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

Demonstration of nitric oxide synthase activity in crustacean hemocytes and anti-microbial activity of hemocyte-derived nitric oxide.

PubMed

Yeh, Feng-Ching; Wu, Su-Hua; Lai, Chi-Yung; Lee, Chi-Ying

2006-05-01

We determined the biochemical characteristics of nitric oxide synthase (NOS) in hemocytes of the crayfish Procambarus clarkii and investigated the roles of hemocyte-derived NO in host defense. Biochemical analysis indicated the presence of a Ca2+ -independent NOS activity, which was elevated by lipopolysaccharide (LPS) treatment. When bacteria (Staphylococcus aureus) and hemocytes were co-incubated, adhesion of bacteria to hemocytes was observed. NO donor sodium nitroprusside (SNP) significantly increased the numbers of hemocytes to which bacteria adhered. Similarly, LPS elicited bacterial adhesion and the LPS-induced adhesion was prevented by NOS inhibitor NG-monomethyl-L-arginine (L-NMMA). Finally, plate count assay demonstrated that addition of LPS to the hemocytes/bacteria co-incubation resulted in a significant decrease in bacterial colony forming unit (CFU), and that L-NMMA reversed the decreasing effect of LPS on CFU. The combined results demonstrate the presence of a Ca2+ -independent LPS-inducible NOS activity in crayfish hemocytes and suggest that hemocyte-derived NO is involved in promoting bacterial adhesion to hemocytes and enhancing bactericidal activity of hemocytes.

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

Hematopoiesis in larval Pseudoplusia includens and Spodoptera frugiperda.

PubMed

Gardiner, E M; Strand, M R

2000-04-01

Maintenance of circulating hemocytes in larval Lepidoptera has been attributed to both mitosis of hemocytes already in circulation and the release of hemocytes from hematopoietic organs. In this study, we compared hematopoiesis in the noctuids Pseudoplusia includens and Spodoptera frugiperda. For both species, hemocyte densities per microl of blood increased with instar. Differential hemocyte counts indicated that plasmatocytes were the most abundant hemocyte type during early instars but granular cells were the most abundant hemocyte type in the last instar. Hematopoietic organs were located in the meso- and metathorax of S. Frugiperda and P. Includens. These organs contained large numbers of hemocytes in S. Frugiperda, but contained few hemocytes in P. Includens. The majority of the hemocytes recovered from hematopoietic organs were identified as plasmatocytes. Using hemocyte type-specific markers and bromodeoxyuridine (BrdU) incorporation experiments, we determined that all hemocyte types with the exception of oenocytoids synthesize DNA. BrdU labeling indices for both species also fluctuated with the molting cycle. Ligation experiments suggested that hematopoietic organs are an important source of circulating plasmatocytes in S. Frugiperda but not in P. Includens. Injection of heat killed bacteria into larvae induced higher levels of BrdU labeling than injection of sterile saline, suggesting that infection and wounding induce different levels of hemocyte proliferation. Arch. Copyright 2000 Wiley-Liss, Inc.

Mitotic activity of the hemocytes in the tick Ixodes ricinus (Acari; Ixodidae).

PubMed

Kuhn, K H

1996-01-01

The blood cells, or hemocytes, of Ixodes ricinus have been shown to recognize, attack, and phagocytose microorganisms invading the body cavity, or hemocoel, of this tick. Regulated proliferation and differentiation of hemocytes, also referred to as immunocytes, is basic to an effective immune response to invading microorganisms. Therefore, this study dealt with hemopoiesis in I. ricinus, the vector tick of the Lyme disease spirochete Borrelia burgdorferi. Histological evidence for the presence of hemopoietic tissue, a preferential proliferation site of hemocytes, is presented. Mainly the mitotic activity of free-floating hemocytes was examined. By means of microscopical photometry and flow cytometry, all three types of hemocytes in engorging female I. ricinus were found in different stages of the cell cycle. In the engorging tick, up to 40% of the hemocytes counted were in the S phase or the G2/M phase. From this study we conclude that the differentiated hemocyte types do not differentiate from stem cells in the adult tick. Moreover, microorganisms entering the hemocoel of engorging ticks are confronted with high numbers of hemocytes and, therefore, with an effective cellular immune response.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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

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.

Hemocyte–hemocyte adhesion and nodulation reactions of the greater wax moth, Galleria mellonella are influenced by cholera toxin and its B-subunit

PubMed Central

Lapointe, Jason F.; Dunphy, Gary B.; Mandato, Craig A.

2012-01-01

Nodulation, the lepidopteran insect immune response to large numbers of microbes in the blood (hemolymph) consists of the coordination of the blood cell (hemocyte) types the granular cells and plasmatocytes in terms of granular cell–bacteria adhesion and hemocyte–hemocyte adhesion (microaggregation). Hemocyte–microbe adhesion is influenced by the secondary messenger, cAMP, and cAMP-dependent protein kinase A. In the present study, cholera toxin, an AB5 protein known to indirectly stimulate adenylate cyclase, is used to examine the hemocyte responses to glass, bacteria and hemocyte–hemocyte microaggregates. In vitro, this toxin induces a bimodal hemocyte adhesion response that varies with the holotoxin concentration in terms of the individual and aggregated hemocyte adhesion responses: the lower CTX concentration (1.2 nM) increases microaggregate adhesion and decreases individual hemocyte binding to glass, as does higher concentrations (6–120 nM), however microaggregates induced by lower concentrations do not adhere to glass. Cholera toxin-induced microaggregation is inhibited by RGDS, suggestive of integrin involvement. In vivo, cholera toxin (1.2–120 nM) injected into larvae induces also a bimodal hemocytic response: low levels (1.2–6 nM) cause reduced hemocyte adhesion, while high levels (12–120 nM) increase hemocyte release or mobilization of adhesive hemocyte counts in the hemolymph. Increasing levels of cholera toxin concomitantly injected with the non-pathogenic bacterium, Bacillus subtilis produces a bimodal pattern in bacterial removal from the hemolymph which correlates with nodule frequency in larvae injected with cholera toxin only. The effects of higher concentrations of cholera toxin in vitro (6–120 nM) and in vivo (12–120 nM) are mediated by the B-subunit, whereas the isolated A-subunit has no effect on hemocyte activity. Cholera toxin and its individual subunits did not detectably alter levels of intracellular cAMP in the hemocytes, suggesting a cAMP-independent mechanism stimulating the nodulation response. PMID:24371567

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.

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.

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.

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.

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.

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 hemocytes from the mosquitoes Anopheles gambiae and Aedes aegypti.

PubMed

Castillo, J C; Robertson, A E; Strand, M R

2006-12-01

Hemocytes are an essential component of the mosquito immune system but current knowledge of the types of hemocytes mosquitoes produce, their relative abundance, and their functions is limited. Addressing these issues requires improved methods for collecting and maintaining mosquito hemocytes in vitro, and comparative data that address whether important vector species produce similar or different hemocyte types. Toward this end, we conducted a comparative study with Anopheles gambiae and Aedes aegypti. Collection method greatly affected the number of hemocytes and contaminants obtained from adult females of each species. Using a collection method called high injection/recovery, we concluded that hemolymph from An. gambiae and Ae. aegypti adult females contains three hemocyte types (granulocytes, oenocytoids and prohemocytes) that were distinguished from one another by a combination of morphological and functional markers. Significantly more hemocytes were recovered from An. gambiae females than Ae. aegypti. However, granulocytes were the most abundant cell type in both species while oenocytoids and prohemocytes comprised less than 10% of the total hemocyte population. The same hemocyte types were collected from larvae, pupae and adult males albeit the absolute number and proportion of each hemocyte type differed from adult females. The number of hemocytes recovered from sugar fed females declined with age but blood feeding transiently increased hemocyte abundance. Two antibodies tested as potential hemocyte markers (anti-PP06 and anti-Dox-A2) also exhibited alterations in staining patterns following immune challenge with the bacterium Escherichia coli.

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.

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.

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.

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.

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.

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.

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.

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

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.

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.

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.

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

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.

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.

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.

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.

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.

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.

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.

The two origins of hemocytes in Drosophila.

PubMed

Holz, Anne; Bossinger, Barbara; Strasser, Thomas; Janning, Wilfried; Klapper, Robert

2003-10-01

As in many other organisms, the blood of Drosophila consists of several types of hemocytes, which originate from the mesoderm. By lineage analyses of transplanted cells, we specified two separate anlagen that give rise to different populations of hemocytes: embryonic hemocytes and lymph gland hemocytes. The anlage of the embryonic hemocytes is restricted to a region within the head mesoderm between 70 and 80% egg length. In contrast to all other mesodermal cells, the cells of this anlage are already determined as hemocytes at the blastoderm stage. Unexpectedly, these hemocytes do not degenerate during late larval stages, but have the capacity to persist through metamorphosis and are still detectable in the adult fly. A second anlage, which gives rise to additional hemocytes at the onset of metamorphosis, is located within the thoracic mesoderm at 50 to 53% egg length. After transplantation within this region, clones were detected in the larval lymph glands. Labeled hemocytes are released by the lymph glands not before the late third larval instar. The anlage of these lymph gland-derived hemocytes is not determined at the blastoderm stage, as indicated by the overlap of clones with other tissues. Our analyses reveal that the hemocytes of pupae and adult flies consist of a mixture of embryonic hemocytes and lymph gland-derived hemocytes, originating from two distinct anlagen that are determined at different stages of development.

Immunochemical identification of insect hemocyte populations: monoclonal antibodies distinguish four major hemocyte types in manduca sexta.

PubMed

Willott, E; Trenczek, T; Thrower, L W; Kanost, M R

1994-12-01

We have made 140 monoclonal antibodies to hemocytes (insect blood cells) from Manduca sexta. Four of these antibodies, when used in immunofluorescent microscopy of fixed hemocytes, distinguish the four main morphologically distinct hemocyte types. Plasmatocytes, granular cells, and oenocytoids are each recognized by a unique antibody specific to that type; spherulocytes are recognized by an antibody that also binds to plasmatocytes. When used in flow cytometry with nonfixed hemocytes, three of the four antibodies bind their respective cells; the oenocytoid marker failed to bind to any hemocytes. This set of four monoclonal antibodies may be useful for labeling individual cell types and for separating the different hemocyte types for further study of hemocyte functions.

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.

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.

Tissue-enriched expression profiles in Aedes aegypti identify hemocyte-specific transcriptome responses to infection

PubMed Central

Choi, Young-Jun; Fuchs, Jeremy F.; Mayhew, George F.; Yu, Helen E.; Christensen, Bruce M.

2012-01-01

Hemocytes are integral components of mosquito immune mechanisms such as phagocytosis, melanization, and production of antimicrobial peptides. However, our understanding of hemocyte-specific molecular processes and their contribution to shaping the host immune response remains limited. To better understand the immunophysiological features distinctive of hemocytes, we conducted genome-wide analysis of hemocyte-enriched transcripts, and examined how tissue-enriched expression patterns change with the immune status of the host. Our microarray data indicate that the hemocyte-enriched trascriptome is dynamic and context-dependent. Analysis of transcripts enriched after bacterial challenge in circulating hemocytes with respect to carcass added a dimension to evaluating infection-responsive genes and immune-related gene families. We resolved patterns of transcriptional change unique to hemocytes from those that are likely shared by other immune responsive tissues, and identified clusters of genes preferentially induced in hemocytes, likely reflecting their involvement in cell type specific functions. In addition, the study revealed conserved hemocyte-enriched molecular repertoires which might be implicated in core hemocyte function by cross-species meta-analysis of microarray expression data from Anopheles gambiae and Drosophila melanogaster. PMID:22796331

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.

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.

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.

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.

Hemocyte-mediated phagocytosis differs between honey bee (Apis mellifera) worker castes

PubMed Central

Salmela, Heli; Amdam, Gro Vang; Münch, Daniel

2017-01-01

Honey bees as other insects rely on the innate immune system for protection against diseases. The innate immune system includes the circulating hemocytes (immune cells) that clear pathogens from hemolymph (blood) by phagocytosis, nodulation or encapsulation. Honey bee hemocyte numbers have been linked to hemolymph levels of vitellogenin. Vitellogenin is a multifunctional protein with immune-supportive functions identified in a range of species, including the honey bee. Hemocyte numbers can increase via mitosis, and this recruitment process can be important for immune system function and maintenance. Here, we tested if hemocyte mediated phagocytosis differs among the physiologically different honey bee worker castes (nurses, foragers and winter bees), and study possible interactions with vitellogenin and hemocyte recruitment. To this end, we adapted phagocytosis assays, which—together with confocal microscopy and flow cytometry—allow qualitative and quantitative assessment of hemocyte performance. We found that nurses are more efficient in phagocytic uptake than both foragers and winter bees. We detected vitellogenin within the hemocytes, and found that winter bees have the highest numbers of vitellogenin-positive hemocytes. Connections between phagocytosis, hemocyte-vitellogenin and mitosis were worker caste dependent. Our results demonstrate that the phagocytic performance of immune cells differs significantly between honey bee worker castes, and support increased immune competence in nurses as compared to forager bees. Our data, moreover, provides support for roles of vitellogenin in hemocyte activity. PMID:28877227

Hemocyte-mediated phagocytosis differs between honey bee (Apis mellifera) worker castes.

PubMed

Hystad, Eva Marit; Salmela, Heli; Amdam, Gro Vang; Münch, Daniel

2017-01-01

Honey bees as other insects rely on the innate immune system for protection against diseases. The innate immune system includes the circulating hemocytes (immune cells) that clear pathogens from hemolymph (blood) by phagocytosis, nodulation or encapsulation. Honey bee hemocyte numbers have been linked to hemolymph levels of vitellogenin. Vitellogenin is a multifunctional protein with immune-supportive functions identified in a range of species, including the honey bee. Hemocyte numbers can increase via mitosis, and this recruitment process can be important for immune system function and maintenance. Here, we tested if hemocyte mediated phagocytosis differs among the physiologically different honey bee worker castes (nurses, foragers and winter bees), and study possible interactions with vitellogenin and hemocyte recruitment. To this end, we adapted phagocytosis assays, which-together with confocal microscopy and flow cytometry-allow qualitative and quantitative assessment of hemocyte performance. We found that nurses are more efficient in phagocytic uptake than both foragers and winter bees. We detected vitellogenin within the hemocytes, and found that winter bees have the highest numbers of vitellogenin-positive hemocytes. Connections between phagocytosis, hemocyte-vitellogenin and mitosis were worker caste dependent. Our results demonstrate that the phagocytic performance of immune cells differs significantly between honey bee worker castes, and support increased immune competence in nurses as compared to forager bees. Our data, moreover, provides support for roles of vitellogenin in hemocyte activity.

