is the interval of time between one take-over and the next, by qRT-PCR. In addition, we investigated the effects of cadmium (Cd), an inducer of oxidative stress, on gene transcription. Our results indicated that i) antioxidant gene expression is modulated in the course of the blastogenetic cycle and upon exposure to Cd, and ii) hemocytes synthesize both enzymatic and nonenzymatic antioxidants, in line with the idea that they represent a major detoxification system for ascidians.

Conserved hemopoietic transcription factor Cg-SCL delineates hematopoiesis of Pacific oyster Crassostrea gigas.

PubMed

Song, Xiaorui; Wang, Hao; Chen, Hao; Sun, Mingzhe; Liang, Zhongxiu; Wang, Lingling; Song, Linsheng

2016-04-01

Hemocytes are the effective immunocytes in bivalves, which have been reported to be derived from stem-like cells in gill epithelium of oyster. In the present work, a conserved haematopoietic transcription factor Tal-1/Scl (Stem Cell Leukemia) was identified in Pacific oyster (Cg-SCL), and it was evolutionarily close to the orthologs in deuterostomes. Cg-SCL was highly distributed in the hemocytes as well as gill and mantle. The hemocyte specific genes Integrin, EcSOD and haematopoietic transcription factors GATA3, C-Myb, c-kit, were down-regulated when Cg-SCL was interfered by dsRNA. During the larval developmental stages, the mRNA transcripts of Cg-SCL gradually increased after fertilization and peaked at early trochophore larvae stage (10 hpf, hours post fertilization), then sharply decreased in late trochophore larvae stage (15 hpf) before resuming in umbo larvae (120 hpf). Whole-mount immunofluorescence assay further revealed that the immunoreactivity of Cg-SCL appeared in blastula larvae with two approximate symmetric spots, and this expression pattern lasted in gastrula larvae. By trochophore, the immunoreactivity formed a ring around the dorsal region and then separated into two remarkable spots at the dorsal side in D-veliger larvae. After bacterial challenge, the mRNA expression levels of Cg-SCL were significantly up-regulated in the D-veliger and umbo larvae, indicating the available hematopoietic regulation in oyster larvae. These results demonstrated that Cg-SCL could be used as haematopoietic specific marker to trace potential developmental events of hematopoiesis during ontogenesis of oyster, which occurred early in blastula stage and maintained until D-veliger larvae. Copyright © 2016 Elsevier Ltd. All rights reserved.

A novel siglec (CgSiglec-1) from the Pacific oyster (Crassostrea gigas) with broad recognition spectrum and inhibitory activity to apoptosis, phagocytosis and cytokine release.

PubMed

Liu, Conghui; Jiang, Shuai; Wang, Mengqiang; Wang, Lingling; Chen, Hao; Xu, Jiachao; Lv, Zhao; Song, Linsheng

2016-08-01

Sialic acid binding immunoglobulin-type lectin (siglec) belongs to the immunoglobulin superfamily (IgSF), which acts as regulator involved in glycan recognition and signal transduction in the immune and nervous systems. In the present study, a siglec gene (designated CgSiglec-1) was characterized from the Pacific oyster, Crassostrea gigas. The cDNA of CgSiglec-1 was of 1251 bp encoding a predicted polypeptide of 416 amino acids. CgSiglec-1 was composed of two I-set immunoglobulin (Ig) domains, one transmembrane (TM) domain and two ITIM motifs, sharing a sequence similarity with vertebrate CD22 homologs. The mRNA expression of CgSiglec-1 could be detected in all the selected tissues, with the highest level in hemocytes and labial palps. The confocal analysis revealed that CgSiglec-1 mainly distributed on the cytoplasmic membrane of the oyster hemocytes. In addition, the mRNA transcripts of CgSiglec-1 in hemocytes increased significantly (4.29-fold to that of control group, p < 0.05) after Vibrio splendidus stimulation. The recombinant CgSiglec-1 protein (rCgSiglec-1) could bind to poly sialic acid (pSIAS), lipopolysaccharides (LPS) and peptidoglycan (PGN) in a dose-dependent manner. The blockade of CgSiglec-1 by specific polyclonal antibodies could enhance the LPS-induced cell apoptosis, phagocytosis towards V. splendidus and the release of cytokines, such as CgTNF-1, CgIFNLP and CgIL-17. The results collectively indicated that CgSiglec-1 could act as a bridge molecule between invader recognition and signal transduction cascade, and modulate the immune response by inhibiting various important processes of immunity in oyster. Copyright © 2016 Elsevier Ltd. All rights reserved.

Synthesis methods influence characteristics, behaviour and toxicity of bare CuO NPs compared to bulk CuO and ionic Cu after in vitro exposure of Ruditapes philippinarum hemocytes.

PubMed

Volland, Moritz; Hampel, Miriam; Katsumiti, Alberto; Yeste, María Pilar; Gatica, José Manuel; Cajaraville, Miren; Blasco, Julián

2018-06-01

Copper oxide (CuO) nanoparticles (NPs) are increasingly investigated, developed and produced for a wide range of industrial and consumer products. Notwithstanding their promising novel applications, concern has been raised that their increased use and disposal could consequently increase their release into marine systems and potentially affect species within. To date the understanding of factors and mechanisms of CuO (nano-) toxicity to marine invertebrates is still limited. Hence, we studied the characteristics and behaviour of two commercially available CuO NPs of similar size, but produced employing distinct synthesis methods, under various environmentally and experimentally relevant conditions. In addition, cell viability and DNA damage, as well as gene expression of detoxification, oxidative stress, inflammatory response, DNA damage repair and cell death mediator markers were studied in primary cultures of hemocytes from the marine clam Ruditapes philippinarum and, where applicable, compared to bulk CuO and ionic Cu (as CuSO 4 ) behaviour and effects. We found that the synthesis method can influence particle characteristics and behaviour, as well as the toxicity of CuO NPs to Ruditapes philippinarum hemocytes. Our results further indicate that under the tested conditions aggregating behaviour influences the toxicity of CuO NPs by influencing their rate of extra- and intracellular dissolution. In addition, gene expression analysis identified similar transcriptional de-regulation for all tested copper treatments for the here measured suite of genes. Finally, our work highlights various differences in the aggregation and dissolution kinetics of CuO particles under environmental (marine) and cell culture exposure conditions that need consideration when extrapolating in vitro findings. Copyright © 2018 Elsevier B.V. All rights reserved.

Age, pathogen exposure, but not maternal care shape offspring immunity in an insect with facultative family life.

PubMed

Vogelweith, Fanny; Körner, Maximilian; Foitzik, Susanne; Meunier, Joël

2017-03-07

To optimize their resistance against pathogen infection, individuals are expected to find the right balance between investing into the immune system and other life history traits. In vertebrates, several factors were shown to critically affect the direction of this balance, such as the developmental stage of an individual, its current risk of infection and/or its access to external help such as parental care. However, the independent and/or interactive effects of these factors on immunity remain poorly studied in insects. Here, we manipulated maternal presence and pathogen exposure in families of the European earwig Forficula auricularia to measure whether and how the survival rate and investment into two key immune parameters changed during offspring development. The pathogen was the entomopathogenic fungus Metarhizium brunneum and the immune parameters were hemocyte concentration and phenol/pro-phenoloxidase enzyme activity (total-PO). Our results surprisingly showed that maternal presence had no effect on offspring immunity, but reduced offspring survival. Pathogen exposure also lowered the survival of offspring during their early development. The concentration of hemocytes and the total-PO activity increased during development, to be eventually higher in adult females compared to adult males. Finally, pathogen exposure overall increased the concentration of hemocytes-but not the total-PO activity-in adults, while it had no effect on these measures in offspring. Our results show that, independent of their infection risk and developmental stage, maternal presence does not shape immune defense in young earwigs. This reveals that pathogen pressure is not a universal evolutionary driver of the emergence and maintenance of post-hatching maternal care in insects.

The Encapsidated Genome of Microplitis demolitor Bracovirus Integrates into the Host Pseudoplusia includens ▿ ‡

PubMed Central

Beck, Markus H.; Zhang, Shu; Bitra, Kavita; Burke, Gaelen R.; Strand, Michael R.

2011-01-01

Polydnaviruses (PDVs) are symbionts of parasitoid wasps that function as gene delivery vehicles in the insects (hosts) that the wasps parasitize. PDVs persist in wasps as integrated proviruses but are packaged as circularized and segmented double-stranded DNAs into the virions that wasps inject into hosts. In contrast, little is known about how PDV genomic DNAs persist in host cells. Microplitis demolitor carries Microplitis demolitor bracovirus (MdBV) and parasitizes the host Pseudoplusia includens. MdBV infects primarily host hemocytes and also infects a hemocyte-derived cell line from P. includens called CiE1 cells. Here we report that all 15 genomic segments of the MdBV encapsidated genome exhibited long-term persistence in CiE1 cells. Most MdBV genes expressed in hemocytes were persistently expressed in CiE1 cells, including members of the glc gene family whose products transformed CiE1 cells into a suspension culture. PCR-based integration assays combined with cloning and sequencing of host-virus junctions confirmed that genomic segments J and C persisted in CiE1 cells by integration. These genomic DNAs also rapidly integrated into parasitized P. includens. Sequence analysis of wasp-viral junction clones showed that the integration of proviral segments in M. demolitor was associated with a wasp excision/integration motif (WIM) known from other bracoviruses. However, integration into host cells occurred in association with a previously unknown domain that we named the host integration motif (HIM). The presence of HIMs in most MdBV genomic DNAs suggests that the integration of each genomic segment into host cells occurs through a shared mechanism. PMID:21880747

Isolation, gene cloning and expression profile of a pathogen recognition protein: a serine proteinase homolog (Sp-SPH) involved in the antibacterial response in the crab Scylla paramamosain.

PubMed

Liu, Hai-peng; Chen, Rong-yuan; Zhang, Min; Wang, Ke-jian

2010-07-01

To identify the frontline defense molecules against microbial infection in the crab Scylla paramamosain, a live crab pathogenic microbe, Vibrio parahaemolyticus, was recruited as an affinity matrix to isolate innate immune factors from crab hemocytes lysate. Interestingly, a serine proteinase homolog (Sp-SPH) was obtained together with an antimicrobial peptide-antilipopolysaccharide factor (Sp-ALF). We then determined the full-length cDNA sequence of Sp-SPH, which contained 1298bp with an open reading frame of 1107bp encoding 369 amino acid residues. Multiple alignment analysis showed that the deduced amino acid sequences of Sp-SPH shared overall identity (83.8%) with those of SPH-containing proteins from other crab species. Tissue distribution analysis indicated that the Sp-SPH transcripts were present in various tissues including eye stalk, subcuticular epidermis, gill, hemocyte, stomach, thorax ganglion, brain and muscle of S. paramamosain. The Sp-SPH was highly expressed in selected different development stages including embryo (I, II, III and V), zoea (I), megalopa, and juvenile. Importantly, the prophenoloxidase was also present in the embryos, zoea, juvenile and adult crabs, but relatively lower in megalopa compared to those of other stages. Furthermore, the Sp-SPH mRNA expression showed a statistically significant increase (P<0.05) in both hemocyte and subcuticular epidermis at 24h, and in gill at 96h after challenge of V. parahaemolyticus determined by quantitative real-time PCR. Taken together, the live-bacterial-binding activity and the acute-phase response against bacterial infection of Sp-SPH suggested that it might function as an innate immune recognition molecule and play a key role in host defense against microbe invasion in the crab S. paramamosain. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

GREATER HEMOCYTE BACTERICIDAL ACTIVITY IN OYSTERS (CRASSOSTREA VIRGINICA) FROM A RELATIVELY CONTAMINATED SITE IN PENSACOLA BAY, FLORIDA.

EPA Science Inventory

Bivalve mollusks such as Crassostrea virginica inhabiting polluted estuaries and coastal areas may bioaccumulate high concentrations of contaminants without apparent ill effects. However, changes in putative internal defense activities have been associated with contaminant accumu...

DIFFERENTIAL EFFECTS OF OYSTER (CRASSOSTREA VIRGINICA) DEFENSES ON CLINICAL AND ENVIRONMENTAL ISOLATES OF VIBRIO PARAHEMOLYTICUS

EPA Science Inventory

Three clinical (2030, 2062, and 2107) and three environmental (1094, 1163, and ATCC 17802) isolates of Vibrio parahaemolyticus were exposed to hemocytes and plasma collected from oysters (Crassostrea virginica) to determine their susceptibility to putative oyster defenses. Clinic...

Persistence of enteric viruses within oysters (Crassostrea virginica)

USDA-ARS?s Scientific Manuscript database

It is well known that water-borne enteric viruses are concentrated by bivalves. Why these viruses are selectively retained and remain infectious within shellfish tissues for extended periods is unknown. Our current hypothesis is that phagocytic hemocytes (blood cells) are a site of virus persiste...

Prostaglandin-mediated recovery from bacteriosemia delays larval development in fall armyworms, Spodoptera frugiperda

USDA-ARS?s Scientific Manuscript database

Insect immunity includes a surveillance system that detects and signals infections, coupled with hemocytic and humoral immune functions. These functions are signaled and coordinated by several biochemicals, including biogenic amines, insect cytokines, peptides and prostaglandins (PGs). The actions o...

BRICHOS domain-containing leukocyte cell-derived chemotaxin 1-like cDNA from disk abalone Haliotis discus discus.

PubMed

Kim, Yucheol; De Zoysa, Mahanama; Lee, Youngdeuk; Whang, Ilson; Lee, Jehee

2010-11-01

A BRICHOS domain-containing leukocyte cell-derived chemotaxin 1-like cDNA was cloned from the disk abalone (Haliotis discus discus) and designated as AbLECT-1. A full-length (705 bp) of AbLECT-1 cDNA was composed of a 576 bp open reading frame that translates into a putative peptide of 192 amino acids. Deduced amino acid sequence of AbLECT-1 had 15.5- and 27.8% identity and similarity to human LECT-1, respectively. Quantitative real-time PCR analysis results showed that the mRNA of AbLECT-1 was constitutively expressed in abalone hemocytes, gills, mantle, muscle, digestive tract and hepatopancreas in a tissue-specific manner. Moreover, the AbLECT-1 transcription level was induced in hemocytes after challenge with Vibrio alginolyticus, Vibrio parahemolyticus, and Listeria monocytogenes suggesting that it may be involved in immune response reactions in abalone. Copyright 2010 Elsevier Ltd. All rights reserved.

Reduced plant nutrition under elevated CO2 depresses the immunocompetence of cotton bollworm against its endoparasite

NASA Astrophysics Data System (ADS)

Yin, Jin; Sun, Yucheng; Ge, Feng

2014-04-01

Estimating the immunocompetence of herbivore insects under elevated CO2 is an important step in understanding the effects of elevated CO2 on crop-herbivore-natural enemy interactions. Current study determined the effect of elevated CO2 on the immune response of Helicoverpa armigera against its parasitoid Microplitis mediator. H. armigera were reared in growth chambers with ambient or elevated CO2, and fed wheat grown in the concentration of CO2 corresponding to their treatment levels. Our results showed that elevated CO2 decreases the nutritional quality of wheat, and reduces the total hemocyte counts and impairs the capacity of hemocyte spreading of hemolymph of cotton bollworm larvae, fed wheat grown in the elevated CO2, against its parasitoid; however, this effect was insufficient to change the development and parasitism traits of M. mediator. Our results suggested that lower plant nutritional quality under elevated CO2 could decrease the immune response of herbivorous insects against their parasitoid natural enemies.

Maturation of the immune system of the male house cricket, Acheta domesticus.

PubMed

Piñera, Angelica V; Charles, Heather M; Dinh, Tracy A; Killian, Kathleen A

2013-08-01

The immune system functions to counteract the wide range of pathogens an insect may encounter during its lifespan, ultimately maintaining fitness and increasing the likelihood of survival to reproductive maturity. In this study, we describe the maturation of the innate immune system of the male house cricket Acheta domesticus during the last two nymphal stages, and during early and late adulthood. Total hemolymph phenoloxidase enzyme activity, lysozyme-like enzyme activity, the number of circulating hemocytes, and encapsulation ability were all determined for each developmental stage or age examined. The number of circulating hemocytes and lysozyme-like enzyme activity were similar for all developmental stages examined. Nymphs and newly molted adult males, however, had significantly lower total phenoloxidase activity than later adult stages, yet nymphs were able to encapsulate a nylon thread just as well as adults. Encapsulation ability would thus appear to be independent of total phenoloxidase activity. Copyright © 2013 Elsevier Ltd. All rights reserved.

Assessment of the fitness of the mussel Mytilus galloprovincialis two years after the Hebei Spirit oil spill.

PubMed

Donaghy, Ludovic; Hong, Hyun-Ki; Kim, Moonkoo; Park, Heung-Sik; Choi, Kwang-Sik

2016-12-15

In December 2007, >150km of the West coast of Korea were heavily polluted by crude oil leaked from the oil tanker Hebei Spirit, leading to mass mortality of bivalve mollusks on the intertidal areas. Two years after, mussels Mytilus galloprovincialis were collected from two impacted sites to investigate sub-lethal effects of the oil spill. Tissue content in polycyclic aromatic hydrocarbons (PAHs), hemocyte parameters, reproductive status and energetic reserves were analyzed. PAHs in tissues of mussels as well as hemocyte parameters were not different between impacted and control sites. Energetic reserves were altered in mussels from the impacted sites. Glycogen content remained low at polluted sites, whatever the season. Two years after the Hebei Spirit oil spill, mussels then presented altered energetic metabolism. Further investigations are thus warranted to monitor the sustainability of mussel populations on the oil spilled West coast of Korea. Copyright © 2016 Elsevier Ltd. All rights reserved.

Drosophila sessile hemocyte clusters are true hematopoietic tissues that regulate larval blood cell differentiation

PubMed Central

Leitão, Alexandre B; Sucena, Élio

2015-01-01

Virtually all species of coelomate animals contain blood cells that display a division of labor necessary for homeostasis. This functional partition depends upon the balance between proliferation and differentiation mostly accomplished in the hematopoietic organs. In Drosophila melanogaster, the lymph gland produces plasmatocytes and crystal cells that are not released until pupariation. Yet, throughout larval development, both hemocyte types increase in numbers. Mature plasmatocytes can proliferate but it is not known if crystal cell numbers increase by self-renewal or by de novo differentiation. We show that new crystal cells in third instar larvae originate through a Notch-dependent process of plasmatocyte transdifferentiation. This process occurs in the sessile clusters and is contingent upon the integrity of these structures. The existence of this hematopoietic tissue, relying on structure-dependent signaling events to promote blood homeostasis, creates a new paradigm for addressing outstanding questions in Drosophila hematopoiesis and establishing further parallels with vertebrate systems. DOI: http://dx.doi.org/10.7554/eLife.06166.001 PMID:25650737

viking: identification and characterization of a second type IV collagen in Drosophila.

PubMed

Yasothornsrikul, S; Davis, W J; Cramer, G; Kimbrell, D A; Dearolf, C R

1997-10-01

We have taken an enhancer trap approach to identify genes that are expressed in hematopoietic cells and tissues of Drosophila. We conducted a molecular analysis of two P-element insertion strains that have reporter gene expression in embryonic hemocytes, strain 197 and vikingICO. This analysis has determined that viking encodes a collagen type IV gene, alpha2(IV). The viking locus is located adjacent to the previously described DCg1, which encodes collagen alpha1(IV), and in the opposite orientation. The alpha2(IV) and alpha1(IV) collagens are structurally very similar to one another, and to vertebrate type IV collagens. In early development, viking and DCg1 are transcribed in the same tissue-specific pattern, primarily in the hemocytes and fat body cells. Our results suggest that both the alpha1 and alpha2 collagen IV chains may contribute to basement membranes in Drosophila. This work also provides the foundation for a more complete genetic dissection of collagen type IV molecules and their developmental function in Drosophila.

Application of the morpho-functional analysis of hydrobionts ( Anadara sp. cf. Anadara inaequivalvis Bivalvia) to environmental monitoring

NASA Astrophysics Data System (ADS)

Kolyuchkina, G. A.; Miljutin, D. M.

2013-03-01

The population dynamics and morphological and functional characteristics (the concentration of hemocytes in the hemolymph and the level of histopathology) of the bivalve Anadara sp. cf. Anadara inaequivalvis were studied in 2005-2007 on the North Caucasian coast of the Black Sea. A drastic decline in the abundance of the bivalves simultaneously with cadmium's exceedence in their soft tissues and bottom sediments were recorded in May of 2007. Six months before the manifestation of the population effects, morphological and functional changes (a decrease in the content of hemocytes in the hemolymph and an increase in the content of brown cells in the connective tissue and lipofuscin-like pigment granules in the digestive gland epithelium) in the bivalves were detected that were the consequences of an external effect (presumably, cadmium intoxication). Thus, the morphological and functional changes of the individual Anadaras may be a promising tool for the early detection of the impact of pollutants on benthic ecosystems.

Effect of Nitrogen Compounds on Shrimp Litopenaeus vannamei: Histological Alterations of the Antennal Gland.

PubMed

Fregoso-López, Marcela G; Morales-Covarrubias, María S; Franco-Nava, Miguel A; Ponce-Palafox, Jesús T; Fierro-Sañudo, Juan F; Ramírez-Rochín, Javier; Páez-Osuna, Federico

2018-06-01

Two experimental modules with different stocking densities (M1 = 70 and M2 = 120 shrimp m -2 ) were examined weekly during 72-day culture cycle at low-salinity water (1.9 g L -1 ) and zero-water exchange to examine the effects of water quality deterioration on the antennal gland (AG) of shrimp. Results showed survival rates of 87.7% and 11.9% in M1 and M2, respectively. Water temperature, pH, dissolved oxygen, and chlorophyll a were not significantly different between modules but the concentrations of the nitrogen compounds were significantly different between modules with the exception of nitrite-N, showing a higher histological alteration index in M2 (32 ± 10) than M1 (22 ± 0) with a strong correlation with the nitrogen compounds. During the last weeks was evidenced in M1 inflammation and hemocytic and hemolymph infiltration, while in M2, melanization, hemocytic melanized nodules and cells with kariorrexis.

Lysosomal membrane stability of the mussel, Mytilus galloprovincialis (L.), as a biomarker of tributyltin exposure.

PubMed

Okoro, Hussein K; Snyman, Reinette G; Fatoki, Olalekan S; Adekola, Folahan A; Ximba, Bhekumusa J; Slabber, Michelle Y

2015-05-01

The effect of tributyltin (TBT) on the stability of hemocytic lysosome membranes of the mussel, Mytilus galloprovincialis, and the use thereof as a biomarker of TBT-induced stress, was investigated. Mussels were exposed to 0.1 and 1.0 µg/L tributyltin respectively for 4 weeks. Lysosomal membrane stability of hemocytes was tested weekly by means of the neutral red retention time (NRRT) assay, after which the mussel samples were analyzed for TBT content. The two exposed groups exhibited significantly increased (p < 0.05) whole body TBT concentrations with concomitant significant decreases (p < 0.05) in NRRT (R(2) values of 0.85 and 0.971 for lower and higher exposure groups, respectively). The higher exposure group showed a typical dose-response curve. For the control, no TBT was detected and NRRT remained stable. It was concluded that the NRRT assay could be considered as a useful technique, and lysosomal membrane destabilization a useful early warning and cellular biomarker of stress due to TBT exposure in M. galloprovincialis.

Molecular cloning and functional analysis of the fatty acid-binding protein (Sp-FABP) gene in the mud crab (Scylla paramamosain).

PubMed

Zeng, Xianglan; Ye, Haihui; Yang, Ya'nan; Wang, Guizhong; Huang, Huiyang

2013-03-01

Intracellular fatty acid-binding proteins (FABPs) are multifunctional cytosolic lipid-binding proteins found in vertebrates and invertebrates. In this work, we used RACE to obtain a full-length cDNA of Sp-FABP from the mud crab Scylla paramamosain. The open reading frame of the full length cDNA (886 bp) encoded a 136 amino acid polypeptide that showed high homology with related genes from other species. Real-time quantitative PCR identified variable levels of Sp-FABP transcripts in epidermis, eyestalk, gill, heart, hemocytes, hepatopancreas, muscle, ovary, stomach and thoracic ganglia. In ovaries, Sp-FABP expression increased gradually from stage I to stage IV of development and decreased in stage V. Sp-FABP transcripts in the hepatopancreas and hemocytes were up-regulated after a bacterial challenge with Vibrio alginnolyficus. These results suggest that Sp-FABP may be involved in the growth, reproduction and immunity of the mud crab.

Structural and biological characterization of one antibacterial acylpolyamine isolated from the hemocytes of the spider Acanthocurria gomesiana

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pereira, Lourivaldo S.; Silva, Pedro I.; Miranda, M. Teresa M.

2007-01-26

We have isolated a 417 Da antibacterial molecule, named mygalin, from the hemocytes of the spider Acanthoscurria gomesiana. The structure of mygalin was elucidated by tandem mass spectrometry (MS/MS) and by two spectroscopic techniques, nuclear magnetic resonance (NMR) and ultraviolet (UV) spectroscopy. Mygalin was identified as bis-acylpolyamine N1,N8-bis(2,5-dihydroxybenzoyl)spermidine, in which the primary amino groups of the spermidine are acylated with the carboxyl group of the 2,5-dihydroxybenzoic acid. Mygalin was active against Escherichia coli at 85 {mu}M, being this activity inhibited completely by catalase. Therefore, the antibacterial activity of mygalin was attributed to its production of hydrogen peroxide (H{sub 2}O{sub 2}).more » The putative mechanisms of formation of H{sub 2}O{sub 2} from mygalin are discussed. To our knowledge this is the first report of one bis-acylpolyamine with antibacterial activity purified from animal source.« less

MODULATION OF EASTERN OYSTER HEMOCYTE ACTIVITIES BY PERKINSUS MARINUS EXTRACELLULAR PROTEINS

EPA Science Inventory

The oyster pathogen Perkinsus marinusproduces many extracellular proteins (ECP) in vitro. Analysis of this ECP revealed a battery of hydrolytic enzymes. Some of these enzymes are known to modulate the activity of host defense cells. Although information on the effects of P. marin...

Immunotoxicity and genotoxicity testing for in-flight experiments under microgravity

NASA Astrophysics Data System (ADS)

Hansen, Peter-Diedrich; Hansen, Peter-Diedrich; Unruh, Eckehardt

Life Sciences as Related to Space (F) Influence of Spaceflight Environment on Biological Systems (F44) Immunotoxicity and genotoxicity testing for In-flight experiments under microgravity Sensing approaches for ecosystem and human health Author: Peter D. Hansen Technische Universit¨t Berlin, Faculty VI - Planen, Bauen, Umwelt, a Institute for Ecological Research and Technology, Department for Ecotoxicology, Berlin, Germany Peter-diedrich.hansen@tu-berlin.de Eckehardt Unruh Technische Universit¨t Berlin, Faculty VI - Planen, Bauen, Umwelt, Institute a for Ecological Research and Technology, Department for Ecotoxicology, Berlin, Germany An immune response by mussel hemocytes is the selective reaction to particles which are identified as foreign by its immune system shown by phagocytosis. Phagocytotic activity is based on the chemotaxis and adhesion, ingestion and phagosome formation. The attachment at the surface of the hemocytes and consequently the uptake of the particles or bacteria can be directly quantified in the format of a fluorescent assay. Another relevant endpoint of phagocytosis is oxidative burst measured by luminescence. Phagocytosis-related production of ROS will be stimulated with opsonised zymosan. The hemocytes will be stored frozen at -80oC and reconstituted in-flight for the experiment. The assay system of the TRIPLELUX-B Experiment has been performed with a well-defined quantification and evaluation of the immune function phagocytosis. The indicator cells are the hemocytes of blue mussels (Mytilus edulis). The signals of the immuno cellular responses are translated into luminescence as a rapid optical reporter system. The results expected will determine whether the observed responses are caused by microgravity and/or radiation (change in permeability, endpoints in genotoxicity: DNA unwinding). The samples for genotoxicity will be processed after returning to earth. The immune system of invertebrates has not been studied so far in space. The choice of phagocytes from invertebrates is justified to study the universal validity of innate immune responses. The TRIPLELUX-B Experiment contributes to risk assessment concerning immunotoxicity under space flight conditions. The components of the phagocytosis test system for the BIOLAB are now established and the technical realization of the TRIPLELUX- B Experiment is in final progess. The components of the fully automated AEC (Advanced Experimental Containment) will be demonstrated in the poster. There will be two AECs for reference measurements at 1xg and 0xg. The AEC of the TRIPLELUX-B experiment will contribute to a real time operational monitoring for immunotoxicity testing on earth. The AEC will allow "real time monitoring" providing automated observations of immunotoxicity in coastal and inland waters.

Molecular and functional analyses of novel anti-lipopolysaccharide factors in giant river prawn (Macrobrachium rosenbergii, De Man) and their expression responses under pathogen and temperature exposure.

PubMed

Srisapoome, Prapansak; Klongklaew, Nawanith; Areechon, Nontawith; Wongpanya, Ratree

2018-06-15

Anti-lipopolysaccharide factor (ALF) is an immune-related protein that is crucially involved in immune defense mechanisms against invading pathogens in crustaceans. In the current study, three different ALFs of giant river prawn (Mr-ALF3, Mr-ALF8 and Mr-ALF9) were discovered. Based on sequence analysis, Mr-ALF3 and Mr-ALF9 were identified as new members of ALFs in crustaceans (groups F and G, respectively). Structurally, each newly identified Mr-ALF contained three α-helices packed against a four-stranded β-sheet bearing the LPS-binding motif, which usually binds to the cell wall components of bacteria. Tissue expression analysis using quantitative real-time RT-PCR (qRT-PCR) demonstrated that Mr-ALF3 was expressed in most tissues, and the highest expression was in the heart and hemocytes. The Mr-ALF8 gene was highly expressed in the heart, hemocytes, midgut, hepatopacreas and hindgut, respectively, while the Mr-ALF9 gene was modestly expressed in the heart and hemocytes, respectively. The transcriptional responses of the Mr-ALFs to Aeromonas hydrophila and hot/cold temperatures were investigated by qRT-PCR in the gills, hepatopancreas and hemocytes. We found that all Mr-ALFs were clearly suppressed in all tested tissues when the experimental prawns were exposed to extreme temperatures (25 and 35 °C). Moreover, the expression levels of these genes were significantly induced in all examined tissues by 2 different concentrations of A. hydrophila (1 × 10 6 and 1 × 10 9  CFU/ml), particularly 12 and 96 h after the injection. Finally, binding activity analysis of LPS-motif peptides of each Mr-ALF revealed that the LPS peptide of Mr-ALF3 exhibited the strongest adhesion to two pathogenic Gram-negative bacteria, A. hydrophila and Vibrio harveyi, and the non-pathogenic Gram-positive Bacillus megaterium. The results also showed that the Mr-ALF8 and Mr-ALF9 peptides had mild antimicrobial effects against similar tested bacteria. Based on information obtained in this study, novel ALF genes were clearly identified. Analyses of their responses under pathogenic and temperature stresses demonstrated the binding and antimicrobial activities of these ALFs and the consequent physiological effects, indicating their crucial functional roles in the prawn immune system. Copyright © 2018 Elsevier Ltd. All rights reserved.

Retention of Enteric Viruses by the Hemocytes of the Eastern Oyster (Crassostrea virginica)

USDA-ARS?s Scientific Manuscript database

Shellfish are an important vector for transmission of enteric pathogens. Interventions, such as depuration, do not adequately clear enteric viruses, while fecal bacteria levels are significantly reduced. Why viruses are retained in the bivalve flesh is not well understood. We hypothesize that phagoc...

Eicosanoids up-regulate production of reactive oxygen species by NADPH-dependent oxidase in Spodoptera exigua phagocytic hemocytes

USDA-ARS?s Scientific Manuscript database

Eicosanoids mediate cellular immune responses in insects, including phagocytosis of invading microbes. Phagocytosis entails two major steps, the internalization of microbes and the subsequent killing of them via formation of reactive oxygen species (ROS). Here, we posed the hypothesis that eicosanoi...

Rapid detection of hepatitis A virus and murine norovirus in hemocytes of contaminated oysters

USDA-ARS?s Scientific Manuscript database

The human enteric pathogens, hepatitis A virus and human norovirus, have been shown to contaminate molluscan shellfish and cause foodborne disease in consumers. Rapid viral extraction methods are needed to replace current time consuming methods, which use whole oysters or dissected tissues. In our ...

Parasitization by Cotesia chilonis influences gene expression in fatbody and hemocytes of Chilo suppressalis

USDA-ARS?s Scientific Manuscript database

During oviposition many parasitoid wasps inject various factors along with eggs that manipulate the physiology and development of their hosts. These manipulations are thought to benefit the parasites. However, the detailed mechanisms of host-parasitoid interactions are not fully understood. We posed...

Chinese mitten crab (Eriocheir sinensis) iron-sulphur cluster assembly protein 2 (EsIscA2) is differentially regulated after immune and oxidative stress challenges.

PubMed

Zhang, Peng; Liu, Yu; Wang, Min; Dong, Miren; Liu, Zhaoqun; Jia, Zhihao; Wang, Weilin; Zhang, Anguo; Wang, Lingling; Song, Linsheng

2018-07-01

Iron-sulphur clusters (ISCs), one of the oldest and most versatile cofactors of proteins, are involved in catalysis reactions, electron transport reactions, regulation processes as well as sensing of ambient conditions. Iron-sulphur cluster assembly protein (IscA) is a scaffold protein member of ISC formation system, which plays a significant role in the assembly and maturation process of ISC proteins. In the present study, the cDNA sequence of iron-sulphur cluster assembly protein 2 (designated as EsIscA2) was cloned from Eriocheir sinensis. The open reading frame (ORF) of EsIscA2 was of 507 bp, encoding a peptide of 168 amino acids with a typically conserved Fe-S domain. A tetrameric form was predicated by the SWISS-MODEL prediction algorithm, and three conserved cysteine residues (Cys-93, Cys-158, Cys-160) from each IscA monomer were predicted to form a 'cysteine pocket'. The deduced amino acid sequence of EsIscA2 shared over 50% similarity with that of other IscAs. EsIscA2 was clustered with IscA2 proteins from invertebrates and vertebrates, indicating that the protein was highly conservative in the evolution. rEsIscA2 exhibited a high iron binding affinity in the concentration ranging from 2 to 200 μM. EsIscA2 transcripts were detected in all the tested tissues including gonad, hemocytes, gill, muscle, heart, hepatopancreas and eyestalk, and EsIscA2 protein was detected in the mitochondria of hemocytes. The highest mRNA expression level of EsIscA2 was detected in muscle and hepatopancreas, which was about 34.66-fold (p < 0.05) and 27.07-fold (p < 0.05) of that in hemocytes, respectively. After Aeromonas hydrophila and lipopolysaccharide (LPS) stimulations, the mRNA expression of EsIscA2 in hemocytes was down-regulated and reached the lowest level at 24 h (0.31-fold, p < 0.05) and 48 h (0.29-fold, p < 0.05) compared to control group, respectively. And the expression of EsIscA2 mRNA in hepatopancreas was repressed from 6 h to 48 h post stimulation (p < 0.05). When the primary cultured crab hemocytes were incubated with different concentrations of H 2 O 2 for 15 min, the expression level of EsIscA2 mRNA was significantly repressed to the 0.34-0.44-fold of that in the control group. After A. hydrophila stimulation, the mRNA expression of EsGrx2 was up-regulated at 3 h (3.22-fold compared to control group, p < 0.05) and reached the peak at 12 h (4.88-fold, p < 0.05). All these results suggested that EsIscA2 had iron-binding capabilities as observed in IscA proteins from other organisms, supporting the role of EsIscA2 as a mitochondrial iron donor for ISC synthesis in Chinese mitten crab. Its differential mRNA expression after immune and oxidative stress challenges suggested the adaptations of ISC synthesis rates to these stress conditions. Copyright © 2018 Elsevier Ltd. All rights reserved.

Wound Signaling: Monkeywrenching Macrophage Migration with Microscopes, Movies, and Math.

PubMed

Galko, Michael J

2016-08-08

Drosophila hemocytes (blood cells) have emerged as a powerful system to image wound-induced inflammatory responses in vivo. New work reveals that layering mathematical modeling on top of imaging may be the most powerful tool yet for determining the properties of wound-induced signals. Copyright © 2016 Elsevier Ltd. All rights reserved.

From Embryo to Adult: Hematopoiesis along the Drosophila Life Cycle.

PubMed

Ramond, Elodie; Meister, Marie; Lemaitre, Bruno

2015-05-26

Studies on Drosophila hematopoiesis have thus far focused on the embryonic and larval origin of hemocytes, the fly blood cells. In this issue of Developmental Cell, Ghosh et al. (2015) identify adult hematopoietic hubs containing progenitors that can differentiate into different blood cell types. Copyright © 2015 Elsevier Inc. All rights reserved.

Dopamine modulates hemocyte phagocytosis via a D1-like receptor in the rice stem borer, Chilo suppressalis

USDA-ARS?s Scientific Manuscript database

Dopamine (DA) is a signal moiety bridging the nervous and immune systems. DA dysregulation is linked to serious human diseases, including addiction, schizophrenia, and Parkinson's disease. However, DA actions in the immune system remain incompletely understood. In this study, we found that DA modula...