Spatial and temporal in vivo analysis of circulating and sessile immune cells in mosquitoes: hemocyte mitosis following infection

PubMed Central

2013-01-01

Background Mosquitoes respond to infection by mounting immune responses. The primary regulators of these immune responses are cells called hemocytes, which kill pathogens via phagocytosis and via the production of soluble antimicrobial factors. Mosquito hemocytes are circulated throughout the hemocoel (body cavity) by the swift flow of hemolymph (blood), and data show that some hemocytes also exist as sessile cells that are attached to tissues. The purpose of this study was to create a quantitative physical map of hemocyte distribution in the mosquito, Anopheles gambiae, and to describe the cellular immune response in an organismal context. Results Using correlative imaging methods we found that the number of hemocytes in a mosquito decreases with age, but that regardless of age, approximately 75% of the hemocytes occur in circulation and 25% occur as sessile cells. Infection induces an increase in the number of hemocytes, and tubulin and nuclear staining showed that this increase is primarily due to mitosis and, more specifically, autonomous cell division, by circulating granulocytes. The majority of sessile hemocytes are present on the abdominal wall, although significant numbers of hemocytes are also present in the thorax, head, and several of the appendages. Within the abdominal wall, the areas of highest hemocyte density are the periostial regions (regions surrounding the valves of the heart, or ostia), which are ideal locations for pathogen capture as these are areas of high hemolymph flow. Conclusions These data describe the spatial and temporal distribution of mosquito hemocytes, and map the cellular response to infection throughout the hemocoel. PMID:23631603

Hemiuroid trematode sporocysts are undetected by hemocytes of their intermediate host, the ark cockle Anadara trapezia: potential role of surface carbohydrates in successful parasitism.

PubMed

Kawasaki, Minami; Delamare-Deboutteville, Jerome; Dang, Cecile; Barnes, Andrew C

2013-12-01

In order to establish a successful relationship with their hosts, parasites must subvert or evade immune defences. Cockle Anadara trapezia and Sydney Rock oyster (SRO) Saccostrea glomerata live in the same location but only ark cockles are infected by sporocysts of hemiuroid trematode. This provides an opportunity to explore differing interactions between the parasite and the immune system of susceptible and refractive hosts. Rapid migration and encapsulation of sporocysts was observed by SRO hemocytes but not by cockle hemocytes. This migration/encapsulation was inhibited by N-acetylglucosamine or N-acetylgalactosamine but not by the other sugars, implicating specific surface carbohydrates in immune detection. Effector responses of hemocytes were investigated in vitro in terms of production of reactive oxygen production (ROS). Hemocytes of both species strongly reacted to Zymosan, but only SRO hemocytes responded to live sporocysts. Neither species' hemocytes produced ROS in the presence of dead/fixed sporocysts, and there was no suppression of Zymosan-induced respiratory burst by sporocysts. This suggests that immune escape is mediated by avoiding encapsulation, perhaps through molecular mimicry. Membrane-shaving with proteases indicated that sporocyst surface proteins are not a key factors in hemocytic detection. Surface carbohydrates of SRO and cockle hemocytes and of sporocysts were profiled with a panel of biotinylated lectins. This revealed substantial differences between cockle and SRO hemocytes, but greater similarity between cockle hemocytes and sporocysts. Results suggest that surface carbohydrates play an integral role in hemocyte immunorecognition and that surface carbohydrate molecular mimicry is a potential strategy for immune evasion in cockles by hemiuroid trematode sporocysts. Crown Copyright © 2013. Published by 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.

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.

[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.

Development, characterization, and lethal effect of monoclonal antibodies against hemocytes in an adult female tick, Ornithodoros moubata (Acari: Argasidae).

PubMed

Matsuo, T; Tsukamoto, D; Inoue, N; Fujisaki, K

2003-12-01

In the present study, 19 monoclonal antibodies (mAbs) against adult Ornithodoros moubata hemocytes were established, and the reactivity of the hemocytes to these mAbs was examined by an indirect fluorescent antibody test (IFAT), Western blot and immunoprecipitation analyses. It was shown that the reactivities of the hemocytes to the mAbs varied among morphologically similar hemocyte types, and most mAbs produced in the present study showed the multiple band reactivity. However, the presence of shared epitopes among peptide subunits of the same protein or entirely different proteins are not common, so their reactivity could not be explained in detail. These results suggest that there are morphologically similar but functionally differentiated hemocytes. Therefore, in addition to morphological classification, the molecular-based classification of the hemocytes is also required. In order to assess the lethal effect of blood meal containing each mAb, artificial feeding was performed. The OmHC 31 showed the strongest lethal effect on adult female O. moubata. In conclusion, anti-hemocyte mAbs produced in this study are useful not only for the immunological classification of hemocytes but also for the immunological control of the tick.

[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.

In vitro cultivation and cryopreservation of Babesia bigemina sporokinetes in hemocytes of Rhipicephalus microplus.

PubMed

de Rezende, Jania; Rangel, Charles P; McIntosh, Douglas; Silveira, Júlia A G; Cunha, Nathalie C; Ramos, Carlos A N; Fonseca, Adivaldo H

2015-09-15

Cultures of tick hemocytes represent alternative cell lines for the isolation and cultivation of a variety of hemoparasites. The present study reports the development and evaluation of methods for the in vitro culture and maintenance of sporokinetes of Babesia bigemina in association with hemocytes of the tick Rhipicephalus microplus. Hemolymph, from engorged females infected with B. bigemina sporokinetes, was incubated at 28 °C in L15 culture medium supplemented with 40% fetal bovine serum. Adherence of hemocytes to flask surfaces and the development of B. bigemina sporokinetes commenced on the first day of cultivation. The protozoa demonstrated clear motility and the capacity to adhere to hemocyte membranes for up to 25 days, at which time the hemocytes began to show signs of degeneration. Examination of Giemsa stained hemocyte cultures, revealed the presence of pyriformis forms, as well as mature and immature sporokinetes with dark red nuclei, centralized or near the apical extremities. Sporokinetes harvested from culture supernatants were cryopreserved in liquid nitrogen. Inoculation of parasite-free hemocyte cultures with defrosted sporokinetes, demonstrated the viability and interaction of the protozoa with the hemocytes over 21 days. Cultured hemocytes of R. microplus hold potential for development as a tool in the study of host parasite interactions and as a substrate for the in vitro maintenance of B. bigemina sporokinetes. Copyright © 2015 Elsevier B.V. All rights reserved.

Activation of Drosophila hemocyte motility by the ecdysone hormone

PubMed Central

Sampson, Christopher J.; Amin, Unum; Couso, Juan-Pablo

2013-01-01

Summary Drosophila hemocytes compose the cellular arm of the fly's innate immune system. Plasmatocytes, putative homologues to mammalian macrophages, represent ∼95% of the migratory hemocyte population in circulation and are responsible for the phagocytosis of bacteria and apoptotic tissues that arise during metamorphosis. It is not known as to how hemocytes become activated from a sessile state in response to such infectious and developmental cues, although the hormone ecdysone has been suggested as the signal that shifts hemocyte behaviour from quiescent to migratory at metamorphosis. Here, we corroborate this hypothesis by showing the activation of hemocyte motility by ecdysone. We induce motile behaviour in larval hemocytes by culturing them with 20-hydroxyecdysone ex vivo. Moreover, we also determine that motile cell behaviour requires the ecdysone receptor complex and leads to asymmetrical redistribution of both actin and tubulin cytoskeleton. PMID:24285708

Immune challenge differentially affects transcript abundance of three antimicrobial peptides in hemocytes from the moth Pseudoplusia includens.

PubMed

Lavine, M D; Chen, G; Strand, M R

2005-12-01

Inducible expression of antimicrobial peptides and other humoral immune factors by the insect fat body is well documented. Hemocytes comprise the second essential arm of the insect immune system but it is unclear whether antimicrobial peptide genes are expressed by all or only some types of hemocytes. Here we report the cloning of cecropin A (Pi-cecA), lebocin (Pi-leb) and lysozyme (Pi-lys) homologs from the moth Pseudoplusia includens. Relative-quantitative real-time PCR (rq-rtPCR) indicated that transcript abundance for each antimicrobial gene increased in fat body and hemocytes following immune challenge with the Gram-negative bacterium Escherichia coli. Relative transcript abundance of Pi-cecA was much higher in fat body than hemocytes. In contrast, transcript levels of Pi-leb were three-fold lower in hemocytes than fat body while transcript levels of Pi-lys were three-fold higher. Estimates for the overall contribution of the fat body and hemocytes to antimicrobial peptide expression suggested that hemocytes contribute significantly to Pi-lys transcript levels in larvae but produce much smaller amounts of Pi-cecA and Pi-leb compared to the fat body. Each antimicrobial peptide was also inducibly expressed in hemocytes following challenge with the Gram-positive bacterium Micrococcus luteus or when hemocytes formed capsules around chromatography beads. Analysis of hemocyte types indicated that granulocytes and plasmatocytes expressed all three antimicrobial peptides, whereas spherule cells and oenocytoids expressed only lysozyme. Transcriptional profiles of these antimicrobial genes were similar in granulocytes and plasmatocytes in vivo but were very different in vitro.

Definition of Drosophila hemocyte subsets by cell-type specific antigens.

PubMed

Kurucz, Eva; Váczi, B; Márkus, R; Laurinyecz, Barbara; Vilmos, P; Zsámboki, J; Csorba, Kinga; Gateff, Elisabeth; Hultmark, D; Andó, I

2007-01-01

We analyzed the heterogeneity of Drosophila hemocytes on the basis of the expression of cell-type specific antigens. The antigens characterize distinct subsets which partially overlap with those defined by morphological criteria. On the basis of the expression or the lack of expression of blood cell antigens the following hemocyte populations have been defined: crystal cells, plasmatocytes, lamellocytes and precursor cells. The expression of the antigens and thus the different cell types are developmentally regulated. The hemocytes are arranged in four main compartments: the circulating blood cells, the sessile tissue, the lymph glands and the posterior hematopoietic tissue. Each hemocyte compartment has a specific and characteristic composition of the various cell types. The described markers represent the first successful attempt to define hemocyte lineages by immunological markers in Drosophila and help to define morphologically, functionally, spatially and developmentally distinct subsets of hemocytes.

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

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

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

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

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.

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

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.

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.

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.

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.

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

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.

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.

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

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.

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.

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

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.

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.

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.

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

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.

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.

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

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.

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.

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.

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

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.

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.

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

Effects of waterborne nickel on the physiological and immunological parameters of the Pacific abalone Haliotis discus hannai during thermal stress.

PubMed

Min, Eun Young; Cha, Yong-Joo; Kang, Ju-Chan

2015-09-01

In this study, the 96-h LC50 at 22 and 26 °C values was 28.591 and 11.761 mg/L, respectively, for NiCl2 exposure in the abalone. The alteration of physiological and immune-toxicological parameters such as the total hemocyte count (THC), lysozyme, phenoloxidase (PO), and phagocytosis activity was measured in the abalone exposed to nickel (200 and 400 μg/L) under thermal stress for 96 h. In this study, Mg and THC decreased, while Ca, lysozyme, PO, and phagocytosis activity increased in the hemolymph of Pacific abalone exposed to NiCl2 when compared to a control at both 22 and 26 °C. However, these parameters were not affected by a rise in temperature from 22 to 26 °C in non-exposed groups. Our results showed that NiCl2 below 400 μg/L was able to stimulate immune responses in abalone. However, complex stressors, thermal changes, or NiCl2 can modify the immunological response and lead to changes in the physiology of host-pollutant interactions in the abalone.

Modelling interactions of acid–base balance and respiratory status in the toxicity of metal mixtures in the American oyster Crassostrea virginica

PubMed Central

Macey, Brett M.; Jenny, Matthew J.; Williams, Heidi R.; Thibodeaux, Lindy K.; Beal, Marion; Almeida, Jonas S.; Cunningham, Charles; Mancia, Annalaura; Warr, Gregory W.; Burge, Erin J.; Holland, A. Fred; Gross, Paul S.; Hikima, Sonomi; Burnett, Karen G.; Burnett, Louis; Chapman, Robert W.

2010-01-01

Heavy metals, such as copper, zinc and cadmium, represent some of the most common and serious pollutants in coastal estuaries. In the present study, we used a combination of linear and artificial neural network (ANN) modelling to detect and explore interactions among low-dose mixtures of these heavy metals and their impacts on fundamental physiological processes in tissues of the Eastern oyster, Crassostrea virginica. Animals were exposed to Cd (0.001–0.400 µM), Zn (0.001–3.059 µM) or Cu (0.002–0.787 µM), either alone or in combination for 1 to 27 days. We measured indicators of acid–base balance (hemolymph pH and total CO2), gas exchange (Po2), immunocompetence (total hemocyte counts, numbers of invasive bacteria), antioxidant status (glutathione, GSH), oxidative damage (lipid peroxidation; LPx), and metal accumulation in the gill and the hepatopancreas. Linear analysis showed that oxidative membrane damage from tissue accumulation of environmental metals was correlated with impaired acid–base balance in oysters. ANN analysis revealed interactions of metals with hemolymph acid–base chemistry in predicting oxidative damage that were not evident from linear analyses. These results highlight the usefulness of machine learning approaches, such as ANNs, for improving our ability to recognize and understand the effects of subacute exposure to contaminant mixtures. PMID:19958840

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.

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.

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.

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.

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

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 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

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.

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.

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

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.

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.

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...

White Shrimp Litopenaeus vannamei That Have Received Gracilaria tenuistipitata Extract Show Early Recovery of Immune Parameters after Ammonia Stressing.

PubMed

Chen, Yu-Yuan; Chen, Jiann-Chu; Lin, Yong-Chin; Yeh, Su-Tuen; Huang, Chien-Lun

2015-06-05

White shrimp Litopenaeus vannamei immersed in seawater (35‰) containing Gracilaria tenuistipitata extract (GTE) at 0 (control), 400, and 600 mg/L for 3 h were exposed to 5 mg/L ammonia-N (ammonia as nitrogen), and immune parameters including hyaline cells (HCs), granular cells (GCs, including semi-granular cells), total hemocyte count (THC), phenoloxidase (PO) activity, respiratory bursts (RBs), superoxide dismutase (SOD) activity, lysozyme activity, and hemolymph protein level were examined 24~120 h post-stress. The immune parameters of shrimp immersed in 600 mg/L GTE returned to original values earlier, at 96~120 h post-stress, whereas in control shrimp they did not. In another experiment, shrimp were immersed in seawater containing GTE at 0 and 600 mg/L for 3 h and examined for transcript levels of immune-related genes at 24 h post-stress. Transcript levels of lipopolysaccharide and β-1,3-glucan binding protein (LGBP), peroxinectin (PX), cytMnSOD, mtMnSOD, and HSP70 were up-regulated at 24 h post-stress in GTE receiving shrimp. We concluded that white shrimp immersed in seawater containing GTE exhibited a capability for maintaining homeostasis by regulating cellular and humoral immunity against ammonia stress as evidenced by up-regulated gene expression and earlier recovery of immune parameters.