Pathways of nucleopolyhedrosis virus infection in the gypsy moth, Lymantria dispar

Treesearch

K. S. Shields

1985-01-01

Gypsy moth nucleopolyhedrosis virus polyhedral inclusion bodies dissolve slowly in host digestive fluids, in vitro. Infectious viral material is in the hemocoel two hours after ingestion of inclusion bodies. Hemocytes produce and release nucleocapsids throughout the course of infection, but in the fat body, nearly all nucleocapsids are enveloped and...

Development of rapid hemocyte-based extraction methods for detection of hepatitis A virus and murine norovirus in contaminated oysters

USDA-ARS?s Scientific Manuscript database

The human enteric pathogens, hepatitis A virus and human norovirus, have been shown to contaminate molluscan shellfish and cause foodborne disease in consumers. Rapid viral extraction methods are needed to replace current time consuming methods, which use whole oysters or dissected tissues. In our ...

Histological Alterations in Pacific Oysters Crassostrea gigas that Survived a Summer Mortality Event in Baja California, Mexico.

PubMed

Cáceres-Martínez, Jorge; Vásquez-Yeomans, Rebeca; Danigo, Philippe; Reyes-Roel, Carlos

2018-03-01

A mortality episode (>90%) of triploid and diploid Pacific oysters Crassostrea gigas cultured in Baja California Sur occurred during summer 2012, coinciding with a thermal anomaly, an algal bloom, and low oxygen values. To help explain the cause of the mortalities, histological analyses and molecular tests for specific pathogens (ostreid herpesvirus 1 [OsHV-1] and Perkinsus marinus) were performed on oysters surviving at the end of the episode. Triploid oysters showed a high percentage of males (43%) and hermaphrodites (30%); 93% of these oysters were in the gonadic reabsorption stage, and in some cases, hemocytes completely filled the lumen of the gonadic follicles. Oysters presented large areas with severe hemocyte infiltration that extended toward the digestive gland. Diploid oysters showed similar gonad alterations. None of samples showed histological or molecular evidence of OsHV-1 or P. marinus. Histological alterations can be related to physiological disorders caused by the mechanism driving summer mortality. This is the first case history of a summer mortality episode among Pacific oysters in Mexico. © 2017 American Fisheries Society.

Cationic antimicrobial peptides in penaeid shrimp.

PubMed

Tassanakajon, Anchalee; Amparyup, Piti; Somboonwiwat, Kunlaya; Supungul, Premruethai

2011-08-01

Penaeid shrimp aquaculture has been consistently affected worldwide by devastating diseases that cause a severe loss in production. To fight a variety of harmful microbes in the surrounding environment, particularly at high densities (of which intensive farming represents an extreme example), shrimps have evolved and use a diverse array of antimicrobial peptides (AMPs) as part of an important first-line response of the host defense system. Cationic AMPs in penaeid shrimps composed of penaeidins, crustins, and anti-lipopolysaccharide factors are comprised of multiple classes or isoforms and possess antibacterial and antifungal activities against different strains of bacteria and fungi. Shrimp AMPs are primarily expressed in circulating hemocytes, which is the main site of the immune response, and hemocytes expressing AMPs probably migrate to infection sites to fight against pathogen invasion. Indeed, most AMPs are produced as early as the nauplii developmental stage to protect shrimp larvae from infections. In this review, we discuss the sequence diversity, expression, gene structure, and antimicrobial activities of cationic AMPs in penaeid shrimps. The information available on antimicrobial activities indicates that these shrimp AMPs have potential therapeutic applications in the control of disease problems in aquaculture.

A single Photorhabdus gene, makes caterpillars floppy (mcf), allows Escherichia coli to persist within and kill insects

PubMed Central

Daborn, P. J.; Waterfield, N.; Silva, C. P.; Au, C. P. Y.; Sharma, S.; ffrench-Constant, R. H.

2002-01-01

Photorhabdus luminescens, a bacterium with alternate pathogenic and symbiotic phases of its lifestyle, represents a source of novel genes associated with both virulence and symbiosis. This entomopathogen lives in a “symbiosis of pathogens” with nematodes that invade insects. Thus the bacteria are symbiotic with entomopathogenic nematodes but become pathogenic on release from the nematode into the insect blood system. Within the insect, the bacteria need to both avoid the peptide- and cellular- (hemocyte) mediated immune response and also to kill the host, which then acts as a reservoir for bacterial and nematode reproduction. However, the mechanisms whereby Photorhabdus evades the insect immune system and kills the host are unclear. Here we show that a single large Photorhabdus gene, makes caterpillars floppy (mcf), is sufficient to allow Esherichia coli both to persist within and kill an insect. The predicted high molecular weight Mcf toxin has little similarity to other known protein sequences but carries a BH3 domain and triggers apoptosis in both insect hemocytes and the midgut epithelium. PMID:12136122

A single Photorhabdus gene, makes caterpillars floppy (mcf), allows Escherichia coli to persist within and kill insects.

PubMed

Daborn, P J; Waterfield, N; Silva, C P; Au, C P Y; Sharma, S; Ffrench-Constant, R H

2002-08-06

Photorhabdus luminescens, a bacterium with alternate pathogenic and symbiotic phases of its lifestyle, represents a source of novel genes associated with both virulence and symbiosis. This entomopathogen lives in a "symbiosis of pathogens" with nematodes that invade insects. Thus the bacteria are symbiotic with entomopathogenic nematodes but become pathogenic on release from the nematode into the insect blood system. Within the insect, the bacteria need to both avoid the peptide- and cellular- (hemocyte) mediated immune response and also to kill the host, which then acts as a reservoir for bacterial and nematode reproduction. However, the mechanisms whereby Photorhabdus evades the insect immune system and kills the host are unclear. Here we show that a single large Photorhabdus gene, makes caterpillars floppy (mcf), is sufficient to allow Esherichia coli both to persist within and kill an insect. The predicted high molecular weight Mcf toxin has little similarity to other known protein sequences but carries a BH3 domain and triggers apoptosis in both insect hemocytes and the midgut epithelium.

Origins and activation of prophenoloxidases in the digestive tract of the cricket, Gryllus bimaculatus.

PubMed

Joseph, Woodring

2014-10-01

The function of Phenoloxidases (POs) in sclerotization and defense in insects is well understood, but little is known concerning their occurrence, origins, and function in the digestive tract. In Gyrllus bimaculatus gut all of the PO activity is found in the lumen of the digestive tract, and no detectible activity is found in homogenates of the gut epithelium or secretions from incubated epithelial tissues. Prophenoloxidases (PPOs) are synthesized in the hemocytes of  Bombyx mori and are transported into the cuticle. It is suggested that the PPOs in the caecal lumen of G. bimaculatus likewise are synthesized in hemocytes and are transported by unknown means into the caecal lumen, where they are activated to POs by trypsin. Peristalsis transports the POs both forward into the crop and posterior within the peritrophic membrane into the hind gut. The PPOs in the hemolymph consist of a trimer (270-280 kDa) and a tetramer (340-370 kDa). The active POs in the gut lumen consist of a monomer (85-95 kDa) in addition to an activated trimer and tetramer. © 2014 Wiley Periodicals, Inc.

Transcriptome analysis of eyestalk and hemocytes in the ridgetail white prawn Exopalaemon carinicauda: assembly, annotation and marker discovery.

PubMed

Li, Jitao; Li, Jian; Chen, Ping; Liu, Ping; He, Yuying

2015-01-01

The ridgetail white prawn Exopalaemon carinicauda is one of major economic mariculture species in eastern China. The deficiency of genomic and transcriptomic data is becoming the bottleneck of further researches on its good traits. In the present study, 454 pyrosequencing was undertaken to investigate the transcriptome profiles of E. carinicauda. A collection of 1,028,710 sequence reads (459.59 Mb) obtained from cDNA prepared from eyestalk and hemocytes was assembled into 162,056 expressed sequence tags (ESTs). Of these, 29.88 % of 48,428 contigs and 70.12 % of 113,628 singlets possessed high similarities to sequences in the GenBank non-redundant database, with most significant (E value <1e(-10)) unigenes matches occurring with crustacean and insect sequences. KEGG analysis of unigenes identified putative members of biological pathways related to growth and immunity. In addition, we obtained a total of putative 125,112 SNPs and 13,467 microsatellites. These results will contribute to the understanding of the genome makeup and provide useful information for future functional genomic research in E. carinicauda.

Isolation and identification of a plasmatocyte-spreading peptide from the hemolymph of the lepidopteran insect Pseudoplusia includens.

PubMed

Clark, K D; Pech, L L; Strand, M R

1997-09-12

Insect blood cells (hemocytes) play an essential role in defense against parasites and other pathogenic organisms that infect insects. A key class of hemocytes involved in insect cellular immunity is plasmatocytes. Here we describe the isolation and identification of a peptide from the moth Pseudoplusia includens that mediates the spreading of plasmatocytes to foreign surfaces. This peptide, designated plasmatocyte-spreading peptide (PSP1), contains 23 amino acid residues in the following sequence: H-ENFNGGCLAGYMRTADGRCKPTF-OH. In vitro assays using the synthetic peptide at concentrations >/=2 nM induced plasmatocytes from P. includens to spread on the surface of culture dishes. Injection of this peptide into P. includens larvae caused a transient depletion of plasmatocytes from circulation. Labeling studies indicated that this peptide induced 75% of plasmatocytes that were double-labeled by the monoclonal antibodies 49G3A3 and 43E9A8 to spread, whereas plasma induced significantly more plasmatocytes to spread. This suggests that only a certain subpopulation of plasmatocytes responds to the peptide and that other peptidyl factors mediate plasmatocyte adhesion responses.

Bombyx mori Serpin6 regulates prophenoloxidase activity and the expression of antimicrobial proteins.

PubMed

Li, Bing; Yu, Hai-Zhong; Ye, Chong-Jun; Ma, Yan; Li, Xing; Fan, Tao; Chen, Fu-Sheng; Xu, Jia-Ping

2017-04-30

Serpins are a family of serine protease inhibitors that are found widely in insects. They play an important role in insect physiological responses, such as innate immunity and development. In this study, we obtained the Bombyx mori serpin6 (BmSerpin6) sequence from National Center for Biotechnology Information (NCBI) and the silkworm genome database (SilkDB). Reverse transcription PCR (RT-PCR) results showed that BmSerpin6 was expressed highly in hemocytes, the midgut, and the fat body. After challenging with Micrococcus luteus (Mi) and Serratia marcescens (Sm), the BmSerpin6 expression level was induced significantly. Transcript levels of gloverin2 and prophenoloxidase (PPO) activity were reduced significantly in the fat body and hemocytes after injecting the recombinant BmSerpin6 protein into silkworm larvae. A BmSerpin6 recombinant plasmid (BmSerpin6-pAC 5.1) was constructed successfully and transfected into Drosophila S2 cells, which resulted in significantly reduced expression of the drosomycin protein. These results indicated that BmSerpin6 might regulate silkworm immune responses. Copyright © 2017 Elsevier B.V. All rights reserved.

Reaction of the mussel Mytilus galloprovincialis (Bivalvia) to Eugymnanthea inquilina (Cnidaria) and Urastoma cyprinae (Turbellaria) concurrent infestation.

PubMed

Mladineo, Ivona; Petrić, Mirela; Hrabar, Jerko; Bočina, Ivana; Peharda, Melita

2012-05-01

In total 480 individuals of Mytilus galloprovincialis were sampled monthly from October 2009 to September 2010, at the shellfish farm in the Mali Ston Bay, south Adriatic Sea (Croatia) in order to assess the extent of pathology imposed by two parasites, Eugymnanthea inquilina (Cnidaria) and Urastoma cyprinae (Turbellaria). Although a deteriorating impact on host reproduction or condition index was lacking, we evidenced ultrastructural and functional alteration in host cells at the attachment site. Ultrastructural changes included hemocytic encapsulation of the turbellarian and cell desquamation in medusoid infestation. Caspase positive reaction inferred by immunohistochemistry (IHC) was triggered in cases of turbellarian infestation, in contrast with hydroids, suggesting that the former exhibits more complex host-parasite interaction, reflected in the persistent attempts of the parasite to survive bivalve reaction. We have evidenced that both organisms trigger specific host reaction that although not costly in terms of host reproductive cycle or growth, results in mild tissue destruction and hemocyte activation. A lower degree of tissue reaction was observed in cases of hydroid infestation, compared to turbellarian. Copyright © 2012 Elsevier Inc. All rights reserved.

Characterization of a novel anti-lipopolysaccharide factor isoform (SpALF5) in mud crab, Scylla paramamosain.

PubMed

Sun, Wanwei; Wan, Weisong; Zhu, Shuo; Wang, Shasha; Wang, Shuqi; Wen, Xiaobo; Zheng, Huaiping; Zhang, Yueling; Li, Shengkang

2015-04-01

Anti-lipopolysaccharide factors (ALFs), the potential antimicrobial peptides that bind and neutralize lipopolysaccharide (LPS), are common effectors of innate immunity in crustaceans. In this study, a novel isoform of ALFs (SpALF5) was isolated from the hemocytes of mud crab Scylla paramamosain. The full-length 975bp SpALF5 contains a 375bp open reading frame (ORF) encoding 125 amino acids. Although SpALF5 exhibits a low degree of nucleotide homology with other reported ALFs, it contains the conserved amino acid sequence with a signal peptide and a LPS-binding domain including two conservative cysteine residues. The genomic organization of SpALF5 consists of four exons and three introns, with each intron containing one or more tandem repeats. Unlike most of ALFs mainly distributed in crab hemocytes, SpALF5 transcript was predominantly observed in the brain, muscle and skin, while barely detected in the hemocytes in our study. In situ hybridization assay also showed that SpALF5 mRNA was localized in brain, muscle and skin tissues of mud crab. Further, SpALF5 transcript was significantly up-regulated after challenge with LPS, polyinosinic polycytidylic acid (PolyI:C) (with the except of that in brain), Vibrio parahemolyticus or white spot syndrome virus (WSSV). The recombinant SpALF5 protein showed a varying degree of binding activity towards bacteria and fungus. Moreover, in vitro, the recombinant SpALF5 revealed a strong antimicrobial activity against Gram-negative bacteria (V. parahemolyticus, Vibrio alginolyticus, Escherichia coli, Aeromonas hydrophila) and fungus (Sacchromyces cerevisiae), but could only inhibited the growth of some Gram-positive bacteria like Staphylococcus aureus. The results suggest that SpALF5 is a potent immune protector and plays an important role in immune defense against invading pathogens in S. paramamosain. Copyright © 2014 Elsevier Ltd. All rights reserved.

Effects of low-dose exposure to pesticide mixture on physiological responses of the Pacific oyster, Crassostrea gigas.

PubMed

Geret, F; Burgeot, T; Haure, J; Gagnaire, B; Renault, T; Communal, P Y; Samain, J F

2013-12-01

This study investigated the effects on the physiology of Pacific oyster, Crassostrea gigas, of a mixture of pesticides containing 0.8 μg L(-1) alachlor, 0.6 μg L(-1) metolachlor, 0.7 μg L(-1) atrazine, 0.6 μg L(-1) terbuthylazine, 0.5 μg L(-1) diuron, 0.6 μg L(-1) fosetyl aluminum, 0.05 μg L(-1) carbaryl, and 0.7 μg L(-1) glyphosate for a total concentration of 4.55 μg L(-1) . The total nominal concentration of pesticides mixture corresponds to the pesticide concentrations in the shellfish culture area of the Marennes-Oleron basin. Two varieties of C. gigas were selected on the foreshore, based on their characteristics in terms of resistance to summer mortality, to assess the effects of the pesticide mixture after 7 days of exposure under controlled conditions. The early effects of the mixture were assessed using enzyme biomarkers of nitrogen metabolism (GS, glutamine synthetase), detoxification metabolism (GST, glutathione S-transferase), and oxidative stress (CAT, catalase). Sublethal effects on hemocyte parameters (phagocytosis and esterase activity) and DNA damages (DNA adducts) were also measured. Changes in metabolic activities were characterized by increases in GS, GST, and CAT levels on the first day of exposure for the "resistant" oysters and after 3-7 days of exposure for the "susceptible" oysters. The formation of DNA adducts was detected after 7 days of exposure. The percentage of hemocyte esterase-positive cells was reduced in the resistant oysters, as was the hemocyte phagocytic capacity in both oyster varieties after 7 days of exposure to the pesticide mixture. This study highlights the need to consider the low doses and the mixture of pesticides to evaluate the effects of these molecules on organisms. Copyright © 2011 Wiley Periodicals, Inc.

Immune response of Exopalaemon carinicauda infected with an AHPND-causing strain of Vibrio parahaemolyticus.

PubMed

Ge, Qianqian; Li, Jian; Li, Jitao; Wang, Jiajia; Li, Zhengdao

2018-03-01

To investigate the immune response of Exopalaemon carinicauda infected with an AHPND-causing strain of Vibrio parahaemolyticus (VP AHPND ), three-generation breeding of shrimp selected for their survival to VP AHPND infection was applied to explore the relationship between immune parameters and AHPND-resistant capacity of E. carinicauda. In this study, the LD 50 dose of 48 h and survival rates at 144 h of shrimp to VP AHPND increased from 10 6.0 to 10 6.6  cfu ml -1 and from 26.67% to 36.67% by three successive generations selection, respectively, while there was no significant difference between the first and second generation (p > .05). Then the immune parameters including vibrio density, total hemocyte counts (THCs), hemocyanin (HEM) concentration, antibacterial activity, activities of four immune enzymes, and expressions of eight immune-related genes were determined in the shrimp of the first (G1) and the third selective generation (G3). The results showed that the shrimp in G1 and G3 generation cleared most of VP AHPND infecting hepatopancreas during 24 h and 6 h post injection, respectively. The levels of THCs, HEM concentration, antibacterial activity, immune enzymes including lysozyme (LZM) activity, alkaline phosphatase (AKP) activity in cell-free hemolymph, and the expression levels of Tollip, ALF, cathepsin B in hemocytes and hepatopancreas, crustin, LZM, SR in hepatopancreas and LGBP in hemocytes were higher in G3 generation than in G1 generation after infection with VP AHPND , suggesting that these parameters may serve as potential disease-resistant indicators for evaluating the physiological status and disease-resistant capability of shrimp when infected with VP AHPND . To further test the role of above genes in the shrimp immune response, RNAi was used to suppress their expressions and a significant decrease in survival was observed in knockdown shrimp infected with VP AHPND as compared to controls. Copyright © 2017 Elsevier Ltd. All rights reserved.

A novel junctional adhesion molecule A (CgJAM-A-L) from oyster (Crassostrea gigas) functions as pattern recognition receptor and opsonin.

PubMed

Liu, Conghui; Wang, Mengqiang; Jiang, Shuai; Wang, Lingling; Chen, Hao; Liu, Zhaoqun; Qiu, Limei; Song, Linsheng

2016-02-01

Junctional adhesion molecule (JAM), a subfamily of immunoglobulin superfamily (IgSF) with a couple of immunoglobulin domains, can act as regulator in homeostasis and inflammation of vertebrates. In the present study, a structural homolog of JAM-A (designated CgJAM-A-L) was screened out from oyster, Crassostrea gigas, through a search of JAM-A D1 domain (N-terminal Ig domain in JAM-A). The cDNA of CgJAM-A-L was of 1188 bp encoding a predicted polypeptide of 395 amino acids. The immunoreactive area of CgJAM-A-L mainly distributed over the plasma membrane of hemocytes. After Vibro splendidus or tumor necrosis factor (CgTNF-1) stimulation, the mRNA transcripts of CgJAM-A-L in hemocytes increased significantly by 4.46-fold and 9.00-fold (p < 0.01) of those in control group, respectively. The recombinant CgJAM-A-L protein (rCgJAM-A-L) could bind multiple PAMPs including lipopolysaccharides (LPS), peptidoglycan (PGN), lipoteichoic acid (LTA), mannose (MAN), β-glucan (GLU) and poly(I:C), and various microorganisms including Micrococcus luteus, Staphylococcus aureus, Escherichia coli, Vibro anguillarum, V. splendidus, Pastoris pastoris and Yarrowia lipolytica. The phagocytic rates of oyster hemocytes towards Gram-negative bacteria V. anguillarum and yeast P. pastoris were significantly enhanced after the incubation of rCgJAM-A-L, and even increased more significantly after the pre-incubation of rCgJAM-A-L with microbes (p < 0.01). The results collectively indicated that CgJAM-A-L functioned as an important pattern recognition receptor (PRR) and opsonin in the immune defense against invading pathogen in oyster. Moreover, as the most primitive specie with homolog of JAMs, the information of CgJAM-A-L in oyster would provide useful clues for the evolutionary study of JAMs and immunoglobulins. Copyright © 2015 Elsevier Ltd. All rights reserved.

Respiratory and Metabolic Impacts of Crustacean Immunity: Are there Implications for the Insects?

PubMed

Burnett, Karen G; Burnett, Louis E

2015-11-01

Extensive similarities in the molecular architecture of the crustacean immune system to that of insects give credence to the current view that the Hexapoda, including Insecta, arose within the clade Pancrustacea. The crustacean immune system is mediated largely by hemocytes, relying on suites of pattern recognition receptors, effector functions, and signaling pathways that parallel those of insects. In crustaceans, as in insects, the cardiovascular system facilitates movement of hemocytes and delivery of soluble immune factors, thereby supporting immune surveillance and defense along with other physiological functions such as transport of nutrients, wastes, and hormones. Crustaceans also rely heavily on their cardiovascular systems to mediate gas exchange; insects are less reliant on internal circulation for this function. Among the largest crustaceans, the decapods have developed a condensed heart and a highly arteriolized cardiovascular system that supports the metabolic demands of their often large body size. However, recent studies indicate that mounting an immune response can impair gas exchange and metabolism in their highly developed vascular system. When circulating hemocytes detect the presence of potential pathogens, they aggregate rapidly with each other and with the pathogen. These growing aggregates can become trapped in the microvasculature of the gill where they are melanized and may be eliminated at the next molt. Prior to molting, trapped aggregates of hemocytes also can impair hemolymph flow and oxygenation at the gill. Small shifts to anaerobic metabolism only partially compensate for this decrease in oxygen uptake. The resulting metabolic depression is likely to impact other energy-expensive cellular processes and whole-animal performance. For crustaceans that often live in microbially-rich, but oxygen-poor aquatic environments, there appear to be distinct tradeoffs, based on the gill's multiple roles in respiration and immunity. Insects have developed a separate tracheal system for the delivery of oxygen to tissues, so this particular tradeoff between oxygen transport and immune function is avoided. Few studies in crustaceans or insects have tested whether mounting an immune response might impact other functions of the cardiovascular system or alter integrity of the gut, respiratory, and reproductive epithelia where processes of the attack on pathogens, defense by the host, and physiological functions play out. Such tradeoffs might be fruitfully addressed by capitalizing on the ease of molecular and genetic manipulation in insects. Given the extensive similarities between the insect and the crustacean immune systems, such models of epithelial infection could benefit our understanding of the physiological consequences of immune defense in all of the Pancrustacea. © The Author 2015. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.

A theoretical individual-based model of Brown Ring Disease in Manila clams, Venerupis philippinarum

NASA Astrophysics Data System (ADS)

Paillard, Christine; Jean, Fred; Ford, Susan E.; Powell, Eric N.; Klinck, John M.; Hofmann, Eileen E.; Flye-Sainte-Marie, Jonathan

2014-08-01

An individual-based mathematical model was developed to investigate the biological and environmental interactions that influence the prevalence and intensity of Brown Ring Disease (BRD), a disease, caused by the bacterial pathogen, Vibrio tapetis, in the Manila clam (Venerupis (= Tapes, = Ruditapes) philippinarum). V. tapetis acts as an external microparasite, adhering at the surface of the mantle edge and its secretion, the periostracal lamina, causing the symptomatic brown deposit. Brown Ring Disease is atypical in that it leaves a shell scar that provides a unique tool for diagnosis of either live or dead clams. The model was formulated using laboratory and field measurements of BRD development in Manila clams, physiological responses of the clam to the pathogen, and the physiology of V. tapetis, as well as theoretical understanding of bacterial disease progression in marine shellfish. The simulation results obtained for an individual Manila clam were expanded to cohorts and populations using a probability distribution that prescribed a range of variability for parameters in a three dimensional framework; assimilation rate, clam hemocyte activity rate (the number of bacteria ingested per hemocyte per day), and clam calcification rate (a measure of the ability to recover by covering over the symptomatic brown ring deposit), which sensitivity studies indicated to be processes important in determining BRD prevalence and intensity. This approach allows concurrent simulation of individuals with a variety of different physiological capabilities (phenotypes) and hence by implication differing genotypic composition. Different combinations of the three variables provide robust estimates for the fate of individuals with particular characteristics in a population that consists of mixtures of all possible combinations. The BRD model was implemented using environmental observations from sites in Brittany, France, where Manila clams routinely exhibit BRD signs. The simulated annual cycle of BRD prevalence and intensity agrees with observed disease cycles in cultured clam populations from this region, with maximum disease prevalence and intensity occurring from December to April. Sensitivity analyses of modeled physiological processes showed that the level of hemocyte activity is the primary intrinsic determinant of recovery of infected clams. Simulations designed to investigate environmental effects on BRD suggested that the outcome of the host-parasite interaction is dependent on food supply (high values being favorable for the host) and temperature. Results of simulations illustrate the complex interaction of temperature effects on propagation and viability of the bacterium, on the phagocytic activity of the hemocytes, and on other physiological processes of the host clam. Simulations using 1 °C and 2 °C increases in temperature generally favored disease development, indicating that climate warming might favor the spread of BRD.

Molecular cloning, characterization, and expression analysis of a heat shock protein (HSP) 70 gene from Paphia undulata.

PubMed

Wu, Xiangwei; Tan, Jing; Cai, Mingyi; Liu, Xiande

2014-06-15

In this study, a full-length HSP70 cDNA from Paphia undulata was cloned using reverse transcriptase polymerase chain reaction (RT-PCR) coupled with rapid amplification of cDNA ends (RACE). The full-length cDNA is 2,351 bp, consisting of a 5'-untranslated region (UTR) of 83 bp, a 3'-UTR of 315 bp, and an open reading frame (ORF) of 1,953 bp. This cDNA encodes 650 amino acids with an estimated molecular weight of 71.3 kDa and an isoelectric point of 5.51. Based on the amino acid sequence analysis and phylogenetic analysis, this HSP70 gene was identified as a member of the cytoplasmic HSP70 family, being the constitutive expression, and it was designated as PuHSC70. The distribution of PuHSC70 mRNA in the mantle, digestive gland, adductor muscle, gonad, gill, heart, and hemocytes suggested that PuHSC70 is ubiquitously expressed. The mRNA levels of PuHSC70 under high temperature and high salinity stresses were analyzed by real-time PCR. Under high temperature stress of 32°C, PuHSC70 mRNA in the mantle, digestive gland, gill, and heart was significantly up-regulated at 1h and 2h, and it was then progressively down-regulated. In the adductor muscle, the level of PuHSC70 mRNA gradually increased throughout the study period; the mRNA levels in the gonad and hemocytes increased significantly at 4h and 8h (P<0.05) and then decreased at 8h and 14 h, respectively, however they increased again afterwards, reaching the highest levels at 50h. Under high salinity (32 ‰) stress, the mRNA levels of PuHSC70 in the mantle and gonad were increased significantly only at 24h and 48 h (P<0.05), and at the rest of the study period they were slightly elevated. Compared with the pretreatment level, the levels of expression in the digestive gland and gill were unchanged or reduced throughout the study period. The levels of PuHSC70 mRNA in the adductor muscle, hemocytes, and heart were significantly increased, reaching a maximum at 24h, and then they gradually decreased; moreover, in the heart, the mRNA expression recovered to the pretreatment level at 50h; while in the adductor muscle and hemocytes, the expression level remained higher than that of the control. The cloning and expression analyses of PuHSC70 provide theoretical basis to further study the mechanism of physiological response to thermal and high salinity stresses. Copyright © 2014 Elsevier B.V. All rights reserved.

The neonicotinoids thiacloprid, imidacloprid, and clothianidin affect the immunocompetence of honey bees (Apis mellifera L.).

PubMed

Brandt, Annely; Gorenflo, Anna; Siede, Reinhold; Meixner, Marina; Büchler, Ralph

2016-03-01

A strong immune defense is vital for honey bee health and colony survival. This defense can be weakened by environmental factors that may render honey bees more vulnerable to parasites and pathogens. Honey bees are frequently exposed to neonicotinoid pesticides, which are being discussed as one of the stress factors that may lead to colony failure. We investigated the sublethal effects of the neonicotinoids thiacloprid, imidacloprid, and clothianidin on individual immunity, by studying three major aspects of immunocompetence in worker bees: total hemocyte number, encapsulation response, and antimicrobial activity of the hemolymph. In laboratory experiments, we found a strong impact of all three neonicotinoids. Thiacloprid (24h oral exposure, 200 μg/l or 2000 μg/l) and imidacloprid (1 μg/l or 10 μg/l) reduced hemocyte density, encapsulation response, and antimicrobial activity even at field realistic concentrations. Clothianidin had an effect on these immune parameters only at higher than field realistic concentrations (50-200 μg/l). These results suggest that neonicotinoids affect the individual immunocompetence of honey bees, possibly leading to an impaired disease resistance capacity. Copyright © 2016 Elsevier Ltd. All rights reserved.

Insect immunology and hematopoiesis.

PubMed

Hillyer, Julián F

2016-05-01

Insects combat infection by mounting powerful immune responses that are mediated by hemocytes, the fat body, the midgut, the salivary glands and other tissues. Foreign organisms that have entered the body of an insect are recognized by the immune system when pathogen-associated molecular patterns bind host-derived pattern recognition receptors. This, in turn, activates immune signaling pathways that amplify the immune response, induce the production of factors with antimicrobial activity, and activate effector pathways. Among the immune signaling pathways are the Toll, Imd, Jak/Stat, JNK, and insulin pathways. Activation of these and other pathways leads to pathogen killing via phagocytosis, melanization, cellular encapsulation, nodulation, lysis, RNAi-mediated virus destruction, autophagy and apoptosis. This review details these and other aspects of immunity in insects, and discusses how the immune and circulatory systems have co-adapted to combat infection, how hemocyte replication and differentiation takes place (hematopoiesis), how an infection prepares an insect for a subsequent infection (immune priming), how environmental factors such as temperature and the age of the insect impact the immune response, and how social immunity protects entire groups. Finally, this review highlights some underexplored areas in the field of insect immunobiology. Copyright © 2015 Elsevier Ltd. All rights reserved.

Transcriptional profiling of immune-related genes in Pacific white shrimp (Litopenaeus vannamei) during ontogenesis.

PubMed

Quispe, Ruth L; Justino, Emily B; Vieira, Felipe N; Jaramillo, Michael L; Rosa, Rafael D; Perazzolo, Luciane M

2016-11-01

We have performed here a gene expression analysis to determine the developmental stage at the main genes involved in crustacean immune response begin to be expressed and their changes in mRNA abundance during shrimp development. By using a quantitative PCR-based approach, we have measured the mRNA abundance of 24 immune-related genes from different functional categories in twelve developmental stages ranging from fertilized eggs to larval and postlarval stages and also in juveniles. We showed for the first time that the main genes from the RNAi-based post-transcriptional pathway involved in shrimp antiviral immunity are transcribed in all developmental stages, but exhibit a diverse pattern of gene expression during shrimp ontogenesis. On the other hand, hemocyte-expressed genes mainly involved in antimicrobial defenses appeared to be transcribed in larval stages, indicating that hematopoiesis initiates early in development. Moreover, transcript levels of some genes were early detected in fertilized eggs at 0-4 h post-spawning, suggesting a maternal contribution of immune-related transcripts to shrimp progeny. Altogether, our results provide important clues regarding the ontogenesis of hemocytes as well the establishment of antiviral and antimicrobial defenses in shrimp. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effects of ocean acidification on immune responses of the Pacific oyster Crassostrea gigas.

PubMed

Wang, Qing; Cao, Ruiwen; Ning, Xuanxuan; You, Liping; Mu, Changkao; Wang, Chunlin; Wei, Lei; Cong, Ming; Wu, Huifeng; Zhao, Jianmin

2016-02-01

Ocean acidification (OA), caused by anthropogenic CO2emissions, has been proposed as one of the greatest threats in marine ecosystems. A growing body of evidence shows that ocean acidification can impact development, survival, growth and physiology of marine calcifiers. In this study, the immune responses of the Pacific oyster Crassostrea gigas were investigated after elevated pCO2 exposure for 28 days. The results demonstrated that OA caused an increase of apoptosis and reactive oxygen species (ROS) production in hemocytes. Moreover, elevated pCO2 had an inhibitory effect on some antioxidant enzyme activities and decreased the GSH level in digestive gland. However, the mRNA expression pattern of several immune related genes varied depending on the exposure time and tissues. After exposure to pCO2 at ∼2000 ppm for 28 days, the mRNA expressions of almost all tested genes were significantly suppressed in gills and stimulated in hemocytes. Above all, our study demonstrated that elevated pCO2 have a significant impact on the immune systems of the Pacific oyster, which may constitute as a potential threat to increased susceptibility of bivalves to diseases. Copyright © 2015 Elsevier Ltd. All rights reserved.

Myeloid leukemia factor functions in anti-WSSV immune reaction of kuruma shrimp, Marsupenaeus japonicus.

PubMed

Feng, Xiao-Wu; Huo, Li-Jie; Sun, Jie-Jie; Xu, Ji-Dong; Niu, Guo-Juan; Wang, Jin-Xing; Shi, Xiu-Zhen

2017-11-01

Myeloid leukemia factor (MLF) plays an important role in development, cell cycle, myeloid differentiation, and regulates the RUNX transcription factors. However, the function of MLF in immunity is still unclear. In this study, an MLF was identified and characterized in kuruma shrimp Marsupenaeus japonicus, and named as MjMLF. The full-length cDNA of MjMLF contained 1111 nucleotides, which had an opening reading frame of 816 bp encoding a protein of 272 amino acids with an MLF1-interacting protein domain. MjMLF could be ubiquitously detected in different tissues of shrimp at the transcriptional level. The expression pattern analysis showed that MjMLF could be upregulated in shrimp hemocytes and hepatopancreas after white spot syndrome virus challenge. The RNA interference and protein injection assay showed that MjMLF could inhibit WSSV replication in vivo. Flow cytometry assay showed that MjMLF could induce hemocytes apoptosis which functioned in the shrimp antiviral reaction. All the results suggested that MjMLF played an important role in the antiviral immune reaction of kuruma shrimp. The research indicated that MjMLF might function as a novel regulator to inhibit WSSV replication in shrimp. Copyright © 2017 Elsevier Ltd. All rights reserved.

HlSRB, a Class B Scavenger Receptor, Is Key to the Granulocyte-Mediated Microbial Phagocytosis in Ticks

PubMed Central

Aung, Kyaw Min; Boldbaatar, Damdinsuren; Umemiya-Shirafuji, Rika; Liao, Min; Tsuji, Naotoshi; Xuenan, Xuan; Suzuki, Hiroshi; Kume, Aiko; Galay, Remil Linggatong; Tanaka, Tetsuya; Fujisaki, Kozo

2012-01-01

Ixodid ticks transmit various pathogens of deadly diseases to humans and animals. However, the specific molecule that functions in the recognition and control of pathogens inside ticks is not yet to be identified. Class B scavenger receptor CD36 (SRB) participates in internalization of apoptotic cells, certain bacterial and fungal pathogens, and modified low-density lipoproteins. Recently, we have reported on recombinant HlSRB, a 50-kDa protein with one hydrophobic SRB domain from the hard tick, Haemaphysalis longicornis. Here, we show that HlSRB plays vital roles in granulocyte-mediated phagocytosis to invading Escherichia coli and contributes to the first-line host defense against various pathogens. Data clearly revealed that granulocytes that up-regulated the expression of cell surface HlSRB are almost exclusively involved in hemocyte-mediated phagocytosis for E. coli in ticks, and post-transcriptional silencing of the HlSRB-specific gene ablated the granulocytes' ability to phagocytose E. coli and resulted in the mortality of ticks due to high bacteremia. This is the first report demonstrating that a scavenger receptor molecule contributes to hemocyte-mediated phagocytosis against exogenous pathogens, isolated and characterized from hematophagous arthropods. PMID:22479406

Parallel and costly changes to cellular immunity underlie the evolution of parasitoid resistance in three Drosophila species.

PubMed

McGonigle, John E; Leitão, Alexandre B; Ommeslag, Sarah; Smith, Sophie; Day, Jonathan P; Jiggins, Francis M

2017-10-01

A priority for biomedical research is to understand the causes of variation in susceptibility to infection. To investigate genetic variation in a model system, we used flies collected from single populations of three different species of Drosophila and artificially selected them for resistance to the parasitoid wasp Leptopilina boulardi, and found that survival rates increased 3 to 30 fold within 6 generations. Resistance in all three species involves a large increase in the number of the circulating hemocytes that kill parasitoids. However, the different species achieve this in different ways, with D. melanogaster moving sessile hemocytes into circulation while the other species simply produce more cells. Therefore, the convergent evolution of the immune phenotype has different developmental bases. These changes are costly, as resistant populations of all three species had greatly reduced larval survival. In all three species resistance is only costly when food is in short supply, and resistance was rapidly lost from D. melanogaster populations when food is restricted. Furthermore, evolving resistance to L. boulardi resulted in cross-resistance against other parasitoids. Therefore, whether a population evolves resistance will depend on ecological conditions including food availability and the presence of different parasite species.

Parallel and costly changes to cellular immunity underlie the evolution of parasitoid resistance in three Drosophila species

PubMed Central

Ommeslag, Sarah; Smith, Sophie; Day, Jonathan P.

2017-01-01

A priority for biomedical research is to understand the causes of variation in susceptibility to infection. To investigate genetic variation in a model system, we used flies collected from single populations of three different species of Drosophila and artificially selected them for resistance to the parasitoid wasp Leptopilina boulardi, and found that survival rates increased 3 to 30 fold within 6 generations. Resistance in all three species involves a large increase in the number of the circulating hemocytes that kill parasitoids. However, the different species achieve this in different ways, with D. melanogaster moving sessile hemocytes into circulation while the other species simply produce more cells. Therefore, the convergent evolution of the immune phenotype has different developmental bases. These changes are costly, as resistant populations of all three species had greatly reduced larval survival. In all three species resistance is only costly when food is in short supply, and resistance was rapidly lost from D. melanogaster populations when food is restricted. Furthermore, evolving resistance to L. boulardi resulted in cross-resistance against other parasitoids. Therefore, whether a population evolves resistance will depend on ecological conditions including food availability and the presence of different parasite species. PMID:29049362

Interactions of cationic polystyrene nanoparticles with marine bivalve hemocytes in a physiological environment: Role of soluble hemolymph proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Canesi, Laura, E-mail: Laura.Canesi@unige.it

The bivalve Mytilus galloprovincialis has proven as a suitable model invertebrate for evaluating the potential impact of nanoparticles (NPs) in the marine environment. In particular, in mussels, the immune system represents a sensitive target for different types of NPs. In environmental conditions, both NP intrinsic properties and those of the receiving medium will affect particle behavior and consequent bioavailability/uptake/toxicity. However, the evaluation of the biological effects of NPs requires additional understanding of how, once within the organism, NPs interact at the molecular level with cells in a physiological environment. In mammalian systems, different NPs associate with serum soluble components, organizedmore » into a “protein corona”, which affects particle interactions with target cells. However, no information is available so far on the interactions of NPs with biological fluids of aquatic organisms. In this work, the influence of hemolymph serum (HS) on the in vitro effects of amino modified polystyrene NPs (PS-NH{sub 2}) on Mytilus hemocytes was investigated. Hemocytes were incubated with PS-NH{sub 2} suspensions in HS (1, 5 and 50 µg/mL) and the results were compared with those obtained in ASW medium. Cell functional parameters (lysosomal membrane stability, oxyradical production, phagocytosis) were evaluated, and morphological changes were investigated by TEM. The activation state of the signalling components involved in Mytilus immune response (p38 MAPK and PKC) was determined. The results show that in the presence of HS, PS-NH{sub 2} increased cellular damage and ROS production with respect to ASW medium. The effects were apparently mediated by disregulation of p38 MAPK signalling. The formation of a PS-NH{sub 2}-protein corona in HS was investigated by centrifugation, and 1D- gel electrophoresis and nano-HPLC-ESI-MS/MS. The results identified the Putative C1q domain containing protein (MgC1q6) as the only component of the PS-NH{sub 2} hard protein corona in Mytilus hemolymph. These data represent the first evidence for the formation of a NP bio-corona in aquatic organisms and underline the importance of the recognizable biological identity of NPs in physiological exposure medium when testing their potential impact environmental model organisms. Although the results obtained in vitro do not entirely reflect a realistic exposure scenario and the more complex formation of a bio-corona that is likely to occur in vivo, these data will contribute to a better understanding of the effects of NPs in marine invertebrates. - Highlights: • The effects of PS-NH2 NPs on Mytilus hemocytes were compared in serum-HS and ASW. • HS increased cellular/lysosomal damage, ROS production and p-p38MAPK levels. • NP-corona proteins in HS were isolated and identified by MS. • NP-protein coronas in biological fluids can affect NP impact in marine species.« less

Oxidative stress induced by chlorine dioxide as an insecticidal factor to the Indian meal moth, Plodia interpunctella.

PubMed

Kumar, Sunil; Park, Jiyeong; Kim, Eunseong; Na, Jahyun; Chun, Yong Shik; Kwon, Hyeok; Kim, Wook; Kim, Yonggyun

2015-10-01

A novel fumigant, chlorine dioxide (ClO2) is a commercial bleaching and disinfection agent. Recent study indicates its insecticidal activity. However, its mode of action to kill insects is yet to be understood. This study set up a hypothesis that an oxidative stress induced by ClO2 is a main factor to kill insects. The Indian meal moth, Plodia interpunctella, is a lepidopteran insect pest infesting various stored grains. Larvae of P. interpunctella were highly susceptible to ClO2 gas, which exhibited an acute toxicity. Physiological damages by ClO2 were observed in hemocytes. At high doses, the larvae of P. interpunctella suffered significant reduction of total hemocytes. At low doses, ClO2 impaired hemocyte behaviors. The cytotoxicity of ClO2 was further analyzed using two insect cell lines, where Sf9 cells were more susceptible to ClO2 than High Five cells. The cells treated with ClO2 produced reactive oxygen species (ROS). The produced ROS amounts increased with an increase of the treated ClO2 amount. However, the addition of an antioxidant, vitamin E, significantly attenuated the cytotoxicity of ClO2 in a dose-dependent manner. To support the oxidative stress induced by ClO2, two antioxidant genes (superoxide dismutase (SOD) and thioredoxin-peroxidase (Tpx)) were identified from P. interpunctella EST library using ortholog sequences of Bombyx mori. Both SOD and Tpx were expressed in larvae of P. interpunctella especially under oxidative stress induced by bacterial challenge. Exposure to ClO2 gas significantly induced the gene expression of both SOD and Tpx. RNA interference of SOD or Tpx using specific double stranded RNAs significantly enhanced the lethality of P. interpunctella to ClO2 gas treatment as well as to the bacterial challenge. These results suggest that ClO2 induces the production of insecticidal ROS, which results in a fatal oxidative stress in P. interpunctella. Copyright © 2015 Elsevier Inc. All rights reserved.

Characterization and pathogenicity assessment of gut-associated microbes of muga silkworm Antheraea assamensis Helfer (Lepidoptera: Saturniidae).

PubMed

Haloi, Kishor; Kalita, Moni Kankana; Nath, Ramesh; Devi, Dipali

2016-07-01

Antheraea assamensis Helfer (muga silkworm) is an economically important endemic insect species of North Eastern Region of India. The silkworm is often susceptible to infection by pathogenic bacteria, leads to a disease commonly known as flacherie which causes 40% loss per annum to the silk industry. In this study, we have reported isolation, characterization and pathogenicity assessment of gut-associated bacteria of healthy and diseased muga silkworms. Thirty five bacterial isolates were screened from the gut of healthy and diseased silkworms by morphological observation and biochemical tests. 11 and 5 strains from healthy and diseased silkworm respectively were identified by 16S rRNA gene sequencing and analyzed. Pseudomonas aeruginosa (DRK1), Ornithinibacillus bavariensis (DRK2), Achromobacter xylosoxidans (KH3) and Staphylococcus aureus (FLG1) strains were commonly found in healthy as well as diseased larvae whereas, Bacillus thuringiensis (MK1) was found only in diseased larvae. Survivability analysis was performed with the identified strains by injection and oral administration (10(4)CFU/ml). The immune response of the silkworm against the pathogen was also studied by phenoloxidase and lysozyme enzyme activity assay, total and differential hemocyte count and phagocytic activity of hemocytes. It was observed that S. aureus, P. aeruginosa and B. thuringiensis significantly reduced the survivability of silkworm (p<0.001) hence found highly pathogenic. The lethal concentrations (LC50) values of the pathogenic strains were calculated at different time intervals (24, 48, 72 and 96h) within the range from 1.38×10(2) to 3.63×10(7)CFU/ml. The pathogenic groups demonstrated inhibition of phenoloxidase activity and decreased in total hemocyte count after 48h of infection. However, the lysozyme activity increased significantly in the pathogenic groups compared to the control (p<0.05). Granulocytes and plasmatocytes showed phagocytosis whereas; other types of cells did not show any phagocytic activity. Increasing granulocytes and plasmatocytes counts corroborates the results of phagocytic activity. The present study might be helpful in understanding the disease prognosis and colonization of bacteria causing the disease in muga silkworm. Copyright © 2016 Elsevier Inc. All rights reserved.

A novel phagocytic receptor (CgNimC) from Pacific oyster Crassostrea gigas with lipopolysaccharide and gram-negative bacteria binding activity.

PubMed

Wang, Weilin; Liu, Rui; Zhang, Tao; Zhang, Ran; Song, Xuan; Wang, Lingling; Song, Linsheng

2015-03-01

Phagocytosis is an evolutionarily conserved process to ingest the invading microbes and apoptotic or necrotic corpses, playing vital roles in defensing invaders and maintenance of normal physiological conditions. In the present study, a new Nimrod family phagocytic receptor with three EGF-like domains was identified in Pacific oyster Crassostrea gigas (designated CgNimC). CgNimC shared homology with other identified multiple EGF-like domain containing proteins. The mRNA transcripts of CgNimC were mainly distributed in mantle and hemocytes. Its relative expression level in hemocytes was significantly (P < 0.01) up-regulated after the injection of bacteria Vibrio anguillarum. Different to the NimC in Drosophila and Anopheles gambiae, the recombinant protein of CgNimC (rCgNimC) could bind directly to two gram-negative bacteria V. anguillarum and Vibrio splendidus, but not to gram-positive bacteria Staphylococci aureus, Micrococcus luteus or fungi Yarrowia lipolytica and Pichia pastoris. The affinity of rCgNimC toward M. luteus and Y. lipolytica was enhanced when the microorganisms were pre-incubated with the cell free hemolymph. rCgNimC exhibited higher affinity to lipopolysaccharide (LPS) and relatively lower affinity to peptidoglycan (PGN), while no affinity to glucan (GLU). After the CgNimC receptor was blocked by anti-rCgNimC antibody in vitro, the phagocytic rate of hemocytes toward two gram-negative bacteria V. anguillarum and V. splendidus was reduced significantly (P < 0.05), but no significant change of phagocytic rate was observed toward M. luteus and Y. lipolytica. All these results implied that CgNimC, with significant binding capability to LPS and gram-negative bacteria, was a novel phagocytic receptor involved in immune response of Pacific oyster. Further, it was speculated that receptors of Nimrod family might function as a phagocytic receptor to recognize PAMPs on the invaders and its recognition could be promoted by opsonization of molecules in hemolymph. Copyright © 2014 Elsevier Ltd. All rights reserved.

HSP70 gene expression in Mytilus galloprovincialis hemocytes is triggered by moderate heat shock and Vibrio anguillarum, but not by V. splendidus or Micrococcus lysodeikticus.

PubMed

Cellura, Cinzia; Toubiana, Mylène; Parrinello, Nicolo; Roch, Philippe

2006-01-01

Complete sequence of HSP70 cDNA from the mussel, Mytilus galloprovincialis was established before quantifying its expression following moderate heat shock or injection of heat-killed bacteria. HSP70 cDNA is comprised of 2378 bp including one ORF of 654 aa, with a predicted 70 bp 5'-UTR and a 343 bp 3'-UTR (GenBank, 18 Jan 05, AY861684). Alignment identity ranged from 89% for Crassostrea ariakensis to 72% for C. virginica. Curiously, HSP70 gene and cDNA sequences from M. galloprovincialis, deposited later (03 and 27 May), show only 73% identity with the present sequence. Meanwhile, characteristic motifs of the HSP70 family were located in conserved positions. Expression of HSP70 gene was quantified on circulating hemocyte mRNA using Q-PCR after RT using random hexaprimers. Housekeeping gene was 28S rRNA. Four stresses were applied: heat shock that consisted of immersing mussels for 90 min at 30 degrees C and returning them to 20 degrees C sea water, one injection of heat-killed Gram-negative bacteria, Vibrio splendidus LGP32, one injection of heat-killed Gram-negative bacteria Vibrio anguillarum, one injection of heat-killed Gram-positive bacteria Micrococcus lysodeikticus. We found no significant modification of 28S rRNA gene expression. Significant increase of 5.2 +/- 0.4 fold the ratio HSP70/28S rRNA was observed 6 h after heat shock and was maximum at 15 h (6.1 +/- 1.1), and still significant after 24 h (1.7 +/- 0.03). Similarly, injecting V. anguillarum resulted in a significant increase of 2.7 +/- 0.1 after 12 h. Expression was maximum after 48 h (5.2 +/- 0.05) and returned to baseline after 72 h. In contrast, injecting V. splendidus or M. lysodeikticus failed to significantly modulate HSP70 gene expression at least during the first 3 days post-injection. Consequently, mussel hemocytes appeared to discriminate between pathogenic and non-pathogenic Vibrios, as well as between Gram-negative and Gram-positive bacteria.

BAX inhibitor-1 silencing suppresses white spot syndrome virus replication in red swamp crayfish, Procambarus clarkii.

PubMed

Du, Zhi-Qiang; Lan, Jiang-Feng; Weng, Yu-Ding; Zhao, Xiao-Fan; Wang, Jin-Xing

2013-07-01

BAX inhibitor-1 (BI-1) was originally described as an anti-apoptotic protein in both animal and plant cells. BI-1 overexpression suppresses ER stress-induced apoptosis in animal cells. Inhibition of BI-1 activity could induce the cell death in mammals and plants. However, the function of BI-1 in crustacean immunity was unclear. In this paper, the full-length cDNA of a BI-1 protein in red swamp crayfish, Procambarus clarkii (PcBI-1) was cloned and its expression profiles in normal and infected crayfish were analyzed. The results showed that PcBI-1 was expressed in hemocytes, heart, hepatopancreas, gills, stomach, and intestines of the crayfish and was upregulated after challenged with Vibrio anguillarum and with white spot syndrome virus (WSSV). To determine the function of PcBI-1 in the innate immunity of the crayfish, the RNA interference against PcBI-1 was performed and the results indicated the hemocyte programmed cell death rate was increased significantly and WSSV replication was declined after PcBI-1 knocked down. Altogether, PcBI-1 plays an anti-apoptotic role, wherein high PcBI-1 expression suppresses programmed cell death, which is beneficial for WSSW replication in crayfish. Copyright © 2013 Elsevier Ltd. All rights reserved.

Immune depression in Rhodnius prolixus by seco-steroids, physalins.

PubMed

Castro, D P; Figueiredo, M B; Ribeiro, I M; Tomassini, T C B; Azambuja, P; Garcia, E S

2008-03-01

A comparative study of the effects of physalins, seco-steroidal substances of Physalis angulata (Solanaceae), on the immune reactions of R. prolixus was carried out. Ecdysis and mortality were not affected by treatment with physalins B, D, F or G (1-10 microg/ml of blood meal). R. prolixus larvae fed with blood containing physalins and inoculated with 1 microl of Enterobacter cloacae beta12 (5 x 10(3)/insect) exhibited mortality rates three times higher than controls. The insects treated with physalin B, and F (1 microg/ml) and inoculated with E. cloacae beta12 showed significant differences on lysozyme activity in the hemolymph compared to untreated insects. Furthermore, physalin D (1 microg/ml) significantly reduced the antibacterial activity. Concerning cellular immune reactions, all insects treated with physalins (1 microg/ml), exhibited drastic reductions in the quantity of yeast cell-hemocyte binding and subsequent internalization. Insects inoculated with bacteria and treated with physalins B, F and G showed reductions of microaggregate formation but physalin D did not. Physalins B and F also reduced total hemocyte count in the hemolymph. These results suggest that, in different ways, probably due to their different chemical structures, physalin B, D, F and G are immunomodulatory substances for the bloodsucking insect, R. prolixus.

Physiological and immunological characterization of Caribbean spiny lobsters Panulirus argus naturally infected with Panulirus argus Virus 1 (PaV1).

PubMed

Pascual Jiménez, Cristina; Huchin-Mian, Juan Pablo; Simões, Nuno; Briones-Fourzán, Patricia; Lozano-Álvarez, Enrique; Sánchez Arteaga, Ariadna; Pérez-Vega, Juan Antonio; Simá-Álvarez, Raúl; Rosas Vazquez, Carlos; Rodríguez-Canul, Rossanna

2012-08-27

The present study compares 13 physiological and immunological variables between a group of healthy Panulirus argus lobsters and a group of lobsters naturally infected with Panulirus argus Virus 1 (PaV1). Viral infection was determined through histopathology and PCR. Ten of the 13 variables differed significantly between the 2 groups. Using these variables, a principal component analysis yielded 2 separate clusters: one corresponding to the healthy group and the other corresponding to the infected group. In particular, infected lobsters exhibited significantly lower levels of osmotic pressure, total hemocyte counts, plasmatic proteins, and total phenoloxidase (PO) activity in plasma, as well as significantly higher levels of cholesterol and acylglycerides. These features are consistent with metabolic wasting, hyperlipidemia, and presumed immune suppression. Infection with PaV1 appears to increase the susceptibility of lobsters to some other opportunistic pathogens, as 61.1% of infected lobsters presented infestations of ciliate epibionts (Epystilis and Zoothamniun) in the gill chamber compared with 11.5% lobsters in the healthy group. Infected lobsters also showed significantly higher levels of total PO activity in degranulated hemocytes and trypsin inhibitor activity, potentially indicating activation of immune response by the PO system during the systemic infection with PaV1.

Identification of an entomopathogenic bacterium, Serratia sp. ANU101, and its hemolytic activity.

PubMed

Kim, Yonggyun; Kim, Keunseob; Seo, Jiae; Shrestha, Sony; Kim, Hosanna H; Nalini, Madanagopal; Yi, Youngkeun

2009-03-01

Four different bacterial colonies were isolated from an old stock of an entomopathogenic nematode, Steinernema monticolum. They all showed entomopathogenicity to final instar larvae of beet armyworm, Spodoptera exigua, by hemocoelic injection. However, they varied in colony form, susceptibility to antibiotics, and postmortem change of the infected host insects. Biolog microbial identification and 16S rDNA sequence analyses indicate that these are four different species classified into different bacterial genera. owing to high entomopathogenicity and a cadaver color of infected insect host, Serratia sp. was selected as a main symbiotic bacterial species and analyzed for its pathogenicity. Although no virulence of Serratia sp. was detected at oral administration, the bacteria gave significant synergistic pathogenicity to fifth instar S. exigua when it was treated along with a spore-forming entomopathogenic bacterium, Bacillus thuringiensis. The synergistic effect was explained by an immunosuppressive effect of Serratia sp. by its high cytotoxic effect on hemocytes of S. exigua, because Serratia sp. caused septicemia of S. exigua when the bacterial cells were injected into S. exigua hemocoel. The cytotoxic factor(s) was present in the culture medium because the sterilized culture broth possessed high potency in the cytotoxicity, which was specific to granular cells and plasmatocytes, two main immune-associated hemocytes in insects.

Use of OmpU porins for attachment and invasion of Crassostrea gigas immune cells by the oyster pathogen Vibrio splendidus

PubMed Central

Duperthuy, Marylise; Schmitt, Paulina; Garzón, Edwin; Caro, Audrey; Rosa, Rafael D.; Le Roux, Frédérique; Lautrédou-Audouy, Nicole; Got, Patrice; Romestand, Bernard; de Lorgeril, Julien; Kieffer-Jaquinod, Sylvie; Bachère, Evelyne; Destoumieux-Garzón, Delphine

2011-01-01

OmpU porins are increasingly recognized as key determinants of pathogenic host Vibrio interactions. Although mechanisms remain incompletely understood, various species, including the human pathogen Vibrio cholera, require OmpU for host colonization and virulence. We have shown previously that OmpU is essential for virulence in the oyster pathogen Vibrio splendidus LGP32. Here, we showed that V. splendidus LGP32 invades the oyster immune cells, the hemocytes, through subversion of host-cell actin cytoskeleton. In this process, OmpU serves as an adhesin/invasin required for β-integrin recognition and host cell invasion. Furthermore, the major protein of oyster plasma, the extracellular superoxide dismutase Cg-EcSOD, is used as an opsonin mediating the OmpU-promoted phagocytosis through its RGD sequence. Finally, the endocytosed bacteria were found to survive intracellularly, evading the host defense by preventing acidic vacuole formation and limiting reactive oxygen species production. We conclude that (i) V. splendidus is a facultative intracellular pathogen that manipulates host defense mechanisms to enter and survive in host immune cells, and (ii) that OmpU is a major determinant of host cell invasion in Vibrio species, used by V. splendidus LGP32 to attach and invade oyster hemocytes through opsonisation by the oyster plasma Cg-EcSOD. PMID:21282662

The Dot/Icm effector SdhA is necessary for virulence of Legionella pneumophila in Galleria mellonella and A/J mice.

PubMed

Harding, Clare R; Stoneham, Charlotte A; Schuelein, Ralf; Newton, Hayley; Oates, Clare V; Hartland, Elizabeth L; Schroeder, Gunnar N; Frankel, Gad

2013-07-01

Legionella pneumophila is an intracellular bacterium that resides within amoebae and macrophages in a specialized compartment termed the Legionella-containing vacuole (LCV). As well as providing an intracellular niche for replication, the LCV helps to prevent the release of bacterial components into the cytoplasm. Recognition of these components as danger signals by the host activates immune responses leading to clearance of the bacterium. Here, we examined the role of two important virulence factors of L. pneumophila, the potent danger signal flagellin and the translocated Dot/Icm type IVB secretion system effector SdhA, which is crucial to maintain LCV integrity, in the Galleria mellonella infection model. We demonstrate that flagellin expression does not contribute to virulence, replication, or induction of clearance mechanisms. Conversely, SdhA expression is important for virulence. We found that in the absence of SdhA, the LCV in hemocytes showed signs of instability and leakage. Furthermore, in contrast to wild-type L. pneumophila, a ΔsdhA mutant caused a transient depletion of hemocytes and reduced mortality. Analysis of the ΔsdhA mutant in the A/J mouse model also showed a significant replication defect. Together, our data underline the crucial importance of SdhA in infection across different model organisms.

Transcriptomic analysis of Ruditapes philippinarum hemocytes reveals cytoskeleton disruption after in vitro Vibrio tapetis challenge.

PubMed

Brulle, Franck; Jeffroy, Fanny; Madec, Stéphanie; Nicolas, Jean-Louis; Paillard, Christine

2012-10-01

The Manila clam, Ruditapes philippinarum, is an economically-important, commercial shellfish; harvests are diminished in some European waters by a pathogenic bacterium, Vibrio tapetis, that causes Brown Ring disease. To identify molecular characteristics associated with susceptibility or resistance to Brown Ring disease, Suppression Subtractive Hybridization (SSH) analyzes were performed to construct cDNA libraries enriched in up- or down-regulated transcripts from clam immune cells, hemocytes, after a 3-h in vitro challenge with cultured V. tapetis. Nine hundred and ninety eight sequences from the two libraries were sequenced, and an in silico analysis identified 235 unique genes. BLAST and "Gene ontology" classification analyzes revealed that 60.4% of the Expressed Sequence Tags (ESTs) have high similarities with genes involved in various physiological functions, such as immunity, apoptosis and cytoskeleton organization; whereas, 39.6% remain unidentified. From the 235 unique genes, we selected 22 candidates based upon physiological function and redundancy in the libraries. Then, Real-Time PCR analysis identified 3 genes related to cytoskeleton organization showing significant variation in expression attributable to V. tapetis exposure. Disruption in regulation of these genes is consistent with the etiologic agent of Brown Ring disease in Manila clams. Copyright © 2012 Elsevier Ltd. All rights reserved.

Bioaccumulation of Heavy Metals in Water, Sediments, and Tissues and Their Histopathological Effects on Anodonta cygnea (Linea, 1876) in Kabul River, Khyber Pakhtunkhwa, Pakistan

PubMed Central

Khan, Muhammad Iftikhar; Gulfam, Naila; Siraj, Muhammad; Zaidi, Farrah; Ahmadullah; Abidullah; Fatima, Syeda Hira; Noreen, Shumaila; Hamidullah; Shah, Zafar Ali; Qadir, Fazli

2018-01-01

The present investigation aimed to assess the concentrations of selected heavy metals in water and sediments and their bioaccumulation in tissues of freshwater mussels and their histopathological effects on the digestive gland, gills, and gonads of Anodonta cygnea. Water, sediments, and freshwater mussel samples were collected at four sites, that is, reference and polluted sites, along the Kabul River, Khyber Pakhtunkhwa. The polluted sites were receiving effluents from the industrial, agricultural, municipal, and domestic sources. The order of metals in the water was Zn > Pb > Ni > Cu > Mn > Fe > Cr > Cd, in sediments the order was Fe > Zn > Cr > Ni > Mn > Pb > Cu > Cd, and in the soft tissues the order was Fe > Zn > Mn > Pb > Cu > Cr > Ni > Cd. Histopathological alterations observed in polluted sites of Kabul River were inflammation, hydropic vacuolation, and lipofuscin pigments (in digestive gland), gill lamellar fusion, dilated hemolymphatic sinus, clumping, and generation of cilia and hemocytic infiltration (in gills), and atresia, necrosis, granulocytoma, hemocytic infiltration, and lipofuscin pigments (in gonads). The histopathological alterations in the organs of Anodonta cygnea can be considered as reliable biomarkers in biomonitoring of heavy metal pollution in aquatic ecosystems. PMID:29693003

Nimrod, a putative phagocytosis receptor with EGF repeats in Drosophila plasmatocytes.

PubMed

Kurucz, Eva; Márkus, Róbert; Zsámboki, János; Folkl-Medzihradszky, Katalin; Darula, Zsuzsanna; Vilmos, Péter; Udvardy, Andor; Krausz, Ildikó; Lukacsovich, Tamás; Gateff, Elisabeth; Zettervall, Carl-Johan; Hultmark, Dan; Andó, István

2007-04-03

The hemocytes, the blood cells of Drosophila, participate in the humoral and cellular immune defense reactions against microbes and parasites [1-8]. The plasmatocytes, one class of hemocytes, are phagocytically active and play an important role in immunity and development by removing microorganisms as well as apoptotic cells. On the surface of circulating and sessile plasmatocytes, we have now identified a protein, Nimrod C1 (NimC1), which is involved in the phagocytosis of bacteria. Suppression of NimC1 expression in plasmatocytes inhibited the phagocytosis of Staphylococcus aureus. Conversely, overexpression of NimC1 in S2 cells stimulated the phagocytosis of both S. aureus and Escherichia coli. NimC1 is a 90-100 kDa single-pass transmembrane protein with ten characteristic EGF-like repeats (NIM repeats). The nimC1 gene is part of a cluster of ten related nimrod genes at 34E on chromosome 2, and similar clusters of nimrod-like genes are conserved in other insects such as Anopheles and Apis. The Nimrod proteins are related to other putative phagocytosis receptors such as Eater and Draper from D. melanogaster and CED-1 from C. elegans. Together, they form a superfamily that also includes proteins that are encoded in the human genome.

A Role for Adenosine Deaminase in Drosophila Larval Development

PubMed Central

Dolezal, Tomas; Dolezelova, Eva; Zurovec, Michal

2005-01-01

Adenosine deaminase (ADA) is an enzyme present in all organisms that catalyzes the irreversible deamination of adenosine and deoxyadenosine to inosine and deoxyinosine. Both adenosine and deoxyadenosine are biologically active purines that can have a deep impact on cellular physiology; notably, ADA deficiency in humans causes severe combined immunodeficiency. We have established a Drosophila model to study the effects of altered adenosine levels in vivo by genetic elimination of adenosine deaminase-related growth factor-A (ADGF-A), which has ADA activity and is expressed in the gut and hematopoietic organ. Here we show that the hemocytes (blood cells) are the main regulator of adenosine in the Drosophila larva, as was speculated previously for mammals. The elevated level of adenosine in the hemolymph due to lack of ADGF-A leads to apparently inconsistent phenotypic effects: precocious metamorphic changes including differentiation of macrophage-like cells and fat body disintegration on one hand, and delay of development with block of pupariation on the other. The block of pupariation appears to involve signaling through the adenosine receptor (AdoR), but fat body disintegration, which is promoted by action of the hemocytes, seems to be independent of the AdoR. The existence of such an independent mechanism has also been suggested in mammals. PMID:15907156

Pathogen recognition of a novel C-type lectin from Marsupenaeus japonicus reveals the divergent sugar-binding specificity of QAP motif.

PubMed

Alenton, Rod Russel R; Koiwai, Keiichiro; Miyaguchi, Kohei; Kondo, Hidehiro; Hirono, Ikuo

2017-04-04

C-type lectins (CTLs) are calcium-dependent carbohydrate-binding proteins known to assist the innate immune system as pattern recognition receptors (PRRs). The binding specificity of CTLs lies in the motif of their carbohydrate recognition domain (CRD), the tripeptide motifs EPN and QPD bind to mannose and galactose, respectively. However, variants of these motifs were discovered including a QAP sequence reported in shrimp believed to have the same carbohydrate specificity as QPD. Here, we characterized a novel C-type lectin (MjGCTL) possessing a CRD with a QAP motif. The recombinant MjGCTL has a calcium-dependent agglutinating capability against both Gram-negative and Gram-positive bacteria, and its sugar specificity did not involve either mannose or galactose. In an encapsulation assay, agarose beads coated with rMjGCTL were immediately encapsulated from 0 h followed by melanization at 4 h post-incubation with hemocytes. These results confirm that MjGCTL functions as a classical CTL. The structure of QAP motif and carbohydrate-specificity of rMjGCTL was found to be different to both EPN and QPD, suggesting that QAP is a new motif. Furthermore, MjGCTL acts as a PRR binding to hemocytes to activate their adherent state and initiate encapsulation.

Pathogen recognition of a novel C-type lectin from Marsupenaeus japonicus reveals the divergent sugar-binding specificity of QAP motif

PubMed Central

Alenton, Rod Russel R.; Koiwai, Keiichiro; Miyaguchi, Kohei; Kondo, Hidehiro; Hirono, Ikuo

2017-01-01

C-type lectins (CTLs) are calcium-dependent carbohydrate-binding proteins known to assist the innate immune system as pattern recognition receptors (PRRs). The binding specificity of CTLs lies in the motif of their carbohydrate recognition domain (CRD), the tripeptide motifs EPN and QPD bind to mannose and galactose, respectively. However, variants of these motifs were discovered including a QAP sequence reported in shrimp believed to have the same carbohydrate specificity as QPD. Here, we characterized a novel C-type lectin (MjGCTL) possessing a CRD with a QAP motif. The recombinant MjGCTL has a calcium-dependent agglutinating capability against both Gram-negative and Gram-positive bacteria, and its sugar specificity did not involve either mannose or galactose. In an encapsulation assay, agarose beads coated with rMjGCTL were immediately encapsulated from 0 h followed by melanization at 4 h post-incubation with hemocytes. These results confirm that MjGCTL functions as a classical CTL. The structure of QAP motif and carbohydrate-specificity of rMjGCTL was found to be different to both EPN and QPD, suggesting that QAP is a new motif. Furthermore, MjGCTL acts as a PRR binding to hemocytes to activate their adherent state and initiate encapsulation. PMID:28374848

Proteomic characterization of the hemolymph of Octopus vulgaris infected by the protozoan parasite Aggregata octopiana.

PubMed

Castellanos-Martínez, Sheila; Diz, Angel P; Álvarez-Chaver, Paula; Gestal, Camino

2014-06-13

The immune system of cephalopods is poorly known to date. The lack of genomic information makes difficult to understand vital processes like immune defense mechanisms and their interaction with pathogens at molecular level. The common octopus Octopus vulgaris has a high economic relevance and potential for aquaculture. However, disease outbreaks provoke serious reductions in production with potentially severe economic losses. In this study, a proteomic approach is used to analyze the immune response of O. vulgaris against the coccidia Aggregata octopiana, a gastrointestinal parasite which impairs the cephalopod nutritional status. The hemocytes and plasma proteomes were compared by 2-DE between sick and healthy octopus. The identities of 12 differentially expressed spots and other 27 spots without significant alteration from hemocytes, and 5 spots from plasma, were determined by mass spectrometry analysis aided by a six reading-frame translation of an octopus hemocyte RNA-seq database and also public databases. Principal component analysis pointed to 7 proteins from hemocytes as the major contributors to the overall difference between levels of infection and so could be considered as potential biomarkers. Particularly, filamin, fascin and peroxiredoxin are highlighted because of their implication in octopus immune defense activity. From the octopus plasma, hemocyanin was identified. This work represents a first step forward in order to characterize the protein profile of O. vulgaris hemolymph, providing important information for subsequent studies of the octopus immune system at molecular level and also to the understanding of the basis of octopus tolerance-resistance to A. octopiana. The immune system of cephalopods is poorly known to date. The lack of genomic information makes difficult to understand vital processes like immune defense mechanisms and their interaction with pathogens at molecular level. The study herein presented is focused to the comprehension of the octopus immune defense against a parasite infection. Particularly, it is centered in the host-parasite relationship developed between the octopus and the protozoan A. octopiana, which induces severe gastrointestinal injuries in octopus that produce a malabsorption syndrome. The common octopus is a commercially important species with a high potential for aquaculture in semi-open systems, and this pathology reduces the condition of the octopus populations on-growing in open-water systems resulting in important economical loses. This is the first proteomic approach developed on this host-parasite relationship, and therefore, the contribution of this work goes from i) ecological, since this particular relationship is tending to be established as a model of host-parasite interaction in natural populations; ii) evolutionary, due to the characterization of immune molecules that could contribute to understand the functioning of the immune defense in these highly evolved mollusks; and iii) to economical view. The results of this study provide an overview of the octopus hemolymph proteome. Furthermore, proteins influenced by the level of infection and implicated in the octopus cellular response are also showed. Consequently, a set of biomarkers for disease resistance is suggested for further research that could be valuable for the improvement of the octopus culture, taken into account their high economical value, the declining of landings and the need for the diversification of reared species in order to ensure the growth of the aquaculture activity. Although cephalopods are model species for biomedical studies and possess potential in aquaculture, their genomes have not been sequenced yet, which limits the application of genomic data to research important biological processes. Similarly, the octopus proteome, like other non-model organisms, is poorly represented in public databases. Most of the proteins were identified from an octopus' hemocyte RNA-seq database that we have performed, which will be the object of another manuscript in preparation. Therefore, the need to increase molecular data from non-model organisms is herein highlighted. Particularly, here is encouraged to expand the knowledge of the genomic of cephalopods in order to increase successful protein identifications. This article is part of a Special Issue entitled: Proteomics of non-model organisms. Copyright © 2013 Elsevier B.V. All rights reserved.

Two types of calmodulin play different roles in Pacific white shrimp (Litopenaeus vannamei) defenses against Vibrio parahaemolyticus and WSSV infection.

PubMed

Ji, Pei-Feng; Yao, Cui-Luan; Wang, Zhi-Yong

2011-08-01

Calmodulin (CaM) plays an important role in calcium-dependent signal transduction pathways. In the present study, two alternative splicing isoforms of CaM (named LvCaM-A and LvCaM-B) cDNA were cloned from the Pacific white shrimp, Litopenaeus vannamei. LvCaM-A was of 1101 bp, including a 5'-terminal untranslated region (UTR) of 70 bp, a 3'-terminal UTR of 581 bp and an open reading frame (ORF) of 450 bp encoding a polypeptide of 149 amino acids with a calculated molecular weight (Mw) of 17 kDa and pI of 4.41. LvCaM-B was 689 bp, including a same 5'-UTR of 70 bp, a 3'-terminal UTR of 109 bp and an ORF of 510 bp encoding a polypeptide of 169 amino acids with a calculated Mw of 19 kDa and pI of 4.36. Both LvCaM-A and LvCaM-B contained 4 conservative EF-hand motifs. Quantitative real-time reverse transcription PCR analysis revealed LvCaM-A to be expressed in most shrimp tissues, with the predominant expression in nerve and the weakest expression in heart. However, LvCaM-B expression level was much weaker than those of LvCaM-A in all the tested tissues with main expression in hepatopancreas. The expression of LvCaM-A and LvCaM-B after challenge with Vibrio parahaemolyticus and WSSV were tested in hemocytes, hepatopancreas and nerve. The results indicated that LvCaM-A and LvCaM-B transcripts could be significantly induced in hemocytes and hepatopancreas respectively by injection with V. parahaemolyticus. The highest expression of LvCaM-A was in the hemocytes with 2.3 times (at 48 h) greater expression than in the control (p < 0.05). However, sharp down-regulation of both LvCaM-A and LvCaM-B were detected in nerve after Vibrio- and WSSV injection (p < 0.05). These results suggested that CaM might play an important role in shrimp's defense against pathogenic infection. Copyright © 2011 Elsevier Ltd. All rights reserved.

The ontogeny of immunity: development of innate immune strength in the honey bee (Apis mellifera).