A Comparative study on the nonspecific immunity of juvenile Litopenaeus vannamei ever inhabiting freshwater and seawater

NASA Astrophysics Data System (ADS)

Jia, Xuying; Ding, Sen; Wang, Fang; Dong, Shuanglin

2014-06-01

A study on the nonspecific immunity of Litopenaeus vannamei ever inhabiting freshwater and seawater was carried out at different molt stages by comparing their total hemocyte count (THC) and respiratory burst (RB) and activity of phenol oxidase (PO), nitric oxide synthase (NOS) and lysozyme (LY). Two-way ANOVA showed that salinity and molt stage independently affected THC and RB and the activity of PO, NOS and LY of juvenile L. vannamei significantly ( P < 0.05). The THC and RB and the activity of NOS gradually increased from the post-molt stages (A and B) to the pre-molt stages (D0-D3), which were common in shrimps inhabiting freshwater and seawater. The activity of PO peaked at the inter-molt stage (C), and touched the lowest at the post-molt stage in freshwater and pre-molt stage in seawater. The activity of LY was stable over the molt cycle. The RB and the activity of PO, NOS and LY of juvenile L. vannamei were significantly lower in freshwater than in seawater; whereas THC was significantly higher in freshwater than in seawater ( P < 0.05). It was concluded that the post-molt stage (especially stage A) was critical to shrimp culture, which should be intensively attended when L. vannamei was cultured in freshwater.

[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.

[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

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.

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.

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

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.

Melatonin Promotes Cheliped Regeneration, Digestive Enzyme Function, and Immunity Following Autotomy in the Chinese Mitten Crab, Eriocheir sinensis

PubMed Central

Zhang, Cong; Yang, Xiao-zhen; Xu, Min-jie; Huang, Gen-yong; Zhang, Qian; Cheng, Yong-xu; He, Long; Ren, Hong-yu

2018-01-01

In the pond culture of juvenile Eriocheir sinensis, a high limb-impairment rate seriously affects the culture success. Therefore, it is particularly important to artificially promote limb regeneration. This study evaluated the effects of melatonin on cheliped regeneration, digestive ability, and immunity, as well as its relationship with the eyestalk. It was found that the injection of melatonin significantly increased the limb regeneration rate compared with the saline group (P < 0.05). The qRT-PCR results of growth-related genes showed that the level of EcR-mRNA (ecdysteroid receptor) and Chi-mRNA (chitinase) expression was significantly increased following the melatonin injection, while the expression of MIH-mRNA (molt-inhibiting hormone) was significantly decreased (P < 0.05). Melatonin significantly increased lipase activity (P < 0.05). We observed that the survival rates of limb-impaired and unilateral eyestalk-ablated crabs were substantially improved following melatonin treatment, whereas the survival of the unilateral eyestalk-ablated crabs was significantly decreased compared with the control group (P < 0.05). Furthermore, the results of serum immune and antioxidant capacity revealed that melatonin significantly increased the total hemocyte counts (THC), hemocyanin content, total antioxidant capacity (T-AOC), acid phosphatase (ACP), and glutathione peroxidase activity (GSH-Px), whereas the immune-related parameters were significantly decreased in eyestalk-ablated crabs (P < 0.05). Therefore, these findings indicate that melatonin exerts a protective effect on organism injury, which could promote limb regeneration by up-regulating the expression of growth-related genes, improve digestive enzyme activity, and strengthen the immune response, particularly antioxidant capacity. PMID:29623051

Effects of ammonia stress in the Amazon river shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae).

PubMed

Pinto, Marcelo R; Lucena, Malson N; Faleiros, Rogério Oliveira; Almeida, Eduardo Alves; McNamara, John C; Leone, Francisco A

2016-01-01

We evaluate the effects of total ammonia nitrogen-N (TAN) exposure for 72h on (Na(+),K(+))- and V(H(+))-ATPase activities and on their subunit expressions in gills of the diadromous freshwater shrimp Macrobrachium amazonicum. Specific (Na(+),K(+))- and V(H(+))-ATPase activities increased roughly 1.5- to 2-fold, respectively, after exposure to 2.0mmolL(-1) TAN. Quantitative RT-PCR analyses revealed a 2.5-fold increase in V(H(+))-ATPase B subunit mRNA expression while (Na(+),K(+))-ATPase α-subunit expression was unchanged. Immunohistochemical analyses of the gill lamellae located the (Na(+),K(+))-ATPase throughout the intralamellar septal cells, independently of TAN concentration, while the V(H(+))-ATPase was located in both the apical pillar cell flanges and pillar cell bodies. Systemic stress parameters like total hemocyte count decreased by 30% after exposure to 2.0mmolL(-1) TAN, accompanied by increased activities of the oxidative stress enzymes superoxide dismutase, glutathione reductase and glucose-6-phosphate dehydrogenase in the gills. The stress responses of M. amazonicum to elevated TAN include increases in gill (Na(+),K(+))- and V(H(+))-ATPase activities that are accompanied by changes in oxidative stress enzyme activities, immune system effects and an increase in gill V(H(+))-ATPase gene expression. These findings likely underpin physiological effects in a crustacean like M. amazonicum that exploits multiple ecosystems during its life cycle, as well as under culture conditions that may significantly impact shrimp production by the aquaculture industry. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

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.

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.

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.

Effects of fluorine-containing usnic acid and fungus Beauveria bassiana on the survival and immune-physiological reactions of Colorado potato beetle larvae.

PubMed

Kryukov, Vadim Yu; Tomilova, Oksana G; Luzina, Olga A; Yaroslavtseva, Olga N; Akhanaev, Yuriy B; Tyurin, Maksim V; Duisembekov, Bahytzhan A; Salakhutdinov, Nariman F; Glupov, Viktor V

2018-03-01

The search for compounds that interact synergistically with entomopathogenic fungi is aimed at enhancing the efficacy and stability of biological products against pest insects, for example, against the Colorado potato beetle (CPB). We hypothesized that fluorine-containing derivatives of usnic acid (FUA) might be candidates for the development of multicomponent bio-insecticides. The aim of this study was to analyze the co-influence of FUA and Beauveria bassiana on the survival and immune-physiological reactions of CPB larvae. Synergy between FUA and B. bassiana was observed after treatment of second, third and fourth larvae instars under laboratory conditions. Furthermore, synergy was observed in field trials in continental climate conditions in southeastern Kazakhstan. In a field experiment, the median lethal time was shortened three-fold, and cumulative mortality for 15 days increased by 36% in the combined treatment compared with a fungal infection alone. FUA treatment delayed larval development, decreased the total hemocyte count, and increased both the phenoloxidase activity in integuments and the detoxification enzyme rate in hemolymph. A combined treatment with fungus and FUA led to increases in the aforementioned changes. Toxicosis caused by FUA provides a stable synergistic effect between FUA and B. bassiana. The combination can be promising for the development of highly efficient products against CPB. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Epigallocatechin-3-gallate protects Kuruma shrimp Marsupeneaus japonicus from white spot syndrome virus and Vibrio alginolyticus.

PubMed

Wang, Zhi; Sun, Baozhen; Zhu, Fei

2018-07-01

Epigallocatechin-3-gallate (EGCG) is the most abundant catechin in green tea and exhibits potential antibacterial and anticancer activities. In this study, EGCG was used in pathogen-challenge experiments in shrimp to discover its effect on the innate immune system of an invertebrate. Kuruma shrimp Marsupeneaus japonicus was used as an experimental model and challenged with white spot syndrome virus (WSSV) and the Gram-negative bacterium Vibrio alginolyticus. Pathogen-challenge experiments showed that EGCG pretreatment significantly delayed and reduced mortality upon WSSV and V. alginolyticus infection, with VP-28 copies of WSSV also reduced. Quantitative reverse transcription polymerase chain reaction revealed the positive influence of EGCG on several innate immune-related genes, including IMD, proPO, QM, myosin, Rho, Rab7, p53, TNF-alpha, MAPK, and NOS, and we observed positive influences on three immune parameters, including total hemocyte count and phenoloxidase and superoxide dismutase activities, by EGCG treatment. Additionally, results showed that EGCG treatment significantly reduced apoptosis upon V. alginolyticus challenge. These results indicated the positive role of EGCG in the shrimp innate immune system as an enhancer of immune parameters and an inhibitor of apoptosis, thereby delaying and reducing mortality upon pathogen challenge. Our findings provide insight into potential therapeutic or preventive functions associated with EGCG to enhance shrimp immunity and protect shrimp from pathogen infection. Copyright © 2018 Elsevier Ltd. All rights reserved.

White Shrimp Litopenaeus vannamei That Have Received Gracilaria tenuistipitata Extract Show Early Recovery of Immune Parameters after Ammonia Stressing

PubMed Central

Chen, Yu-Yuan; Chen, Jiann-Chu; Lin, Yong-Chin; Yeh, Su-Tuen; Huang, Chien-Lun

2015-01-01

White shrimp Litopenaeus vannamei immersed in seawater (35‰) containing Gracilaria tenuistipitata extract (GTE) at 0 (control), 400, and 600 mg/L for 3 h were exposed to 5 mg/L ammonia-N (ammonia as nitrogen), and immune parameters including hyaline cells (HCs), granular cells (GCs, including semi-granular cells), total hemocyte count (THC), phenoloxidase (PO) activity, respiratory bursts (RBs), superoxide dismutase (SOD) activity, lysozyme activity, and hemolymph protein level were examined 24~120 h post-stress. The immune parameters of shrimp immersed in 600 mg/L GTE returned to original values earlier, at 96~120 h post-stress, whereas in control shrimp they did not. In another experiment, shrimp were immersed in seawater containing GTE at 0 and 600 mg/L for 3 h and examined for transcript levels of immune-related genes at 24 h post-stress. Transcript levels of lipopolysaccharide and β-1,3-glucan binding protein (LGBP), peroxinectin (PX), cytMnSOD, mtMnSOD, and HSP70 were up-regulated at 24 h post-stress in GTE receiving shrimp. We concluded that white shrimp immersed in seawater containing GTE exhibited a capability for maintaining homeostasis by regulating cellular and humoral immunity against ammonia stress as evidenced by up-regulated gene expression and earlier recovery of immune parameters. PMID:26058012

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.

Effect of dietary formic acid and astaxanthin on the survival and growth of Pacific white shrimp (Litopenaeus vannamei) and their resistance to Vibrio parahaemolyticus.

PubMed

Chuchird, Niti; Rorkwiree, Phitsanu; Rairat, Tirawat

2015-01-01

A 90-day feeding trial was conducted to evaluate the effects of formic acid (FA) and astaxanthin (AX) on growth, survival, immune parameters, and tolerance to Vibrio infection in Pacific white shrimp. The study was divided into two experiments. In experiment 1, postlarvae-12 were randomly distributed into six groups and then fed four times daily with six experimental diets contained 0.3 % FA, 0.6 % FA, 50 ppm AX, 0.3 % FA + 50 ppm AX, 0.6 % FA + 50 ppm AX, or none of these supplements (control diet). After 60 days of the feeding trials, the body weight of all treatment groups was not significantly different from the control group, although shrimp fed formic acid had significantly lower body weight than shrimp fed 50 ppm AX. However, the 0.6 % FA + 50 ppm AX group had a significantly higher survival rate (82.33 ± 8.32 %) than the control group (64.33 ± 10.12 %). In experiment 2, Vibrio parahaemolyticus was added to each tank to obtain a final concentration of 10(4) colony-forming units/mL. Each treatment group received the aforementioned diets for another 30 days. At the end of this experiment, there was no difference in the weight gain among all experimental groups. However, the survival rate of shrimps whose diet included FA, AX, and their combination (in the range of 45.83-67.50 %) was significantly higher than the control group (20.00 ± 17.32 %). FA-fed shrimps also had significantly lower total intestinal bacteria and Vibrio spp. counts, while immune parameters [total hemocyte count (THC), phagocytosis activity, phenoloxidase (PO) activity, and superoxide dismutase (SOD) activity] of AX-fed groups were significantly improved compared with the other groups. In conclusion, FA, AX, and their combination are useful in shrimp aquaculture.

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

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.

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.

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.

An evaluation of replacing fish meal with fermented soybean meal in the diet of Macrobrachium nipponense: Growth, nonspecific immunity, and resistance to Aeromonas hydrophila.

PubMed

Ding, Zhili; Zhang, Yixiang; Ye, Jinyun; Du, Zhenyu; Kong, Youqin

2015-05-01

Partial or complete replacement of fish meal (FM) with fermented soybean meal (FSM) was examined in Macrobrachium nipponense over an 8-week growth trial. Growth and immune characteristics were evaluated. Fermented soybean meal replaced 0 (FM, control), 25% (R25), 50% (R50), 75% (R75), or 100% of the FM (R100) in five isocaloric and isonitrogenous diets. Each diet was fed to juvenile prawns (mean weight, 0.103 ± 0.0009 g) twice daily to apparent satiation in five replicates. Weight gain and specific growth rate of M. nipponense were significantly higher in prawns fed the R25 diet than that of prawns fed the FM diet. No significant differences were observed among the other treatments. Total hemocyte count and hemolymph phagocytic activity decreased as the proportion of FSM increased. Total antioxidant activity competence and malondialdehyde level in the hepatopancreas were highest in prawns fed the R100 diet. mRNA levels of the antioxidant genes Cu-Zn superoxide dismutase and catalase, heat shock cognate protein 70, and heat shock protein 90 were significantly differentially regulated in the prawn hepatopancreas. In addition, percent mortality increased after challenge with live Aeromonas hydrophila. Percent mortality of prawns fed the R100 diet was significantly higher than that of prawns fed the FM and R25 diets. These findings demonstrate that (1) M. nipponense growth performance was not affected by including a high proportion of FSM in the diet, and the best growth performance was obtained when 25% of the FM was replaced with FSM; (2) nonspecific immunity was impaired when all of the FM was replaced with FSM. Copyright © 2015 Elsevier Ltd. All rights reserved.

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.

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

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

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.

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.

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

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

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.

Effect of silver nanoparticles on the metabolic rate, hematological response, and survival of juvenile white shrimp Litopenaeus vannamei.

PubMed

Juarez-Moreno, Karla; Mejía-Ruiz, Claudio Humberto; Díaz, Fernando; Reyna-Verdugo, Horacio; Re, Ana Denisse; Vazquez-Felix, Edgar F; Sánchez-Castrejón, Edna; Mota-Morales, Josué D; Pestryakov, Alexey; Bogdanchikova, Nina

2017-02-01

White spot syndrome virus (WSSV) is highly lethal and contagious in shrimps; its outbreaks causes an economic crisis for aquaculture. Several attempts have been made to treat this disease; however, to date, there is no effective cure. Because of their antimicrobial activities, silver nanoparticles (AgNPs) are the most studied nanomaterial. Although the antiviral properties of AgNPs have been studied, their antiviral effect against viral infection in aquaculture has not been reported. The AgNPs tested herein are coated with polyvinylpyrrolidone (PVP) and possess multiple international certifications for their use in veterinary and human applications. The aim of this work was to evaluate the survival rate of juvenile white shrimps (Litopenaeus vannamei) after the intramuscular administration of AgNPs. For this, different concentrations of metallic AgNPs and PVP alone were injected into the organisms. After 96 h of administration, shrimp survival was more than 90% for all treatments. The oxygen consumption routine rate and total hemocyte count remained unaltered after AgNP injection, reflecting no stress caused. We evaluated whether AgNPs had an antiviral effect in shrimps infected with WSSV. The results revealed that the survival rate of WSSV-infected shrimps after AgNP administration was 80%, whereas the survival rate of untreated organisms was only 10% 96 h after infection. These results open up the possibility to explore the potential use of AgNPs as antiviral agents for the treatment of diseases in aquaculture organisms, particularly the WSSV in shrimp culture. Copyright © 2016 Elsevier Ltd. All rights reserved.