PubMed

Wilson-Rich, Noah; Dres, Stephanie T; Starks, Philip T

2008-01-01

Honey bees (Apis mellifera) are of vital economic and ecological importance. These eusocial animals display temporal polyethism, which is an age-driven division of labor. Younger adult bees remain in the hive and tend to developing brood, while older adult bees forage for pollen and nectar to feed the colony. As honey bees mature, the types of pathogens they experience also change. As such, pathogen pressure may affect bees differently throughout their lifespan. We provide the first direct tests of honey bee innate immune strength across developmental stages. We investigated immune strength across four developmental stages: larvae, pupae, nurses (1-day-old adults), and foragers (22-30 days old adults). The immune strength of honey bees was quantified using standard immunocompetence assays: total hemocyte count, encapsulation response, fat body quantification, and phenoloxidase activity. Larvae and pupae had the highest total hemocyte counts, while there was no difference in encapsulation response between developmental stages. Nurses had more fat body mass than foragers, while phenoloxidase activity increased directly with honey bee development. Immune strength was most vigorous in older, foraging bees and weakest in young bees. Importantly, we found that adult honey bees do not abandon cellular immunocompetence as has recently been proposed. Induced shifts in behavioral roles may increase a colony's susceptibility to disease if nurses begin foraging activity prematurely.

Immunomodulatory effects and anti-Candida activity of lactobacilli in macrophages and in invertebrate model of Galleria mellonella.

PubMed

de Oliveira, Felipe Eduardo; Rossoni, Rodnei Dennis; de Barros, Patricia Pimentel; Begnini, Barbara Evelyn; Junqueira, Juliana Campos; Jorge, Antonio Olavo Cardoso; Leão, Mariella Vieira Pereira; de Oliveira, Luciane Dias

2017-09-01

Due to the growing number of multi-resistant Candida spp., adjuvant treatments that may help combat these fungal pathogens are relevant and useful. This study evaluated the immunomodulation and anti-Candida activity of Lactobacillus rhamnosus (LR), Lactobacillus acidophilus and Lactobacillus paracasei suspensions, either single- or multiple-strain, in mouse macrophages (RAW 264.7) and Galleria mellonella (GM). Mouse macrophages were activated by different lactobacilli suspensions and challenged with C. albicans (CA). Tumor necrosis factor (TNF)-α, interleukin IL-1β, IL-6 and IL-17 production and cell viability were investigated. LR was the best suspension for stimulating all evaluated cytokines and thus was used in subsequent in vivo assays. Two C. albicans clinical strains, CA21 and CA60, were then added to the GM assays to further confirm the results. LR suspension was injected into the larvae 24 h before challenging with CA. Survival curve, CFU per larva and hemocytes were counted. In the GM, the LR suspension increased the survival rate and hemocyte counts and decreased the CFU per larva counts for all groups. Lactobacilli suspensions presented strain-dependent immunomodulation; however, single suspensions showed better results. Anti-Candida activity was demonstrated by decreased Candida counts in the GM with the use of LR. Copyright © 2017 Elsevier Ltd. All rights reserved.

Characterization of two types of vascular endothelial growth factor from Litopenaeus vannamei and their involvements during WSSV infection.

PubMed

Wang, Zhiwei; Li, Shihao; Li, Fuhua; Yang, Hui; Yang, Fusheng; Xiang, Jianhai

2015-12-01

Vascular endothelial growth factors (VEGFs) are important signaling proteins in VEGF signaling pathway which play key roles in inducing endothelial cell proliferation, migration, angiogenesis, vascular permeability, inhibition of apoptosis and virus infection. In the present study, we isolated and characterized two VEGF genes, LvVEGF1 and LvVEGF2 from Litopenaeus vannamei. The deduced amino acid sequences of both LvVEGF1 and LvVEGF2 contained a signal peptide, a typical PDGF/VEGF domain and a cysteine knot motif (CXCXC). Tissue distribution analysis showed that LvVEGF1 was predominantly expressed in lymphoid organ (Oka) while LvVEGF2 was mainly detected in gill and hemocytes. The transcriptional levels of LvVEGF1 in Oka and LvVEGF2 in gill or hemocytes were apparently up-regulated during WSSV infection. Double-stranded RNA interference was used for further functional studies. The data showed that silencing of LvVEGF1 and LvVEGF2 caused a decrease of the copy numbers of the virus in WSSV infected shrimp and a reduction of the cumulative mortality rate of shrimp during WSSV infection. The present study indicated that LvVEGF1 and LvVEGF2 might facilitate WSSV infection, which provided new evidence to understand the function of VEGF signaling pathway during WSSV infection in shrimp. Copyright © 2015 Elsevier Ltd. All rights reserved.

Isolation, cDNA cloning and gene expression of an antibacterial protein from larvae of the coconut rhinoceros beetle, Oryctes rhinoceros.

PubMed

Yang, J; Yamamoto, M; Ishibashi, J; Taniai, K; Yamakawa, M

1998-08-01

An antibacterial protein, designated rhinocerosin, was purified to homogeneity from larvae of the coconut rhinoceros beetle, Oryctes rhinoceros immunized with Escherichia coli. Based on the amino acid sequence of the N-terminal region, a degenerate primer was synthesized and reverse-transcriptase PCR was performed to clone rhinocerosin cDNA. As a result, a 279-bp fragment was obtained. The complete nucleotide sequence was determined by sequencing the extended rhinocerosin cDNA clone by 5' rapid amplification of cDNA ends. The deduced amino acid sequence of the mature portion of rhinocerosin was composed of 72 amino acids without cystein residues and was shown to be rich in glycine (11.1%) and proline (11.1%) residues. Comparison of the deduced amino acid sequence of rhinocerosin with those of other antibacterial proteins indicated that it has 77.8% and 44.6% identity with holotricin 2 and coleoptrecin, respectively. Rhinocerosin had strong antibacterial activity against E. coli, Streptococcus pyogenes, Staphylococcus aureus but not against Pseudomonas aeruginosa. Results of reverse-transcriptase PCR analysis of gene expression in different tissues indicated that the rhinocerosin gene is strongly expressed in the fat body and the Malpighian tubule, and weakly expressed in hemocytes and midgut. In addition, gene expression was inducible by bacteria in the fat body, the Malpighian tubule and hemocyte but constitutive expression was observed in the midgut.

Cloning and characterization of three suppressors of cytokine signaling (SOCS) genes from the Pacific oyster, Crassostrea gigas.

PubMed

Li, Jun; Zhang, Yang; Zhang, Yuehuan; Liu, Ying; Xiang, Zhiming; Qu, Fufa; Yu, Ziniu

2015-06-01

Members of the suppressor of cytokine signaling (SOCS) family are crucial for the control of a variety of signal transduction pathways that are involved in the immunity, growth and development of organisms. However, in mollusks, the identity and function of SOCS proteins remain largely unclear. In the present study, three SOCS genes, CgSOCS2, CgSOCS5 and CgSOCS7, have been identified by searching and analyzing the Pacific oyster genome. Structural analysis indicated that the CgSOCS share conserved functional domains with their vertebrate counterparts. Phylogenetic analysis showed that the three SOCS genes clustered into two distinct groups, the type I and II subfamilies, indicating that these subfamilies had common ancestors. Tissue-specific expression results showed that the three genes were constitutively expressed in all examined tissues and were highly expressed in immune-related tissues, such as the hemocytes, gills and digestive gland. The expression of CgSOCS can also be induced to varying degrees in hemocytes after challenge with pathogen-associated molecular patterns (PAMPs). Moreover, dual-luciferase reporter assays showed that the over-expression of CgSOCS2 and CgSOCS7, but not CgSOC5, can activate an NF-κB reporter gene. Collectively, these results demonstrated that the CgSOCS might play an important role in the innate immune responses of the Pacific oyster. Copyright © 2015 Elsevier Ltd. All rights reserved.

Molecular cloning and characterization of autophagy-related gene TmATG8 in Listeria-invaded hemocytes of Tenebrio molitor.

PubMed

Tindwa, Hamisi; Jo, Yong Hun; Patnaik, Bharat Bhusan; Lee, Yong Seok; Kang, Sang Sun; Han, Yeon Soo

2015-07-01

Macroautophagy (hereinafter called autophagy) is a highly regulated process used by eukaryotic cells to digest portions of the cytoplasm that remodels and recycles nutrients and disposes of unwanted cytoplasmic constituents. Currently 36 autophagy-related genes (ATG) and their homologs have been characterized in yeast and higher eukaryotes, including insects. In the present study, we identified and functionally characterized the immune function of an ATG8 homolog in a coleopteran insect, Tenebrio molitor (TmATG8). The cDNA of TmATG8 comprises of an ORF of 363 bp that encodes a protein of 120 amino acid residues. TmATG8 transcripts are detected in all the developmental stages analyzed. TmAtg8 protein contains a highly conserved C-terminal glycine residue (Gly116) and shows high amino acid sequence identity (98%) to its Tribolium castaneum homolog, TcAtg8. Loss of function of TmATG8 by RNAi led to a significant increase in the mortality rates of T. molitor larvae against Listeria monocytogenes. Unlike dsEGFP-treated control larvae, TmATG8-silenced larvae failed to turn-on autophagy in hemocytes after injection with L. monocytogenes. These data suggest that TmATG8 play a role in mediating autophagy-based clearance of Listeria in T. molitor. Copyright © 2015 Elsevier Ltd. All rights reserved.

Structural characterization and expression analysis of a beta-thymosin homologue (Tβ) in disk abalone, Haliotis discus discus.

PubMed

Kasthuri, Saranya Revathy; Premachandra, H K A; Umasuthan, Navaneethaiyer; Whang, Ilson; Lee, Jehee

2013-09-15

Repertoires of proteins and small peptides play numerous physiological roles as hormones, antimicrobial peptides, and cellular signaling factors. The beta-thymosins are a group of small acidic peptides involved in processes such as actin sequestration, neuronal development, wound healing, tissue repair, and angiogenesis. Recent characterization of the beta thymosins as immunological regulators in invertebrates led to our identification and characterization of a beta-thymosin homologue (Tβ) from Haliotis discus discus. The cDNA possessed an ORF of 132 bp encoding a protein of 44 amino acids with a molecular mass of 4977 Da. The amino acid sequence shows high identity with another molluskan beta-thymosin and has a characteristic actin binding motif (LKKTET) and glutamyl donors. Phylogenetic analysis showed a close relationship with molluskan homologues, as well as its distinct identity and common ancestral origin. Genomic analysis revealed a 3 exon-2 intron structure similar to the other homologues. In silico promoter analysis also revealed significant transcription factor binding sites, providing evidence for the expression of this gene under different cellular conditions, including stress or pathogenic attack. Tissue distribution profiling revealed a ubiquitous presence in all the examined tissues, but with the highest expression in mantle and hemocyte. Immune challenge with lipopolysaccharide, poly I:C and Vibrio parahemolyticus induced beta-thymosin expression in gill and hemocytes, affirming an immune-related role in invertebrates. Copyright © 2013 Elsevier B.V. All rights reserved.

Three STATs are involved in the regulation of the expression of antimicrobial peptides in the triangle sail mussel, Hyriopsis cumingii.

PubMed

Dai, Yun-Jia; Hui, Kai-Min; Zhang, Ying-Hao; Liu, Yan; Wang, Yu-Qing; Zhao, Li-Juan; Lin, Li; Chai, Lian-Qin; Wei, Shun; Lan, Jiang-Feng

2017-04-01

Janus kinase (Jak) and signal transducers and activators of transcription (STAT) signaling pathway is associated in antiviral and antibacterial immune response. Previous studies primarily investigated the function of STATs in mammals. For most invertebrates, only one STAT was found in each species, such as STAT92E was found in Drosophila melanogaster. The studies, which focus on the functional difference between various STATs in the same species of invertebrate, are limited. In the present study, three STATs (HcSTAT1, HcSTAT2 and HcSTAT3) were identified in triangle shell pearl mussel, Hyriopsis cumingii. Phylogenetic analysis showed that HcSTAT1 and HcSTAT3 were clustered with Homo sapiens STAT5, and HcSTAT2 was clustered with Pinctada fucata STAT and Crassostea gigas STAT6. All three STATs could be detected in all tested tissues (hemocytes, hepatopancreas, gill, mantle and foot), and were induced expression when challenged with Staphylococcus aureus or Aeromonas hydrophilia in hemocytes and hepatopancreas. HcSTAT1 regulated the expression of HcDef, HcWAP, HcThe and HcTNF. The expression of HcWAP and HcTNF was down-regulated in HcSTAT2-RNAi mussel. And HcSTAT3 affected the expression of HcTNF. The study is the first report of different functions in antibacterial immune responses between STATs in mollusks. Copyright © 2017 Elsevier Ltd. All rights reserved.

Trypanosoma rangeli: effects of physalin B on the immune reactions of the infected larvae of Rhodnius prolixus.

PubMed

Garcia, Eloi S; Castro, Daniele P; Ribeiro, Ivone M; Tomassini, Therezinha C B; Azambuja, Patrícia

2006-01-01

Physalins are seco-steroids obtained from plants of the family Solanaceae. Herein, we tested Physalis angulata L purified physalin B as an immunomodulatory compound in 5th-instar larvae of Rhodnius prolixus, which were systemically infected with the H14 Trypanosoma rangeli strain protozoan. In uninfected insects, the effective concentration of physalin B, which inhibited 50% of the blood ingested (ED(50)) volume, was 15.2+/-1.6 microg/ml of the meal. Ecdysis processes and mortality in uninfected larvae, treated orally with physalin B in concentrations ranging from 1 to 10 microg/ml, was similar to that observed in insects not treated with physalin B. However, R. prolixus larvae previously fed on blood containing 1.0, 0.1, and 0.01 microg of physalin B/ml exhibited mortality rates of 78.1, 54.3, and 12.7%, respectively, 6 days after inoculation of T. rangeli (1 x 10(3) parasites/insect), whereas only 7.2% mortality was observed in the control group, injected with sterile culture medium. The insects treated with physalin B (0.1 microg/ml) and inoculated with T. rangeli did not modify the phenoloxidase (PO) activity and total hemocyte count in the hemolymph. However, physalin B treatment caused a reduction in hemocyte micro-aggregation and nitric oxide production and enhanced the parasitemia in the hemolymph. These results demonstrate that physalin B from P. angulata is a potent immunomodulatory substance for the bloodsucking insect, R. prolixus.

Effect of salinity on regulation mechanism of neuroendocrine-immunoregulatory network in Litopenaeus vannamei.

PubMed

Zhao, Qun; Pan, Luqing; Ren, Qin; Wang, Lin; Miao, Jingjing

2016-02-01

The effects of low salinity (transferred from 31‰ to 26‰, 21‰, and 16‰) on the regulation pathways of neuroendocrine-immunoregulatory network were investigated in Litopenaeus vannamei. The results showed that the hormones (corticotrophin-releasing hormone, adrenocorticotropic hormone) and biogenic amines (dopamine, noradrenaline, 5-hydroxytryptamine) concentrations in lower salinity groups increased significantly within 12 h. The gene expression of biogenic amine receptors showed that dopamine receptor D4 and α2 adrenergic receptor in lower salinity groups decreased significantly within 12 h, whereas the 5-HT7 receptor significantly increased within 1d. The second messenger synthetases (adenylyl cyclase, phospholipase C) and the second messengers (cyclic adenosine monophosphate, cyclic guanosine monophosphate) of lower salinity groups shared a similar trend in which adenylyl cyclase and cyclic adenosine monophosphate reached the maximum at 12 h, whereas phospholipase C and cyclic guanosine monophosphate reached the minimum. The immune parameters (total hemocyte count, phenoloxidase activity, phagocytic activity, crustin expression, antibacterial activity, C-type lectin expression, hemagglutinating activity) in lower salinity groups decreased significantly within 12 h. Except for the total hemocyte count, all the parameters recovered to the control levels afterwards. Therefore, it may be concluded that the neuroendocrine-immunoregulatory network plays a principal role in adapting to salinity changes as the main center for sensing the stress and causes immune response in L. vannamei. Copyright © 2016 Elsevier Ltd. All rights reserved.

The receptor tyrosine kinase Pvr promotes tissue closure by coordinating corpse removal and epidermal zippering

PubMed Central

Garlena, Rebecca A.; Lennox, Ashley L.; Baker, Lewis R.; Parsons, Trish E.; Weinberg, Seth M.; Stronach, Beth E.

2015-01-01

A leading cause of human birth defects is the incomplete fusion of tissues, often manifested in the palate, heart or neural tube. To investigate the molecular control of tissue fusion, embryonic dorsal closure and pupal thorax closure in Drosophila are useful experimental models. We find that Pvr mutants have defects in dorsal midline closure with incomplete amnioserosa internalization and epidermal zippering, as well as cardia bifida. These defects are relatively mild in comparison to those seen with other signaling mutants, such as in the JNK pathway, and we demonstrate that JNK signaling is not perturbed by altering Pvr receptor tyrosine kinase activity. Rather, modulation of Pvr levels in the ectoderm has an impact on PIP3 membrane accumulation, consistent with a link to PI3K signal transduction. Polarized PI3K activity influences protrusive activity from the epidermal leading edge and the protrusion area changes in accord with Pvr signaling intensity, providing a possible mechanism to explain Pvr mutant phenotypes. Tissue-specific rescue experiments indicate a partial requirement in epithelial tissue, but confirm the essential role of Pvr in hemocytes for embryonic survival. Taken together, we argue that inefficient removal of the internalizing amnioserosa tissue by mutant hemocytes coupled with impaired midline zippering of mutant epithelium creates a situation in some embryos whereby dorsal midline closure is incomplete. Based on these observations, we suggest that efferocytosis (corpse clearance) could contribute to proper tissue closure and thus might underlie some congenital birth defects. PMID:26293306

Analysis on the expression and function of syndecan in the Pacific white shrimp Litopenaeus vannamei.

PubMed

Yang, Hui; Li, Shihao; Li, Fuhua; Wen, Rong; Xiang, Jianhai

2015-08-01

Syndecan is considered to be a multifunctional protein which functions as a cell surface receptor involved in cell adhesion, migration, cytoskeleton organization and differentiation. Previous bioinformatic analysis has revealed that syndecan in shrimp might interact with white spot syndrome virus (WSSV). In the present study, we experimentally studied the function of syndecan in shrimp immunity. The syndecan from Litopenaeus vannamei (LvSDC) was cloned and analyzed. The full-length cDNA of LvSDC was 1005 bp, consisting of 59 bp 5'-UTR, 253 bp 3'-UTR, and 693 bp open reading frame encoding 230 amino acids. LvSDC consisted of an extracellular domain (ED), a transmembrane domain (TM) and a cytoplasmic domain (CD). TM and CD shared high similarities with those of syndecan proteins from other species. LvSDC was ubiquitously expressed in all tested tissues, with the highest level in Oka. After WSSV challenge, the transcription level of LvSDC in Oka was apparently up-regulated. Recombinant LvSDC protein and its rabbit polyclonal antibody were prepared for detecting the location of LvSDC in hemocytes using immunocytochemistry approach. Data showed that LvSDC mainly located at the cell membrane and the cytoplasm of hemocytes. After silencing of LvSDC with siRNA, the WSSV copy numbers and mortality of shrimp after WSSV infection were both significantly decreased. These data provide useful information for understanding the immune mechanism of shrimp to WSSV infection. Copyright © 2015 Elsevier Ltd. All rights reserved.

Inactivation of the budded virus of Autographa californica M nucleopolyhedrovirus by gloverin

PubMed Central

Moreno-Habel, Daniela A.; Biglang-awa, Ivan M.; Dulce, Angelica; DeeLuu, Dee; Garcia, Peter; Weers, Paul M. M.; Haas-Stapleton, Eric J.

2012-01-01

Antimicrobial peptides are generated in insects exposed to pathogens for combating infection. Gloverin is a small cationic antibacterial protein whose expression is induced in the hemocytes and fat body cells of Trichoplusia ni larvae exposed to bacteria. The purpose of this study was to determine the role of gloverin during baculovirus infection. We found that gloverin expression is induced in T. ni systemically infected with the baculovirus Autographa californica M nucleopolyhedrovirus (AcMNPV). Two gloverin genes were cloned using RNA isolated from the hemocytes of T. ni larvae that were systemically infected AcMNPV budded virus (BV) and C-terminal 6x-His and V5 epitope tags were incorporated to facilitate gloverin isolation, detection and functional studies. The supernatants of Sf9 cells stably transfected with the two gloverin expression plasmids and affinity purified gloverin proteins reduced the quantity of infectious AcMNPV BV as measured in vitro by plaque assay with untransfected Sf9 cells. Nanomolar concentrations of affinity column purified gloverin protein caused calcein to be rapidly released from unilamellar vesicles comprised of phosphatidylglycerol, but not from vesicles made up of phosphatidylcholine, suggesting that gloverin interaction with membranes is rapid and affected by membrane charge. Both the BV inactivation and calcein release activities of gloverin increased with higher concentrations of gloverin. These results demonstrate that gloverin is an antiviral protein that interacts with vesicle membranes to cause the contents to be released. PMID:22401766

Effects of several immunostimulants on phenoloxidase and hemocytes of the crab Charybdis japonica

NASA Astrophysics Data System (ADS)

Fan, Tingjun; Yu, Miaomiao; Yang, Lingling; Shi, Zhenping; Sun, Wenjie; Cong, Rishan; Yang, Xiuxia; Jiang, Guojian

2009-09-01

To investigate the stimulating effects of immunostimulants on the autogenous immunocompetence of crabs and the possible mechanisms involved, the immunostimulating effects of β-1,3-glucan, lipopolysaccharide (LPS), inactivated Vibrio harveyi and Vibrio anguillarum on phenoloxidase (PO) and hemocytes of Charybdis japonica were investigated in this study. It was found that the yields and the enzymatic activities of purified PO in C. japonica increased significantly after the crabs were treated with immunostimulants, while the unit enzymatic activities remained almost the same. After treatment with β-1,3-glucan and LPS, the amount of rough endoplasmic reticulum (RER) and the number of mitochondria in both semigranular cells and granular cells increased greatly, and the number of cytoplasmic granules decreased but with enlarged volume. However, the corresponding characteristics of hyaline cells remained almost the same. On the other hand, the number of granules in semigranular cells decreased greatly, and the number of mitochondria of hyaline cells increased greatly, after treatment with inactivated vibrios. It may be concluded that the effect of polysaccharide immunostimulants on the innate immune system of C. japonica is different from that of inactivated vibrio immunostimulants. The immunity-enhancing mechanism of polysaccharides in crab autogenous immunocompetence is probably accomplished by the increased yields of PO and total PO activities, while that of inactivated vibrios is probably accomplished by the partially increased yields of PO and total PO activities as well as the significantly improved phagocytotic abilities of semigranular cells and hyaline cells.

Sequence analysis, expression profiles and function of thioredoxin 2 and thioredoxin reductase 1 in resistance to nucleopolyhedrovirus in Helicoverpa armigera

PubMed Central

Zhang, Songdou; Li, Zhen; Nian, Xiaoge; Wu, Fengming; Shen, Zhongjian; Zhang, Boyu; Zhang, Qingwen; Liu, Xiaoxia

2015-01-01

The thioredoxin system, including NADPH, thioredoxin (Trx), and thioredoxin reductase (TrxR), plays significant roles in maintaining intracellular redox homeostasis and protecting organisms against oxidative damage. In this study, the characteristics and functions of H. armigera HaTrx2 and HaTrxR1 were identified. Sequence analysis showed that HaTrx2 and HaTrxR1 were both highly conserved and shared high sequence identity with other insect counterparts. The mRNA of HaTrx2 was expressed the highest in 5th instar 96 h and was mainly detected in heads and epidermis. The expression of HaTrxR1 was highly concentrated in 5th instar 72 h and 96 h, and higher in malpighian tube, midgut and hemocyte than other examined tissues. HaTrx2 and HaTrxR1 were markedly induced by various types of stress. HaTrx2- or HaTrxR1-knockdown increased ROS production in hemocytes and also increased the lipid damage in NPV infected H. armigera larvae. Furthermore, interference with expression of HaTrx2 or HaTrxR1 transcripts in H. armigera larvae resulted in increased sensitivity to NPV infection and shortened LT50 values. Our findings indicated that HaTrx2 and HaTrxR1 contribute to the susceptibility of H. armigera to NPV and also provided the theoretical basis for the in-depth study of insect thioredoxin system. PMID:26502992

Identification and Characterization of Pathogen-Response Genes (repat) in Spodoptera frugiperda (Lepidoptera: Noctuidae).

PubMed

Machado, Vilmar; Serrano, Jose; Galián, Jose

2016-01-01

The fall armyworm (Spodoptera frugiperda, Noctuidae, Lepidoptera) is one of the most important crop pests in the Americas, causing significant damage to maize, rice and sorghum. The mechanisms that determine its defences against pathogens are particularly relevant for the development of management and control strategies. We used an in silico approach to identify and characterize pathogen response genes (repat) present in different tissue libraries of S. fugiperda. The analyses revealed complete cDNA for nine repat genes; of these, repat15 and repat39 were found in libraries from a specific tissue--the midgut of larvae fed with xenobiotic substances. High expression levels of some genes were found in different libraries: 39 hits in repat30 in challenged hemocytes, 16 hits in repat31 in fat body, 10 hits in repat32 in fat body and 10 in challenged hemocytes, and 10 hits in repat38 in midgut of non-treated larvae and midgut of larvae fed with natural and xenobiotic substances. The genes corresponded to two ontology categories, stress response and immune response, and their phylogenetic relationships, nucleotide similarity, number of amino acid residues and molecular weights agree with what has been described for repat genes. It is noteworthy that proteins encoded by the repat genes of S. frugiperda have important defence functions in other tissues beyond midgut and that their functional categories are likely diverse, as they are related to cell envelope structure, energy metabolism, transport and binding.

ABCB1 and ABCC1-like transporters in immune system cells from sea urchins Echinometra lucunter and Echinus esculentus and oysters Crassostrea gasar and Crassostrea gigas.

PubMed

Marques-Santos, Luis Fernando; Hégaret, Hélène; Lima-Santos, Leonardo; Queiroga, Fernando Ramos; da Silva, Patricia Mirella

2017-11-01

ABC transporters activity and expression have been associated with the multixenobiotic resistance phenotype (MXR). The activity of these proteins leads to a reduction in the intracellular concentration of several xenobiotics, thus reducing their toxicity. However, little attention has been given to the expression of ABC transporters in marine invertebrates and few studies have investigated their role in immune system cells of sea urchins and shellfish bivalves. The aim of the present study was to investigate the activity of the ABC transporters ABCB1 and ABCC1 in immune system cells of sea urchins (coelomocytes) and oysters (hemocytes) from different climatic regions (Brazil and France). Sea urchins and oysters were collected at Paraíba coast; Brazil (Echinometra lucunter and Crassostrea gasar) and Rade of Brest; France (Echinus esculentus and Crassostrea gigas). Coelomocytes and hemocytes were stained with the ABC transporter substrate calcein-AM and dye accumulation analyzed under flow cytometry. Reversin 205 (ABCB1 transporter blocker) and MK571 (ABCC1 transporter blocker) were used as pharmacological tools to investigate ABC transporter activity. A different pattern of calcein accumulation was observed in coelomocytes: phagocytes > colorless spherulocytes > vibrate cells > red spherulocytes. The treatment with MK571 increased calcein fluorescence levels in coelomocytes from both species. However, reversin 205 treatment was not able to increase calcein fluorescence in E. esculentus coelomocytes. These data suggest that ABCC1-like transporter activity is present in both sea urchin species, but ABCB1-like transporter activity might only be present in E. lucunter coelomocytes. The activity of ABCC1-like transporter was observed in all cell types from both bivalve species. However, reversin 205 only increased calcein accumulation in hyalinocytes of the oyster C. gasar, suggesting the absence of ABCB1-like transporter activity in all other cell types, including hyalinocytes from the oyster C. gigas. Additionally, our results showed that C. gigas exhibited higher activity of ABCC1-like transporter in all hemocyte types than C. gasar. The present work is the first to characterize ABCB1 and ABCC1-like transporter activity in the immune system cells of sea urchins E. lucunter and E. esculentus and oysters. Our findings encourage the performing studies regarding ABC transporters activity/expression in immune system cells form marine invertebrates under stress conditions and the possible use of ABC transporters as biomarkers. Copyright © 2017 Elsevier Ltd. All rights reserved.

Interactions of cationic polystyrene nanoparticles with marine bivalve hemocytes in a physiological environment: Role of soluble hemolymph proteins.

PubMed

Canesi, Laura; Ciacci, Caterina; Fabbri, Rita; Balbi, Teresa; Salis, Annalisa; Damonte, Gianluca; Cortese, Katia; Caratto, Valentina; Monopoli, Marco P; Dawson, Kenneth; Bergami, Elisa; Corsi, Ilaria

2016-10-01

The bivalve Mytilus galloprovincialis has proven as a suitable model invertebrate for evaluating the potential impact of nanoparticles (NPs) in the marine environment. In particular, in mussels, the immune system represents a sensitive target for different types of NPs. In environmental conditions, both NP intrinsic properties and those of the receiving medium will affect particle behavior and consequent bioavailability/uptake/toxicity. However, the evaluation of the biological effects of NPs requires additional understanding of how, once within the organism, NPs interact at the molecular level with cells in a physiological environment. In mammalian systems, different NPs associate with serum soluble components, organized into a "protein corona", which affects particle interactions with target cells. However, no information is available so far on the interactions of NPs with biological fluids of aquatic organisms. In this work, the influence of hemolymph serum (HS) on the in vitro effects of amino modified polystyrene NPs (PS-NH2) on Mytilus hemocytes was investigated. Hemocytes were incubated with PS-NH2 suspensions in HS (1, 5 and 50µg/mL) and the results were compared with those obtained in ASW medium. Cell functional parameters (lysosomal membrane stability, oxyradical production, phagocytosis) were evaluated, and morphological changes were investigated by TEM. The activation state of the signalling components involved in Mytilus immune response (p38 MAPK and PKC) was determined. The results show that in the presence of HS, PS-NH2 increased cellular damage and ROS production with respect to ASW medium. The effects were apparently mediated by disregulation of p38 MAPK signalling. The formation of a PS-NH2-protein corona in HS was investigated by centrifugation, and 1D- gel electrophoresis and nano-HPLC-ESI-MS/MS. The results identified the Putative C1q domain containing protein (MgC1q6) as the only component of the PS-NH2 hard protein corona in Mytilus hemolymph. These data represent the first evidence for the formation of a NP bio-corona in aquatic organisms and underline the importance of the recognizable biological identity of NPs in physiological exposure medium when testing their potential impact environmental model organisms. Although the results obtained in vitro do not entirely reflect a realistic exposure scenario and the more complex formation of a bio-corona that is likely to occur in vivo, these data will contribute to a better understanding of the effects of NPs in marine invertebrates. Copyright © 2016 Elsevier Inc. All rights reserved.

Accumulation and Effects of Organotin Compounds in Oysters and Mussels: Correlation with Serum Biochemical and Cytological Factors and Tissue Burdens

DTIC Science & Technology

1988-01-01

metallothioneins in the serum hemocytes of either bivalve. Responses by these animals to fatal or near fatal doses of TBT were thus very different from responses...Diego, CA 92110. USA N " IL -_ Dvsters and mussels exposed to a concentration of OTppb (g/liter) tributyltin from painted panels in fwin-g secivater accuu...doses of TBT were thus very different from responses to copper that we have reported elsewhere. " 2 We have been studying the cellular and biochemical

Sepsis and Hemocyte Loss in Honey Bees (Apis mellifera) Infected with Serratia marcescens Strain Sicaria.

PubMed

Burritt, Nancy L; Foss, Nicole J; Neeno-Eckwall, Eric C; Church, James O; Hilger, Anna M; Hildebrand, Jacob A; Warshauer, David M; Perna, Nicole T; Burritt, James B

2016-01-01

Global loss of honey bee colonies is threatening the human food supply. Diverse pathogens reduce honey bee hardiness needed to sustain colonies, especially in winter. We isolated a free-living Gram negative bacillus from hemolymph of worker honey bees (Apis mellifera) found separated from winter clusters. In some hives, greater than 90% of the dying bees detached from the winter cluster were found to contain this bacterium in their hemolymph. Throughout the year, the same organism was rarely found in bees engaged in normal hive activities, but was detected in about half of Varroa destructor mites obtained from colonies that housed the septic bees. Flow cytometry of hemolymph from septic bees showed a significant reduction of plasmatocytes and other types of hemocytes. Interpretation of the16S rRNA sequence of the bacterium indicated that it belongs to the Serratia genus of Gram-negative Gammaproteobacteria, which has not previously been implicated as a pathogen of adult honey bees. Complete genome sequence analysis of the bacterium supported its classification as a novel strain of Serratia marcescens, which was designated as S. marcescens strain sicaria (Ss1). When compared with other strains of S. marcescens, Ss1 demonstrated several phenotypic and genetic differences, including 65 genes not previously found in other Serratia genomes. Some of the unique genes we identified in Ss1 were related to those from bacterial insect pathogens and commensals. Recovery of this organism extends a complex pathosphere of agents which may contribute to failure of honey bee colonies.

Cloning and localization of MCdef, a defensin from Manila clams (Ruditapes philippinarum).

PubMed

Adhya, Mausumi; Jeung, Hee-Do; Kang, Hyun-Sil; Choi, Kwang-Sik; Lee, Dong Sun; Cho, Moonjae

2012-01-01

A defensin-like peptide was previously detected in hemocytes of Manila clams (Ruditapes philippinarum). In the current study, we cloned and characterized this defensin, designated MCdef. Cloning produced a full-length gene sequence of 201 bp predicted to encode a 66-amino-acid precursor protein maturing to a 44-amino-acid residue. Amino acid sequence analysis showed that MCdef is similar to defensins from marine mollusks and ticks. Phylogenetic analysis suggested that MCdef is closely related to defensins from Mytilus galloprovincialis (Mediterranean mussel) and Crassostrea gigas (Pacific cupped oyster). The three-dimensional structure of MCdef was modeled using the solution structure of C. gigas defensin as a template. With the exception of three variable loop areas, the modeled structure of MCdef was identical to that of C. gigas defensin. MCdef antiserum was raised against a synthetic MCdef peptide and verified by Western blotting using recombinant MCdef. RT-PCR analysis demonstrated high levels of MCdef mRNA in hemocytes and adductor, foot, gill, mantle, palp, and siphon tissues of Vibrio tapetis-infected Manila clams, whereas in V. tapetis-uninfected Manila clams, the level of MCdef mRNA was low in adductor, palp, and siphon tissues and even lower in the other tested tissues. Immunohistochemical analysis revealed high MCdef expression was detected in the gill, the mantle, and the digestive tubules of the diverticulum of V. tapetis-infected Manila clams. Minimum inhibitory concentration (MIC) of the purified rMCdef was determined. MCdef showed highest activity against Streptococcus iniae and Staphylococcus aureus. Copyright © 2011 Elsevier Inc. All rights reserved.

Drosophila E-Cadherin Functions in Hematopoietic Progenitors to Maintain Multipotency and Block Differentiation

PubMed Central

Gao, Hongjuan; Wu, Xiaorong; Fossett, Nancy

2013-01-01

A fundamental question in stem cell biology concerns the regulatory strategies that control the choice between multipotency and differentiation. Drosophila blood progenitors or prohemocytes exhibit key stem cell characteristics, including multipotency, quiescence, and niche dependence. As a result, studies of Drosophila hematopoiesis have provided important insights into the molecular mechanisms that control these processes. Here, we show that E-cadherin is an important regulator of prohemocyte fate choice, maintaining prohemocyte multipotency and blocking differentiation. These functions are reminiscent of the role of E-cadherin in mammalian embryonic stem cells. We also show that mis-expression of E-cadherin in differentiating hemocytes disrupts the boundary between these cells and undifferentiated prohemocytes. Additionally, upregulation of E-cadherin in differentiating hemocytes increases the number of intermediate cell types expressing the prohemocyte marker, Patched. Furthermore, our studies indicate that the Drosophila GATA transcriptional co-factor, U-shaped, is required for E-cadherin expression. Consequently, E-cadherin is a downstream target of U-shaped in the maintenance of prohemocyte multipotency. In contrast, we showed that forced expression of the U-shaped GATA-binding partner, Serpent, repressed E-cadherin expression and promoted lamellocyte differentiation. Thus, U-shaped may maintain E-cadherin expression by blocking the inhibitory activity of Serpent. Collectively, these observations suggest that GATA:FOG complex formation regulates E-cadherin levels and, thereby, the choice between multipotency and differentiation. The work presented in this report further defines the molecular basis of prohemocyte cell fate choice, which will provide important insights into the mechanisms that govern stem cell biology. PMID:24040319

Identification of a Serine Proteinase Homolog (Sp-SPH) Involved in Immune Defense in the Mud Crab Scylla paramamosain

PubMed Central

Zhang, Qiu-xia; Liu, Hai-peng; Chen, Rong-yuan; Shen, Kai-li; Wang, Ke-jian

2013-01-01

Clip domain serine proteinase homologs are involved in many biological processes including immune response. To identify the immune function of a serine proteinase homolog (Sp-SPH), originally isolated from hemocytes of the mud crab, Scylla paramamosain, the Sp-SPH was expressed recombinantly and purified for further studies. It was found that the Sp-SPH protein could bind to a number of bacteria (including Aeromonas hydrophila, Escherichia coli, Staphylococcus aureus, Vibrio fluvialis, Vibrio harveyi and Vibrio parahemolyticus), bacterial cell wall components such as lipopolysaccharide or peptidoglycan (PGN), and β-1, 3-glucan of fungus. But no direct antibacterial activity of Sp-SPH protein was shown by using minimum inhibitory concentration or minimum bactericidal concentration assays. Nevertheless, the Sp-SPH protein was found to significantly enhance the crab hemocyte adhesion activity (paired t-test, P<0.05), and increase phenoloxidase activity if triggered by PGN in vitro (paired t-test, P<0.05). Importantly, the Sp-SPH protein was demonstrated to promote the survival rate of the animals after challenge with A. hydrophila or V. parahemolyticus which were both recognized by Sp-SPH protein, if pre-incubated with Sp-SPH protein, respectively. Whereas, the crabs died much faster when challenged with Vibrio alginolyiicus, a pathogenic bacterium not recognized by Sp-SPH protein, compared to those of crabs challenged with A. hydrophila or V. parahemolyticus when pre-coated with Sp-SPH protein. Taken together, these data suggested that Sp-SPH molecule might play an important role in immune defense against bacterial infection in the mud crab S. paramamosain. PMID:23724001

Identification of a serine proteinase homolog (Sp-SPH) involved in immune defense in the mud crab Scylla paramamosain.

PubMed

Zhang, Qiu-xia; Liu, Hai-peng; Chen, Rong-yuan; Shen, Kai-li; Wang, Ke-jian

2013-01-01

Clip domain serine proteinase homologs are involved in many biological processes including immune response. To identify the immune function of a serine proteinase homolog (Sp-SPH), originally isolated from hemocytes of the mud crab, Scylla paramamosain, the Sp-SPH was expressed recombinantly and purified for further studies. It was found that the Sp-SPH protein could bind to a number of bacteria (including Aeromonas hydrophila, Escherichia coli, Staphylococcus aureus, Vibrio fluvialis, Vibrio harveyi and Vibrio parahemolyticus), bacterial cell wall components such as lipopolysaccharide or peptidoglycan (PGN), and β-1, 3-glucan of fungus. But no direct antibacterial activity of Sp-SPH protein was shown by using minimum inhibitory concentration or minimum bactericidal concentration assays. Nevertheless, the Sp-SPH protein was found to significantly enhance the crab hemocyte adhesion activity (paired t-test, P<0.05), and increase phenoloxidase activity if triggered by PGN in vitro (paired t-test, P<0.05). Importantly, the Sp-SPH protein was demonstrated to promote the survival rate of the animals after challenge with A. hydrophila or V. parahemolyticus which were both recognized by Sp-SPH protein, if pre-incubated with Sp-SPH protein, respectively. Whereas, the crabs died much faster when challenged with Vibrio alginolyiicus, a pathogenic bacterium not recognized by Sp-SPH protein, compared to those of crabs challenged with A. hydrophila or V. parahemolyticus when pre-coated with Sp-SPH protein. Taken together, these data suggested that Sp-SPH molecule might play an important role in immune defense against bacterial infection in the mud crab S. paramamosain.

Identification and characterization of the related immune-enhancing proteins in crab Scylla paramamosain stimulated with rhubarb polysaccharides.

PubMed

Cao, Jingsong; Wang, Zehuan; Zhang, Yueling; Qu, Fengliang; Guo, Lingling; Zhong, Mingqi; Li, Shengkang; Zou, Haiying; Chen, Jiehui; Wang, Xiuying

2014-02-01

Recently, considerable interest has been focused on immunostimulants to reduce diseases in crab aquaculture. However, information regarding to the related immune-enhancing proteins in crabs is not available yet. In this study, rhubarb polysaccharides were tested for enhancement of the immune activity in crab Scylla paramamosain. Compared with those in the control group, values of, phenoloxidase (PO), alkaline phosphatase (AKP) and alkaline phosphatasein (ACP) activity in the, experimental group were improved significantly 4 d after the treatment. Furthermore, 15 and 17 altered proteins from haemocytes and hepatopancreas, respectively, were found in rhubarb polysaccharide-treated crabs using 2-DE approach. Of these, hemocyanin, chymotrypsin, cryptocyanin, C-type lectin receptor, and ferritin protein were identified by mass spectrometry. In addition, RT-PCR, analysis showed that the mRNA levels of hemocyanin and chymotrypsin increased about 2.4- and 1.4-fold in the experiment group. Moreover, the hemocyanin gene in S. paramamosain (SpHMC) was, cloned and characterized. SpHMC contains one open reading frame of 2022 bp and encodes a polypeptide of 673 amino acids. It is clustered into one branch along with crab hemocyanin in a phylogenetic tree. The mRNA transcripts of SpHMC were detected mainly in the tissues of, hepatopancreas, hemocyte and intestines, and its levels were up-regulated significantly in hemocytes, of S. paramamosain treated with Vibrio parahemolyticus, Beta streptococcus or poly I:C for 6-48 h. Taken together, these studies found 5 related immune-enhancing proteins and a novel heomcyanin homologue with potential pathogen-resistant activities in crab. Copyright © 2013 Elsevier Ltd. All rights reserved.

Galleria mellonella larvae are capable of sensing the extent of priming agent and mounting proportionatal cellular and humoral immune responses.

PubMed

Wu, Gongqing; Xu, Li; Yi, Yunhong

2016-06-01

Larvae of Galleria mellonella are useful models for studying the innate immunity of invertebrates or for evaluating the virulence of microbial pathogens. In this work, we demonstrated that prior exposure of G. mellonella larvae to high doses (1×10(4), 1×10(5) or 1×10(6) cells/larva) of heat-killed Photorhabdus luminescens TT01 increases the resistance of larvae to a lethal dose (50 cells/larva) of viable P. luminescens TT01 infection administered 48h later. We also found that the changes in immune protection level were highly correlated to the changes in levels of cellular and humoral immune parameters when priming the larvae with different doses of heat-killed P. luminescens TT01. Priming the larvae with high doses of heat-killed P. luminescens TT01 resulted in significant increases in the hemocytes activities of phagocytosis and encapsulation. High doses of heat-killed P. luminescens TT01 also induced an increase in total hemocyte count and a reduction in bacterial density within the larval hemocoel. Quantitative real-time PCR analysis showed that genes coding for cecropin and gallerimycin and galiomycin increased in expression after priming G. mellonella with heat-killed P. luminescens TT01. All the immune parameters changed in a dose-dependent manner. These results indicate that the insect immune system is capable of sensing the extent of priming agent and mounting a proportionate immune response. Copyright © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Sepsis and Hemocyte Loss in Honey Bees (Apis mellifera) Infected with Serratia marcescens Strain Sicaria

PubMed Central

Burritt, Nancy L.; Foss, Nicole J.; Neeno-Eckwall, Eric C.; Church, James O.; Hildebrand, Jacob A.; Warshauer, David M.; Perna, Nicole T.; Burritt, James B.

2016-01-01

Global loss of honey bee colonies is threatening the human food supply. Diverse pathogens reduce honey bee hardiness needed to sustain colonies, especially in winter. We isolated a free-living Gram negative bacillus from hemolymph of worker honey bees (Apis mellifera) found separated from winter clusters. In some hives, greater than 90% of the dying bees detached from the winter cluster were found to contain this bacterium in their hemolymph. Throughout the year, the same organism was rarely found in bees engaged in normal hive activities, but was detected in about half of Varroa destructor mites obtained from colonies that housed the septic bees. Flow cytometry of hemolymph from septic bees showed a significant reduction of plasmatocytes and other types of hemocytes. Interpretation of the16S rRNA sequence of the bacterium indicated that it belongs to the Serratia genus of Gram-negative Gammaproteobacteria, which has not previously been implicated as a pathogen of adult honey bees. Complete genome sequence analysis of the bacterium supported its classification as a novel strain of Serratia marcescens, which was designated as S. marcescens strain sicaria (Ss1). When compared with other strains of S. marcescens, Ss1 demonstrated several phenotypic and genetic differences, including 65 genes not previously found in other Serratia genomes. Some of the unique genes we identified in Ss1 were related to those from bacterial insect pathogens and commensals. Recovery of this organism extends a complex pathosphere of agents which may contribute to failure of honey bee colonies. PMID:28002470

Expression of matrix metalloproteinase genes during basement membrane degradation in the metamorphosis of Bombyx mori.

PubMed

Kawasaki, Hideki; Manickam, Asaithambi; Shahin, Rima; Ote, Manabu; Iwanaga, Masashi

2018-01-05

The present study was conducted to clarify the involvement of the basement membrane (BM) in insect metamorphosis through analysis of the expression profile of two types of metalloproteinase (MMP and ADAMTS) genes in several organs, their ecdysone involvement, and the histological change of BM. BM was observed around wing sac and in the wing cavity and around fat bodies at the W0 stage but disappeared after the W3 stage, and wing discs evaginated and fat body cells scattered after the W3 stage. The disappearance of the BM of midgut and silk glands was not observed after the W3 stage, but degenerated epithelium cells in the midgut and shrunken cells in the silk gland were observed after the W3 stage. BmMMP1 showed a peak at P0 in the wing discs, fat bodies, midgut, and silk gland. BmMMP2 showed a broad peak around pupation in the wing discs, fat bodies, midgut, and silk gland. BmADAMTS-1 showed enhanced expression at W2 in the wing discs, fat bodies, midgut, and hemocyte, while BmADAMTS-L showed enhanced expression at W3 in the fat bodies, midgut, silk gland, and hemocyte. After pupation, they showed a different expression in different organs. All of four genes were induced by 20-hydroxyecdysone in wing discs in vitro. The present results suggested the involvement of MMPs and ADAMTS in the BM digestion and the morphogenesis of organs during Bombyx metamorphosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Short-term heat shock affects the course of immune response in Galleria mellonella naturally infected with the entomopathogenic fungus Beauveria bassiana.

PubMed

Vertyporokh, Lidiia; Taszłow, Paulina; Samorek-Pieróg, Małgorzata; Wojda, Iwona

2015-09-01

We aimed to investigate how exposition of infected insects to short-term heat shock affects the biochemical and molecular aspects of their immune response. Galleria mellonella larvae were exposed to 43°C for 15min, at the seventy second hour after natural infection with entomopathogenic fungus Beauveria bassiana. As a result, both qualitative and quantitative changes in hemolymph protein profiles, and among them infection-induced changes in the amount of apolipophorin III (apoLp-III), were observed. Heat shock differently affects the expression of the tested immune-related genes. It transiently inhibits expression of antifungal peptides gallerimycin and galiomicin in both the fat body and hemocytes of infected larvae. The same, although to a lesser extent, concerned apoLp-III gene expression and was observed directly after heat shock. Nevertheless, in larvae that had recovered from heat shock, apoLp-III expression was higher in comparison to unshocked larvae in the fat body but not in hemocytes, which was consistent with the higher amount of this protein detected in the hemolymph of the infected, shocked larvae. Furthermore, lysozyme-type activity was higher directly after heat shock, while antifungal activity was significantly higher also in larvae that had recovered from heat shock, in comparison to the respective values in their non-shocked, infected counterparts. These results show how changes in the external temperature modulate the immune response of G. mellonella suffering from infection with its natural pathogen B. bassiana. Copyright © 2015 Elsevier Inc. All rights reserved.

Influence of Salmonella enterica Serovar Typhimurium ssrB on Colonization of Eastern Oysters (Crassostrea virginica) as Revealed by a Promoter Probe Screen

PubMed Central

Cox, Clayton E.; Wright, Anita C.; McClelland, Michael

2015-01-01

Although Salmonella has been isolated from 7.4 to 8.6% of domestic raw oysters, representing a significant risk for food-borne illness, little is known about the factors that influence their initial colonization by Salmonella. This study tested the hypothesis that specific regulatory changes enable a portion of the invading Salmonella population to colonize oysters. An in vivo promoter probe library screen identified 19 unique regions as regulated during colonization. The mutants in the nearest corresponding downstream genes were tested for colonization defects in oysters. Only one mutation, in ssrB, resulted in a significantly reduced ability to colonize oysters compared to that of wild-type Salmonella. Because ssrB regulates Salmonella pathogenicity island 2 (SPI-2)-dependent infections in vertebrate macrophages, the possibility that ssrB mediated colonization of oyster hemocytes in a similar manner was examined. However, no difference in hemocyte colonization was observed. The complementary hypothesis that signal exchange between Salmonella and the oyster's native microbial community aids colonization was also tested. Signals that triggered responses in quorum sensing (QS) reporters were shown to be produced by oyster-associated bacteria and present in oyster tissue. However, no evidence for signal exchange was observed in vivo. The sdiA reporter responded to salinity, suggesting that SdiA may also have a role in environmental sensing. Overall, this study suggests the initial colonization of live oysters by Salmonella is controlled by a limited number of regulators, including ssrB. PMID:26497459

The first invertebrate NFIL3 transcription factor with role in immune defense identified from the Hong Kong oyster, Crassostrea hongkongensis.

PubMed

Li, Jun; Zhang, Yang; Zhang, Yuehuan; Mao, Fan; Xiang, Zhiming; Xiao, Shu; Ma, Haitao; Yu, Ziniu

2017-11-01

NFIL3 (nuclear factor interleukin 3-regulated) is a basic leucine zipper type transcription factor that mediates a variety of immune responses in vertebrates. However, the sequence information and function of NFIL3 homologs in invertebrates, especially mollusks, remains unknown. In the present study, the first NFIL3 homolog was identified in a marine mollusk, Crassostrea hongkongensis (designated as ChNFIL3), followed by its functional characterization. The full-length cDNA of ChNFIL3 is 2221 bp and consists of an open reading frame (ORF) of 1536 bp that encodes a polypeptide of 551 amino acids. Simple Modular Architecture Research Tool (SMART) analysis indicated that ChNFIL3 has two basic leucin zipper domains, similar to the other known NFIL3 family proteins. Tissue distribution analysis of NFIL3 in this mollusk revealed high expression in digestive glands and hemocytes. A significant induction in the mRNA level of ChNFIL3 was observed following bacterial stimulation. ChNFIL3 was found to be localized in the nucleus and over expression of ChNIFL3 led to upregulation of transcriptional activity of an NF-κB reporter gene in HEK 293T cells, indicating its role in innate immunity. Furthermore, addition of exogenous recombinant ChNFIL3 proteins resulted in enhanced mRNA level of hemocyte interleukin 17 in vitro. In conclusion, our findings revealed that NFIL3 in molluscs, plays a conserved role in host defense, similar to its mammalian homolog. Copyright © 2017 Elsevier Ltd. All rights reserved.

Pattern Recognition Protein Binds to Lipopolysaccharide and β-1,3-Glucan and Activates Shrimp Prophenoloxidase System*

PubMed Central

Amparyup, Piti; Sutthangkul, Jantiwan; Charoensapsri, Walaiporn; Tassanakajon, Anchalee

2012-01-01

The prophenoloxidase (proPO) system is activated upon recognition of pathogens by pattern recognition proteins (PRPs), including a lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP). However, shrimp LGBPs that are involved in the proPO system have yet to be clarified. Here, we focus on characterizing the role of a Penaeus monodon LGBP (PmLGBP) in the proPO system. We found that PmLGBP transcripts are expressed primarily in the hemocytes and are increased at 24 h after pathogenic bacterium Vibrio harveyi challenge. The binding studies carried out using ELISA indicated that recombinant (r)PmLGBP binds to β-1,3-glucan and LPS with a dissociation constant of 6.86 × 10−7 m and 3.55 × 10−7 m, respectively. Furthermore, we found that rPmLGBP could enhance the phenoloxidase (PO) activity of hemocyte suspensions in the presence of LPS or β-1,3-glucan. Using dsRNA interference-mediated gene silencing assay, we further demonstrated that knockdown of PmLGBP in shrimp in vivo significantly decreased the PmLGBP transcript level but had no effect on the expression of the other immune genes tested, including shrimp antimicrobial peptides (AMPs). However, suppression of proPO expression down-regulated PmLGBP, proPO-activating enzyme (PmPPAE2), and AMPs (penaeidin and crustin). Such PmLGBP down-regulated shrimp showed significantly decreased total PO activity. We conclude that PmLGBP functions as a pattern recognition protein for LPS and β-1,3-glucan in the shrimp proPO activating system. PMID:22235126

New polymorphic microsatellite markers derived from hemocyte cDNA library of Manila clam Ruditapes philippinarum challenged by the protozoan parasite Perkinsus olseni

NASA Astrophysics Data System (ADS)

Kang, Hyun-Sil; Hong, Hyun-Ki; Park, Kyung-Il; Cho, Moonjae; Youn, Seok-Hyun; Choi, Kwang-Sik

2017-03-01

Manila clam Ruditapes philippinarum is one of the most important benthic animals in the coastal north Pacific region, where clam populations have been mixed genetically through trade and aquaculture activities. Accordingly, identification of the genetically different clam populations has become one of the most important issues to manage interbreeding of the local and introduced clam populations. To identify genetically different populations of clam populations, we developed 11 expressed sequence tag (EST)-microsatellite loci (i.e., simple sequence repeat, SSR) from 1,128 clam hemocyte cDNA clones challenged by the protozoan parasite Perkinsus olseni. Genotype analysis using the markers developed in this study demonstrated that clams from a tidal flat on the west coast contained 6 to 19 alleles per locus, and a population from Jeju Island had 4 to 20 alleles per locus. The expected heterozygosity of the 2 clam populations ranged from 0.472 to 0.919 for clams from the west coast, and 0.494 to 0.919 for clams from Jeju Island, respectively. Among the 11 loci discovered in this study, 7 loci significantly deviated from the Hardy-Weinberg equilibrium after Bonferroni correction. The 5 loci developed in this study also successfully amplified the SSRs of R. variegatus, a clam species taxonomically very close to R. philippinarum, from Hong Kong and Jeju Island. We believe that the 11 novel polymorphic SSR developed in this study can be utilized successfully in Manila clam genetic diversity analysis, as well as in genetic discrimination of different clam populations.

Phenoloxidase activity among developmental stages and pupal cell types of the ground beetle Carabus (Chaetocarabus) lefebvrei (Coleoptera, Carabidae).

PubMed

Giglio, Anita; Giulianini, Piero Giulio

2013-04-01

In ecological immunology is of great importance the study of the immune defense plasticity as response to a variable environment. In holometabolous insects the fitness of each developmental stage depends on the capacity to mount a response (i.e. physiological, behavioral) under environmental pressure. The immune response is a highly dynamic trait closely related to the ecology of organism and the variation in the expression of an immune system component may affect another fitness relevant trait of organism (i.e. growth, reproduction). The present research quantified immune function (total and differential number of hemocytes, phagocytosis in vivo and activity of phenoloxidase) in the pupal stage of Carabus (Chaetocarabus) lefebvrei. Moreover, the cellular and humoral immune function was compared across the larval, pupal and adult stages to evaluate the changes in immunocompetence across the developmental stages. Four types of circulating hemocytes were characterized via transmission electron microscopy in the pupal stage: prohemocytes, plasmatocytes, granulocytes and oenocytoids. The artificial non-self-challenge treatments performed in vivo have shown that plasmatocytes and granulocytes are responsible for phagocytosis. The level of active phenoloxidase increases with the degree of pigmentation of the cuticle in each stage. In C. lefebvrei, there are different strategies in term of immune response to enhance the fitness of each life stage. The results have shown that the variation in speed and specificity of immune function across the developmental stages is correlated with differences in infection risk, life expectancy and biological function of the life cycle. Copyright © 2013 Elsevier Ltd. All rights reserved.

Ulcerative enteritis in Homarus americanus: case report and molecular characterization of intestinal aerobic bacteria of apparently healthy lobsters in live storage.

PubMed

Battison, Andrea L; Després, Béatrice M; Greenwood, Spencer J

2008-10-01

An intermoult male American lobster, Homarus americanus, with severe intestinal lesions was encountered while collecting samples of aerobic intestinal bacteria from lobsters held in an artificial sea-water recirculation aquarium system. Grossly, the intestine was firm, thickened, and white. Histologic examination revealed a severe, diffuse, ulcerative enteritis which spared the chitin-lined colon, somewhat similar to hemocytic enteritis of shrimp. The bacterial isolates from this lobster were compared to 11 other lobsters lacking gross intestinal lesions. Two organisms, one identified as Vibrio sp. and another most similar to an uncultured proteobacterium (98.9%), clustering with Rhanella and Serratia species using 16S rDNA PCR, were isolated from the intestines of the 11, grossly normal, lobsters and the affected lobster. An additional two intestinal isolates were cultured only from the lobster with ulcerative enteritis. One, a Flavobacterium, similar to Lutibacter litoralis (99.3%), possibly represented a previously described commensal of the distal intestine. The second, a Vibrio sp., was unique to the affected animal. While the etiology of the ulcerative enteritis remains undetermined, this report represents the first description of gross and histologic findings in H. americanus of a condition which has morphologic similarities to hemocytic enteritis of shrimp. An additional observation was a decrease in the number of intestinal isolates recovered from the 11 apparently healthy lobsters compared to that previously reported for recently harvested lobster. More comprehensive studies of the relationship between the health of lobsters, gut microbial flora and the husbandry and environment maintained within holding units are warranted.

Dietary nano-selenium relieves hypoxia stress and, improves immunity and disease resistance in the Chinese mitten crab (Eriocheir sinensis).

PubMed

Qin, Fenju; Shi, Miaomiao; Yuan, Hongxia; Yuan, Linxi; Lu, Wenhao; Zhang, Jie; Tong, Jian; Song, Xuehong

2016-07-01

Hypoxia is a relevant physiological challenge for crab culture, and the hemolymph plays a crucial role in response to the hypoxia. In a 60 d feeding trial, Chinese mitten crabs (Eriocheir sinensis) fed a diet containing 0.2 mg/kg nano-selenium (nanoSe) showed a significantly increased weight gain rate (WGR) and a reduced feed coefficient (FC) compared to those fed diets with 0, 0.1, 0.4, 0.8, and 1.6 mg/kg nanoSe. Another 90 d feeding trial was conducted to determine the influence of dietary nanoSe on the immune response in juvenile Chinese mitten crabs kept under the condition of hypoxia. The results showed that hypoxia stress resulted in significantly increased hemocyte counts (THC, LGC, SGC, and HC), expression levels of the hemocyanin gene and protein, lactic acid level, and antioxidant capacity (T-AOC activities, SOD activities, GSH-Px and GSH content) in hemolymph supernatant. When these crabs were infected with Aeromonas hydrophila bacteria, hypoxia exposure increased mortality, but it was alleviated by a diet supplemented with 0.2 mg/kg nanoSe. The up-regulative effects of nanoSe (0.2 mg/kg) on antioxidant capacity, hemocyte counts, and hemocyanin expression under hypoxia exposure were further strengthened throughout, whereas lactic acid levels induced by hypoxia stress were restored. Thus, the observations in this study indicate that the level of dietary nanoSe is important in regulating immunity and disease resistance in crabs kept under hypoxia stress. Copyright © 2016 Elsevier Ltd. All rights reserved.

Size distribution effects of cadmium tellurium quantum dots (CdS/CdTe) immunotoxicity on aquatic organisms.

PubMed

Bruneau, A; Fortier, M; Gagne, F; Gagnon, C; Turcotte, P; Tayabali, A; Davis, T L; Auffret, M; Fournier, M

2013-03-01

The increasing use of products derived from nanotechnology has raised concern about their potential toxicity to aquatic life. This study sought to examine the comparative immunotoxicity of capped cadmium sulphide/cadmium telluride (CdS/CdTe) quantum dots (QDs) and possible impact of particle/aggregate size on two bivalves (Mytilus edulis and Elliptio complanata) and a fish (Oncorhynchus mykiss). The QDs were dispersed in sterile water and fractionated using a series of micro/ultrafiltration membranes of decreasing pore size: 450 nm, 100 nm, 50 nm, 25 nm, 100 kDa (6.8 nm), 30 kDa (4.6 nm), 10 kDa (3.2 nm) and 1 kDa (1.5 nm). The total concentrations of cadmium and tellurium were determined for the filtered material and for that retained on the filters (retentate). The immunotoxicity was determined by measuring cell viability and phagocytosis. Results revealed that nanoparticles retained on the ultrafilters had a higher Cd/Te ratio compared to the permeate fraction (ratio of 5 and 2 respectively) which could indicate that the CdS core was not associated with the permeable fraction of Cd. Our results demonstrate that the toxicity of CdS/CdTe QDs was concentration and size dependent. Large CdS/CdTe QD aggregates (25 nm < size < 100 nm) reduced phagocytosis more than did smaller nanoparticles (<25 nm). Moreover, our results revealed that the different species responded differently to these fractions. Mytilus edulis hemocytes were less sensitive to CdS/CdTe QDs than the Oncorhynchus mykiss macrophage and Elliptio complanata hemocytes.

Relative Roles of the Cellular and Humoral Responses in the Drosophila Host Defense against Three Gram-Positive Bacterial Infections

PubMed Central

Cho, Ju Hyun; Lee, Janice; Lafarge, Marie-Céline; Kocks, Christine; Ferrandon, Dominique

2011-01-01

Background Two NF-kappaB signaling pathways, Toll and immune deficiency (imd), are required for survival to bacterial infections in Drosophila. In response to septic injury, these pathways mediate rapid transcriptional activation of distinct sets of effector molecules, including antimicrobial peptides, which are important components of a humoral defense response. However, it is less clear to what extent macrophage-like hemocytes contribute to host defense. Methodology/Principal Findings In order to dissect the relative importance of humoral and cellular defenses after septic injury with three different Gram-positive bacteria (Micrococcus luteus, Enterococcus faecalis, Staphylococcus aureus), we used latex bead pre-injection to ablate macrophage function in flies wildtype or mutant for various Toll and imd pathway components. We found that in all three infection models a compromised phagocytic system impaired fly survival – independently of concomitant Toll or imd pathway activation. Our data failed to confirm a role of the PGRP-SA and GNBP1 Pattern Recognition Receptors for phagocytosis of S. aureus. The Drosophila scavenger receptor Eater mediates the phagocytosis by hemocytes or S2 cells of E. faecalis and S. aureus, but not of M. luteus. In the case of M. luteus and E. faecalis, but not S. aureus, decreased survival due to defective phagocytosis could be compensated for by genetically enhancing the humoral immune response. Conclusions/Significance Our results underscore the fundamental importance of both cellular and humoral mechanisms in Drosophila immunity and shed light on the balance between these two arms of host defense depending on the invading pathogen. PMID:21390224

New Functions of Arthropod Bursicon: Inducing Deposition and Thickening of New Cuticle and Hemocyte Granulation in the Blue Crab, Callinectes sapidus

PubMed Central

Chung, J. Sook; Katayama, Hidekazu; Dircksen, Heinrich

2012-01-01

Arthropod growth requires molt-associated changes in softness and stiffness of the cuticle that protects from desiccation, infection and injury. Cuticle hardening in insects depends on the blood-borne hormone, bursicon (Burs), although it has never been determined in hemolymph. Whilst also having Burs, decapod crustaceans reiterate molting many more times during their longer life span and are encased in a calcified exoskeleton, which after molting undergoes similar initial cuticle hardening processes as in insects. We investigated the role of homologous crustacean Burs in cuticular changes and growth in the blue crab, Callinectes sapidus. We found dramatic increases in size and number of Burs cells during development in paired thoracic ganglion complex (TGC) neurons with pericardial organs (POs) as neurohemal release sites. A skewed expression of Burs β/Burs α mRNA in TGC corresponds to protein contents of identified Burs β homodimer and Burs heterodimer in POs. In hemolymph, Burs is consistently present at ∼21 pM throughout the molt cycle, showing a peak of ∼89 pM at ecdysis. Since initial cuticle hardness determines the degree of molt-associated somatic increment (MSI), we applied recombinant Burs in vitro to cuticle explants of late premolt or early ecdysis. Burs stimulates cuticle thickening and granulation of hemocytes. These findings demonstrate novel cuticle-associated functions of Burs during molting, while the unambiguous and constant presence of Burs in cells and hemolymph throughout the molt cycle and life stages may implicate further functions of its homo- and heterodimer hormone isoforms in immunoprotective defense systems of arthropods. PMID:23029467

Insights into the toxicity of iron oxides nanoparticles in land snails.

PubMed

Sidiropoulou, Eirini; Feidantsis, Konstantinos; Kalogiannis, Stavros; Gallios, George P; Kastrinaki, Georgia; Papaioannou, Eleni; Václavíková, Miroslava; Kaloyianni, Martha

2018-04-01

The use of manufactured nanoparticles (NPs) is spreading rapidly across technology and medicine fields, posing concerns about their consequence on ecosystems and human health. The present study aims to assess the biological responses triggered by iron oxide NPs (IONPs) and iron oxide NPs incorporated into zeolite (IONPZ) in relation to oxidative stress on the land snail Helix aspersa in order to investigate its use as a biomarker for terrestrial environments. Morphology and structure of both NPs were characterized. Snail food was supplemented with a range of concentrations of IONPs and IONPZ and values of the hemocyte lysosomal membranes' destabilization by 50% were estimated by the neutral red retention (NRRT50) assay. Subsequently, snails were fed with NPs concentrations equal to half of the NRRT50 values, 0.05 mg L -1 for IONPs and 1 mg L -1 for IONPZ, for 1, 5, 10 and 20 days. Both effectors induced oxidative stress in snails' hemocytes compared to untreated animals. The latter was detected by NRRT changes, reactive oxygen species (ROS) production, lipid peroxidation estimation, DNA integrity loss, measurement of protein carbonyl content by an enzyme-linked immunoabsorbent assay (ELISA), determination of ubiquitin conjugates and cleaved caspases conjugates levels. The results showed that the simultaneous use of the parameters tested could constitute possible reliable biomarkers for the evaluation of NPs toxicity. However, more research is required in order to enlighten the disposal and toxic impact of iron oxide NPs on the environment to ensure their safe use in the future. Copyright © 2018 Elsevier Inc. All rights reserved.

MANF silencing, immunity induction or autophagy trigger an unusual cell type in metamorphosing Drosophila brain.

PubMed

Stratoulias, Vassilis; Heino, Tapio I

2015-05-01

Glia are abundant cells in the brain of animals ranging from flies to humans. They perform conserved functions not only in neural development and wiring, but also in brain homeostasis. Here we show that by manipulating gene expression in glia, a previously unidentified cell type appears in the Drosophila brain during metamorphosis. More specifically, this cell type appears in three contexts: (1) after the induction of either immunity, or (2) autophagy, or (3) by silencing of neurotrophic factor DmMANF in glial cells. We call these cells MANF immunoreactive Cells (MiCs). MiCs are migratory based on their shape, appearance in brain areas where no cell bodies exist and the nuclear localization of dSTAT. They are labeled with a unique set of molecular markers including the conserved neurotrophic factor DmMANF and the transcription factor Zfh1. They possess the nuclearly localized protein Relish, which is the hallmark of immune response activation. They also express the conserved engulfment receptor Draper, therefore indicating that they are potentially phagocytic. Surprisingly, they do not express any of the common glial and neuronal markers. In addition, ultrastructural studies show that MiCs are extremely rich in lysosomes. Our findings reveal critical molecular and functional components of an unusual cell type in the Drosophila brain. We suggest that MiCs resemble macrophages/hemocytes and vertebrate microglia based on their appearance in the brain upon genetically challenged conditions and the expression of molecular markers. Interestingly, macrophages/hemocytes or microglia-like cells have not been reported in the fly nervous system before.

Comparative study on the toxic effects of red tide flagellates Heterocapsa circularisquama and Chattonella marina on the short-necked clam (Ruditapes philippinarum).

PubMed

Kim, Daekyung; Choi, Kwang-Sik; Hong, Hyun-Ki; Jiang, Zedong; Zou, Yanan; Choi, Kyu-Sung; Yamasaki, Yasuhiro; Matsuyama, Yukihiko; Yamaguchi, Kenichi; Oda, Tatsuya

2011-01-01

Heterocapsa circularisquama showed much higher toxic effects on short-necked clams than Chattonella marina. Clams exposed to H. circularisquama exhibited morphological changes concomitant with an accumulation of mucus-like substances in the gills, a profound reduction in filtration activity, and lysosomal destabilization in hemocytes. Chattonella marina was less effective than H. circularisquama, and Heterocapsa triquetra was almost harmless in all these criteria. These results suggest that H. circularisquama exerted its lethal effect on short-necked clams through gill tissue damage and subsequent induction of physiological stress.

An experimental heat wave changes immune defense and life history traits in a freshwater snail.

PubMed

Leicht, Katja; Jokela, Jukka; Seppälä, Otto

2013-12-01

The predicted increase in frequency and severity of heat waves due to climate change is expected to alter disease dynamics by reducing hosts' ability to resist infections. This could take place via two different mechanisms: (1) through general reduction in hosts' performance under harsh environmental conditions and/or (2) through altered resource allocation that reduces expression of defense traits in order to maintain other traits. We tested these alternative hypotheses by measuring the effect of an experimental heat wave (25 vs. 15°C) on the constitutive level of immune defense (hemocyte concentration, phenoloxidase [PO]-like activity, antibacterial activity of hemolymph), and life history traits (growth and number of oviposited eggs) of the great pond snail Lymnaea stagnalis. We also manipulated the exposure time to high temperature (1, 3, 5, 7, 9, or 11 days). We found that if the exposure to high temperature lasted <1 week, immune function was not affected. However, when the exposure lasted longer than that, the level of snails' immune function (hemocyte concentration and PO-like activity) was reduced. Snails' growth and reproduction increased within the first week of exposure to high temperature. However, longer exposures did not lead to a further increase in cumulative reproductive output. Our results show that short experimental heat waves do not alter immune function but lead to plastic responses that increase snails' growth and reproduction. Thus, although the relative expression of traits changes, short experimental heat waves do not impair snails' defenses. Negative effects on performance get pronounced when the heat waves are prolonged suggesting that high performance cannot be maintained over long time periods. This ultimately reduces the levels of defense traits.

Identification of clam plasma proteins that bind its pathogen Quahog Parasite Unknown.

PubMed

Hartman, Rachel; Pales Espinosa, Emmanuelle; Allam, Bassem

2018-06-01

The hard clam (Mercenaria mercenaria) is among the most economically-important marine species along the east coast of the United States, representing the first marine resource in several Northeastern states. The species is rather resilient to infections and the only important disease of hard clams results from an infection caused by Quahog Parasite Unknown (QPX), a protistan parasite that can lead to significant mortality events in wild and aquacultured clam stocks. Though the presence of QPX disease has been documented since the 1960s, little information is available on cellular and molecular interactions between the parasite and the host. This study examined the interactions between the clam immune system and QPX cells. First, the effect of clam plasma on the binding of hemocytes to parasite cells was evaluated. Second, clam plasma proteins that bind QPX cells were identified through proteomic (LC-MS/MS) analyses. Finally, the effect of prior clam exposure to QPX on the abundance of QPX-reactive proteins in the plasma was evaluated. Results showed that plasma factors enhance the attachment of hemocytes to QPX. Among the proteins that specifically bind to QPX cells, several lectins were identified, as well as complement component proteins and proteolytic enzymes. Furthermore, results showed that some of these lectins and complement-related proteins are inducible as their abundance significantly increased following QPX challenge. These results shed light on plasma proteins involved in the recognition and binding of parasite cells and provide molecular targets for future investigations of factors involved in clam resistance to the disease, and ultimately for the selection of resistant clam stocks. Copyright © 2018 Elsevier Ltd. All rights reserved.

The glucose transporter 1 -GLUT1- from the white shrimp Litopenaeus vannamei is up-regulated during hypoxia.

PubMed

Martínez-Quintana, José A; Peregrino-Uriarte, Alma B; Gollas-Galván, Teresa; Gómez-Jiménez, Silvia; Yepiz-Plascencia, Gloria

2014-12-01

During hypoxia the shrimp Litopenaeus vannamei accelerates anaerobic glycolysis to obtain energy; therefore, a correct supply of glucose to the cells is needed. Facilitated glucose transport across the cells is mediated by a group of membrane embedded integral proteins called GLUT; being GLUT1 the most ubiquitous form. In this work, we report the first cDNA nucleotide and deduced amino acid sequences of a glucose transporter 1 from L. vannamei. A 1619 bp sequence was obtained by RT-PCR and RACE approaches. The 5´ UTR is 161 bp and the poly A tail is exactly after the stop codon in the mRNA. The ORF is 1485 bp and codes for 485 amino acids. The deduced protein sequence has high identity to GLUT1 proteins from several species and contains all the main features of glucose transporter proteins, including twelve transmembrane domains, the conserved motives and amino acids involved in transport activity, ligands binding and membrane anchor. Therefore, we decided to name this sequence, glucose transporter 1 of L. vannamei (LvGLUT1). A partial gene sequence of 8.87 Kbp was also obtained; it contains the complete coding sequence divided in 10 exons. LvGlut1 expression was detected in hemocytes, hepatopancreas, intestine gills, muscle and pleopods. The higher relative expression was found in gills and the lower in hemocytes. This indicates that LvGlut1 is ubiquitously expressed but its levels are tissue-specific and upon short-term hypoxia, the GLUT1 transcripts increase 3.7-fold in hepatopancreas and gills. To our knowledge, this is the first evidence of expression of GLUT1 in crustaceans.