First report of the pituitary adenylate cyclase activating polypeptide (PACAP) in crustaceans: conservation of its functions as growth promoting factor and immunomodulator in the white shrimp Litopenaeus vannamei.

PubMed

Lugo, Juana María; Carpio, Yamila; Morales, Reynold; Rodríguez-Ramos, Tania; Ramos, Laida; Estrada, Mario Pablo

2013-12-01

The high conservation of the pituitary adenylate cyclase activating polypeptide (PACAP) sequence indicates that this peptide fulfills important biological functions in a broad spectrum of organisms. However, in invertebrates, little is known about its presence and its functions remain unclear. Up to now, in non-mammalian vertebrates, the majority of studies on PACAP have focused mainly on the localization, cloning and structural evolution of this peptide. As yet, little is known about its biological functions as growth factor and immunomodulator in lower vertebrates. Recently, we have shown that PACAP, apart from its neuroendocrine role, influences immune functions in larval and juvenile fish. In this work, we isolated for the first time the cDNA encoding the mature PACAP from a crustacean species, the white shrimp Litopenaeus vannamei, corroborating its high degree of sequence conservation, when compared to sequences reported from tunicates to mammalian vertebrates. Based on this, we have evaluated the effects of purified recombinant Clarias gariepinus PACAP administrated by immersion baths on white shrimp growth and immunity. We demonstrated that PACAP improves hemocyte count, superoxide dismutase, lectins and nitric oxide synthase derived metabolites in treated shrimp related with an increase in total protein concentration and growth performance. From our results, PACAP acts as a regulator of shrimp growth and immunity, suggesting that in crustaceans, as in vertebrate organisms, PACAP is an important molecule shared by both the endocrine and the immune systems. Copyright © 2013 Elsevier Ltd. All rights reserved.

Total motile sperm count has a superior predictive value over the WHO 2010 cut-off values for the outcomes of intracytoplasmic sperm injection cycles.

PubMed

Borges, E; Setti, A S; Braga, D P A F; Figueira, R C S; Iaconelli, A

2016-09-01

The objective of this study was to compare (i) the intracytoplasmic sperm injection outcomes among groups with different total motile sperm count ranges, (ii) the intracytoplasmic sperm injection outcomes between groups with normal and abnormal total motile sperm count, and (iii) the predictive values of WHO 2010 cut-off values and pre-wash total motile sperm count for the intracytoplasmic sperm injection outcomes, in couples with male infertility. This study included data from 518 patients undergoing their first intracytoplasmic sperm injection cycle as a result of male infertility. Couples were divided into five groups according to their total motile sperm count: Group I, total motile sperm count <1 × 10(6) ; group II, total motile sperm count 1-5 × 10(6) ; group III, total motile sperm count 5-10 × 10(6) ; group IV, total motile sperm count 10-20 × 10(6) ; and group V, total motile sperm count >20 × 10(6) (which was considered a normal total motile sperm count value). Then, couples were grouped into an abnormal and normal total motile sperm count group. The groups were compared regarding intracytoplasmic sperm injection outcomes. The predictive values of WHO 2010 cut-off values and total motile sperm count for the intracytoplasmic sperm injection outcomes were also investigated. The fertilization rate was lower in total motile sperm count group I compared to total motile sperm count group V (72.5 ± 17.6 vs. 84.9 ± 14.4, p = 0.011). The normal total motile sperm count group had a higher fertilization rate (84.9 ± 14.4 vs. 81.1 ± 15.8, p = 0.016) and lower miscarriage rate (17.9% vs. 29.5%, p = 0.041) compared to the abnormal total motile sperm count group. The total motile sperm count was the only parameter that demonstrated a predictive value for the formation of high-quality embryos on D2 (OR: 1.18, p = 0.013), formation of high-quality embryos on D3 (OR: 1.12, p = 0.037), formation of blastocysts on D5 (OR: 1.16, p = 0.011), blastocyst expansion grade on D5 (OR: 1.27, p = 0.042), and the odds of miscarriage (OR: 0.52, p < 0.045). The total motile sperm count has a greater predictive value than the WHO 2010 cut-off values for laboratory results and pregnancy outcomes in couples undergoing intracytoplasmic sperm injection as a result of male infertility. © 2016 American Society of Andrology and European Academy of Andrology.

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.

NASA Space Radiation Program Integrative Risk Model Toolkit

NASA Technical Reports Server (NTRS)

Kim, Myung-Hee Y.; Hu, Shaowen; Plante, Ianik; Ponomarev, Artem L.; Sandridge, Chris

2015-01-01

NASA Space Radiation Program Element scientists have been actively involved in development of an integrative risk models toolkit that includes models for acute radiation risk and organ dose projection (ARRBOD), NASA space radiation cancer risk projection (NSCR), hemocyte dose estimation (HemoDose), GCR event-based risk model code (GERMcode), and relativistic ion tracks (RITRACKS), NASA radiation track image (NASARTI), and the On-Line Tool for the Assessment of Radiation in Space (OLTARIS). This session will introduce the components of the risk toolkit with opportunity for hands on demonstrations. The brief descriptions of each tools are: ARRBOD for Organ dose projection and acute radiation risk calculation from exposure to solar particle event; NSCR for Projection of cancer risk from exposure to space radiation; HemoDose for retrospective dose estimation by using multi-type blood cell counts; GERMcode for basic physical and biophysical properties for an ion beam, and biophysical and radiobiological properties for a beam transport to the target in the NASA Space Radiation Laboratory beam line; RITRACKS for simulation of heavy ion and delta-ray track structure, radiation chemistry, DNA structure and DNA damage at the molecular scale; NASARTI for modeling of the effects of space radiation on human cells and tissue by incorporating a physical model of tracks, cell nucleus, and DNA damage foci with image segmentation for the automated count; and OLTARIS, an integrated tool set utilizing HZETRN (High Charge and Energy Transport) intended to help scientists and engineers study the effects of space radiation on shielding materials, electronics, and biological systems.

The effects of high voltage transmission lines on honey bees. Interim report. [765 kV transmission line

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

Greenberg, B.; Kunich, J.C.; Bindokas, V.P.

1978-10-01

Results of the first year's field study of possible effects on honey bees of a 765 kV transmission line are reported. Conventional hives and metal-free hives, shielded and unshielded, were placed under the line (E-field, ca. 7 kV/m) and in a control area (E-field, ca. 10 V/m) about 400 m away. Bees in unshielded conventional hives under the line weighed less, stored little honey whose moisture content was subnormal (hive weight gain was essentially zero), propolyzed hive entrances excessively but not completely, produced fewer pupae but normal numbers of eggs and larvae, and failed to survive the winter. Unshielded metal-freemore » hives under the line had the following normal features: bee weight; hive weight gain; honey moisture content; and number of eggs, larvae, and pupae. Their abnormal features were: propolization of hive entrances, but at a slower rate and to a lesser extent than conventional hives; aggressive clusters of bees at lower front hive corners; poor overwintering survival; and possibly higher hemocyte counts.« less

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.

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.

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.

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.

Effect of Shilajit enriched diet on immunity, antioxidants, and disease resistance in Macrobrachium rosenbergii (de Man) against Aeromonas hydrophila.

PubMed

Musthafa, Mohamed Saiyad; Jawahar Ali, Abdul Rahman; Hyder Ali, Abdul Rahuman; Mohamed, Mohamed Jamal; War, Mehrajuddin; Naveed, Mohamed Saquib; Al-Sadoon, Mohammad K; Paray, Bilal Ahmad; Rani, Kuppusamy Umaa; Arockiaraj, Jesu; Balasundaram, Chellam; Harikrishnan, Ramasamy

2016-10-01

The effect of diet supplemented with Shilajit, a multi-component natural mineral substance on the antioxidant activity, immune response, and disease resistance in freshwater prawn, Macrobrachium rosenbergii (de Man) against Aeromonas hydrophila is reported. The total hemocyte count (THC) and phagocytic activity significantly increased with 2 g kg(-1) supplemented diet on first week and with other enriched diets on weeks 2 and 4. The respiratory burst (RB) activity and glutathione peroxidase (GPx) activity were significantly increased with 2 g kg(-1) supplemented diet on weeks 1 and 2 whereas 2 and 4 g kg(-1) diets on week 4. The phenoloxidase (PO) activity increased significantly with 2 g kg(-1) diet only on second week and with other enriched diets only on fourth week. The superoxide dismutase (SOD) activity increased significantly with any enriched diet during the experimental period except with 6 g kg(-1) diets on first week. However, the glutathione reductase (GR) activity was enhanced significantly only with 2 g kg(-1) enriched diets on weeks 2 and 4. The cumulative mortality of the prawn fed with 2 and 4 g kg(-1) enriched diets was 10% and 15% whereas with 6 g kg(-1) diet the mortality was 20%. The results suggest that diet enriched with Shilajit at 2 g kg(-1) or 4 g kg(-1) positively enhances the antioxidant activity, immunity, and disease resistance in M. rosenbergii against A. hydrophila. Copyright © 2016 Elsevier Ltd. All rights reserved.

Total lymphocyte count and subpopulation lymphocyte counts in relation to dietary intake and nutritional status of peritoneal dialysis patients.

PubMed

Grzegorzewska, Alicja E; Leander, Magdalena

2005-01-01

Dietary deficiency causes abnormalities in circulating lymphocyte counts. For the present paper, we evaluated correlations between total and subpopulation lymphocyte counts (TLC, SLCs) and parameters of nutrition in peritoneal dialysis (PD) patients. Studies were carried out in 55 patients treated with PD for 22.2 +/- 11.4 months. Parameters of nutritional status included total body mass, lean body mass (LBM), body mass index (BMI), and laboratory indices [total protein, albumin, iron, ferritin, and total iron binding capacity (TIBC)]. The SLCs were evaluated using flow cytometry. Positive correlations were seen between TLC and dietary intake of niacin; TLC and CD8 and CD16+56 counts and energy delivered from protein; CD4 count and beta-carotene and monounsaturated fatty acids 17:1 intake; and CD19 count and potassium, copper, vitamin A, and beta-carotene intake. Anorexia negatively influenced CD19 count. Serum albumin showed correlations with CD4 and CD19 counts, and LBM with CD19 count. A higher CD19 count was connected with a higher red blood cell count, hemoglobin, and hematocrit. Correlations were observed between TIBC and TLC and CD3 and CD8 counts, and between serum Fe and TLC and CD3 and CD4 counts. Patients with a higher CD19 count showed a better clinical-laboratory score, especially less weakness. Patients with a higher CD4 count had less expressed insomnia. Quantities of ingested vitamins and minerals influence lymphocyte counts in the peripheral blood of PD patients. Evaluation of TLC and SLCs is helpful in monitoring the effectiveness of nutrition in these patients.

A THUMBNAIL HISTORY OF HETEROTROPHIC PLATE COUNT (HPC) METHODOLOGY IN THE UNITED STATES

EPA Science Inventory

Over the past 100 years, the method of determining the number of bacteria in water, foods or other materials has been termed variously as: bacterial plate count, total plate count, total viable plate count, aerobic plate count, standard plate cound and more recently, heterotrophi...

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.

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

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.

Forecasting models for sugi (Cryptomeria japonica D. Don) pollen count showing an alternate dispersal rhythm.

PubMed

Ito, Yukiko; Hattori, Reiko; Mase, Hiroki; Watanabe, Masako; Shiotani, Itaru

2008-12-01

Pollen information is indispensable for allergic individuals and clinicians. This study aimed to develop forecasting models for the total annual count of airborne pollen grains based on data monitored over the last 20 years at the Mie Chuo Medical Center, Tsu, Mie, Japan. Airborne pollen grains were collected using a Durham sampler. Total annual pollen count and pollen count from October to December (OD pollen count) of the previous year were transformed to logarithms. Regression analysis of the total pollen count was performed using variables such as the OD pollen count and the maximum temperature for mid-July of the previous year. Time series analysis revealed an alternate rhythm of the series of total pollen count. The alternate rhythm consisted of a cyclic alternation of an "on" year (high pollen count) and an "off" year (low pollen count). This rhythm was used as a dummy variable in regression equations. Of the three models involving the OD pollen count, a multiple regression equation that included the alternate rhythm variable and the interaction of this rhythm with OD pollen count showed a high coefficient of determination (0.844). Of the three models involving the maximum temperature for mid-July, those including the alternate rhythm variable and the interaction of this rhythm with maximum temperature had the highest coefficient of determination (0.925). An alternate pollen dispersal rhythm represented by a dummy variable in the multiple regression analysis plays a key role in improving forecasting models for the total annual sugi pollen count.

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

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.

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.

Relationship between shift work and peripheral total and differential leukocyte counts in Chinese steel workers.

PubMed

Lu, Li-Fen; Wang, Chao-Ping; Tsai, I-Ting; Hung, Wei-Chin; Yu, Teng-Hung; Wu, Cheng-Ching; Hsu, Chia-Chang; Lu, Yung-Chuan; Chung, Fu-Mei; Jean, Mei-Chu Yen

2016-01-01

Even though shift work has been suspected to be a risk factor for cardiovascular disease, little research has been done to determine the logical underlying inflammation mechanisms. This study investigated the association between shift work and circulating total and differential leukocyte counts among Chinese steel workers. The subjects were 1,654 line workers in a steel plant, who responded to a cross-sectional survey with a questionnaire on basic attributes, life style, and sleep. All workers in the plant received a periodic health checkup. Total and differential leukocytes counts were also examined in the checkup. Shift workers had higher rates of alcohol use, smoking, poor sleep, poor physical exercise, and obesity than daytime workers. In further analysis, we found that the peripheral total WBC, monocyte, neutrophil, and lymphocyte counts were also greater in shift workers than in daytime workers. When subjects were divided into quartiles according to total WBC, neutrophil, monocyte, and lymphocyte counts, increased leukocyte count was associated with shift work. Using stepwise linear regression analysis, smoking, obesity, and shift work were independently associated with total WBC, monocyte, neutrophil, and lymphocyte counts. This study indicates that peripheral total and differential leukocyte counts are significantly higher in shift workers, which suggests that shift work may be a risk factor of cardiovascular disease. Applicable intervention strategies are needed for prevention of cardiovascular disease for shift workers.

Increment of absolute neutrophil count in the third trimester and increased risk of small-for-gestational-age birth: Hirakata Risk Associated with Pregnancy Assessment Research (HIRAPAR).

PubMed

Harita, Nobuko; Kariya, Masatoshi; Hayashi, Tomoshige; Sato, Kyoko Kogawa; Nakamura, Kimihiko; Endo, Ginji; Narimoto, Katsuhiko

2012-09-01

Small-for-gestational-age (SGA) infants, who have growth restriction, have higher perinatal morbidity and mortality. Excessive inflammatory reaction such as neutrophil activation has been observed in pregnant women whose offspring had restricted fetal growth, but the association between white blood cell (WBC) counts and SGA birth has not yet been assessed. We therefore examined the association of WBC count and its change with the risk of SGA birth. We enrolled 2356 pregnant women who had full-term singleton delivery at a private maternity hospital in Hirakata, Japan. SGA was defined as under the 10th percentile of birthweight for gestational age, baby sex, and mother's parity according to the Japanese neonatal anthropometric charts renewed in 2010. Blood samples were measured in the first and third trimesters. We performed multiple logistic regression analysis to assess associations between total and differential WBC counts and SGA birth. Women with SGA birth tended to have higher total WBC count in the third trimester compared with women who did not have SGA birth. This tendency was not observed for total WBC count in the first trimester. After adjustment for age, height, body mass index at entry, smoking habit, weekly gestational weight gain, and pregnancy-induced hypertension, higher total WBC count in the third trimester was associated with an increased risk of SGA birth. Total WBC count in the first trimester did not show any significant association with SGA birth. The ratio of total WBC count in the third trimester to that in the first trimester was associated with SGA birth; the odds ratio for 1 unit increase was 3.02 (95% CI: 1.54-5.92). Regarding differential WBC counts in the third trimester, neutrophil count but not lymphocyte count was associated positively with SGA birth. Higher total WBC and absolute neutrophil counts in the third trimester were associated with SGA birth. In addition, greater ratio of increase in total WBC counts during pregnancy showed a positive association with the incidence of SGA birth. These associations may reflect a vicious cycle of inflammation and placental dysfunction as a cause of fetal growth restriction. Copyright © 2012 Elsevier Ireland 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.

Testing the Accuracy of Aerial Surveys for Large Mammals: An Experiment with African Savanna Elephants (Loxodonta africana).

PubMed

Schlossberg, Scott; Chase, Michael J; Griffin, Curtice R

2016-01-01

Accurate counts of animals are critical for prioritizing conservation efforts. Past research, however, suggests that observers on aerial surveys may fail to detect all individuals of the target species present in the survey area. Such errors could bias population estimates low and confound trend estimation. We used two approaches to assess the accuracy of aerial surveys for African savanna elephants (Loxodonta africana) in northern Botswana. First, we used double-observer sampling, in which two observers make observations on the same herds, to estimate detectability of elephants and determine what variables affect it. Second, we compared total counts, a complete survey of the entire study area, against sample counts, in which only a portion of the study area is sampled. Total counts are often considered a complete census, so comparing total counts against sample counts can help to determine if sample counts are underestimating elephant numbers. We estimated that observers detected only 76% ± SE of 2% of elephant herds and 87 ± 1% of individual elephants present in survey strips. Detectability increased strongly with elephant herd size. Out of the four observers used in total, one observer had a lower detection probability than the other three, and detectability was higher in the rear row of seats than the front. The habitat immediately adjacent to animals also affected detectability, with detection more likely in more open habitats. Total counts were not statistically distinguishable from sample counts. Because, however, the double-observer samples revealed that observers missed 13% of elephants, we conclude that total counts may be undercounting elephants as well. These results suggest that elephant population estimates from both sample and total counts are biased low. Because factors such as observer and habitat affected detectability of elephants, comparisons of elephant populations across time or space may be confounded. We encourage survey teams to incorporate detectability analysis in all aerial surveys for mammals.

Testing the Accuracy of Aerial Surveys for Large Mammals: An Experiment with African Savanna Elephants (Loxodonta africana)

PubMed Central

Schlossberg, Scott; Chase, Michael J.; Griffin, Curtice R.

2016-01-01

Accurate counts of animals are critical for prioritizing conservation efforts. Past research, however, suggests that observers on aerial surveys may fail to detect all individuals of the target species present in the survey area. Such errors could bias population estimates low and confound trend estimation. We used two approaches to assess the accuracy of aerial surveys for African savanna elephants (Loxodonta africana) in northern Botswana. First, we used double-observer sampling, in which two observers make observations on the same herds, to estimate detectability of elephants and determine what variables affect it. Second, we compared total counts, a complete survey of the entire study area, against sample counts, in which only a portion of the study area is sampled. Total counts are often considered a complete census, so comparing total counts against sample counts can help to determine if sample counts are underestimating elephant numbers. We estimated that observers detected only 76% ± SE of 2% of elephant herds and 87 ± 1% of individual elephants present in survey strips. Detectability increased strongly with elephant herd size. Out of the four observers used in total, one observer had a lower detection probability than the other three, and detectability was higher in the rear row of seats than the front. The habitat immediately adjacent to animals also affected detectability, with detection more likely in more open habitats. Total counts were not statistically distinguishable from sample counts. Because, however, the double-observer samples revealed that observers missed 13% of elephants, we conclude that total counts may be undercounting elephants as well. These results suggest that elephant population estimates from both sample and total counts are biased low. Because factors such as observer and habitat affected detectability of elephants, comparisons of elephant populations across time or space may be confounded. We encourage survey teams to incorporate detectability analysis in all aerial surveys for mammals. PMID:27755570

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

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.

A comparison of manual and electronic counting for total nucleated cell counts on synovial fluid from canine stifle joints.

PubMed

Atilola, M A; Lumsden, J H; Rooke, F

1986-04-01

Synovial fluids collected from the stifle joints of 20 physically normal adult dogs were subjected to cytological examination. A total nucleated cell count was performed on each sample using both an electronic cell counter and a hemocytometer. The mean of the total counts done with the electronic counter was significantly higher (1008 cells/microL) than that obtained manually with the hemocytometer (848 cells/microL).

[Endotoxin Contamination and Correlation with Other Water Quality Parameters of Groundwater from Self-Contained Wells in Beijing].

PubMed

Zhang, Can; Liu, Wen-jun; Ao, Lu; Shi, Yun; An, Dai-zhi; Liu, Zhi-ping

2015-12-01

A survey of endotoxin activity in groundwater from 14 self-contained wells in PLA units stationed in Beijing was conducted by the kinetic-turbid assay of Tachypleus Amebocyte Lysate (TAL). Bacteriological parameters, including total cell counts detected by flow cytometry, heterotrophic plate counts (HPC), standard plate counts and total coliforms were analyzed. Additionally, suspended particles, turbidity, dissolved organic carbon (DOC), and UV₂₅₄ were investigated. Total endotoxin activities ranged from 0. 15 to 13.20 EU · mL⁻¹, free endotoxin activities ranged from 0.10 to 5.29 EU · mL⁻¹ and bound endotoxin activities ranged from 0.01 to 8.60 EU · mL⁻¹. Most of the endotoxins in heavily contaminated groundwater existed as bound endotoxins. As for total endotoxins, the sequence of correlation coefficients with other parameters was total cell counts (r = 0.88 ) > HPC (r = 0.79) > DOC (r = 0.77) > UV₂₅₄ (r = 0.57) > total coliforms (r = 0.50) > standard plate counts (r = 0.49) = turbidity (r = 0. 49) > total particles (r = 0.41). The sequence of correlations of the bound endotoxins with other parameters was total cell counts (r = 0.81) > HPC (r = 0.66) > total coliforms (r = 0.65) > turbidity (r = 0.62) > total particles (r = 0.58) > standard plate counts (r = 0.22). Free endotoxins were correlated with DOC and UV₂₅₄, r = 0.58 and 0.26, respectively. Result showed free endotoxins had a higher correlation with DOC, and a lower correlation with UV₂₅₄.

Total airborne mold particle sampling: evaluation of sample collection, preparation and counting procedures, and collection devices.

PubMed

Godish, Diana; Godish, Thad

2008-02-01

This study was conducted to evaluate (i) procedures used to collect, prepare, and count total airborne mold spore/particle concentrations, and (ii) the relative field performance of three commercially available total airborne mold spore/particle sampling devices. Differences between factory and laboratory airflow calibration values of axial fan-driven sampling instruments (used in the study) indicated a need for laboratory calibration using a mass flow meter to ensure that sample results were accurately calculated. An aniline blue-amended Calberla's solution adjusted to a pH of 4.2-4.4 provided good sample mounting/counting results using Dow Corning high vacuum grease, Dow Corning 280A adhesive, and Dow Corning 316 silicone release spray for samples collected using mini-Burkard and Allergenco samplers. Count variability among analysts was most pronounced in 5% counts of relatively low mold particle deposition density samples and trended downward with increased count percentage and particle deposition density. No significant differences were observed among means of 5, 10, and 20% counts and among analysts; a significant interaction effect was observed between analysts' counts and particle deposition densities. Significantly higher mini-Burkard and Air-O-Cell total mold spore/particle counts for 600x vs. 400x (1.9 and 2.3 x higher, respectively), 1000x vs. 600x (1.9 and 2.2 x higher, respectively) and 1000x vs. 400x (3.6 and 4.6 x higher, respectively) comparisons indicated that 1000x magnification counts best quantified total airborne mold spore/particles using light microscopy, and that lower magnification counts may result in unacceptable underreporting of airborne mold spore/particle concentrations. Modest but significantly higher (1.2x) total mold spore concentrations were observed with Allergenco vs. mini-Burkard samples collected in co-located, concurrently operated sampler studies; moderate but significantly higher mini-Burkard count values (1.4x) were observed in similar studies with Air-O-Cell samplers. These count differences were relatively small compared with the large differences observed among three count magnifications.

[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.

Innovative application of classic and newer techniques for the characterization of haemocytes in the New Zealand black-footed abalone (Haliotis iris).

PubMed

Grandiosa, Roffi; Mérien, Fabrice; Pillay, Krish; Alfaro, Andrea

2016-01-01

Haemocytes play an important role in innate immune responses within invertebrate organisms. However, identification and quantification of different types of haemocytes can be extremely challenging, and has led to numerous inconsistencies and misinterpretations within the literature. As a step to rectify this issue, we present a comprehensive and detailed approach to characterize haemocytes using a combination of classical (cytochemical and phagocytosis assays with optical microscopy) and novel (flow cytometry with Sysmex XN-1000 and Muse(®) Cell analyser) techniques. The Sysmex XN-1000 is an innovative fluorescent flow cytometric analyser that can effectively detect, identify and count haemocytes, while the Muse(®) Cell analyser provides accurate and rapid haemocyte cell counts and viability. To illustrate this approach, we present the first report on morphological and functional features of New Zealand black-footed abalone (Haliotis iris) haemocyte cells. Two types of haemocytes were identified in this study, including type I (monocyte-like) and type II (lymphocyte-like) cells. Granular cells, which have been reported in other molluscan species, were not detected in H. iris. Cell types were categorized based on shape, size, internal structures and function. The lymphocyte-like haemocytes were the most abundant hemocytes in the haemolymph samples, and they had large nuclei and basic cytoplasms. Monocyte-like cells generally were larger cells compared to lymphocyte-like cells, and had low nucleus-cytoplasm ratios. Monocyte-like cells showed higher phagocytic activity when encountering Zymosan A particles compared to lymphocyte-like cells. The present study provides a comprehensive and accurate new approach to identify and quantify haemocyte cells for future comparative studies on the immune system of abalone and other molluscan species. Copyright © 2015 Elsevier Ltd. All rights reserved.

Platelet count and total and cause-specific mortality in the Women's Health Initiative.

PubMed

Kabat, Geoffrey C; Kim, Mimi Y; Verma, Amit K; Manson, JoAnn E; Lin, Juan; Lessin, Lawrence; Wassertheil-Smoller, Sylvia; Rohan, Thomas E

2017-04-01

We used data from the Women's Health Initiative to examine the association of platelet count with total mortality, coronary heart disease (CHD) mortality, cancer mortality, and non-CHD/noncancer mortality. Platelet count was measured at baseline in 159,746 postmenopausal women and again in year 3 in 75,339 participants. Participants were followed for a median of 15.9 years. Cox proportional hazards models were used to estimate the relative mortality hazards associated with deciles of baseline platelet count and of the mean of baseline + year 3 platelet count. Low and high deciles of both baseline and mean platelet count were positively associated with total mortality, CHD mortality, cancer mortality, and non-CHD/noncancer mortality. The association was robust and was not affected by adjustment for a number of potential confounding factors, exclusion of women with comorbidity, or allowance for reverse causality. Low- and high-platelet counts were associated with all four outcomes in never smokers, former smokers, and current smokers. In this large study of postmenopausal women, both low- and high-platelet counts were associated with total and cause-specific mortality. Copyright © 2017 Elsevier Inc. All rights reserved.

Use of an activity monitor to detect response to treatment in dogs with osteoarthritis.

PubMed

Brown, Dorothy Cimino; Boston, Raymond C; Farrar, John T

2010-07-01

To determine whether an activity monitor (AM) could be used to detect changes in activity in dogs with osteoarthritis treated with carprofen or a placebo. Randomized controlled trial. 70 dogs with no clinically important abnormalities other than osteoarthritis for which they were not currently being treated. Dogs wore an AM continuously for 21 days. On days 8 through 21, the dogs were treated with carprofen (n = 35) or a placebo (35). Total activity counts for days 1 through 7 (baseline) were compared with total activity counts for days 15 through 21 (endpoint). The change in total activity count from baseline to endpoint was assessed within each treatment group as well as between groups. Linear regression analysis was performed to test for an association between treatment and percentage change in activity counts while controlling for other variables. For placebo-treated dogs, median baseline total activity count was not significantly different from median endpoint total activity count (1,378,408 vs 1,310,112, respectively). For dogs receiving carprofen, there was a significant increase in median activity count from baseline to endpoint (1,276,427 vs 1,374,133). When age and baseline activity counts were controlled for, dogs in the carpofen-treated group had a 20% increase in activity counts, compared with placebo-treated dogs (95% confidence interval, 10% to 26%). Results suggested that the AM used in the present study may be a valid outcome assessment tool for documenting improved activity associated with treatment in dogs with osteoarthritis.

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).

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.

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.

Total mesophilic counts underestimate in many cases the contamination levels of psychrotrophic lactic acid bacteria (LAB) in chilled-stored food products at the end of their shelf-life.

PubMed

Pothakos, Vasileios; Samapundo, Simbarashe; Devlieghere, Frank

2012-12-01

The major objective of this study was to determine the role of psychrotrophic lactic acid bacteria (LAB) in spoilage-associated phenomena at the end of the shelf-life of 86 various packaged (air, vacuum, modified-atmosphere) chilled-stored retail food products. The current microbiological standards, which are largely based on the total viable mesophilic counts lack discriminatory capacity to detect psychrotrophic LAB. A comparison between the total viable counts on plates incubated at 30 °C (representing the mesophiles) and at 22 °C (indicating the psychrotrophs) for 86 food samples covering a wide range - ready-to-eat vegetable salads, fresh raw meat, cooked meat products and composite food - showed that a consistent underestimation of the microbial load occurs when the total aerobic mesophilic counts are used as a shelf-life parameter. In 38% of the samples, the psychrotrophic counts had significantly higher values (+0.5-3 log CFU/g) than the corresponding total aerobic mesophilic counts. A total of 154 lactic acid bacteria, which were unable to proliferate at 30 °C were isolated. In addition, a further 43 with a poor recovery at this temperature were also isolated. This study highlights the potential fallacy of the total aerobic mesophilic count as a reference shelf-life parameter for chilled food products as it can often underestimate the contamination levels at the end of the shelf-life. Copyright © 2012 Elsevier Ltd. All rights reserved.