Functional Conservation of the Glide/Gcm Regulatory Network Controlling Glia, Hemocyte, and Tendon Cell Differentiation in Drosophila

PubMed Central

Cattenoz, Pierre B.; Popkova, Anna; Southall, Tony D.; Aiello, Giuseppe; Brand, Andrea H.; Giangrande, Angela

2016-01-01

High-throughput screens allow us to understand how transcription factors trigger developmental processes, including cell specification. A major challenge is identification of their binding sites because feedback loops and homeostatic interactions may mask the direct impact of those factors in transcriptome analyses. Moreover, this approach dissects the downstream signaling cascades and facilitates identification of conserved transcriptional programs. Here we show the results and the validation of a DNA adenine methyltransferase identification (DamID) genome-wide screen that identifies the direct targets of Glide/Gcm, a potent transcription factor that controls glia, hemocyte, and tendon cell differentiation in Drosophila. The screen identifies many genes that had not been previously associated with Glide/Gcm and highlights three major signaling pathways interacting with Glide/Gcm: Notch, Hedgehog, and JAK/STAT, which all involve feedback loops. Furthermore, the screen identifies effector molecules that are necessary for cell-cell interactions during late developmental processes and/or in ontogeny. Typically, immunoglobulin (Ig) domain–containing proteins control cell adhesion and axonal navigation. This shows that early and transiently expressed fate determinants not only control other transcription factors that, in turn, implement a specific developmental program but also directly affect late developmental events and cell function. Finally, while the mammalian genome contains two orthologous Gcm genes, their function has been demonstrated in vertebrate-specific tissues, placenta, and parathyroid glands, begging questions on the evolutionary conservation of the Gcm cascade in higher organisms. Here we provide the first evidence for the conservation of Gcm direct targets in humans. In sum, this work uncovers novel aspects of cell specification and sets the basis for further understanding of the role of conserved Gcm gene regulatory cascades. PMID:26567182

Differential immunosuppression by Campoletis chlorideae eggs and ichnovirus in larvae of Helicoverpa armigera and Spodoptera exigua.

PubMed

Han, Li-Bin; Yin, Li-Hong; Huang, Ling-Qiao; Wang, Chen-Zhu

2015-09-01

The ichneumonid wasp, Campoletis chlorideae Uchida, successfully develops in the cotton bollworm Helicoverpa armigera (Hübner), but rarely survives in the beet armyworm Spodoptera exigua (Hübner) due to the encapsulation by host immunity. In this study, we investigated the role of C. chlorideae ichnovirus (CcIV) and eggs in the evasion of the host immune system. Washed eggs of different types, immature, mature, newly laid, or pretreated with protease K, were injected alone or with the calyx fluid containing CcIV into the larvae of H. armigera and S. exigua. In H. armigera, when injected with washed eggs alone, only 9.5% of the mature eggs were encapsulated at 24h post-injection. This is much lower than that of the immature eggs (100%), mature eggs pretreated with protease K (100%) and newly laid eggs (54.4%). No encapsulation was observed when the washed eggs were co-injected with calyx fluid at 24h post-injection. Conversely, the eggs in all treatments were encapsulated in S. exigua. Electron microscopic observations of parasitoid eggs showed structural differences between the surfaces of the mature and other kinds of eggs. The injected CcIV decreased the numbers of host hemocytes and suppressed the spreading ability of plasmatocytes and granulocytes in H. armigera, but had little effect on the hemocytes from S. exigua. In conclusion, the C. chlorideae egg provides a passive protection against encapsulation by itself, and CcIV supplies an active protection through disrupting host immune responses. These coordinated protections are host-specific, implying their role in host range determination. Copyright © 2015 Elsevier Inc. All rights reserved.

A Genetic Screen Reveals an Unexpected Role for Yorkie Signaling in JAK/STAT-Dependent Hematopoietic Malignancies in Drosophila melanogaster

PubMed Central

Anderson, Abigail M.; Bailetti, Alessandro A.; Rodkin, Elizabeth; De, Atish; Bach, Erika A.

2017-01-01

A gain-of-function mutation in the tyrosine kinase JAK2 (JAK2V617F) causes human myeloproliferative neoplasms (MPNs). These patients present with high numbers of myeloid lineage cells and have numerous complications. Since current MPN therapies are not curative, there is a need to find new regulators and targets of Janus kinase/Signal transducer and activator of transcription (JAK/STAT) signaling that may represent additional clinical interventions . Drosophila melanogaster offers a low complexity model to study MPNs as JAK/STAT signaling is simplified with only one JAK [Hopscotch (Hop)] and one STAT (Stat92E). hopTumorous-lethal (Tum-l) is a gain-of-function mutation that causes dramatic expansion of myeloid cells, which then form lethal melanotic tumors. Through an F1 deficiency (Df) screen, we identified 11 suppressors and 35 enhancers of melanotic tumors in hopTum-l animals. Dfs that uncover the Hippo (Hpo) pathway genes expanded (ex) and warts (wts) strongly enhanced the hopTum-l tumor burden, as did mutations in ex, wts, and other Hpo pathway genes. Target genes of the Hpo pathway effector Yorkie (Yki) were significantly upregulated in hopTum-l blood cells, indicating that Yki signaling was increased. Ectopic hematopoietic activation of Yki in otherwise wild-type animals increased hemocyte proliferation but did not induce melanotic tumors. However, hematopoietic depletion of Yki significantly reduced the hopTum-l tumor burden, demonstrating that Yki is required for melanotic tumors in this background. These results support a model in which elevated Yki signaling increases the number of hemocytes, which become melanotic tumors as a result of elevated JAK/STAT signaling. PMID:28620086

Molecular cloning, characterization and expression profiles of thioredoxin 1 and thioredoxin 2 genes in Mytilus galloprovincialis

NASA Astrophysics Data System (ADS)

Wang, Qing; Ning, Xuanxuan; Pei, Dong; Zhao, Jianmin; You, Liping; Wang, Chunyan; Wu, Huifeng

2013-05-01

Thioredoxin (Trx) proteins are involved in many biological processes especially the regulation of cellular redox homeostasis. In this study, two Trx cDNAs were cloned from the mussel Mytilus galloprovincialis using rapid amplifi cation of cDNA ends-polymerase chain reaction (RACE-PCR). The two cDNAs were named MgTrx1 and MgTrx2, respectively. The open reading frames of MgTrx1 and MgTrx2 were 318 and 507 base pairs (bp) and they encoded proteins of 105 and 168 amino acids with estimated molecular masses of 11.45 and 18.93 kDa, respectively. Sequence analysis revealed that both proteins possessed the conserved active site dithiol motif Cys-Gly-Pro-Cys. In addition, MgTrx2 also possessed a putative mitochondrial targeting signal suggesting that it is located in the mitochondria. Quantitative real-time polymerase chain reaction (qPCR) revealed that both MgTrx1 and MgTrx2 were constitutively expressed in all tissues examined. The MgTrx1 transcript was most abundant in hemocytes and gills, whereas the MgTrx2 transcript was most abundant in gonad, hepatopancreas, gill and hemocytes. Following Vibrio anguillarum challenge, the expression of MgTrx1 was up-regulated and reached its peak, at a value 10-fold the initial value, at 24 h. Subsequently, expression returned back to the original level. In contrast, the expression level of MgTrx2 was down-regulated following bacterial stimulation, with one fi fth of the control level evident at 12 h post challenge. These results suggest that MgTrx1 and MgTrx2 may play important roles in the response of M. galloprovincialis to bacterial challenge.

Structural and functional characterization of a novel molluskan ortholog of TRAF and TNF receptor-associated protein from disk abalone (Haliotis discus discus).

PubMed

Lee, Youngdeuk; Elvitigala, Don Anushka Sandaruwan; Whang, Ilson; Lee, Sukkyoung; Kim, Hyowon; Zoysa, Mahanama De; Oh, Chulhong; Kang, Do-Hyung; Lee, Jehee

2014-09-01

Immune signaling cascades have an indispensable role in the host defense of almost all the organisms. Tumor necrosis factor (TNF) signaling is considered as a prominent signaling pathway in vertebrate as well as invertebrate species. Within the signaling cascade, TNF receptor-associated factor (TRAF) and TNF receptor-associated protein (TTRAP) has been shown to have a crucial role in the modulation of immune signaling in animals. Here, we attempted to characterize a novel molluskan ortholog of TTRAP (AbTTRAP) from disk abalone (Haliotis discus discus) and analyzed its expression levels under pathogenic stress. The complete coding sequence of AbTTRAP consisted of 1071 nucleotides, coding for a 357 amino acid peptide, with a predicted molecular mass of 40 kDa. According to our in-silico analysis, AbTTRAP resembled the typical TTRAP domain architecture, including a 5'-tyrosyl DNA phosphodiesterase domain. Moreover, phylogenetic analysis revealed its common ancestral invertebrate origin, where AbTTRAP was clustered with molluskan counterparts. Quantitative real time PCR showed universally distributed expression of AbTTRAP in selected tissues of abalone, from which more prominent expression was detected in hemocytes. Upon stimulation with two pathogen-derived mitogens, lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly I:C), transcript levels of AbTTRAP in hemocytes and gill tissues were differentially modulated with time. In addition, the recombinant protein of AbTTRAP exhibited prominent endonuclease activity against abalone genomic DNA, which was enhanced by the presence of Mg(2+) in the medium. Collectively, these results reinforce the existence of the TNF signaling cascade in mollusks like disk abalone, further implicating the putative regulatory behavior of TTRAP in invertebrate host pathology. Copyright © 2014 Elsevier Ltd. All rights reserved.

Morphological and histological studies on freshwater prawn Macrobrachium rosenbergii (de man) irradiated with (60)Co gamma radiation.

PubMed

Stalin, A; Broos, K V; Sadiq Bukhari, A; Syed Mohamed, H E; Singhal, R K; Venu-Babu, P

2013-11-15

This study was framed to investigate the (60)Co gamma radiation induced morphological and histological variations in freshwater prawn Macrobrachium rosenbergii. The LD50 value of (60)Co gamma irradiated M. rosenbergii observed (by probit analysis) at 30 Gy. Prawns were irradiated to four different dose levels (3 mGy, 30 mGy, 300 mGy and 3,000 mGy) using Theratron Phoenix TeleCobalt Unit [P-33] and one control group (without irradiation) maintained separately. Irradiated groups exhibited several morphological variations such as discoloration; damaged rostrum; opaque coloration in cephalothorax; black bands and dot formation in abdomen; deformed uropods and telson in tail regions when compared with control group. The Hepato Somatic Index reflected the severity of radiation on hepatopancreas. Histological variations in gills, hepatopancreas and muscles of irradiated groups were observed. In gills, structural changes such as swollen and fused lamellae, abnormal gill tips, hyperplasic, necrotic and clavate-globate lamellae were observed in gamma irradiated prawns. Accumulation of hemocytes in hemocoelic space, interstitial sinuses filled with abnormal infiltrated hemocytes, the tubular epithelium with ruptured basal laminae, abnormal and coagulated lumen, necrotic tubules, thickened basal laminae, tissue debris, necrotic hepatocytes were observed in irradiated prawn hepatopancreas. In muscle, shrinkage of muscular fiber and necrotic musculature were observed in irradiated prawns. These structural alterations of the organs it is felt could affect the vital physiological functions such as respiration, osmotic and ionic regulation in gills and muscles; absorption, storage and secretion of the hepatopancreas which in turn could adversely affect the growth and survival of freshwater prawn M. rosenbergii. Copyright © 2013 Elsevier B.V. All rights reserved.

Immunoevasive protein (IEP)-containing surface layer covering polydnavirus particles is essential for viral infection.

PubMed

Furihata, Shunsuke; Tanaka, Kohjiro; Ryuda, Masasuke; Ochiai, Masanori; Matsumoto, Hitoshi; Csikos, Gyorge; Hayakawa, Yoichi

2014-01-01

Polydnaviruses (PDVs) are unique symbiotic viruses associated with parasitoid wasps: PDV particles are injected into lepidopteran hosts along with the wasp eggs and express genes that interfere with aspects of host physiology such as immune defenses and development. Recent comparative genomic studies of PDVs have significantly improved our understanding of their origin as well as the genome organization. However, the structural features of functional PDV particles remain ambiguous. To clear up the structure of Cotesia kariyai PDV (CkPDV) particles, we focused on immunoevasive protein (IEP), which is a mediator of immunoevasion by the wasp from the encapsulation reaction of the host insect's hemocytes, because it has been demonstrated to be present on the surface of the virus particle. We discovered that IEP tends to polymerize and constitutes a previously unidentified thin surface layer covering CkPDV particles. This outermost surface layer looked fragile and was easily removed from CkPVD particles by mechanical stressors such as shaking, which prevented CkPDV from expressing the encoded genes in the host target tissues such as fat body or hemocytes. Furthermore, we detected IEP homologue gene expression in the wasp's venom reservoirs, implying IEP has another unknown biological function in the wasp or parasitized hosts. Taken together, the present results demonstrated that female C. kariyai wasps produce the fragile thin layer partly composed of IEP to cover the outer surfaces of CkPDV particles; otherwise, they cannot function as infectious agents in the wasp's host. The fact that IEP family proteins are expressed in both venom reservoirs and oviducts suggests an intimate relationship between both tissues in the development of the parasitism strategy of the wasp. Copyright © 2013 Elsevier Inc. All rights reserved.

A three-domain Kazal-type serine proteinase inhibitor exhibiting domain inhibitory and bacteriostatic activities from freshwater crayfish Procambarus clarkii.

PubMed

Li, Xin-Cang; Wang, Xian-Wei; Wang, Zong-Heng; Zhao, Xiao-Fan; Wang, Jin-Xing

2009-12-01

In crustaceans, Kazal-type serine proteinase inhibitors in hemolymph are believed to function as regulators of the host-defense reactions or inhibitors against proteinases from microorganisms. In this study, we report a Kazal-type serine proteinase inhibitor, named hcPcSPI1, from freshwater crayfish (Procambarus clarkii). We found that hcPcSPI1 is composed of a putative signal peptide, an RGD motif, and three tandem Kazal-type domains with the domain P1 residues L, L and E, respectively. Mainly, hcPcSPI1 was detected in hemocytes as well as in the heart, gills, and intestine at both the mRNA and protein levels. Quantitative real-time PCR analysis showed that hcPcSPI1 in hemocytes was upregulated by the stimulation of Esherichia coli (8099) or became decreased after a white spot syndrome virus (WSSV) challenge. In addition, hcPcSPI1 and its three independent domains were overexpressed and purified to explore their potential functions. All four proteins inhibited subtilisin A and proteinase K, but not alpha-chymotypsin or trypsin. Recombinant hcPcSPI1 could firmly attach to Gram-negative bacteria E. coli and Klebsiella pneumoniae; Gram-positive bacteria Bacillus subtilis, Bacillus thuringiensis and Staphylococcus aureus; fungi Candida albicans and Saccharomyce cerevisiae, and only domain 1 was responsible for the binding to E. coli and S. aureus. In addition, recombinant hcPcSPI1 was also found to possess bacteriostatic activity against the B. subtilis and B. thuringiensis. Domains 2 and 3 contributed mainly to these bacteriostatic activities. All results suggested that hcPcSPI1 might play important roles in the innate immunity of crayfish.

The novel white spot syndrome virus-induced gene, PmERP15, encodes an ER stress-responsive protein in black tiger shrimp, Penaeus monodon.

PubMed

Leu, Jiann-Horng; Liu, Kuan-Fu; Chen, Kuan-Yu; Chen, Shu-Hwa; Wang, Yu-Bin; Lin, Chung-Yen; Lo, Chu-Fang

2015-04-01

By microarray screening, we identified a white spot syndrome virus (WSSV)-strongly induced novel gene in gills of Penaeus monodon. The gene, PmERP15, encodes a putative transmembrane protein of 15 kDa, which only showed some degree of similarity (54-59%) to several unknown insect proteins, but had no hits to shrimp proteins. RT-PCR showed that PmERP15 was highly expressed in the hemocytes, heart and lymphoid organs, and that WSSV-induced strong expression of PmERP15 was evident in all tissues examined. Western blot analysis likewise showed that WSSV strongly up-regulated PmERP15 protein levels. In WSSV-infected hemocytes, immunofluorescence staining showed that PmERP15 protein was colocalized with an ER enzyme, protein disulfide isomerase, and in Sf9 insect cells, PmERP15-EGFP fusion protein colocalized with ER -Tracker™ Red dye as well. GRP78, an ER stress marker, was found to be up-regulated in WSSV-infected P. monodon, and both PmERP15 and GRP78 were up-regulated in shrimp injected with ER stress inducers tunicamycin and dithiothreitol. Silencing experiments showed that although PmERP15 dsRNA-injected shrimp succumbed to WSSV infection more rapidly, the WSSV copy number had no significant changes. These results suggest that PmERP15 is an ER stress-induced, ER resident protein, and its induction in WSSV-infected shrimp is caused by the ER stress triggered by WSSV infection. Furthermore, although PmERP15 has no role in WSSV multiplication, its presence is essential for the survival of WSSV-infected shrimp. Copyright © 2014 Elsevier Ltd. All rights reserved.

Effect of the plant Cupressus macro-carpa (Cupressacea) on some haematolo-gical and biochemical parameters of Biomphalaria alexandrina snails.

PubMed

El-Sayed, Kamelia A

2006-12-01

The dry powder of the plant aereal part; Cupressus macro-carpa (Cupressacea) was tested against Biomphalaria alexandrina. LC50 & LC90 values were 59.5 & 98.8 ppm, respec-tively. Exposure of B. alexandrina to sublethal concentrations (LC0, LC10 & LC25) of C. macrocarpa for three weeks signi-ficantly decreased the number of circulating hemocytes. The magnitude of reduction was increased with increasing of the tested concentration. The main type of cell in the hemolymph of B. alexandrina was the granulocyte (71.8%), followed by large round cells or hyalinocytes (19.0%) and small round cells or undifferentiate cells (9.2%). The percentage of different hemocyte categories was changed in treated snails. In snails maintained at LC25, showed significantly higher percentages of small round cells than controls, 56.2% & 9.2% respectively. Maintainence of B. alexandrina in sublethal concentrations for three weeks significantly reduced protein & hemoglobin content in the hemolymph. Reduction in enzyme activities occurred in the hemolymph and tissues of treated snails. The enzymes were pyruvate kinase (PK), lactat dehydrogenase (LDH), hexokinase (HK) and phosphoenol pyruvate carboxy kinase (PEPCK) which are very important in metabolism of the protein and carbohydrate. The infectivity of Schistosoma mansoni miracidia was greatly reduced by exposure to the sublethal concentrations (LC0, LC10 & LC25) of Cupressus. Infection rate of B. alexandrina reached to 54.5%, 37.5% & 16.7%, respectively compared to control (81.25%). Duration of cercarial shedding and the total periodic cercarial production/snail showed significant reduction while the parasite incubation period was significantly longer (p<0.05).

Low-cost motility tracking system (LOCOMOTIS) for time-lapse microscopy applications and cell visualisation.

PubMed

Lynch, Adam E; Triajianto, Junian; Routledge, Edwin

2014-01-01

Direct visualisation of cells for the purpose of studying their motility has typically required expensive microscopy equipment. However, recent advances in digital sensors mean that it is now possible to image cells for a fraction of the price of a standard microscope. Along with low-cost imaging there has also been a large increase in the availability of high quality, open-source analysis programs. In this study we describe the development and performance of an expandable cell motility system employing inexpensive, commercially available digital USB microscopes to image various cell types using time-lapse and perform tracking assays in proof-of-concept experiments. With this system we were able to measure and record three separate assays simultaneously on one personal computer using identical microscopes, and obtained tracking results comparable in quality to those from other studies that used standard, more expensive, equipment. The microscopes used in our system were capable of a maximum magnification of 413.6×. Although resolution was lower than that of a standard inverted microscope we found this difference to be indistinguishable at the magnification chosen for cell tracking experiments (206.8×). In preliminary cell culture experiments using our system, velocities (mean µm/min ± SE) of 0.81 ± 0.01 (Biomphalaria glabrata hemocytes on uncoated plates), 1.17 ± 0.004 (MDA-MB-231 breast cancer cells), 1.24 ± 0.006 (SC5 mouse Sertoli cells) and 2.21 ± 0.01 (B. glabrata hemocytes on Poly-L-Lysine coated plates), were measured and are consistent with previous reports. We believe that this system, coupled with open-source analysis software, demonstrates that higher throughput time-lapse imaging of cells for the purpose of studying motility can be an affordable option for all researchers.

Low-Cost Motility Tracking System (LOCOMOTIS) for Time-Lapse Microscopy Applications and Cell Visualisation

PubMed Central

Lynch, Adam E.; Triajianto, Junian; Routledge, Edwin

2014-01-01

Direct visualisation of cells for the purpose of studying their motility has typically required expensive microscopy equipment. However, recent advances in digital sensors mean that it is now possible to image cells for a fraction of the price of a standard microscope. Along with low-cost imaging there has also been a large increase in the availability of high quality, open-source analysis programs. In this study we describe the development and performance of an expandable cell motility system employing inexpensive, commercially available digital USB microscopes to image various cell types using time-lapse and perform tracking assays in proof-of-concept experiments. With this system we were able to measure and record three separate assays simultaneously on one personal computer using identical microscopes, and obtained tracking results comparable in quality to those from other studies that used standard, more expensive, equipment. The microscopes used in our system were capable of a maximum magnification of 413.6×. Although resolution was lower than that of a standard inverted microscope we found this difference to be indistinguishable at the magnification chosen for cell tracking experiments (206.8×). In preliminary cell culture experiments using our system, velocities (mean µm/min ± SE) of 0.81±0.01 (Biomphalaria glabrata hemocytes on uncoated plates), 1.17±0.004 (MDA-MB-231 breast cancer cells), 1.24±0.006 (SC5 mouse Sertoli cells) and 2.21±0.01 (B. glabrata hemocytes on Poly-L-Lysine coated plates), were measured and are consistent with previous reports. We believe that this system, coupled with open-source analysis software, demonstrates that higher throughput time-lapse imaging of cells for the purpose of studying motility can be an affordable option for all researchers. PMID:25121722

Transcriptomics of In Vitro Immune-Stimulated Hemocytes from the Manila Clam Ruditapes philippinarum Using High-Throughput Sequencing

PubMed Central

Moreira, Rebeca; Balseiro, Pablo; Planas, Josep V.; Fuste, Berta; Beltran, Sergi; Novoa, Beatriz; Figueras, Antonio

2012-01-01

Background The Manila clam (Ruditapes philippinarum) is a worldwide cultured bivalve species with important commercial value. Diseases affecting this species can result in large economic losses. Because knowledge of the molecular mechanisms of the immune response in bivalves, especially clams, is scarce and fragmentary, we sequenced RNA from immune-stimulated R. philippinarum hemocytes by 454-pyrosequencing to identify genes involved in their immune defense against infectious diseases. Methodology and Principal Findings High-throughput deep sequencing of R. philippinarum using 454 pyrosequencing technology yielded 974,976 high-quality reads with an average read length of 250 bp. The reads were assembled into 51,265 contigs and the 44.7% of the translated nucleotide sequences into protein were annotated successfully. The 35 most frequently found contigs included a large number of immune-related genes, and a more detailed analysis showed the presence of putative members of several immune pathways and processes like the apoptosis, the toll like signaling pathway and the complement cascade. We have found sequences from molecules never described in bivalves before, especially in the complement pathway where almost all the components are present. Conclusions This study represents the first transcriptome analysis using 454-pyrosequencing conducted on R. philippinarum focused on its immune system. Our results will provide a rich source of data to discover and identify new genes, which will serve as a basis for microarray construction and the study of gene expression as well as for the identification of genetic markers. The discovery of new immune sequences was very productive and resulted in a large variety of contigs that may play a role in the defense mechanisms of Ruditapes philippinarum. PMID:22536348

Effects of tributyltin and benzo[a]pyrene on the immune-associated activities of hemocytes and recovery responses in the gastropod abalone, Haliotis diversicolor.

PubMed

Gopalakrishnan, Singaram; Huang, Wei-Bin; Wang, Qiang-Wei; Wu, Man-Li; Liu, Jie; Wang, Ke-Jian

2011-08-01

Our previous study reports that short-term exposure to sublethal concentrations of benzo[a]pyrene (BaP) induces immunomodulation in the gastropod abalone, Haliotis diversicolor. In the present study, it was further observed that long-term chronic exposure to sublethal concentrations of BaP modulated the immunocompetence of abalones in terms of the change in activity of the antioxidant and immune associated parameters tested. In addition, the effect of tributyltin (TBT), another important genotoxicant in the aquatic environment, was investigated. Exposure of abalones to sublethal concentrations of TBT and BaP for 21 days resulted in significant decrease of total hemocyte count, phagocytosis, membrane stability and lysozyme activity. Conversely induction of extra and intra cellular superoxide generation, nitric oxide, nitric oxide synthase and myeloperoxidase activity was present when the abalones were exposed to TBT and BaP. Most of the immune associated parameters tested showed clear time dependent response to both toxicants. Within 14 days after the 21 day exposure to BaP, recovery was observed as evidenced by most of the parameters returning to their normal level. However, no recovery was observed within 14 days after the 21 day exposure to TBT as evidenced by continued elevation of intra cellular superoxide and nitrite production and decrease in THC, membrane stability and lysozyme activity. This suggested a prolonged TBT-induced impact on the immune reaction and possibly more damage than that caused by BaP. Overall the results suggest that chronic exposure to sublethal concentrations of TBT or BaP causes modulations in the immunocompetence of abalones with most of the immune associated parameters tested being stimulated, and this might be harmful to the host. Copyright © 2011 Elsevier Inc. All rights reserved.

Sequencing and de novo analysis of the hemocytes transcriptome in Litopenaeus vannamei response to white spot syndrome virus infection.

PubMed

Xue, Shuxia; Liu, Yichen; Zhang, Yichen; Sun, Yan; Geng, Xuyun; Sun, Jinsheng

2013-01-01

White spot syndrome virus (WSSV) is a causative pathogen found in most shrimp farming areas of the world and causes large economic losses to the shrimp aquaculture. The mechanism underlying the molecular pathogenesis of the highly virulent WSSV remains unknown. To better understand the virus-host interactions at the molecular level, the transcriptome profiles in hemocytes of unchallenged and WSSV-challenged shrimp (Litopenaeus vannamei) were compared using a short-read deep sequencing method (Illumina). RNA-seq analysis generated more than 25.81 million clean pair end (PE) reads, which were assembled into 52,073 unigenes (mean size = 520 bp). Based on sequence similarity searches, 23,568 (45.3%) genes were identified, among which 6,562 and 7,822 unigenes were assigned to gene ontology (GO) categories and clusters of orthologous groups (COG), respectively. Searches in the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) mapped 14,941 (63.4%) unigenes to 240 KEGG pathways. Among all the annotated unigenes, 1,179 were associated with immune-related genes. Digital gene expression (DGE) analysis revealed that the host transcriptome profile was slightly changed in the early infection (5 hours post injection) of the virus, while large transcriptional differences were identified in the late infection (48 hpi) of WSSV. The differentially expressed genes mainly involved in pattern recognition genes and some immune response factors. The results indicated that antiviral immune mechanisms were probably involved in the recognition of pathogen-associated molecular patterns. This study provided a global survey of host gene activities against virus infection in a non-model organism, pacific white shrimp. Results can contribute to the in-depth study of candidate genes in white shrimp, and help to improve the current understanding of host-pathogen interactions.

Involvement of the Anopheles gambiae Nimrod gene family in mosquito immune responses.

PubMed

Estévez-Lao, Tania Y; Hillyer, Julián F

2014-01-01

Insects fight infection using a variety of signaling pathways and immune effector proteins. In Drosophila melanogaster, three members of the Nimrod gene family (draper, nimC1 and eater) bind bacteria, and this binding leads to phagocytosis by hemocytes. The Nimrod gene family has since been identified in other insects, but their function in non-drosophilids remains unknown. The purpose of this study was to identify the members of the Nimrod gene family in the malaria mosquito, Anopheles gambiae, and to assess their role in immunity. We identified and sequenced three members of this gene family, herein named draper, nimrod and eater, which are the orthologs of D. melanogaster draper, nimB2 and eater, respectively. The three genes are preferentially expressed in hemocytes and their peak developmental expression is in pupae and young adults. Infection induces the transcriptional upregulation of all three genes, but the magnitude of this upregulation becomes more attenuated as mosquitoes become older. RNAi-based knockdown of eater, but not draper or nimrod, decreased a mosquito's ability to kill Escherichia coli in the hemocoel. Knockdown of draper, eater, or any combination of Nimrod family genes rendered mosquitoes more likely to die from Staphylococcus epidermidis. Finally, knockdown of Nimrod family genes did not impact mRNA levels of the antimicrobial peptides defensin (def1), cecropin (cecA) or gambicin (gam1), but eater knockdown led to a decrease in mRNA levels of nitric oxide synthase. Together, these data show that members of the A. gambiae Nimrod gene family are positive regulators of the mosquito antibacterial response. Copyright © 2013 Elsevier Ltd. All rights reserved.

Characterization of a serine proteinase homologous (SPH) in Chinese mitten crab Eriocheir sinensis.

PubMed

Qin, Chuanjie; Chen, Liqiao; Qin, Jian G; Zhao, Daxian; Zhang, Hao; Wu, Ping; Li, Erchao

2010-01-01

The serine protease homologous (SPH) is an important cofactor of prophenoloxidase-activating enzyme (PPAE). The gene of SPH of Chinese mitten crab Eriocheir sinensis (EsSPH) in hemocytes was cloned and characterized using reverse transcript polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The SPH cDNA consisted of 1386 bp with an open reading frame (ORF) encoded a protein of 378 amino acids, 154 bp 5'-untranslated region, and 95 bp 3'-untranslated region. Sequence comparisons against the GenBank database showed that EsSPH deduced amino acids had an overall identity to the gene of serine protease family from 41% to 70% of 15 invertebrate species. The protein had the structural characteristics of SPH, including the conserved six cysteine residues in the N-terminal clip domain and the functional activity (His157, Asp209, Gly311) in the C-terminal serine proteinase-like domain. To analyze the role of EsSPH in an acute infection, the temporal expression of the EsSPH gene after the Aeromonas hydrophila challenge was measured by real-time RT-PCR. The EsSPH transcripts in hemocytes significantly increased at 6 h, 12 h and 48 h over time after the A. hydrophila injection. This expression pattern shows that EsSPH has the potential to defend against invading microorganisms. The mRNA transcripts of EsSPH were detected in all tissues with the highest in the hepatopancreas. Interestingly, the mRNA transcripts of EsSPH and proPO were found in ova and expressed in oosperms, suggesting that the maternal transfer of EsSPH and proPO may exit in crab, but this warrants confirmation in further research.

FmLC5, a putative galactose-binding C-type lectin with two QPD motifs from the hemocytes of Fenneropenaeus merguiensis participates in shrimp immune defense.

PubMed

Senghoi, Wilaiwan; Runsaeng, Phanthipha; Utarabhand, Prapaporn

2017-11-01

Crustaceans are deficient in adaptive immune system. They depend completely on an innate immunity to protect themselves from invading microorganisms. One kind of pattern recognition receptors that contribute roles in the innate immunity is lectin. A new C-type lectin gene designated as FmLC5 was isolated from Fenneropenaeus merguiensis. Its full-length cDNA is composed of 1526bp and one open reading frame of 852bp encoding a peptide of 284 amino acids. The deduced amino acid sequence of FmLC5 comprises a signal peptide of 20 contiguous amino acids with a molecular mass of 31.47kDa and an isoelectric point of 4.35. The primary structure of FmLC5 consists of two similar carbohydrate recognition domains (CRDs), each CRD contains a Ca 2+ binding site-2 and a QPD motif specific for galactose-binding. The FmLC5 transcripts were detected only in the hemocytes analyzed by RT-PCR and in situ hybridization. The FmLC5 expression was significantly up-regulated after challenge with Vibrio harveyi, white spot syndrome virus (WSSV) or lipopolysaccharide. RNAi-based silencing with co-injection by V. harveyi or WSSV resulted in critical suppression of the FmLC5 expression, increasing in mortality and reduction of the median lethal time. These results conclude that FmLC5 is unique putative galactose-binding C-type lectin in F. merguiensis that may contribute as receptor molecule in the immune response to defend the shrimp from pathogenic bacteria and viruses. Copyright © 2017 Elsevier Inc. All rights reserved.

DNA extracellular traps are part of the immune repertoire of Periplaneta americana.

PubMed

Nascimento, M T C; Silva, K P; Garcia, M C F; Medeiros, M N; Machado, E A; Nascimento, S B; Saraiva, E M

2018-07-01

Extracellular traps (ETs), web-like structures composed of DNA and histones, are released by innate immune cells in a wide range of organisms. ETs capture microorganisms, thereby avoiding their spread, and also concentrate antimicrobial molecules, which helps to kill microbes. Although vertebrate innate immune systems share homology with the insect immune system, ETosis have yet to be characterized in insects. Here, we report that the hemocytes of the hemimetabolous insect Periplaneta americana release ETs upon in vitro stimulation. We further discuss the relationship between ETs and nodulation and in controlling bacterial spread in vivo. Copyright © 2018 Elsevier Ltd. All rights reserved.

Dietary supplementation of probiotic Bacillus PC465 isolated from the gut of Fenneropenaeus chinensis improves the health status and resistance of Litopenaeus vannamei against white spot syndrome virus.

PubMed

Chai, Peng-Cheng; Song, Xiao-Ling; Chen, Guo-Fu; Xu, Hua; Huang, Jie

2016-07-01

This study conducted a 30-day feeding trial and a subsequent 20-day anti-virus infection trial to determine the effects of probiotic Bacillus PC465 on the growth, health status, and disease resistance of Litopenaeus vannamei. Shrimp samples were fed with three practical diets prepared from shrimp feed containing varying probiotic doses [0 (control), 10(7), and 10(9) CFU g(-1)]. Probiotic supplementation significantly increased the weight gain and survival of L. vannamei (p < 0.05). The effect of 10(9) CFU g(-1) on the growth rate was higher than that of 10(7) CFU g(-1). Compared with those in the control group, the activities of digestive enzymes, such as amylase, protease, and lipase, in the shrimp mid-gut significantly increased in the probiotic-fed groups on days 15 and 30, except lipase on day 30. The influence of 10(9) CFU g(-1) on enzyme activities was also greater than that of 10(7) CFU g(-1). Scanning electron microscopy revealed folds and large ravines across the interior surface of the mid-gut, and the number of these folds and ravines increased significantly after the probiotic was administered. The probiotic treatment significantly (p < 0.05) enhanced the transcription of penaeidin 3a (Pen-3a), peroxinectin, C-type lectin 3 (Lec-3), and thioredoxin (Trx) in the hemocytes of L. vannamei. Likewise, probiotic treatment increased the transcription of hemocyanin in the hepatopancreas of L. vannamei. The probiotic treatment also significantly increased the transcription of prophenoloxidase (proPO) but decreased the transcription of crustin in hemocytes. By contrast, the same treatment failed to increase the transcription of Ras-related protein (Rab-6) in hemocytes. The number of species and biomass of Bacillus in the mid-gut were higher in the probiotic-fed group than in the control group. The total biomass of microbes was higher in the shrimp fed with 10(7) CFU g(-1) than in the shrimp fed with 10(9) CFU g(-1) and the control group on days 15 and 30 post-feeding. In two white spot syndrome virus (WSSV) infections, the weight gain, survival, and WSSV copies within the gills of the probiotic-treated shrimp significantly differed (p < 0.05) from those of the control group. Relatively efficient protection was associated with probiotic feeding. Results suggested that Bacillus PC465 feeding improves the growth performance, survival, digestion, and nutrient absorption of L. vannamei. Probiotic treatment also enhances the microbial structures in the gut, promotes the immune status of shrimp, and provides protection against viral infection. The supplementation with 10(9) CFU g(-1) can also improve the growth and survival of L. vannamei. Copyright © 2016 Elsevier Ltd. All rights reserved.

Hemocytes from Pediculus humanus humanus are hosts for human bacterial pathogens

PubMed Central

Coulaud, Pierre-Julien; Lepolard, Catherine; Bechah, Yassina; Berenger, Jean-Michel; Raoult, Didier; Ghigo, Eric

2015-01-01

Pediculus humanus humanus is an human ectoparasite which represents a serious public health threat because it is vector for pathogenic bacteria. It is important to understand and identify where bacteria reside in human body lice to define new strategies to counterstroke the capacity of vectorization of the bacterial pathogens by body lice. It is known that phagocytes from vertebrates can be hosts or reservoirs for several microbes. Therefore, we wondered if Pediculus humanus humanus phagocytes could hide pathogens. In this study, we characterized the phagocytes from Pediculus humanus humanus and evaluated their contribution as hosts for human pathogens such as Rickettsia prowazekii, Bartonella Quintana, and Acinetobacter baumannii. PMID:25688336

Microplitis demolitor bracovirus genome segments vary in abundance and are individually packaged in virions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beck, Markus H.; Inman, Ross B.; Strand, Michael R.