Methods for analyzing matched designs with double controls: excess risk is easily estimated and misinterpreted when evaluating traffic deaths.

PubMed

Redelmeier, Donald A; Tibshirani, Robert J

2018-06-01

To demonstrate analytic approaches for matched studies where two controls are linked to each case and events are accumulating counts rather than binary outcomes. A secondary intent is to clarify the distinction between total risk and excess risk (unmatched vs. matched perspectives). We review past research testing whether elections can lead to increased traffic risks. The results are reinterpreted by analyzing both the total count of individuals in fatal crashes and the excess count of individuals in fatal crashes, each time accounting for the matched double controls. Overall, 1,546 individuals were in fatal crashes on the 10 election days (average = 155/d), and 2,593 individuals were in fatal crashes on the 20 control days (average = 130/d). Poisson regression of total counts yielded a relative risk of 1.19 (95% confidence interval: 1.12-1.27). Poisson regression of excess counts yielded a relative risk of 3.22 (95% confidence interval: 2.72-3.80). The discrepancy between analyses of total counts and excess counts replicated with alternative statistical models and was visualized in graphical displays. Available approaches provide methods for analyzing count data in matched designs with double controls and help clarify the distinction between increases in total risk and increases in excess risk. Copyright © 2018 Elsevier Inc. All rights reserved.

Influence of protein deposition on bacterial adhesion to contact lenses.

PubMed

Subbaraman, Lakshman N; Borazjani, Roya; Zhu, Hua; Zhao, Zhenjun; Jones, Lyndon; Willcox, Mark D P

2011-08-01

The aim of the study is to determine the adhesion of Gram positive and Gram negative bacteria onto conventional hydrogel (CH) and silicone hydrogel (SH) contact lens materials with and without lysozyme, lactoferrin, and albumin coating. Four lens types (three SH-balafilcon A, lotrafilcon B, and senofilcon A; one CH-etafilcon A) were coated with lysozyme, lactoferrin, or albumin (uncoated lenses acted as controls) and then incubated in Staphylococcus aureus (Saur 31) or either of two strains of Pseudomonas aeruginosa (Paer 6294 and 6206) for 24 h at 37 °C. The total counts of the adhered bacteria were determined using the H-thymidine method and viable counts by counting the number of colony-forming units on agar media. All three strains adhered significantly lower to uncoated etafilcon A lenses compared with uncoated SH lenses (p < 0.05). Lysozyme coating on all four lens types increased binding (total and viable counts) of Saur 31 (p < 0.05). However, lysozyme coating did not influence P. aeruginosa adhesion (p > 0.05). Lactoferrin coating on lenses increased binding (total and viable counts) of Saur 31 (p < 0.05). Lactoferrin-coated lenses showed significantly higher total counts (p < 0.05) but significantly lower viable counts (p < 0.05) of adhered P. aeruginosa strains. There was a significant difference between the total and viable counts (p < 0.05) that were bound to lactoferrin-coated lenses. Albumin coating of lenses increased binding (total and viable counts) of all three strains (p < 0.05). Lysozyme deposited on contact lenses does not possess antibacterial activity against certain bacterial strains, whereas lactoferrin possess an antibacterial effect against strains of P. aeruginosa.

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.

Reference values for maternal total and differential leukocyte counts in different trimesters of pregnancy and the initial postpartum period in western Turkey.

PubMed

Sanci, Muzaffer; Töz, Emrah; Ince, Onur; Özcan, Aykut; Polater, Kevser; Inan, Abdurrahman Hamdi; Beyan, Emrah; Akkaya, Emrah

2017-07-01

The aim of this study was to investigate alterations in the leukocyte and differential leukocyte counts in different trimesters of pregnancy and the initial postpartum period. The study population consisted of 40,325 pregnant women. A full blood count and automated differential leukocyte count were performed and all the haemogram results in the different trimesters of pregnancy were recorded. Percentiles were calculated using statistical software. A total of 82,786 complete blood count evaluations were performed in 40,325 subjects from the 6th to 41st week of pregnancy and in the initial postpartum period. The leukocyte counts increased from the 1st to the 3rd trimester and peaked in the initial postpartum period. Our reference values for the total and differential leukocyte counts may assist clinicians in distinguishing between leukocytosis and pathological elevation of the white blood cell count during pregnancy and the initial postpartum period. Impact statement Pregnancy requires profound adaptation by multiple systems to accommodate the demands of the developing foetus. Similar to all other systems, many haematological changes occur during pregnancy. Studies of normal variation in leukocyte counts were insufficient to distinguish normal from abnormal leukocyte counts during pregnancy and in the initial postpartum period, due to small numbers of patients and a lack of differential leukocyte counts. Without reference leukocyte levels, infections may be more difficult to assess during pregnancy and in the postpartum period. In this study, we report the 3rd, 5th, 10th, 50th, 95th and 99th percentile values for the total and differential leukocyte counts according to trimester in normal pregnancy and the initial postpartum period. Our reference values for the total and differential leukocyte counts in each trimester and the initial postpartum period may assist clinicians in distinguishing between normal leukocytosis and pathological elevation of the white blood cell count during pregnancy and the initial postpartum period. Our results may prevent misdiagnosis of physiological elevated leukocytes as bacterial infection that leads to unnecessary medication use that may compromise the foetus.

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...

Use of an activity monitor to detect response to treatment in dogs with osteoarthritis

PubMed Central

Brown, Dorothy Cimino; Boston, Raymond C.; Farrar, John T.

2010-01-01

Objective To determine whether an activity monitor (AM) could be used to detect changes in activity in dogs with osteoarthritis treated with carprofen or a placebo. Design Randomized controlled trial. Animals 70 dogs with no clinically important abnormalities other than osteoarthritis for which they were not currently being treated. Procedures Dogs wore an AM continuously for 21 days. On days 8 through 21, the dogs were treated with carprofen (n = 35) or a placebo (35). Total activity counts for days 1 through 7 (baseline) were compared with total activity counts for days 15 through 21 (endpoint). The change in total activity count from baseline to endpoint was assessed within each treatment group as well as between groups. Linear regression analysis was performed to test for an association between treatment and percentage change in activity counts while controlling for other variables. Results For placebo-treated dogs, median baseline total activity count was not significantly different from median endpoint total activity count (1,378,408 vs 1,310,112, respectively). For dogs receiving carprofen, there was a significant increase in median activity count from baseline to endpoint (1,276,427 vs 1,374,133). When age and baseline activity counts were controlled for, dogs in the carpofen-treated group had a 20% increase in activity counts, compared with placebo-treated dogs (95% confidence interval, 10% to 26%). Conclusions and Clinical Relevance Results suggested that the AM used in the present study may be a valid outcome assessment tool for documenting improved activity associated with treatment in dogs with osteoarthritis. PMID:20590496

White Blood Cell Count and Total and Cause-Specific Mortality in the Women's Health Initiative.

PubMed

Kabat, Geoffrey C; Kim, Mimi Y; Manson, JoAnn E; Lessin, Lawrence; Lin, Juan; Wassertheil-Smoller, Sylvia; Rohan, Thomas E

2017-07-01

White blood cell (WBC) count appears to predict total mortality and coronary heart disease (CHD) mortality, but it is unclear to what extent the association reflects confounding by smoking, underlying illness, or comorbid conditions. We used data from the Women's Health Initiative to examine the associations of WBC count with total mortality, CHD mortality, and cancer mortality. WBC count was measured at baseline in 160,117 postmenopausal women and again in year 3 in 74,375 participants. Participants were followed for a mean of 16 years. Cox proportional hazards models were used to estimate the relative mortality hazards associated with deciles of baseline WBC count and of the mean of baseline + year 3 WBC count. High deciles of both baseline and mean WBC count were positively associated with total mortality and CHD mortality, whereas the association with cancer mortality was weaker. The association of WBC count with mortality was independent of smoking and did not appear to be influenced by previous disease history. The potential clinical utility of this common laboratory test in predicting mortality risk warrants further study. © The Author 2017. Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Evaluating remotely sensed plant count accuracy with differing unmanned aircraft system altitudes, physical canopy separations, and ground covers

NASA Astrophysics Data System (ADS)

Leiva, Josue Nahun; Robbins, James; Saraswat, Dharmendra; She, Ying; Ehsani, Reza

2017-07-01

This study evaluated the effect of flight altitude and canopy separation of container-grown Fire Chief™ arborvitae (Thuja occidentalis L.) on counting accuracy. Images were taken at 6, 12, and 22 m above the ground using unmanned aircraft systems. Plants were spaced to achieve three canopy separation treatments: 5 cm between canopy edges, canopy edges touching, and 5 cm of canopy edge overlap. Plants were placed on two different ground covers: black fabric and gravel. A counting algorithm was trained using Feature Analyst®. Total counting error, false positives, and unidentified plants were reported for images analyzed. In general, total counting error was smaller when plants were fully separated. The effect of ground cover on counting accuracy varied with the counting algorithm. Total counting error for plants placed on gravel (-8) was larger than for those on a black fabric (-2), however, false positive counts were similar for black fabric (6) and gravel (6). Nevertheless, output images of plants placed on gravel did not show a negative effect due to the ground cover but was impacted by differences in image spatial resolution.

Avian leucocyte counting using the hemocytometer

USGS Publications Warehouse

Dein, F.J.; Wilson, A.; Fischer, D.; Langenberg, P.

1994-01-01

Automated methods for counting leucocytes in avian blood are not available because of the presence of nucleated erythrocytes and thrombocytes. Therefore, total white blood cell counts are performed by hand using a hemocytometer. The Natt and Herrick and the Unopette methods are the most common stain and diluent preparations for this procedure. Replicate hemocytometer counts using these two methods were performed on blood from four birds of different species. Cells present in each square of the hemocytometer were counted. Counting cells in the corner, side, or center hemocytometer squares produced statistically equivalent results; counting four squares per chamber provided a result similar to that obtained by counting nine squares; and the Unopette method was more precise for hemocytometer counting than was the Natt and Herrick method. The Unopette method is easier to learn and perform but is an indirect process, utilizing the differential count from a stained smear. The Natt and Herrick method is a direct total count, but cell identification is more difficult.

A case-control study of domestic kitchen microbiology and sporadic Salmonella infection.

PubMed

Parry, S M; Slader, J; Humphrey, T; Holmes, B; Guildea, Z; Palmer, S R

2005-10-01

The microbiology of domestic kitchens in the homes of subjects who had suffered sporadic Salmonella infection (cases) was compared with control domestic kitchens. Case and control dishcloths and refrigerator swabs were examined for the presence of Salmonella spp., total Enterobacteriaceae counts and total aerobic colony counts. Salmonella spp. were isolated from both case and control dishcloths and refrigerators but there were no significant differences between the two groups. Colony counts were similar in case and control dishcloths and refrigerator swabs. There was no relationship between the total counts and presence of Salmonella . There was no evidence that cases of Salmonella infection were more likely to have kitchens which were contaminated with these bacteria or have higher bacterial counts than controls. Total bacterial counts were poor indicators of Salmonella contamination of the domestic kitchen environment. Further factors which could not be identified by a study of this design may increase risk of Salmonella food poisoning. These factors may include individual susceptibility of the patient. Alternatively, sporadic cases of Salmonella food poisoning may arise from food prepared outside the home.

Effect of various Na/K ratios in low-salinity well water on growth performance and physiological response of Pacific white shrimp Litopenaeus vannamei

NASA Astrophysics Data System (ADS)

Liu, Hongyu; Tan, Beiping; Yang, Jinfang; Lin, Yingbo; Chi, Shuyan; Dong, Xiaohui; Yang, Qihui

2014-09-01

To investigate the influence of sodium to potassium (Na/K) ratios on the growth performance and physiological response of the Pacific white shrimp ( Litopenaeus vananmei), various concentrations of KCl were added to low-salinity well water (salinity 4) in an 8-week culture trial. Six treatments with Na/K ratios of 60:1, 42:1, 33:1, 23:1, 17:1, and 14:1 were replicated in triplicate. The highest weight-gain rate (3 506±48)% and survival rate (89.38±0.88)% was observed in well water with Na/K ratios of 23:1 and 42:1, respectively, while the feed conversion ratio (1.02±0.01), oxygen consumption, and ammonia-N excretion rate was the lowest in the medium with a Na/K ratio of 23:1. Gill Na+-K+-ATPase activity, as an indicator of osmoregulation, peaked in the treatment where the Na/K ratio was 17:1. The total hemocyte count, respiratory burst, and immune-related enzyme activities (ALP, LSZ, PO, and SOD) of L. vananmei were affected significantly by Na/K ratios ( P<0.05). After challenged with Vibrio harveyi, the cumulative mortality of shrimp reared in a Na/K ratio of 23:1 (30±14.14)% was significantly lower than the control (75±7.07)%. In conclusion, the addition of K+ to low-salinity well water in L. vannamei cultures is feasible. Na/K ratios ranging from 23:1 to 33:1 might improve survival and growth. Immunity and disease resistance are also closely related to the Na/K ratio of the low-salinity well water. The findings may contribute to the development of more efficient K + remediation strategies for L. vananmei culture in low-salinity well water.

Development of Meteorus pulchricornis and regulation of its noctuid host, Pseudaletia separata.

PubMed

Suzuki, M; Tanaka, T

2007-10-01

The solitary endoparasitoid Meteorus pulchricornis can parasitize many lepidopteran host species successfully. In the case of parasitization of Pseudaletia separata, developmental duration of M. pulchricornis was 8-9 days from egg to larval emergence and 6 days from prepupa to adult emergence. Successful parasitism by M. pulchricornis decreased with host age. Following parasitization of day-0 4th host instar, the parasitoid embryo, whilst still enclosed in serosal cell membrane, hatched out of the egg chorion 2 days after oviposition. Subsequently, the 1st instar parasitoid emerged from the surrounding serosal cell membrane. Serosal cells dissociated and developed as teratocytes 3.5 days after oviposition. One embryo of M. pulchricornis gave rise to approximately 1200 teratocytes, a number that remained constant until 6 days after parasitization, but decreased drastically to 200 at 7 days post-oviposition. The teratocytes of M. pulchricornis were round- or oval-shaped and grew from 65 microm at 4 days to 200 microm in the long axis at 6 days post-parasitization. At 4 days post-parasitization, many cells or cell clusters with lipid particles were observed in the hemocoels of parasitized hosts. In addition, paraffin sections of parasitized hosts revealed that many teratocytes were attached to the host's fat body and contributed to disrupting the fat body tissue. Further, examination of the total hemocyte count (THC) during parasitization revealed that THC was maintained at low levels. Surprisingly, a temporal decrease followed by restoration of THC was observed in hosts injected with virus-like particles of M. pulchricornis (MpVLPs) plus venom, which contrasts with the constant THC suppression seen in parasitized hosts. This indicates that MpVLP function is temporal and is involved in regulation of the host during early parasitism. Therefore, teratocytes, a host regulation factor in late parasitism, could be involved in keeping THC at a low level.

Identifying the function of LvPI3K during the pathogenic infection of Litopenaeus vannamei by Vibrio alginolyticus.

PubMed

Kong, Jing-Rong; Wei, Wei; Liang, Qing-Jian; Qiao, Xue-Li; Kang, Huan; Liu, Yuan; Wang, Wei-Na

2018-05-01

It is well known that PI3K regulates various processes in mammalian cells by generating a secondary messenger that later activates AKT. However, its innate immune function in crustaceans remains unclear. We report the characterization of Litopenaeus vannamei PI3K (LvPI3K) for investigating how PI3K participates in the innate immunity of crustaceans. Full-length LvPI3K cDNA was 3357 bp long, with a 3222 bp open reading frame (ORF) that encodes a putative protein of 1292 amino acids. The PI3K catalytic domain (PI3Kc) of LvPI3K was found to be rather conserved when the PI3Ks from other species were analyzed. The LvPI3K protein was shown to be localized to the cytoplasm of Drosophila S2 cells, while LvPI3K mRNA was ubiquitously expressed in healthy L. vannamei, with the highest expression found in hemolymph. A dual luciferase reporter gene assay demonstrated that LvPI3K overexpression activated the promoter of antibacterial peptide LvPEN4 in a dose-dependent manner. However, the addition of PDTC, a specific inhibitor of NF-κB, suppressed the LvPI3K-induced LvPEN4 promoter activation. Moreover, Vibrio alginolyticus challenge induced a rapid up-regulation of LvPI3K expression. Further experiments showed that LvPI3K silencing in shrimp challenged with V. alginolyticus significantly increased Vibrio number, ROS production and DNA damage in the hemolymph, as well as significantly decreased total hemocyte count. The mRNA levels of certain molecules related to LvPI3K signaling, such as LvAKT and LvPEN4, also decreased following LvPI3K silencing. Taken together, these results suggest that LvPI3K regulates the downstream signal component LvPEN4 and functions in V. alginolyticus resistance. Copyright © 2018 Elsevier Ltd. All rights reserved.

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...

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

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.

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.

Detection of Salmonella sp., Vibrio sp. and total plate count bacteria on blood cockle (Anadara granosa)

NASA Astrophysics Data System (ADS)

Ekawati, ER; Yusmiati, S. N. H.

2018-01-01

Blood cockle (Anadara granosa) has high level of zinc and protein, which is beneficial for therapeutic function for malnourished particularly stunting case in children. Zinc in animal foods is more absorbable than that from vegetable food. Blood cockle (Anadara granosa) is rich in nutrient and an excellent environment for the growth of microorganisms. This research aimed to identify the contamination of Salmonella sp., Vibrio sp. and total plate count bacteria on blood cockle (Anadara granosa). This was observation research with laboratory analysis. Salmonella sp. and Vibrio sp. were detected from blood cockle. Total plate count was determine of the total amount of the bacteria. Results detected from 20 samples of blood cockle showed that all samples were negative of Salmonella sp. and 1 sample positive Vibrio sp. The result of total plate count bacteria was < 5 x 105 colony/g sample.

Association of peripheral differential leukocyte counts with dyslipidemia risk in Chinese patients with hypertension: insight from the China Stroke Primary Prevention Trial.

PubMed

Liu, Yanhong; Kong, Xiangyi; Wang, Wen; Fan, Fangfang; Zhang, Yan; Zhao, Min; Wang, Yi; Wang, Yupeng; Wang, Yu; Qin, Xianhui; Tang, Genfu; Wang, Binyan; Xu, Xiping; Hou, Fan Fan; Gao, Wei; Sun, Ningling; Li, Jianping; Venners, Scott A; Jiang, Shanqun; Huo, Yong

2017-01-01

The aim of the present study was to examine the association between peripheral differential leukocyte counts and dyslipidemia in a Chinese hypertensive population. A total of 10,866 patients with hypertension were enrolled for a comprehensive assessment of cardiovascular risk factors using data from the China Stroke Primary Prevention Trial. Plasma lipid levels and total leukocyte, neutrophil, and lymphocyte counts were determined according to standard methods. Peripheral differential leukocyte counts were consistently and positively associated with serum total cholesterol (TC), LDL cholesterol (LDL-C), and TG levels (all P < 0.001 for trend), while inversely associated with HDL cholesterol levels (P < 0.05 for trend). In subsequent analyses where serum lipids were dichotomized (dyslipidemia/normolipidemia), we found that patients in the highest quartile of total leukocyte count (≥7.6 × 10 9 cells/l) had 1.64 times the risk of high TG [95% confidence interval (CI): 1.46, 1.85], 1.34 times the risk of high TC (95% CI: 1.20, 1.50), and 1.24 times the risk of high LDL-C (95% CI: 1.12, 1.39) compared with their counterparts in the lowest quartile of total leukocyte count. Similar patterns were also observed with neutrophils and lymphocytes. In summary, these findings indicate that elevated differential leukocyte counts are directly associated with serum lipid levels and increased odds of dyslipidemia. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

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.

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

Association of peripheral total and differential leukocyte counts with obesity-related complications in young adults.

PubMed

Yoshimura, Aya; Ohnishi, Shunsuke; Orito, Chieko; Kawahara, Yukako; Takasaki, Hiroyo; Takeda, Hiroshi; Sakamoto, Naoya; Hashino, Satoshi

2015-01-01

Obesity has been demonstrated to be associated with elevated leukocytes in adults and children. This study assessed the associations between peripheral total and differential leukocyte counts and obesity-related complications in young adults. 12 obese (median age 21.5 (range 19-28) years, median BMI 35.7 (range 32.0-44.9) kg/m(2)) and 11 normal (median age 23 (range 18-27) years, median BMI 19.5 (range 18.1-21.7) kg/m(2)) adults were enrolled. Complete blood count and serum levels of liver enzymes, fasting blood glucose, insulin and lipids were measured, and the homeostasis model assessment of insulin resistance was calculated. Fat mass was calculated using a bioimpedance analysis device, and ultrasonography was performed to measure fat thickness and to detect fatty change of the liver. Total leukocyte and monocyte counts were significantly increased in obese young adults. Total leukocyte count was associated with liver enzyme levels, insulin resistance as well as visceral and subcutaneous fat thickness. Neutrophil count was associated with insulin resistance. Lymphocyte count was associated with serum liver enzymes, insulin resistance, and dyslipidemia. Monocyte count was associated with serum liver enzyme, insulin resistance, visceral and subcutaneous fat thickness, body fat mass, and percentage body fat. The results of this study suggest that chronic low-grade systemic inflammation is associated with obesity-related complications such as nonalcoholic fatty liver disease, insulin resistance, and dyslipidemia in young adults. © 2015 S. Karger GmbH, Freiburg.

Predictive factors for long-term engraftment of autologous blood stem cells.

PubMed

Duggan, P R; Guo, D; Luider, J; Auer, I; Klassen, J; Chaudhry, A; Morris, D; Glück, S; Brown, C B; Russell, J A; Stewart, D A

2000-12-01

Data from 170 consecutive patients aged 19-66 years (median age 46 years) who underwent unmanipulated autologous blood stem cell transplant (ASCT) were analyzed to determine if total CD34+ cells/kg infused, CD34+ subsets (CD34+41+, CD34+90+, CD34+33-, CD34+38-, CD34+38-DR-), peripheral blood CD34+ cell (PBCD34+) count on first apheresis day, or various clinical factors were associated with low blood counts 6 months post ASCT. Thirty-four patients were excluded from analysis either because of death (n = 17) or re-induction chemotherapy prior to 6 months post ASCT (n = 13), or because of lack of follow-up data (n = 4). Of the remaining 136 patients, 46% had low WBC ( < 4 x 10(9)/l), 41% low platelets (<150 x 10(9)/l), and 34% low hemoglobin ( < 120 g/l) at a median of 6 months following ASCT. By Spearman's rank correlation, both the total CD34+ cell dose/kg and the PBCD34+ count correlated with 6 month blood counts better than any subset of CD34+ cells or any clinical factor. The PBCD34+ count was overall a stronger predictor of 6 month blood counts than was the total CD34+ cells/kg infused. Both factors retained their significance in multivariate analysis, controlling for clinical factors. In conclusion, subsets of CD34+ cells and clinical factors are inferior to the total CD34+ cell dose/kg and PBCD34+ count in predicting 6 month blood counts following ASCT.

Obesity History and Daily Patterns of Physical Activity at Age 60-64 Years: Findings From the MRC National Survey of Health and Development.

PubMed

Cooper, Rachel; Huang, Lei; Hardy, Rebecca; Crainiceanu, Adina; Harris, Tamara; Schrack, Jennifer A; Crainiceanu, Ciprian; Kuh, Diana

2017-10-01

The aim of this study was to investigate associations of current body mass index (BMI) and obesity history with daily patterns of physical activity. At age 60-64, participants from a British birth cohort study wore accelerometers for 5 days. Accelerometry counts were log-transformed and mean log-counts were used to derive a summary variable indicating total daily log-activity counts. Among those with complete data (n = 1,388) the associations of current BMI and age of first obesity were examined with: (a) total daily log-activity counts and (b) total log-activity counts in four segments of the day. Higher current BMI and younger age at obesity were strongly associated with lower levels of total daily activity at age 60-64 even after adjustment for sex, socioeconomic factors, and health status. The fully-adjusted mean difference in total daily log-activity counts was -581.7 (95% confidence interval: -757.2, -406.3) when comparing BMI ≥35 kg/m2 with <25 kg/m2, representing an 18.4% difference. Participants who had been obese since early adulthood had the lowest levels of activity (mean difference in total daily log-activity counts was -413.1 (-638.1, -188.2) when comparing those who were obese by age 26 or 36 with those who were never obese, representing a 13.1% difference). Obese older adults may require targeted interventions and additional support to improve their daily activity levels. As younger generations with greater lifetime exposure to obesity reach old age the proportion of adults achieving sufficient levels of activity to realize its associated health benefits is likely to decline. © The Author 2017. Published by Oxford University Press on behalf of The Gerontological Society of America.

Obesity History and Daily Patterns of Physical Activity at Age 60–64 Years: Findings From the MRC National Survey of Health and Development

PubMed Central

Cooper, Rachel; Huang, Lei; Hardy, Rebecca; Crainiceanu, Adina; Harris, Tamara; Schrack, Jennifer A; Crainiceanu, Ciprian; Kuh, Diana

2017-01-01

Abstract Background The aim of this study was to investigate associations of current body mass index (BMI) and obesity history with daily patterns of physical activity. Methods At age 60–64, participants from a British birth cohort study wore accelerometers for 5 days. Accelerometry counts were log-transformed and mean log-counts were used to derive a summary variable indicating total daily log-activity counts. Among those with complete data (n = 1,388) the associations of current BMI and age of first obesity were examined with: (a) total daily log-activity counts and (b) total log-activity counts in four segments of the day. Results Higher current BMI and younger age at obesity were strongly associated with lower levels of total daily activity at age 60–64 even after adjustment for sex, socioeconomic factors, and health status. The fully-adjusted mean difference in total daily log-activity counts was −581.7 (95% confidence interval: −757.2, −406.3) when comparing BMI ≥35 kg/m2 with <25 kg/m2, representing an 18.4% difference. Participants who had been obese since early adulthood had the lowest levels of activity (mean difference in total daily log-activity counts was −413.1 (−638.1, −188.2) when comparing those who were obese by age 26 or 36 with those who were never obese, representing a 13.1% difference). Conclusions Obese older adults may require targeted interventions and additional support to improve their daily activity levels. As younger generations with greater lifetime exposure to obesity reach old age the proportion of adults achieving sufficient levels of activity to realize its associated health benefits is likely to decline. PMID:28329086

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.

Biogeographical drivers of ragweed pollen concentrations in Europe

NASA Astrophysics Data System (ADS)

Matyasovszky, István; Makra, László; Tusnády, Gábor; Csépe, Zoltán; Nyúl, László G.; Chapman, Daniel S.; Sümeghy, Zoltán; Szűcs, Gábor; Páldy, Anna; Magyar, Donát; Mányoki, Gergely; Erostyák, János; Bodnár, Károly; Bergmann, Karl-Christian; Deák, Áron József; Thibaudon, Michel; Albertini, Roberto; Bonini, Maira; Šikoparija, Branko; Radišić, Predrag; Gehrig, Regula; Rybníček, Ondřej; Severova, Elena; Rodinkova, Victoria; Prikhodko, Alexander; Maleeva, Anna; Stjepanović, Barbara; Ianovici, Nicoleta; Berger, Uwe; Seliger, Andreja Kofol; Weryszko-Chmielewska, Elżbieta; Šaulienė, Ingrida; Shalaboda, Valentina; Yankova, Raina; Peternel, Renata; Ščevková, Jana; Bullock, James M.

2017-06-01

The drivers of spatial variation in ragweed pollen concentrations, contributing to severe allergic rhinitis and asthma, are poorly quantified. We analysed the spatiotemporal variability in 16-year (1995-2010) annual total (66 stations) and annual total (2010) (162 stations) ragweed pollen counts and 8 independent variables (start, end and duration of the ragweed pollen season, maximum daily and calendar day of the maximum daily ragweed pollen counts, last frost day in spring, first frost day in fall and duration of the frost-free period) for Europe (16 years, 1995-2010) as a function of geographical coordinates. Then annual total pollen counts, annual daily peak pollen counts and date of this peak were regressed against frost-related variables, daily mean temperatures and daily precipitation amounts. To achieve this, we assembled the largest ragweed pollen data set to date for Europe. The dependence of the annual total ragweed pollen counts and the eight independent variables against geographical coordinates clearly distinguishes the three highly infected areas: the Pannonian Plain, Western Lombardy and the Rhône-Alpes region. All the eight variables are sensitive to longitude through its temperature dependence. They are also sensitive to altitude, due to the progressively colder climate with increasing altitude. Both annual total pollen counts and the maximum daily pollen counts depend on the start and the duration of the ragweed pollen season. However, no significant changes were detected in either the eight independent variables as a function of increasing latitude. This is probably due to a mixed climate induced by strong geomorphological inhomogeneities in Europe.

Determination of the volume activity concentration of alpha artificial radionuclides with alpha spectrometer.