2007-03-01

Polydnaviruses (PDVs) are distinguished by their unique association with parasitoid wasps and their segmented, double-stranded (ds) DNA genomes that are non-equimolar in abundance. Relatively little is actually known, however, about genome packaging or segment abundance of these viruses. Here, we conducted electron microscopy (EM) and real-time polymerase chain reaction (PCR) studies to characterize packaging and segment abundance of Microplitis demolitor bracovirus (MdBV). Like other PDVs, MdBV replicates in the ovaries of females where virions accumulate to form a suspension called calyx fluid. Wasps then inject a quantity of calyx fluid when ovipositing into hosts. The MdBV genome consists of 15more » segments that range from 3.6 (segment A) to 34.3 kb (segment O). EM analysis indicated that MdBV virions contain a single nucleocapsid that encapsidates one circular DNA of variable size. We developed a semi-quantitative real-time PCR assay using SYBR Green I. This assay indicated that five (J, O, H, N and B) segments of the MdBV genome accounted for more than 60% of the viral DNAs in calyx fluid. Estimates of relative segment abundance using our real-time PCR assay were also very similar to DNA size distributions determined from micrographs. Analysis of parasitized Pseudoplusia includens larvae indicated that copy number of MdBV segments C, B and J varied between hosts but their relative abundance within a host was virtually identical to their abundance in calyx fluid. Among-tissue assays indicated that each viral segment was most abundant in hemocytes and least abundant in salivary glands. However, the relative abundance of each segment to one another was similar in all tissues. We also found no clear relationship between MdBV segment and transcript abundance in hemocytes and fat body.« less

Real-Time In Vivo Imaging of Butterfly Wing Development: Revealing the Cellular Dynamics of the Pupal Wing Tissue

PubMed Central

Iwata, Masaki; Ohno, Yoshikazu; Otaki, Joji M.

2014-01-01

Butterfly wings are covered with regularly arranged single-colored scales that are formed at the pupal stage. Understanding pupal wing development is therefore crucial to understand wing color pattern formation. Here, we successfully employed real-time in vivo imaging techniques to observe pupal hindwing development over time in the blue pansy butterfly, Junonia orithya. A transparent sheet of epithelial cells that were not yet regularly arranged was observed immediately after pupation. Bright-field imaging and autofluorescent imaging revealed free-moving hemocytes and tracheal branches of a crinoid-like structure underneath the epithelium. The wing tissue gradually became gray-white, epithelial cells were arranged regularly, and hemocytes disappeared, except in the bordering lacuna, after which scales grew. The dynamics of the epithelial cells and scale growth were also confirmed by fluorescent imaging. Fluorescent in vivo staining further revealed that these cells harbored many mitochondria at the surface of the epithelium. Organizing centers for the border symmetry system were apparent immediately after pupation, exhibiting a relatively dark optical character following treatment with fluorescent dyes, as well as in autofluorescent images. The wing tissue exhibited slow and low-frequency contraction pulses with a cycle of approximately 10 to 20 minutes, mainly occurring at 2 to 3 days postpupation. The pulses gradually became slower and weaker and eventually stopped. The wing tissue area became larger after contraction, which also coincided with an increase in the autofluorescence intensity that might have been caused by scale growth. Examination of the pattern of color development revealed that the black pigment was first deposited in patches in the central areas of an eyespot black ring and a parafocal element. These results of live in vivo imaging that covered wide wing area for a long time can serve as a foundation for studying the cellular dynamics of living wing tissues in butterflies. PMID:24586829

Genomic characterization and expression profiles upon bacterial infection of a novel cystatin B homologue from disk abalone (Haliotis discus discus).

PubMed

Premachandra, H K A; Wan, Qiang; Elvitigala, Don Anushka Sandaruwan; De Zoysa, Mahanama; Choi, Cheol Young; Whang, Ilson; Lee, Jehee

2012-12-01

Cystatins are a large family of cysteine proteinase inhibitors which are involved in diverse biological and pathological processes. In the present study, we identified a gene related to cystatin superfamily, AbCyt B, from disk abalone Haliotis discus discus by expressed sequence tag (EST) analysis and BAC library screening. The complete cDNA sequence of AbCyt B is comprised of 1967 nucleotides with a 306 bp open reading frame (ORF) encoding for 101 amino acids. The amino acid sequence consists of a single cystatin-like domain, which has a cysteine proteinase inhibitor signature, a conserved Gly in N-terminal region, QVVAG motif and a variant of PW motif. No signal peptide, disulfide bonds or carbohydrate side chains were identified. Analysis of deduced amino acid sequence revealed that AbCyt B shares up to 44.7% identity and 65.7% similarity with the cystatin B genes from other organisms. The genomic sequence of AbCyt B is approximately 8.4 Kb, consisting of three exons and two introns. Phylogenetic tree analysis showed that AbCyt B was closely related to the cystatin B from pacific oyster (Crassostrea gigas) under the family 1.Functional analysis of recombinant AbCyt B protein exhibited inhibitory activity against the papain, with almost 84% inhibition at a concentration of 3.5 μmol/L. In tissue expression analysis, AbCyt B transcripts were expressed abundantly in the hemocyte, gill, mantle, and digestive tract, while weakly in muscle, testis, and hepatopancreas. After the immune challenge with Vibrio parahemolyticus, the AbCyt B showed significant (P<0.05) up-regulation of relative mRNA expression in gill and hemocytes at 24 and 6 h of post infection, respectively. These results collectively suggest that AbCyst B is a potent inhibitor of cysteine proteinases and is also potentially involved in immune responses against invading bacterial pathogens in abalone. Copyright © 2012 Elsevier Ltd. All rights reserved.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) participates in anti-lipopolysaccharide factors (ALFs) gene expression in mud crab.

PubMed

Sun, Wan-Wei; Zhang, Xin-Xu; Wan, Wei-Song; Wang, Shu-Qi; Wen, Xiao-Bo; Zheng, Huai-Ping; Zhang, Yue-Ling; Li, Sheng-Kang

2017-02-01

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a key cytoplasm signal adaptor that mediates signals activated by tumor necrosis factor receptor (TNFR) superfamily and the Interleukin-1 receptor/Toll-like receptor (IL-1/TLR) superfamily. The full-length 2492 bp TRAF6 (Sp-TRAF6) from Scylla paramamosain contains 1800 bp of open reading frame (ORF) encoding 598 amino acids, including an N-terminal RING-type zinc finger, two TRAF-type zinc fingers and a conserved C-terminal meprin and TRAF homology (MATH) domain. Multiple alignment analysis shows that the putative amino acid sequence of Sp-TRAf6 has highest identity of 88% with Pt-TRAF6 from Portunus trituberculatus, while the similarity of Sp-TRAF6 with other crustacean sequences was 54-55%. RT-PCR analysis indicated that Sp-TRAF6 transcripts were predominantly expressed in the hepatopancreas and stomach, whereas it was barely detected in the heart and hemocytes in our study. Moreover, Sp-TRAF6 transcripts were significantly up-regulated after Vibrio parahemolyticus and LPS challenges. RNA interference assay was carried out used by siRNA to investigate the genes expression patterns regulated by Sp-TRAF6. The qRT-PCR results showed that silencing Sp-TRAF6 gene could inhibit SpALF1, SpALF2, SpALF5 and SpALF6 expression in hemocytes, while inhibit SpALF1, SpALF3, SpALF4, SpALF5 and SpALF6 expression in hepatopancreas. Taken together, the acute-phase response to immune challenges and the inhibition of SpALFs gene expression indicate that Sp-TRAF6 plays an important role in host defense against pathogen invasions via regulation of ALF gene expression in S. paramamosain. Copyright © 2016. Published by Elsevier Ltd.

Responses to a warming world: Integrating life history, immune investment, and pathogen resistance in a model insect species.

PubMed

Laughton, Alice M; O'Connor, Cian O; Knell, Robert J

2017-11-01

Environmental temperature has important effects on the physiology and life history of ectothermic animals, including investment in the immune system and the infectious capacity of pathogens. Numerous studies have examined individual components of these complex systems, but little is known about how they integrate when animals are exposed to different temperatures. Here, we use the Indian meal moth ( Plodia interpunctella ) to understand how immune investment and disease resistance react and potentially trade-off with other life-history traits. We recorded life-history (development time, survival, fecundity, and body size) and immunity (hemocyte counts, phenoloxidase activity) measures and tested resistance to bacterial ( E. coli ) and viral ( Plodia interpunctella granulosis virus) infection at five temperatures (20-30°C). While development time, lifespan, and size decreased with temperature as expected, moths exhibited different reproductive strategies in response to small changes in temperature. At cooler temperatures, oviposition rates were low but tended to increase toward the end of life, whereas warmer temperatures promoted initially high oviposition rates that rapidly declined after the first few days of adult life. Although warmer temperatures were associated with strong investment in early reproduction, there was no evidence of an associated trade-off with immune investment. Phenoloxidase activity increased most at cooler temperatures before plateauing, while hemocyte counts increased linearly with temperature. Resistance to bacterial challenge displayed a complex pattern, whereas survival after a viral challenge increased with rearing temperature. These results demonstrate that different immune system components and different pathogens can respond in distinct ways to changes in temperature. Overall, these data highlight the scope for significant changes in immunity, disease resistance, and host-parasite population dynamics to arise from small, biologically relevant changes to environmental temperature. In light of global warming, understanding these complex interactions is vital for predicting the potential impact of insect disease vectors and crop pests on public health and food security.

Host plant associated enhancement of immunity and survival in virus infected caterpillars.

PubMed

Smilanich, Angela M; Langus, Tara C; Doan, Lydia; Dyer, Lee A; Harrison, Joshua G; Hsueh, Jennifer; Teglas, Mike B

2018-01-01

Understanding the interaction between host plant chemistry, the immune response, and insect pathogens can shed light on host plant use by insect herbivores. In this study, we focused on how interactions between the insect immune response and plant secondary metabolites affect the response to a viral pathogen. Based upon prior research, we asked whether the buckeye caterpillar, Junonia coenia (Nymphalidae), which specializes on plants containing iridoid glycosides (IGs), is less able to resist the pathogenic effects of a densovirus infection when feeding on plants with high concentrations of IGs. In a fully factorial design, individuals were randomly assigned to three treatments, each of which had two levels: (1) exposed to the densovirus versus control, (2) placed on a plant species with high concentrations of IGs (Plantago lanceolata, Plantaginaceae) versus low concentrations of IGs (P. major), and (3) control versus surface sterilized to exclude surface microbes that may contribute to viral resistance. We measured phenoloxidase (PO) activity, hemocyte counts, and gut bacterial diversity (16S ribosomal RNA) during the fourth larval instar, as well as development time, pupal weight, and survival to adult. Individuals infected with the virus were immune-suppressed (as measured by PO response and hemocyte count) and developed significantly faster than virus-free individuals. Contrary to our predictions,mortality was significantly less for virus challengedindividuals reared on the high IG plant compared to the low IG plant.This suggests that plant secondary metabolites can influence survival from viral infection and may be associated with activation of PO. Removing egg microbes did not affect the immune response or survival of the larvae. In summary, these results suggest that plant secondary metabolites are important for survival against a viral pathogen. Even though the PO response was better on the high IG plant, the extent to which this result contributes to survival against the virus needs further investigation. Copyright © 2017 Elsevier Inc. All rights reserved.

Structural, functional, and evolutionary aspects of galectins in aquatic mollusks: From a sweet tooth to the Trojan horse

PubMed Central

Vasta, GR; Feng, C; Bianchet, MA; Bachvaroff, TR; Tasumi, S

2015-01-01

Galectins constitute a conserved and widely distributed lectin family characterized by their binding affinity for β-galactosides and a unique binding site sequence motif in the carbohydrate recognition domain (CRD). In spite of their structural conservation, galectins display a remarkable functional diversity, by participating in developmental processes, cell adhesion and motility, regulation of immune homeostasis, and recognition of glycans on the surface of viruses, bacteria and protozoan parasites. In contrast with mammals, and other vertebrate and invertebrate taxa, the identification and characterization of bona fide galectins in aquatic mollusks has been relatively recent. Most of the studies have focused on the identification and domain organization of galectin-like transcripts or proteins in diverse tissues and cell types, including hemocytes, and their expression upon environmental or infectious challenge. Lectins from the eastern oyster Crassostrea virginica, however, have been characterized in their molecular, structural and functional aspects and some notable features have become apparent in the galectin repertoire of aquatic mollusks. These including less diversified galectin repertoires and different domain organizations relative to those observed in vertebrates, carbohydrate specificity for blood group oligosaccharides, and up regulation of galectin expression by infectious challenge, a feature that supports their proposed role(s) in innate immune responses. Although galectins from some aquatic mollusks have been shown to recognize microbial pathogens and parasites and promote their phagocytosis, they can also selectively bind to phytoplankton components, suggesting that they also participate in uptake and intracellular digestion of microalgae. In addition, the experimental evidence suggests that the protozoan parasite Perkinsus marinus has co-evolved with the oyster host to be selectively recognized by the oyster hemocyte galectins over algal food or bacterial pathogens, thereby subverting the oyster’s innate immune/feeding recognition mechanisms to gain entry into the host cells. PMID:25982395

Transcript profiles of mitochondrial and cytoplasmic manganese superoxide dismutases in Exopalaemon carinicauda under ammonia stress

NASA Astrophysics Data System (ADS)

Ren, Hai; Li, Jian; Li, Jitao; Liu, Ping; Liang, Zhongxiu; Wu, Jianhua

2015-05-01

Superoxide dismutase (SOD) is one of the most important antioxidant defense enzymes, and is considered as the first line against oxidative stress. In this study, we cloned a mitochondrial manganese (Mn) SOD ( mMnSOD) cDNA from the ridgetail white prawn Exopalaemon carinicauda by using rapid amplification of cDNA ends (RACE) methods. The full-length cDNA for mMnSOD was 1 014-bp long, containing a 5'-untranslated region (UTR) of 37-bp, a 3'-UTR of 321-bp with a poly (A) tail, and included a 657-bp open reading frame encoding a protein of 218 amino acids with a 16-amino-acid signal peptide. The protein had a calculated molecular weight of 23.87 kDa and a theoretical isoelectric point of 6.75. The mMnSOD sequence included two putative N-glycosylation sites (NHT and NLS), the MnSOD signature sequence 180DVWEHAYY187, and four putative Mn binding sites (H48, H96, D180, and H184). Sequence comparison showed that the mMnSOD deduced amino acid sequence of E. carinicauda shared 97%, 95%, 89%, 84%, 82%, 72%, and 69% identity with that of Macrobrachium rosenbergii, Macrobrachium nipponense, Fenneropeneaus chinensis, Callinectes sapidus, Perisesarma bidens, Danio rerio, and Homo sapiens, resectively. Quantitative real-time RT-PCR analysis showed that mMnSOD transcripts were present in all E. carinicauda tissues examined, with the highest levels in the hepatopancreas. During an ammonia stress treatment, the transcript levels of mMnSOD and cMnSOD were up-regulated at 12 h in hemocytes and at 24 h in the hepatopancreas. As the duration of the ammonia stress treatment extended to 72 h, the transcript levels of mMnSOD and cMnSOD significantly decreased both in hemocytes and hepatopancreas. These findings indicate that the SOD system is induced to respond to acute ammonia stress, and may be involved in environmental stress responses in E. carinicauda.

Identification and transcriptional analysis of two types of lectins (SgCTL-1 and SgGal-1) from mollusk Solen grandis.

PubMed

Wei, Xiumei; Yang, Jianmin; Liu, Xiangquan; Yang, Dinglong; Xu, Jie; Fang, Jinghui; Wang, Weijun; Yang, Jialong

2012-08-01

C-type lectin and galectin are two types of animal carbohydrate-binding proteins which serve as pathogen recognition molecules and play crucial roles in the innate immunity of invertebrates. In the present study, a C-type lectin (designated as SgCTL-1) and galectin (designated as SgGal-1) were identified from mollusk Solen grandis, and their expression patterns, both in tissues and toward three pathogen-associated molecular patterns (PAMPs) stimulation were characterized. The full-length cDNA of SgCTL-1 and SgGal-1 was 1280 and 1466 bp, containing an open reading frame (ORF) of 519 and 1218 bp, respectively. Their deduced amino acid sequences showed high similarity to other members of C-type lectin and galectin superfamily, respectively. SgCTL-1 encoded a single carbohydrate-recognition domain (CRD), and the motif of Ca(2+)-binding site 2 was EPN (Glu(135)-Pro(136)-Asn(137)). While SgGal-1 encoded two CRDs, and the amino acid residues constituted the carbohydrate-binding motifs were well conserved in CRD1 but partially conserved in CRD2. Although SgCTL-1 and SgGal-1 exhibited different tissue expression pattern, they were both constitutively expressed in all tested tissues, including hemocytes, gonad, mantle, muscle, gill and hepatopancreas, and they were both highly expressed in hepatopancreas and gill. Furthermore, the mRNA expression of two lectins in hemocytes was significantly (P < 0.01) up-regulated with different levels after S. grandis were stimulated by lipopolysaccharide (LPS), peptidoglycan (PGN) or β-1,3-glucan. Our results suggested that SgCTL-1 and SgGal-1 from razor clam were two novel members of animal lectins, and they might function as pattern recognition receptors (PRRs) taking part in the process of pathogen recognition. Copyright © 2012 Elsevier Ltd. All rights reserved.

The genomic features of parasitism, Polyembryony and immune evasion in the endoparasitic wasp Macrocentrus cingulum.

PubMed

Yin, Chuanlin; Li, Meizhen; Hu, Jian; Lang, Kun; Chen, Qiming; Liu, Jinding; Guo, Dianhao; He, Kang; Dong, Yipei; Luo, Jiapeng; Song, Zhenkun; Walters, James R; Zhang, Wenqing; Li, Fei; Chen, Xuexin

2018-05-30

Parasitoid wasps are well-known natural enemies of major agricultural pests and arthropod borne diseases. The parasitoid wasp Macrocentrus cingulum (Hymenoptera: Braconidae) has been widely used to control the notorious insect pests Ostrinia furnacalis (Asian Corn Borer) and O. nubilalis (European corn borer). One striking phenomenon exhibited by M. cingulum is polyembryony, the formation of multiple genetically identical offspring from a single zygote. Moreover, M. cingulum employs a passive parasitic strategy by preventing the host's immune system from recognizing the embryo as a foreign body. Thus, the embryos evade the host's immune system and are not encapsulated by host hemocytes. Unfortunately, the mechanism of both polyembryony and immune evasion remains largely unknown. We report the genome of the parasitoid wasp M. cingulum. Comparative genomics analysis of M. cingulum and other 11 insects were conducted, finding some gene families with apparent expansion or contraction which might be linked to the parasitic behaviors or polyembryony of M. cingulum. Moreover, we present the evidence that the microRNA miR-14b regulates the polyembryonic development of M. cingulum by targeting the c-Myc Promoter-binding Protein 1 (MBP-1), histone-lysine N-methyltransferase 2E (KMT2E) and segmentation protein Runt. In addition, Hemomucin, an O-glycosylated transmembrane protein, protects the endoparasitoid wasp larvae from being encapsulated by host hemocytes. Motif and domain analysis showed that only the hemomucin in two endoparasitoids, M. cingulum and Venturia canescens, possessing the ability of passive immune evasion has intact mucin domain and similar O-glycosylation patterns, indicating that the hemomucin is a key factor modulating the immune evasion. The microRNA miR-14b participates in the regulation of polyembryonic development, and the O-glycosylation of the mucin domain in the hemomucin confers the passive immune evasion in this wasp. These key findings provide new insights into the polyembryony and immune evasion.

Vaccination with multimeric recombinant VP28 induces high protection against white spot syndrome virus in shrimp.

PubMed

Taengchaiyaphum, Suparat; Nakayama, Hideki; Srisala, Jiraporn; Khiev, Ratny; Aldama-Cano, Diva January; Thitamadee, Siripong; Sritunyalucksana, Kallaya

2017-11-01

To improve the efficacy of WSSV protection, multimeric (tetrameric) recombinant VP28 (4XrVP28) was produced and tested in comparison with those of monomeric VP28 (1XrVP28). In vitro binding of either 1XrVP28 or 4XrVP28 to shrimp hemocyte surface was evident as early as 10 min after protein inoculation. Similar results were obtained in vivo when shrimp were injected with recombinant proteins that the proteins bound to the hemocyte surface could be detected since 5 min after injection. Comparison of the WSSV protection efficiencies of 1XrVP28 or 4XrVP28 were performed by injection the purified 1XrVP28 or 4XrVP28 (22.5 μg/shrimp) and WSSV inoculum (1000 copies/shrimp) into shrimp. At 10 dpi, while shrimp injected with WSSV inoculum reached 100% mortality, shrimp injected with 1XrVP28 + WSSV or 4XrVP28 + WSSV showed relative percent survival (RPS) of 67% and 81%, respectively. PCR quantification revealed high number of WSSV in the moribund shrimp of WSSV- and 1XrVP28+WSSV-injected group. In contrast, lower number of WSSV copies were found in the survivors both from 1XrVP28+WSSV- or 4XrVP28+WSSV- injected groups. Histopathological analysis demonstrated the WSSV infected lesions found in the moribund from WSSV-infected group and 1XrVP28+WSSV-injected group, but less or none in the survivors. ELISA demonstrated that 4XrVP28 exhibited higher affinity binding to rPmRab7, a WSSV binding protein essential for WSSV entry to the cell than 1XrVP28. Taken together, the protection against WSSV in shrimp could be improved by application of multimeric rVP28. Copyright © 2017 Elsevier Ltd. All rights reserved.

A Pathogenic Nematode Targets Recognition Proteins to Avoid Insect Defenses

PubMed Central

Toubarro, Duarte; Avila, Mónica Martinez; Montiel, Rafael; Simões, Nelson

2013-01-01

Steinernema carpocapsae is a nematode pathogenic in a wide variety of insect species. The great pathogenicity of this nematode has been ascribed to its ability to overcome the host immune response; however, little is known about the mechanisms involved in this process. The analysis of an expressed sequence tags (EST) library in the nematode during the infective phase was performed and a highly abundant contig homologous to serine protease inhibitors was identified. In this work, we show that this contig is part of a 641-bp cDNA that encodes a BPTI-Kunitz family inhibitor (Sc-KU-4), which is up-regulated in the parasite during invasion and installation. Recombinant Sc-KU-4 protein was produced in Escherichia coli and shown to inhibit chymotrypsin and elastase activities in a dose-dependent manner by a competitive mechanism with Ki values of 1.8 nM and 2.6 nM, respectively. Sc-KU-4 also inhibited trypsin and thrombin activities to a lesser extent. Studies of the mode of action of Sc-KU-4 and its effects on insect defenses suggest that although Sc-KU-4 did not inhibit the activation of hemocytes or the formation of clotting fibers, it did inhibit hemocyte aggregation and the entrapment of foreign particles by fibers. Moreover, Sc-KU-4 avoided encapsulation and the deposition of clotting materials, which usually occurs in response to foreign particles. We show by protein-protein interaction that Sc-KU-4 targets recognition proteins of insect immune system such as masquerade-like and serine protease-like homologs. The interaction of Sc-KU-4 with these proteins explains the ability of the nematode to overcome host reactions and its large pathogenic spectrum, once these immune proteins are well conserved in insects. The discovery of this inhibitor targeting insect recognition proteins opens new avenues for the development of S . carpocapsae as a biological control agent and provides a new tool to study host-pathogen interactions. PMID:24098715

Real-time in vivo imaging of butterfly wing development: revealing the cellular dynamics of the pupal wing tissue.

PubMed

Iwata, Masaki; Ohno, Yoshikazu; Otaki, Joji M

2014-01-01

Butterfly wings are covered with regularly arranged single-colored scales that are formed at the pupal stage. Understanding pupal wing development is therefore crucial to understand wing color pattern formation. Here, we successfully employed real-time in vivo imaging techniques to observe pupal hindwing development over time in the blue pansy butterfly, Junonia orithya. A transparent sheet of epithelial cells that were not yet regularly arranged was observed immediately after pupation. Bright-field imaging and autofluorescent imaging revealed free-moving hemocytes and tracheal branches of a crinoid-like structure underneath the epithelium. The wing tissue gradually became gray-white, epithelial cells were arranged regularly, and hemocytes disappeared, except in the bordering lacuna, after which scales grew. The dynamics of the epithelial cells and scale growth were also confirmed by fluorescent imaging. Fluorescent in vivo staining further revealed that these cells harbored many mitochondria at the surface of the epithelium. Organizing centers for the border symmetry system were apparent immediately after pupation, exhibiting a relatively dark optical character following treatment with fluorescent dyes, as well as in autofluorescent images. The wing tissue exhibited slow and low-frequency contraction pulses with a cycle of approximately 10 to 20 minutes, mainly occurring at 2 to 3 days postpupation. The pulses gradually became slower and weaker and eventually stopped. The wing tissue area became larger after contraction, which also coincided with an increase in the autofluorescence intensity that might have been caused by scale growth. Examination of the pattern of color development revealed that the black pigment was first deposited in patches in the central areas of an eyespot black ring and a parafocal element. These results of live in vivo imaging that covered wide wing area for a long time can serve as a foundation for studying the cellular dynamics of living wing tissues in butterflies.

Effects of short-term exposure to environmentally relevant concentrations of different pharmaceutical mixtures on the immune response of the pond snail Lymnaea stagnalis.

PubMed

Gust, M; Fortier, M; Garric, J; Fournier, M; Gagné, F

2013-02-15

Pharmaceuticals are pollutants of potential concern in the aquatic environment where they are commonly introduced as complex mixtures via municipal effluents. Many reports underline the effects of pharmaceuticals on immune system of non target species. Four drug mixtures were tested, and regrouped pharmaceuticals by main therapeutic use: psychiatric (venlafaxine, carbamazepine, diazepam), antibiotic (ciprofloxacine, erythromycin, novobiocin, oxytetracycline, sulfamethoxazole, trimethoprim), hypolipemic (atorvastatin, gemfibrozil, benzafibrate) and antihypertensive (atenolol, furosemide, hydrochlorothiazide, lisinopril). Their effects were then compared with a treated municipal effluent known for its contamination, and its effects on the immune response of Lymnaea stagnalis. Adult L. stagnalis were exposed for 3 days to an environmentally relevant concentration of the four mixtures individually and as a global mixture. Effects on immunocompetence (hemocyte viability and count, ROS and thiol levels, phagocytosis) and gene expression were related to the immune response and oxidative stress: catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), Selenium-dependent glutathione peroxidase (SeGPx), two isoforms of the nitric oxide synthetase gene (NOS1 and NOS2), molluscan defensive molecule (MDM), Toll-like receptor 4 (TLR4), allograft inflammatory factor-1 (AIF) and heat-shock protein 70 (HSP70). Immunocompetence was differently affected by the therapeutic class mixtures compared to the global mixture, which increased hemocyte count, ROS levels and phagocytosis, and decreased intracellular thiol levels. TLR4 gene expression was the most strongly increased, especially by psychiatric mixture (19-fold), while AIF-1, GR and CAT genes were downregulated. A decision tree analysis revealed that the immunotoxic responses caused by the municipal effluent were comparable to those obtained with the global pharmaceutical mixture, and the latter shared similarity with the antibiotic mixture. This suggests that pharmaceutical mixtures in municipal effluents represent a risk for gastropods at the immunocompetence levels and the antibiotic group could represent a model therapeutic class for municipal effluent toxicity studies in L. stagnalis. Copyright © 2013 Elsevier B.V. All rights reserved.

Bacterial Infection of Fly Ovaries Reduces Egg Production and Induces Local Hemocyte Activation

PubMed Central

Brandt, Stephanie M.; Schneider, David S.

2009-01-01

Summary Morbidity, the state of being diseased, is an important aspect of pathogenesis that has gone relatively unstudied in fruit flies. Our interest is in characterizing how bacterial pathogenesis affects various physiologies of the fly. We chose to examine the fly ovary because we found bacterial infection had a striking effect on fly reproduction. We observed decreased egg laying after bacterial infection that correlated with increased bacterial virulence. We also found that bacteria colonized the ovary in a previously undescribed manner; bacteria were found in the posterior of the ovary, adjacent to the lateral oviduct. This local infection in the ovary resulted in melanization and activation of the cellular immune response at the site of infection. PMID:17400292

Gene, Immune and Cellular Responses to Single and Combined Space Flight Conditions-B (TripleLux-B):

NASA Image and Video Library

2015-03-31

ISS043E070945 (03/31/2015) --- ESA (European Space Agency) astronaut Samantha Cristoforetti, Expedition 43 flight engineer aboard the International Space Station, is seen working on a science experiment that includes photographic documentation of Cellular Responses to Single and Combined Space Flight Conditions. Some effects of the space environment level appear to act at the cellular level and it is important to understand the underlying mechanisms of these effects. This science project uses invertebrate hemocytes to focus on two aspects of cellular function which may have medical importance. The synergy between the effects of the space radiation environment and microgravity on cellular function is the goal of this experiment along with studying the impairment of immune functions under spaceflight conditions.

JAK/STAT signaling in Drosophila muscles controls the cellular immune response against parasitoid infection.

PubMed

Yang, Hairu; Kronhamn, Jesper; Ekström, Jens-Ola; Korkut, Gül Gizem; Hultmark, Dan

2015-12-01

The role of JAK/STAT signaling in the cellular immune response of Drosophila is not well understood. Here, we show that parasitoid wasp infection activates JAK/STAT signaling in somatic muscles of the Drosophila larva, triggered by secretion of the cytokines Upd2 and Upd3 from circulating hemocytes. Deletion of upd2 or upd3, but not the related os (upd1) gene, reduced the cellular immune response, and suppression of the JAK/STAT pathway in muscle cells reduced the encapsulation of wasp eggs and the number of circulating lamellocyte effector cells. These results suggest that JAK/STAT signaling in muscles participates in a systemic immune defense against wasp infection. © 2015 The Authors. Published under the terms of the CC BY 4.0 license.

Molecular identification of disk abalone (Haliotis discus discus) tetraspanin 33 and CD63: Insights into potent players in the disk abalone host defense system.

PubMed

Priyathilaka, Thanthrige Thiunuwan; Bathige, S D N K; Herath, H M L P B; Lee, Sukkyoung; Lee, Jehee

2017-10-01

Tetraspanins are a superfamily of transmembrane proteins involved in a diverse range of physiological processes including differentiation, adhesion, signal transduction, cell motility, and immune responses. In the present study, two tetraspanins, CD63 and tetraspanin 33 (TSPAN33) from disk abalone (AbCD63 and AbTSPAN33), were identified and characterized at the molecular level. The coding sequences for AbCD63 and AbTSPAN33 encoded polypeptides of 234 and 290 amino acids (aa) with predicted molecular mass of 25.3 and 32.5 kDa, respectively. The deduced AbCD63 and AbTSPAN33 protein sequences were also predicted to have a typical tetraspanin domain architecture, including four transmembrane domains (TM), short N- and C- terminal regions, a short intracellular loop, as well as a large and small extracellular loop. A characteristic CCG motif and cysteine residues, which are highly conserved across CD63 and TSPAN33 proteins of different species, were present in the large extracellular loop of both abalone tetraspanins. Phylogenetic analysis revealed that the AbCD63 and AbTSPAN33 clustered in the invertebrate subclade of tetraspanins, thus exhibiting a close relationship with tetraspanins of other mollusks. The AbCD63 and AbTSPAN33 mRNA transcripts were detected at early embryonic development stages of disk abalone with significantly higher amounts at the trochophore stage, suggesting the involvement of these proteins in embryonic development. Both AbCD63 and AbTSPAN33 were ubiquitously expressed in all the tissues of unchallenged abalones analyzed, with the highest expression levels found in hemocytes. Moreover, significant induction of AbCD63 and AbTSPAN33 mRNA expression was observed in immunologically important tissues, such as hemocytes and gills, upon stimulation with live bacteria (Vibrio parahaemolyticus and Listeria monocytogenes), virus (viral hemorrhagic septicemia virus), and two potent immune stimulators [polyinosinic:polycytidylic acid (poly I:C) and lipopolysaccharide (LPS)]. Collectively, these findings suggest that AbCD63 and AbTSPAN33 are involved in innate immune responses in disk abalone during pathogenic stress. Copyright © 2017 Elsevier Ltd. All rights reserved.

Interactive effects of n-TiO2 and 2,3,7,8-TCDD on the marine bivalve Mytilus galloprovincialis.

PubMed

Canesi, Laura; Frenzilli, Giada; Balbi, Teresa; Bernardeschi, Margherita; Ciacci, Caterina; Corsolini, Simonetta; Della Torre, Camilla; Fabbri, Rita; Faleri, Claudia; Focardi, Silvano; Guidi, Patrizia; Kočan, Anton; Marcomini, Antonio; Mariottini, Michela; Nigro, Marco; Pozo-Gallardo, Karla; Rocco, Lucia; Scarcelli, Vittoria; Smerilli, Arianna; Corsi, Ilaria

2014-08-01

Despite the growing concern over the potential biological impact of nanoparticles (NPs) in the aquatic environment, little is known about their interactions with other pollutants. The bivalve Mytilus sp, largely utilized as a sentinel for marine contamination, has been shown to represent a significant target for different types of NP, including n-TiO2, one of the most widespread in use. In this work, the possible interactive effects of n-TiO2 and 2,3,7,8-TCDD, chosen as models of NP and organic contaminant, respectively, were investigated in Mytilus galloprovincialis. In vitro experiments with n-TiO2 and TCDD, alone and in combination, were carried out in different conditions (concentrations and times of exposure), depending on the target (hemocytes, gill cells and biopsies) and the endpoint measured. Mussels were also exposed in vivo to n-TiO2 (100 μg L(-1)) or to TCDD (0.25 μg L(-1)), alone and in combination, for 96 h. A wide range of biomarkers, from molecular to tissue level, were measured: lysosomal membrane stability and phagocytosis in hemocytes, ATP-binding cassette efflux transporters in gills (gene transcription and efflux activity), several biomarkers of genotoxicity in gill and digestive cells (DNA damage, random amplified polymorphic DNA-RAPD changes), lysosomal biomarkers and transcription of selected genes in the digestive gland. The results demonstrate that n-TiO2 and TCDD can exert synergistic or antagonistic effects, depending on experimental condition, cell/tissue and type of measured response. Some of these interactions may result from a significant increase in TCDD accumulation in whole mussel organisms in the presence of n-TiO2, indicating a Trojan horse effect. The results represent the most extensive data obtained so far on the sub-lethal effects of NPs and organic contaminants in aquatic organisms. Moreover, these data extend the knowledge on the molecular and cellular targets of NPs in bivalves. Copyright © 2013 Elsevier B.V. All rights reserved.

Big defensins, a diverse family of antimicrobial peptides that follows different patterns of expression in hemocytes of the oyster Crassostrea gigas.

PubMed

Rosa, Rafael D; Santini, Adrien; Fievet, Julie; Bulet, Philippe; Destoumieux-Garzón, Delphine; Bachère, Evelyne

2011-01-01

Big defensin is an antimicrobial peptide composed of a highly hydrophobic N-terminal region and a cationic C-terminal region containing six cysteine residues involved in three internal disulfide bridges. While big defensin sequences have been reported in various mollusk species, few studies have been devoted to their sequence diversity, gene organization and their expression in response to microbial infections. Using the high-throughput Digital Gene Expression approach, we have identified in Crassostrea gigas oysters several sequences coding for big defensins induced in response to a Vibrio infection. We showed that the oyster big defensin family is composed of three members (named Cg-BigDef1, Cg-BigDef2 and Cg-BigDef3) that are encoded by distinct genomic sequences. All Cg-BigDefs contain a hydrophobic N-terminal domain and a cationic C-terminal domain that resembles vertebrate β-defensins. Both domains are encoded by separate exons. We found that big defensins form a group predominantly present in mollusks and closer to vertebrate defensins than to invertebrate and fungi CSαβ-containing defensins. Moreover, we showed that Cg-BigDefs are expressed in oyster hemocytes only and follow different patterns of gene expression. While Cg-BigDef3 is non-regulated, both Cg-BigDef1 and Cg-BigDef2 transcripts are strongly induced in response to bacterial challenge. Induction was dependent on pathogen associated molecular patterns but not damage-dependent. The inducibility of Cg-BigDef1 was confirmed by HPLC and mass spectrometry, since ions with a molecular mass compatible with mature Cg-BigDef1 (10.7 kDa) were present in immune-challenged oysters only. From our biochemical data, native Cg-BigDef1 would result from the elimination of a prepropeptide sequence and the cyclization of the resulting N-terminal glutamine residue into a pyroglutamic acid. We provide here the first report showing that big defensins form a family of antimicrobial peptides diverse not only in terms of sequences but also in terms of genomic organization and regulation of gene expression.

Big Defensins, a Diverse Family of Antimicrobial Peptides That Follows Different Patterns of Expression in Hemocytes of the Oyster Crassostrea gigas

PubMed Central

Rosa, Rafael D.; Santini, Adrien; Fievet, Julie; Bulet, Philippe; Destoumieux-Garzón, Delphine; Bachère, Evelyne

2011-01-01

Background Big defensin is an antimicrobial peptide composed of a highly hydrophobic N-terminal region and a cationic C-terminal region containing six cysteine residues involved in three internal disulfide bridges. While big defensin sequences have been reported in various mollusk species, few studies have been devoted to their sequence diversity, gene organization and their expression in response to microbial infections. Findings Using the high-throughput Digital Gene Expression approach, we have identified in Crassostrea gigas oysters several sequences coding for big defensins induced in response to a Vibrio infection. We showed that the oyster big defensin family is composed of three members (named Cg-BigDef1, Cg-BigDef2 and Cg-BigDef3) that are encoded by distinct genomic sequences. All Cg-BigDefs contain a hydrophobic N-terminal domain and a cationic C-terminal domain that resembles vertebrate β-defensins. Both domains are encoded by separate exons. We found that big defensins form a group predominantly present in mollusks and closer to vertebrate defensins than to invertebrate and fungi CSαβ-containing defensins. Moreover, we showed that Cg-BigDefs are expressed in oyster hemocytes only and follow different patterns of gene expression. While Cg-BigDef3 is non-regulated, both Cg-BigDef1 and Cg-BigDef2 transcripts are strongly induced in response to bacterial challenge. Induction was dependent on pathogen associated molecular patterns but not damage-dependent. The inducibility of Cg-BigDef1 was confirmed by HPLC and mass spectrometry, since ions with a molecular mass compatible with mature Cg-BigDef1 (10.7 kDa) were present in immune-challenged oysters only. From our biochemical data, native Cg-BigDef1 would result from the elimination of a prepropeptide sequence and the cyclization of the resulting N-terminal glutamine residue into a pyroglutamic acid. Conclusions We provide here the first report showing that big defensins form a family of antimicrobial peptides diverse not only in terms of sequences but also in terms of genomic organization and regulation of gene expression. PMID:21980497

The effects of stress hormones on immune function may be vital for the adaptive reconfiguration of the immune system during fight-or-flight behavior.

PubMed

Adamo, Shelley A

2014-09-01

Intense, short-term stress (i.e., robust activation of the fight-or-flight response) typically produces a transient decline in resistance to disease in animals across phyla. Chemical mediators of the stress response (e.g., stress hormones) help induce this decline, suggesting that this transient immunosuppression is an evolved response. However, determining the function of stress hormones on immune function is difficult because of their complexity. Nevertheless, evidence suggests that stress hormones help maintain maximal resistance to disease during the physiological changes needed to optimize the body for intense physical activity. Work on insects demonstrates that stress hormones both shunt resources away from the immune system during fight-or-flight responses as well as reconfigure the immune system. Reconfiguring the immune system minimizes the impact of the loss of these resources and reduces the increased costs of some immune functions due to the physiological changes demanded by the fight-or-flight response. For example, during the stress response of the cricket Gryllus texensis, some molecular resources are shunted away from the immune system and toward lipid transport, resulting in a reduction in resistance to disease. However, insects' immune cells (hemocytes) have receptors for octopamine (the insect stress neurohormone). Octopamine increases many hemocyte functions, such as phagocytosis, and these changes would tend to mitigate the decline in immunity due to the loss of molecular resources. Moreover, because the stress response generates oxidative stress, some immune responses are probably more costly when activated during a stress response (e.g., those that produce reactive molecules). Some of these immune responses are depressed during stress in crickets, while others, whose costs are probably not increased during a stress response, are enhanced. Some effects of stress hormones on immune systems may be better understood as examples of reconfiguration rather than as mediating a trade-off. © The Author 2014. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.

Identification, characterization, and functional analysis of Tube and Pelle homologs in the mud crab Scylla paramamosain.

PubMed

Li, Xin-Cang; Zhang, Xiao-Wen; Zhou, Jun-Fang; Ma, Hong-Yu; Liu, Zhi-Dong; Zhu, Lei; Yao, Xiao-Juan; Li, Lin-Gui; Fang, Wen-Hong

2013-01-01

Tube and Pelle are essential components in Drosophila Toll signaling pathway. In this study, we characterized a pair of crustacean homologs of Tube and Pelle in Scylla paramamosain, namely, SpTube and SpPelle, and analyzed their immune functions. The full-length cDNA of SpTube had 2052 bp with a 1578 bp open reading frame (ORF) encoding a protein with 525 aa. A death domain (DD) and a kinase domain were predicted in the deduced protein. The full-length cDNA of SpPelle had 3825 bp with a 3420 bp ORF encoding a protein with 1140 aa. The protein contained a DD and a kinase domain. Two conserved repeat motifs previously called Tube repeat motifs present only in insect Tube or Tube-like sequences were found between these two domains. Alignments and structure predictions demonstrated that SpTubeDD and SpPelleDD significantly differed in sequence and 3D structure. Similar to TubeDD, SpTubeDD contained three common conserved residues (R, K, and R) on one surface that may mediate SpMyD88 binding and two common residues (A and A) on the other surface that may contribute to Pelle binding. By contrast, SpPelleDD lacked similar conservative residues. SpTube, insect Tube-like kinases, and human IRAK4 were found to be RD kinases with an RD dipeptide in the kinase domain. SpPelle, Pelle, insect Pelle-like kinases, and human IRAK1 were found to be non-RD kinases lacking an RD dipeptide. Both SpTube and SpPelle were highly expressed in hemocytes, gills, and hepatopancreas. Upon challenge, SpTube and SpPele were significantly increased in hemocytes by Gram-negative or Gram-positive bacteria, whereas only SpPelle was elevated by White Spot Syndrome Virus. The pull-down assay showed that SpTube can bind to both SpMyD88 and SpPelle. These results suggest that SpTube, SpPelle, and SpMyD88 may form a trimeric complex involved in the immunity of mud crabs against both Gram-negative and Gram-positive bacteria.

Identification, Characterization, and Functional Analysis of Tube and Pelle Homologs in the Mud Crab Scylla paramamosain

PubMed Central

Zhou, Jun-Fang; Ma, Hong-Yu; Liu, Zhi-Dong; Zhu, Lei; Yao, Xiao-Juan; Li, Lin-Gui; Fang, Wen-Hong

2013-01-01

Tube and Pelle are essential components in Drosophila Toll signaling pathway. In this study, we characterized a pair of crustacean homologs of Tube and Pelle in Scylla paramamosain, namely, SpTube and SpPelle, and analyzed their immune functions. The full-length cDNA of SpTube had 2052 bp with a 1578 bp open reading frame (ORF) encoding a protein with 525 aa. A death domain (DD) and a kinase domain were predicted in the deduced protein. The full-length cDNA of SpPelle had 3825 bp with a 3420 bp ORF encoding a protein with 1140 aa. The protein contained a DD and a kinase domain. Two conserved repeat motifs previously called Tube repeat motifs present only in insect Tube or Tube-like sequences were found between these two domains. Alignments and structure predictions demonstrated that SpTubeDD and SpPelleDD significantly differed in sequence and 3D structure. Similar to TubeDD, SpTubeDD contained three common conserved residues (R, K, and R) on one surface that may mediate SpMyD88 binding and two common residues (A and A) on the other surface that may contribute to Pelle binding. By contrast, SpPelleDD lacked similar conservative residues. SpTube, insect Tube-like kinases, and human IRAK4 were found to be RD kinases with an RD dipeptide in the kinase domain. SpPelle, Pelle, insect Pelle-like kinases, and human IRAK1 were found to be non-RD kinases lacking an RD dipeptide. Both SpTube and SpPelle were highly expressed in hemocytes, gills, and hepatopancreas. Upon challenge, SpTube and SpPele were significantly increased in hemocytes by Gram-negative or Gram-positive bacteria, whereas only SpPelle was elevated by White Spot Syndrome Virus. The pull-down assay showed that SpTube can bind to both SpMyD88 and SpPelle. These results suggest that SpTube, SpPelle, and SpMyD88 may form a trimeric complex involved in the immunity of mud crabs against both Gram-negative and Gram-positive bacteria. PMID:24116143

TmSR-C, scavenger receptor class C, plays a pivotal role in antifungal and antibacterial immunity in the coleopteran insect Tenebrio molitor.

PubMed

Kim, Soo Gon; Jo, Yong Hun; Seong, Jeong Hwan; Park, Ki Beom; Noh, Mi Young; Cho, Jun Ho; Ko, Hye Jin; Kim, Chang Eun; Tindwa, Hamisi; Patnaik, Bharat Bhusan; Bang, In Seok; Lee, Yong Seok; Han, Yeon Soo

2017-10-01

Scavenger receptors (SRs) constitute a family of membrane-bound receptors that bind to multiple ligands. The SR family of proteins is involved in removing cellular debris, oxidized low-density lipoproteins, and pathogens. Specifically, class C scavenger receptors (SR-C) have also been reported to be involved in phagocytosis of gram-positive and -negative bacteria in Drosophila and viruses in shrimp. However, reports are unavailable regarding the role of SR-C in antifungal immune mechanisms in insects. In this study, a full-length Tenebrio molitor SR-C (TmSR-C) sequence was obtained by 5'- and 3'-Rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The TmSR-C full-length cDNA comprised 1671 bp with 5'- and 3'-untranslated regions of 23- and 107-bp, respectively. TmSR-C encodes a putative protein of 556 amino acid residues that is constitutively expressed in all tissues of late instar larvae and 2-day-old adults, with the highest transcript levels observed in hemocytes of larvae and adults. TmSR-C mRNA showed a 2.5-fold and 3-fold increase at 24 and 6 h after infection with Candida albicans and β-glucan, respectively. Immunoassay with TmSR-C polyclonal antibody showed induction of the putative protein in the cytosols of hemocytes at 3 h after inoculation of C. albicans. RNA interference (RNAi)-based gene silencing and phagocytosis assays were used to understand the role of TmSR-C in antifungal immunity. Silencing of TmSR-C transcripts reduced the survivability of late instar larvae at 2 days post-inoculation of C. albicans, Escherichia coli, or Staphylococcus aureus. Furthermore, in TmSR-C-silenced larvae, there was a decline in the rate of microorganism phagocytosis. Taken together, results of this study suggest that TmSR-C plays a pivotal role in phagocytosing not only fungi but also gram-negative and -positive bacteria in T. molitor. Copyright © 2017. Published by Elsevier Ltd.

Two C-type lectins from shrimp Litopenaeus vannamei that might be involved in immune response against bacteria and virus.

PubMed

Wei, Xiumei; Liu, Xiangquan; Yang, Jianmin; Fang, Jinghui; Qiao, Hongjin; Zhang, Ying; Yang, Jialong

2012-01-01

C-type lectins play crucial roles in innate immunity to recognize and eliminate pathogens efficiently. In the present study, two C-type lectins from shrimp Litopenaeus vannamei (designated as LvLectin-1 and LvLectin-2) were identified, and their expression patterns, both in tissues and toward pathogen stimulation, were then characterized. The full-length cDNA of LvLectin-1 and LvLectin-2 was 567 and 625 bp, containing an open reading frame (ORF) of 471 and 489 bp, respectively, and deduced amino acid sequences showed high similarity to other members of C-type lectin superfamily. Both two C-type lectins encoded a single carbohydrate-recognition domain (CRD). The motif of Ca(2+) binding site 2 in CRD, which determined carbohydrate-binding specificity, was QPN (Gln(122)-Pro(123)-Asn(124)) in LvLectin-1, but QPD (Gln(128)-Pro(129)-Asp(130)) in LvLectin-2. Two C-type lectins exhibited similar tissue expression pattern, for their mRNA were both constitutively expressed in all tested tissues, including hepatopancreas, muscle, gill, hemocytes, gonad and heart, furthermore they were both mostly expressed in hepatopancreas, though the expression level of LvLectin-2 was much higher than LvLectin-1. The expression level of two C-type lectins mRNA in hemocytes varied greatly after the challenge of Listonella anguillarum or WSSV. After L. anguillarum challenge, the expression of both C-type lectins were significantly (P<0.01) up-regulated compared with blank group, and LvLectin-1 exhibited higher level than LvLectin-2; while after the stimulation of WSSV, the expression of LvLectin-2 was significantly up-regulated at 6 h (P<0.01) and 12 h (P<0.05), but the expression level of LvLectin-1 down-regulated significantly (P<0.01) to 0.4-fold at 6 and 12 h post-stimulation. The results indicated that the two C-type lectins might be involved in immune response toward pathogen infection, and they might perform different recognition specificity toward bacteria or virus. Copyright © 2011 Elsevier Ltd. All rights reserved.

Hemolin-A lepidopteran anti-viral defense factor?

PubMed

Terenius, Olle

2008-01-01

Immunity in insects has largely focused on responses towards bacteria and fungi, but recently the study of immune responses against viral infections has also received attention. In Lepidoptera, phagocytosis and encapsulation mediated by hemocytes, and apoptosis are part of the response against virus infection; however, many studies also suggest the presence of unknown factors involved in the anti-viral defense. An up-regulation of the lepidopteran-specific pattern recognition protein Hemolin after baculovirus infection in the Chinese oak silkmoth and discovery of putative virus responsive elements in the up-stream regions of Hemolin in the Cecropia moth and the Tobacco horn worm could suggest that Hemolin is involved in virus defense. In this paper, a number of studies investigating baculovirus pathogenesis, and others analyzing Hemolin expression have been revisited leading to the speculation that Hemolin could be engaged in several anti-viral processes.

Genotoxic effects induced by the exposure to an environmental mixture of illicit drugs to the zebra mussel.

PubMed

Parolini, Marco; Magni, Stefano; Castiglioni, Sara; Binelli, Andrea

2016-10-01

Despite the growing interest on the presence of illicit drugs in freshwater ecosystems, just recently the attention has been focused on their potential toxicity towards non-target aquatic species. However, these studies largely neglected the effects induced by exposure to complex mixtures of illicit drugs, which could be different compared to those caused by single psychoactive molecules. This study was aimed at investigating the genetic damage induced by a 14-day exposure to a realistic mixture of the most common illicit drugs found in surface waters worldwide (cocaine, benzoylecgonine, amphetamine, morphine and 3,4-methylenedioxymethamphetamine) on the zebra mussel (Dreissena polymorpha). The mixture caused a significant increase of DNA fragmentation and triggered the apoptotic process and micronuclei formation in zebra mussel hemocytes, pointing out its potential genotoxicity towards this bivalve species. Copyright © 2016 Elsevier Inc. All rights reserved.

Sublethal pesticide doses negatively affect survival and the cellular responses in American foulbrood-infected honeybee larvae

NASA Astrophysics Data System (ADS)

López, Javier Hernández; Krainer, Sophie; Engert, Antonia; Schuehly, Wolfgang; Riessberger-Gallé, Ulrike; Crailsheim, Karl

2017-02-01

Disclosing interactions between pesticides and bee infections is of most interest to understand challenges that pollinators are facing and to which extent bee health is compromised. Here, we address the individual and combined effect that three different pesticides (dimethoate, clothianidin and fluvalinate) and an American foulbrood (AFB) infection have on mortality and the cellular immune response of honeybee larvae. We demonstrate for the first time a synergistic interaction when larvae are exposed to sublethal doses of dimethoate or clothianidin in combination with Paenibacillus larvae, the causative agent of AFB. A significantly higher mortality than the expected sum of the effects of each individual stressor was observed in co-exposed larvae, which was in parallel with a drastic reduction of the total and differential hemocyte counts. Our results underline that characterizing the cellular response of larvae to individual and combined stressors allows unmasking previously undetected sublethal effects of pesticides in colony health.

Isolation and characterization of an immunosuppressive protein from venom of the pupa-specific endoparasitoid Pteromalus puparum.

PubMed

Wu, Ma-li; Ye, Gong-yin; Zhu, Jia-ying; Chen, Xue-xin; Hu, Cui

2008-10-01

In hymenopteran parasitoids devoid of symbiotic viruses, venom proteins appear to play a major role in host immune suppression and host regulation. Not much is known about the active components of venom proteins in these parasitoids, especially those that have the functions involved in the suppression of host cellular immunity. Here, we report the isolation and characterization of a venom protein Vn.11 with 24.1 kDa in size from Pteromalus puparum, a pupa-specific endoparasitoid of Pieris rapae. The Vn.11 venom protein is isolated with the combination of ammonium sulfate precipitation and anion exchange chromatography, and its purity is verified using SDS-PAGE analysis. Like crude venom, the Vn.11 venom protein significantly inhibits the spreading behavior and encapsulation ability of host hemocytes in vitro. It is suggested that this protein is an actual component of P. puparum crude venom as host cellular-immune suppressive factor.

The Role of Cytokine PF4 in the Antiviral Immune Response of Shrimp

PubMed Central

Chen, Yulei; Cao, Jiao; Zhang, Xiaobo

2016-01-01

During viral infection in vertebrates, cytokines play important roles in the host defense against the virus. However, the function of cytokines in invertebrates has not been well characterized. In this study, shrimp cytokines involved in viral infection were screened using a cytokine antibody microarray. The results showed that three cytokines, the Fas receptor (Fas), platelet factor 4 (PF4) and interleukin-22 (IL-22), were significantly upregulated in the white spot syndrome virus (WSSV)-challenged shrimp, suggesting that these cytokines played positive regulatory roles in the immune response of shrimp against the virus. Further experiments revealed that PF4 had positive effects on the antiviral immunity of shrimp by enhancing the shrimp phagocytic activity and inhibiting the apoptotic activity of virus-infected hemocytes. Therefore, our study presented a novel mechanism of cytokines in the innate immunity of invertebrates. PMID:27631372

Sublethal pesticide doses negatively affect survival and the cellular responses in American foulbrood-infected honeybee larvae.

PubMed

López, Javier Hernández; Krainer, Sophie; Engert, Antonia; Schuehly, Wolfgang; Riessberger-Gallé, Ulrike; Crailsheim, Karl

2017-02-01

Disclosing interactions between pesticides and bee infections is of most interest to understand challenges that pollinators are facing and to which extent bee health is compromised. Here, we address the individual and combined effect that three different pesticides (dimethoate, clothianidin and fluvalinate) and an American foulbrood (AFB) infection have on mortality and the cellular immune response of honeybee larvae. We demonstrate for the first time a synergistic interaction when larvae are exposed to sublethal doses of dimethoate or clothianidin in combination with Paenibacillus larvae, the causative agent of AFB. A significantly higher mortality than the expected sum of the effects of each individual stressor was observed in co-exposed larvae, which was in parallel with a drastic reduction of the total and differential hemocyte counts. Our results underline that characterizing the cellular response of larvae to individual and combined stressors allows unmasking previously undetected sublethal effects of pesticides in colony health.

PcToll2 positively regulates the expression of antimicrobial peptides by promoting PcATF4 translocation into the nucleus.

PubMed

Lan, Jiang-Feng; Zhao, Li-Juan; Wei, Shun; Wang, Yuan; Lin, Li; Li, Xin-Cang

2016-11-01

Drosophila Toll and mammalian Toll-like receptors (TLRs) are a family of evolutionarily conserved immune receptors that play a crucial role in the first-line defense against intruded pathogens. Activating transcription factor 4 (ATF4), a member of the ATF/CREB transcription factor family, is an important factor that participates in TLR signaling and other physiological processes. However, in crustaceans, whether ATF4 homologs were involved in TLR signaling remains unclear. In the current study, we identified a Toll homolog PcToll2 and a novel ATF4 homolog PcATF4 from Procambarus clarkii, and analyzed the likely regulatory activity of PcATF4 in PcToll2 signaling. The complete cDNA sequence of PcToll2 was 4175 bp long containing an open reading frame of 2820 bp encoding a 939-amino acid protein, and the cDNA sequence of PcATF4 was 2027 bp long with an open reading frame of 1296 bp encoding a 431-amino acid protein. PcToll2 and human TLR4 shared the high identity and they were grouped into a cluster. Furthermore, PcToll2 had a close relationship with other shrimp TLRs that possessed potential antibacterial activity. PcToll2 was highly expressed in the hemocytes, heart and gills, while PcATF4 mainly distributed in gills. Upon challenge with Vibrio parahemolyticus, PcToll2 and PcATF4 together with the antimicrobial peptides of ALF1 and ALF2 were significantly up-regulated in the hemocytes, and the PcATF4 was translocated into the nucleus. After PcToll2 silencing and challenge with Vibrio, the translocation of PcATF4 into the nucleus was inhibited and the expression of ALF1 and ALF2 was reduced, but the expression of PcDorsal and PcSTAT was not affected. Furthermore, after PcATF4 knockdown and challenge with or without Vibrio, the expression of ALF1 and ALF2 was also decreased while the expression of PcToll2 was upregulated. These results suggested that PcToll2 might regulate the expression of ALF1 and ALF2 by promoting the import of PcATF4, instead of the routine transcription factor PcDorsal, into the nucleus participating in the immune defense against Gram-negative bacteria. Copyright © 2016 Elsevier Ltd. All rights reserved.

Molecular and immunological responses of the giant freshwater prawn, Macrobrachium rosenbergii, to the organophosphorus insecticide, trichlorfon.

PubMed

Chang, Chin-Chyuan; Rahmawaty, Atiek; Chang, Zhong-Wen

2013-04-15

Trichlorfon is an organophosphorus (OP) insecticide that is used as an agriculture pesticide to destroy insects, a human medicine to combat internal parasites, and an ectoparasiticide in the livestock and aquaculture industries, but which has caused aquatic toxicity in the prawn industry. The aim of this study was to investigate the effects of trichlorfon on molecular and enzymatic processes of the immunological response of the giant freshwater prawn, Macrobrachium rosenbergii, at 0, 0.2, and 0.4mgL(-1) with 0, 3, 6, 12, and 24h of exposure. The total hemocyte count (THC), respiratory bursts (RBs), phenoloxidase (PO) activity, and superoxide dismutase (SOD) activity were examined to evaluate immunological responses and oxidative stress. Results showed that THCs of the prawn exposed to trichlorfon at both concentrations (0.2 and 0.4mgL(-1)) had increased after 12 and 24h; SOD and PO activities had significantly increased at 3h, whereas production of RBs had dramatically increased as oxidative stress at each sampling time after exposure to trichlorfon compared to the control. A potential biomarker of OPs, acetylcholinesterase (AChE) revealed a significant decrease after exposure for 6h, and showed a time-dependent tendency. Immune gene expressions, including prophenoloxidase (proPO), the lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP), peroxinectin (PE), α2-macroglubulin (α2M), transglutaminase (TG), and copper, zinc (Cu,Zn)-SOD, of prawns exposed to trichlorfon at 0, 0.2, and 0.4mgL(-1) for 0, 6, and 24h were further evaluated. Expressions of all of the immune genes significantly decreased when prawns were exposed to 0.4mgL(-1) trichlorfon for 24h, and among them, an increase in SOD expression was seen after exposure to 0.4mgL(-1) for 6h. Prawns exposed to trichlorfon within 24h exhibited the decrease of circulating hemocytes, and also the induction of oxidative stress, which caused subsequent damage to DNA formation of immune genes. From these results, we concluded that immunological responses and immune gene expressions of prawn exposed to trichlorfon at 0.4mgL(-1) for 24h were perturbed, thus causing a deficiency in immunity and subsequent increased susceptibility to pathogen infections. Copyright © 2013 Elsevier B.V. All rights reserved.

Insights into the innate immunity of the Mediterranean mussel Mytilus galloprovincialis

PubMed Central

2011-01-01

Background Sessile bivalves of the genus Mytilus are suspension feeders relatively tolerant to a wide range of environmental changes, used as sentinels in ecotoxicological investigations and marketed worldwide as seafood. Mortality events caused by infective agents and parasites apparently occur less in mussels than in other bivalves but the molecular basis of such evidence is unknown. The arrangement of Mytibase, interactive catalogue of 7,112 transcripts of M. galloprovincialis, offered us the opportunity to look for gene sequences relevant to the host defences, in particular the innate immunity related genes. Results We have explored and described the Mytibase sequence clusters and singletons having a putative role in recognition, intracellular signalling, and neutralization of potential pathogens in M. galloprovincialis. Automatically assisted searches of protein signatures and manually cured sequence analysis confirmed the molecular diversity of recognition/effector molecules such as the antimicrobial peptides and many carbohydrate binding proteins. Molecular motifs identifying complement C1q, C-type lectins and fibrinogen-like transcripts emerged as the most abundant in the Mytibase collection whereas, conversely, sequence motifs denoting the regulatory cytokine MIF and cytokine-related transcripts represent singular and unexpected findings. Using a cross-search strategy, 1,820 putatively immune-related sequences were selected to design oligonucleotide probes and define a species-specific Immunochip (DNA microarray). The Immunochip performance was tested with hemolymph RNAs from mussels injected with Vibrio splendidus at 3 and 48 hours post-treatment. A total of 143 and 262 differentially expressed genes exemplify the early and late hemocyte response of the Vibrio-challenged mussels, respectively, with AMP trends confirmed by qPCR and clear modulation of interrelated signalling pathways. Conclusions The Mytibase collection is rich in gene transcripts modulated in response to antigenic stimuli and represents an interesting window for looking at the mussel immunome (transcriptomes mediating the mussel response to non-self or abnormal antigens). On this basis, we have defined a new microarray platform, a mussel Immunochip, as a flexible tool for the experimental validation of immune-candidate sequences, and tested its performance on Vibrio-activated mussel hemocytes. The microarray platform and related expression data can be regarded as a step forward in the study of the adaptive response of the Mytilus species to an evolving microbial world. PMID:21269501

The Madagascar hissing cockroach as a novel surrogate host for Burkholderia pseudomallei, B. mallei and B. thailandensis.

PubMed

Fisher, Nathan A; Ribot, Wilson J; Applefeld, Willard; DeShazer, David

2012-06-22

Burkholderia pseudomallei and Burkholderia mallei are gram-negative pathogens responsible for the diseases melioidosis and glanders, respectively. Both species cause disease in humans and animals and have been designated as category B select agents by the Centers for Disease Control and Prevention (CDC). Burkholderia thailandensis is a closely related bacterium that is generally considered avirulent for humans. While it can cause disease in rodents, the B. thailandensis 50% lethal dose (LD50) is typically ≥ 104-fold higher than the B. pseudomallei and B. mallei LD50 in mammalian models of infection. Here we describe an alternative to mammalian hosts in the study of virulence and host-pathogen interactions of these Burkholderia species. Madagascar hissing cockroaches (MH cockroaches) possess a number of qualities that make them desirable for use as a surrogate host, including ease of breeding, ease of handling, a competent innate immune system, and the ability to survive at 37°C. MH cockroaches were highly susceptible to infection with B. pseudomallei, B. mallei and B. thailandensis and the LD50 was <10 colony-forming units (cfu) for all three species. In comparison, the LD50 for Escherichia coli in MH cockroaches was >105 cfu. B. pseudomallei, B. mallei, and B. thailandensis cluster 1 type VI secretion system (T6SS-1) mutants were all attenuated in MH cockroaches, which is consistent with previous virulence studies conducted in rodents. B. pseudomallei mutants deficient in the other five T6SS gene clusters, T6SS-2 through T6SS-6, were virulent in both MH cockroaches and hamsters. Hemocytes obtained from MH cockroaches infected with B. pseudomallei harbored numerous intracellular bacteria, suggesting that this facultative intracellular pathogen can survive and replicate inside of MH cockroach phagocytic cells. The hemolymph extracted from these MH cockroaches also contained multinuclear giant cells (MNGCs) with intracellular B. pseudomallei, which indicates that infected hemocytes can fuse while flowing through the insect's open circulatory system in vivo. The results demonstrate that MH cockroaches are an attractive alternative to mammals to study host-pathogen interactions and may allow the identification of new Burkholderia virulence determinants. The importance of T6SS-1 as a virulence factor in MH cockroaches and rodents suggests that the primary role of this secretion system is to target evasion of the innate immune system.

Drosophila TNF Modulates Tissue Tension in the Embryo to Facilitate Macrophage Invasive Migration.

PubMed

Ratheesh, Aparna; Biebl, Julia; Vesela, Jana; Smutny, Michael; Papusheva, Ekaterina; Krens, S F Gabriel; Kaufmann, Walter; Gyoergy, Attila; Casano, Alessandra Maria; Siekhaus, Daria E

2018-05-07

Migrating cells penetrate tissue barriers during development, inflammatory responses, and tumor metastasis. We study if migration in vivo in such three-dimensionally confined environments requires changes in the mechanical properties of the surrounding cells using embryonic Drosophila melanogaster hemocytes, also called macrophages, as a model. We find that macrophage invasion into the germband through transient separation of the apposing ectoderm and mesoderm requires cell deformations and reductions in apical tension in the ectoderm. Interestingly, the genetic pathway governing these mechanical shifts acts downstream of the only known tumor necrosis factor superfamily member in Drosophila, Eiger, and its receptor, Grindelwald. Eiger-Grindelwald signaling reduces levels of active Myosin in the germband ectodermal cortex through the localization of a Crumbs complex component, Patj (Pals-1-associated tight junction protein). We therefore elucidate a distinct molecular pathway that controls tissue tension and demonstrate the importance of such regulation for invasive migration in vivo. Copyright © 2018 Elsevier Inc. All rights reserved.

In vitro propagation of the microsporidian pathogen Brachiola algerae and studies of its chromosome and ribosomal DNA organization in the context of the complete genome sequencing project.

PubMed

Belkorchia, Abdel; Biderre, Corinne; Militon, Cécile; Polonais, Valérie; Wincker, Patrick; Jubin, Claire; Delbac, Frédéric; Peyretaillade, Eric; Peyret, Pierre

2008-03-01

Brachiola algerae has a broad host spectrum from human to mosquitoes. The successful infection of two mosquito cell lines (Mos55: embryonic cells and Sua 4.0: hemocyte-like cells) and a human cell line (HFF) highlights the efficient adaptive capacity of this microsporidian pathogen. The molecular karyotype of this microsporidian species was determined in the context of the B. algerae genome sequencing project, showing that its haploid genome consists of 30 chromosomal-sized DNAs ranging from 160 to 2240 kbp giving an estimated genome size of 23 Mbp. A contig of 12,269 bp including the DNA sequence of the B. algerae ribosomal transcription unit has been built from initial genomic sequences and the secondary structure of the large subunit rRNA constructed. The data obtained indicate that B. algerae should be an excellent parasitic model to understand genome evolution in relation to infectious capacity.

Gut immunity in Lepidopteran insects.

PubMed

Wu, Kai; Yang, Bing; Huang, Wuren; Dobens, Leonard; Song, Hongsheng; Ling, Erjun

2016-11-01

Lepidopteran insects constitute one of the largest fractions of animals on earth, but are considered pests in their relationship with man. Key to the success of this order of insects is its ability to digest food and absorb nutrition, which takes place in the midgut. Because environmental microorganisms can easily enter Lepidopteran guts during feeding, the innate immune response guards against pathogenic bacteria, virus and microsporidia that can be devoured with food. Gut immune responses are complicated by both resident gut microbiota and the surrounding peritrophic membrane and are distinct from immune responses in the body cavity, which depend on the function of the fat body and hemocytes. Due to their relevance to agricultural production, studies of Lepidopteran insect midgut and immunity are receiving more attention, and here we summarize gut structures and functions, and discuss how these confer immunity against different microorganisms. It is expected that increased knowledge of Lepidopteran gut immunity may be utilized for pest biological control in the future. Copyright © 2016 Elsevier Ltd. All rights reserved.

The early stages of the immune response of the European abalone Haliotis tuberculata to a Vibrio harveyi infection.

PubMed

Cardinaud, Marion; Dheilly, Nolwenn M; Huchette, Sylvain; Moraga, Dario; Paillard, Christine

2015-08-01

Vibrio harveyi is a marine bacterial pathogen responsible for episodic abalone mortalities in France, Japan and Australia. In the European abalone, V. harveyi invades the circulatory system in a few hours after exposure and is lethal after 2 days of infection. In this study, we investigated the responses of European abalone immune cells over the first 24 h of infection. Results revealed an initial induction of immune gene expression including Rel/NF-kB, Mpeg and Clathrin. It is rapidly followed by a significant immuno-suppression characterized by reduced cellular hemocyte parameters, immune response gene expressions and enzymatic activities. Interestingly, Ferritin was overexpressed after 24 h of infection suggesting that abalone attempt to counter V. harveyi infection using soluble effectors. Immune function alteration was positively correlated with V. harveyi concentration. This study provides the evidence that V. harveyi has a hemolytic activity and an immuno-suppressive effect in the European abalone. Copyright © 2015 Elsevier Ltd. All rights reserved.

Mussel micronucleus cytome assay.

PubMed

Bolognesi, Claudia; Fenech, Michael

2012-05-17

The micronucleus (MN) assay is one of the most widely used genotoxicity biomarkers in aquatic organisms, providing an efficient measure of chromosomal DNA damage occurring as a result of either chromosome breakage or chromosome mis-segregation during mitosis. The MN assay is today applied in laboratory and field studies using hemocytes and gill cells from bivalves, mainly from the genera Mytilus. These represent 'sentinel' organisms because of their ability to survive under polluted conditions and to accumulate both organic and inorganic pollutants. Because the mussel MN assay also includes scoring of different cell types, including necrotic and apoptotic cells and other nuclear anomalies, it is in effect an MN cytome assay. The mussel MN cytome (MUMNcyt) assay protocol we describe here reports the recommended experimental design, sample size, cell preparation, cell fixation and staining methods. The protocol also includes criteria and photomicrographs for identifying different cell types and scoring criteria for micronuclei (MNi) and nuclear buds. The complete procedure requires approximately 10 h for each experimental point/sampling station (ten animals).

Modulation of occluding junctions alters the hematopoietic niche to trigger immune activation

PubMed Central

Khadilkar, Rohan J; Vogl, Wayne; Goodwin, Katharine

2017-01-01

Stem cells are regulated by signals from their microenvironment, or niche. During Drosophila hematopoiesis, a niche regulates prohemocytes to control hemocyte production. Immune challenges activate cell-signalling to initiate the cellular and innate immune response. Specifically, certain immune challenges stimulate the niche to produce signals that induce prohemocyte differentiation. However, the mechanisms that promote prohemocyte differentiation subsequent to immune challenges are poorly understood. Here we show that bacterial infection induces the cellular immune response by modulating occluding-junctions at the hematopoietic niche. Occluding-junctions form a permeability barrier that regulates the accessibility of prohemocytes to niche derived signals. The immune response triggered by infection causes barrier breakdown, altering the prohemocyte microenvironment to induce immune cell production. Moreover, genetically induced barrier ablation provides protection against infection by activating the immune response. Our results reveal a novel role for occluding-junctions in regulating niche-hematopoietic progenitor signalling and link this mechanism to immune cell production following infection. PMID:28841136

A comparative analysis of serpin genes in the silkworm genome

PubMed Central

Zou, Zhen; Picheng, Zhao; Weng, Hua; Mita, Kazuei; Jiang, Haobo

2009-01-01

Serine protease inhibitors (serpins) are a superfamily of proteins, most of which control protease-mediated processes by inhibiting their cognate enzymes. Sequencing of the silkworm genome provides an opportunity to investigate serpin structure, function, and evolution at the genome level. There are thirty-four serpin genes in Bombyx mori. Six are highly similar to their Manduca sexta orthologs that regulate innate immunity. Three alternative exons in serpin1 gene and four in serpin28 encode a variable region including the reactive site loop. Splicing of serpin2 pre-mRNA yields variations in serpin2A, 2A′ and 2B. Sequence similarity and intron positions reveal the evolutionary pathway of seven serpin genes in group C. RT-PCR indicates an increase in the mRNA levels of serpin1, 3, 5, 6, 9, 12, 13, 25, 27, 32 and 34 in fat body and hemocytes of larvae injected with bacteria. These results suggest that the silkworm serpins play regulatory roles in defense responses. PMID:19150649

[In life determination of the physiological status of decapod crustaceans (Crustacea: Decapoda) by hematological characteristics].

PubMed

Aleksandrova, E N; Kovacheva, N P

2010-01-01

The application of hematological analysis techniques to detecting the physiological status of the economically valued decapods during their culturing, and in monitoring of the condition of their natural populations, is restrained by the incomplete knowledge of these invertebrates circulatory system and its properties. Scarce data on the use of hematological indicators for determining the physiological status of decapods may be found sporadically in published sources; there is shortage of basic standards needed for interpretation of the analytical results. In this regard the paper considers some data on the major properties of hemolymph and its cellular elements; on methods of their examination; and on the results of application of hematological characteristics to assessing the physiological condition of various species of decapods. The hematological indicators suitable for the analysis of live decapods include: time of coagulation and buffer characteristic of hemolymph; concentration of total proteins, copper, calcium, glucose and lactates in it; total number of hemocytes with the consideration of granulocytes share.

MiniCORVET is a Vps8-containing early endosomal tether in Drosophila.

PubMed

Lőrincz, Péter; Lakatos, Zsolt; Varga, Ágnes; Maruzs, Tamás; Simon-Vecsei, Zsófia; Darula, Zsuzsanna; Benkő, Péter; Csordás, Gábor; Lippai, Mónika; Andó, István; Hegedűs, Krisztina; Medzihradszky, Katalin F; Takáts, Szabolcs; Juhász, Gábor

2016-06-02

Yeast studies identified two heterohexameric tethering complexes, which consist of 4 shared (Vps11, Vps16, Vps18 and Vps33) and 2 specific subunits: Vps3 and Vps8 (CORVET) versus Vps39 and Vps41 (HOPS). CORVET is an early and HOPS is a late endosomal tether. The function of HOPS is well known in animal cells, while CORVET is poorly characterized. Here we show that Drosophila Vps8 is highly expressed in hemocytes and nephrocytes, and localizes to early endosomes despite the lack of a clear Vps3 homolog. We find that Vps8 forms a complex and acts together with Vps16A, Dor/Vps18 and Car/Vps33A, and loss of any of these proteins leads to fragmentation of endosomes. Surprisingly, Vps11 deletion causes enlargement of endosomes, similar to loss of the HOPS-specific subunits Vps39 and Lt/Vps41. We thus identify a 4 subunit-containing miniCORVET complex as an unconventional early endosomal tether in Drosophila.