PubMed

Liu, B; Zhang, Q; Li, Y

1997-12-01

This paper introduces a method to determine the volume activity concentration of alpha and/or beta artificial radionuclides in the environment and radon/thoron progeny background-compensation based on a Si surface-barrier detector. By measuring the alpha peak counts of 218Po and 214Po in two time intervals, the activity concentration of 218Po, 214Pb and 214Bi aerosol particles were determined; meanwhile, the total beta count of 214Pb and 214Bi aerosols was also calculated from their decay scheme. With the average equilibrium factor of thoron progeny in general environment, the alpha and beta counts of thoron progeny were approximately evaluated by 212Po alpha peak counts. The alpha count of transuranic aerosols was determined by subtracting the trail counts of radon/thoron progeny alpha peaks. The total count of beta artificial radionuclides was determined by subtracting the beta counts of radon/thoron progeny aerosol particles. In our preliminary experiments, if the radon progeny concentration is less than 15 Bq m(-3), the lower limit of detection of transuranics concentration is less than 0.1 Bq m(-3). Even if the radon progeny concentration is as high as 75 Bq m(-3), the lower limit of detection of total beta activity concentration of artificial nuclides aerosols is less than 1 Bq m(-3).

Total nucleated cell and leukocyte differential counts in canine pleural and peritoneal fluid and equine synovial fluid samples: comparison of automated and manual methods.

PubMed

Brudvig, Jean M; Swenson, Cheryl L

2015-12-01

Rapid and precise measurement of total and differential nucleated cell counts is a crucial diagnostic component of cavitary and synovial fluid analyses. The objectives of this study included (1) evaluation of reliability and precision of canine and equine fluid total nucleated cell count (TNCC) determined by the benchtop Abaxis VetScan HM5, in comparison with the automated reference instruments ADVIA 120 and the scil Vet abc, respectively, and (2) comparison of automated with manual canine differential nucleated cell counts. The TNCC and differential counts in canine pleural and peritoneal, and equine synovial fluids were determined on the Abaxis VetScan HM5 and compared with the ADVIA 120 and Vet abc analyzer, respectively. Statistical analyses included correlation, least squares fit linear regression, Passing-Bablok regression, and Bland-Altman difference plots. In addition, precision of the total cell count generated by the VetScan HM5 was determined. Agreement was excellent without significant constant or proportional bias for canine cavitary fluid TNCC. Automated and manual differential counts had R(2)  < .5 for individual cell types (least squares fit linear regression). Equine synovial fluid TNCC agreed but with some bias due to the VetScan HM5 overestimating TNCC compared to the Vet abc. Intra-assay precision of the VetScan HM5 in 3 fluid samples was 2-31%. The Abaxis VetScan HM5 provided rapid, reliable TNCC for canine and equine fluid samples. The differential nucleated cell count should be verified microscopically as counts from the VetScan HM5 and also from the ADVIA 120 were often incorrect in canine fluid samples. © 2015 American Society for Veterinary Clinical Pathology.

A side-by-side comparison of Rotorod and Burkard pollen and spore collections.

PubMed

Crisp, Howard C; Gomez, Robert A; White, Kevin M; Quinn, James M

2013-08-01

The Rotorod sampler and Burkard spore trap are 2 devices commonly used to quantify airborne particles. To evaluate the differences in collections between the 2 devices for a wide range of plant pollens and fungal spores. Pollens and spores were collected simultaneously with each device on 167 days during a 1-year period. The Burkard yielded significantly higher total and individual mold spore counts. It yielded statistically higher total grass, total weed, and Urticaceae daily pollen counts, although the absolute differences were small. Daily counts were positively correlated between the 2 devices for the most abundant pollens and mold spores. The Burkard spore trap collects many more mold spores than the Rotorod over a wide variety of species. The Burkard also yielded higher total grass, total weed, and Urticaceae daily pollen counts. Despite these differences, however, either device can be used to follow trends in the most abundant pollen and mold spores. Copyright © 2013 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

[Prediction of the total Japanese cedar pollen counts based on male flower-setting conditions of standard trees].

PubMed

Yuta, Atsushi; Ukai, Kotaro; Sakakura, Yasuo; Tani, Hideshi; Matsuda, Fukiko; Yang, Tian-qun; Majima, Yuichi

2002-07-01

We made a prediction of the Japanese cedar (Cryptomeria japonica) pollen counts at Tsu city based on male flower-setting conditions of standard trees. The 69 standard trees from 23 kinds of clones, planted at Mie Prefecture Science and Technology Promotion Center (Hakusan, Mie) in 1964, were selected. Male flower-setting conditions for 276 faces (69 trees x 4 points of the compass) were scored from 0 to 3. The average of scores and total pollen counts from 1988 to 2000 was analyzed. As the results, the average scores from standard trees and total pollen counts except two mass pollen-scattered years in 1995 and 2000 had a positive correlation (r = 0.914) by linear function. On the mass pollen-scattered years, pollen counts were influenced from the previous year. Therefore, the score of the present year minus that of the previous year were used for analysis. The average scores from male flower-setting conditions and pollen counts had a strong positive correlation (r = 0.994) when positive scores by taking account of the previous year were analyzed. We conclude that prediction of pollen counts are possible based on the male flower-setting conditions of standard trees.

Alternative Fuels Data Center: Alternative Fueling Station Counts by State

Science.gov Websites

charging infrastructure. **Totals by States indicate the total number of stations for all fuel types combined. Individual stations are counted multiple times if the station offers multiple types of fuel. For

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.

Effects of Extended Freezer Storage on the Integrity of Human Milk.

PubMed

Ahrabi, Ali Faraghi; Handa, Deepali; Codipilly, Champa N; Shah, Syed; Williams, Janet E; McGuire, Mark A; Potak, Debra; Aharon, Grace Golda; Schanler, Richard J

2016-10-01

To examine the integrity (pH, bacterial counts, host defense factors, nutrient contents, and osmolality) of freshly expressed and previously refrigerated human milk subjected to long-term freezer storage. Mothers donated 100 mL of freshly expressed milk. Samples were divided into baseline, storage at -20°C (fresh frozen) for 1, 3, 6, and 9 months, and prior storage at +4°C for 72 hours (refrigerated frozen) before storage at -20°C for 1 to 9 months. Samples were analyzed for pH, total bacterial colony count, gram-positive and gram-negative colony counts, and concentrations of total protein, fat, nonesterified fatty acids, lactoferrin, secretory IgA, and osmolality. Milk pH, total bacterial colony count, and Gram-positive colony counts decreased significantly with freezer storage (P < .001); bacterial counts decreased most rapidly in the refrigerated frozen group. The gram-negative colony count decreased significantly over time (P < .001). Nonesterified fatty acid concentrations increased significantly with time in storage (P < .001). Freezing for up to 9 months did not affect total protein, fat, lactoferrin, secretory IgA, or osmolality in either group. Freezer storage of human milk for 9 months at -20°C is associated with decreasing pH and bacterial counts, but preservation of key macronutrients and immunoactive components, with or without prior refrigeration for 72 hours. These data support current guidelines for freezer storage of human milk for up to 9 months for both freshly expressed and refrigerated milk. Copyright © 2016 Elsevier Inc. All rights reserved.

Cytoprotection: Immune and Matrix Modulation of Tissue Repair

DTIC Science & Technology

2012-04-01

bronchoalveolar lavage (BAL) fluid for cytokines, cell counts and eosinophilia, and histological analysis of airway hyper-responsiveness and remodeling...OVA administered intra-nasally on Days 21–25 with or without 0.1% XHA. B) Total leukocyte and eosinophil counts in bronchoalveolar lavage (BAL) fluid...and different doses of HA peptide. The absolute number of Tmr+ and FOXP3+ populations was determined using total cell counts and flow cytometry for Tmr

What is harmful for male fertility: cell phone or the wireless Internet?

PubMed

Yildirim, Mehmet Erol; Kaynar, Mehmet; Badem, Huseyin; Cavis, Mucahıt; Karatas, Omer Faruk; Cimentepe, Ersın

2015-09-01

In this study, we aimed to assess the potential harmful effects of radiofrequency-electromagnetic radiation on sperm parameters. We requested semen for analyses from the male patients coming to our infertility division and also asked them to fill out an anonymous questionnaire. We queried their mobile phone and wireless Internet usage frequencies in order to determine their radiofrequency-electromagnetic radiation exposure. A total of 1082 patients filled the questionnaire but 51 of them were excluded from the study because of azoospermia. There was no significant difference between sperm counts and sperm morphology excluding sperm motility, due to mobile phone usage period, (p = 0.074, p = 0.909, and p = 0.05, respectively). The total motile sperm count and the progressive motile sperm count decreased due to the increase of internet usage (p = 0.032 and p = 0.033, respectively). In line with the total motile sperm count, progressive motile sperm count also decreased with wireless Internet usage compared with the wired Internet connection usage (p = 0.009 and p = 0.018, respectively). There was a negative correlation between wireless Internet usage duration and the total sperm count (r = -0.089, p = 0.039). We have also explored the negative effect of wireless Internet use on sperm motility according to our preliminary results. Copyright © 2015. Published by Elsevier Taiwan.

Changes in total viable count and TVB-N content in marinated chicken breast fillets during storage

NASA Astrophysics Data System (ADS)

Baltić, T.; Ćirić, J.; Velebit, B.; Petronijević, R.; Lakićević, B.; Đorđević, V.; Janković, V.

2017-09-01

Marination is a popular technique for enhancing meat properties. Depending on the marinade type and ingredients added, marination can improve sensory, chemical and microbiological quality of meat products. In this study, the total viable count and total volatile basic nitrogen (TVB-N) content in marinated chicken breast fillets were investigated. The possible correlation between bacterial growth and formation of TVB-N was also tested. Chicken breast fillets were immersed in a solution of table salt (as a control) orthree different marinades,which consisted of table salt, sodium tripolyphosphate and/or sodium citrate, and stored in air for nine days at 4±1°C. Analyses of the total viable count and TVB-N were performed on days0, 3, 6 and 9 day of storage. The total viable count gradually increased in all examined groups, and statistically significant differences (p<0.01 p<0.05) between treatments on days0, 3 and 6 day of storage were established. TVB-N values in marinated chicken were significantly higher (p<0.01 p<0.05) compared to the control. Using the multiple linear regression, a positive correlation between total viable count and formation of TVB-N in chicken marinated with sodium citrate was established (p<0.05), while the intensity of TVB-N formation was lowest in chicken marinated with sodium tripolyphosphate.

Prevalence of Vibrio vulnificus and Vibrio parahaemolyticus in the Maryland Coastal Bays

NASA Astrophysics Data System (ADS)

De Pascuale, V. O.

2016-02-01

The bacterial family of Vibrionaceae is indigenous in the marine estuarine environments such as the Maryland Coastal Bays. Vibrio vulnificus and Vibrio parahaemolyticus are both pathogenic bacteria. Understanding the distribution of Vibrio species is crucial because of the health concerns associated with the bacteria. The aim of this study was to evaluate the overall abundance of bacteria with a focus on Vibrio species in the Maryland Coastal Bays. Seawater samples were collected from 10 different sites that differ with regard to water quality. The total bacteria count (TBC) was determined by two methods: Total plate count and Epifluorescence microscopy. The most-probable-number (MPN) methodology was used to estimate the population of Vibrio parahaemolyticus and Vibrio vulnificus. In addition to the bacteriological analysis, the environmental parameters of temperature and salinity were measured using YSI 6600 multiparameter meter. The average total bacteria count was 2.21 log CFU ml-1. Vibrio vulnificus comprised 5% of the total bacteria count while Vibrio parahaemolyticus comprised only 2% of the total bacteria count. Vibrio vulnificus ranged from 0.30 to 2.48 log MPN ml-1 at the sites tested. Lower Vibrio parahaemolyticus count was observed at the sites with a range of 0.30 to 1.97 log MPN ml-1. There was no significant correlation between the environmental parameters and the Vibrio spp. Since both Vibrio vulnificus and Vibrio parahaemolyticus peak in the summer, there is a potential for a risk of wound infections and gastrointestinal illness based on this data.

Immature germ cells in semen - correlation with total sperm count and sperm motility.

PubMed

Patil, Priya S; Humbarwadi, Rajendra S; Patil, Ashalata D; Gune, Anita R

2013-07-01

Current data regarding infertility suggests that male factor contributes up to 30% of the total cases of infertility. Semen analysis reveals the presence of spermatozoa as well as a number of non-sperm cells, presently being mentioned in routine semen report as "round cells" without further differentiating them into leucocytes or immature germ cells. The aim of this work was to study a simple, cost-effective, and convenient method for differentiating the round cells in semen into immature germ cells and leucocytes and correlating them with total sperm counts and motility. Semen samples from 120 males, who had come for investigation for infertility, were collected, semen parameters recorded, and stained smears studied for different round cells. Statistical analysis of the data was done to correlate total sperm counts and sperm motility with the occurrence of immature germ cells and leucocytes. The average shedding of immature germ cells in different groups with normal and low sperm counts was compared. The clinical significance of "round cells" in semen and their differentiation into leucocytes and immature germ cells are discussed. Round cells in semen can be differentiated into immature germ cells and leucocytes using simple staining methods. The differential counts mentioned in a semen report give valuable and clinically relevant information. In this study, we observed a negative correlation between total count and immature germ cells, as well as sperm motility and shedding of immature germ cells. The latter was statistically significant with a P value 0.000.

[Left ventricular volume determination by first-pass radionuclide angiocardiography using a semi-geometric count-based method].

PubMed

Kinoshita, S; Suzuki, T; Yamashita, S; Muramatsu, T; Ide, M; Dohi, Y; Nishimura, K; Miyamae, T; Yamamoto, I

1992-01-01

A new radionuclide technique for the calculation of left ventricular (LV) volume by the first-pass (FP) method was developed and examined. Using a semi-geometric count-based method, the LV volume can be measured by the following equation: CV = CM/(L/d). V = (CT/CV) x d3 = (CT/CM) x L x d2. (V = LV volume, CV = voxel count, CM = the maximum LV count, CT = the total LV count, L = LV depth where the maximum count was obtained, and d = pixel size.) This theorem was applied to FP LV images obtained in the 30-degree right anterior oblique position. Frame-mode acquisition was performed and the LV end-diastolic maximum count and total count were obtained. The maximum LV depth was obtained as the maximum width of the LV on the FP end-diastolic image, using the assumption that the LV cross-section is circular. These values were substituted in the above equation and the LV end-diastolic volume (FP-EDV) was calculated. A routine equilibrium (EQ) study was done, and the end-diastolic maximum count and total count were obtained. The LV maximum depth was measured on the FP end-diastolic frame, as the maximum length of the LV image. Using these values, the EQ-EDV was calculated and the FP-EDV was compared to the EQ-EDV. The correlation coefficient for these two values was r = 0.96 (n = 23, p less than 0.001), and the standard error of the estimated volume was 10 ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Yeasts from Marine and Estuarine Waters with Different Levels of Pollution in the State of Rio de Janeiro, Brazil

PubMed Central

Hagler, Allen N.; Mendonça-Hagler, Leda C.

1981-01-01

Yeast counts were made at 24 marine and estuarine sites in the vicinity of Rio de Janeiro, Brazil. Mean salinities of estuarine sites ranged from 14.2 to 27.4‰, and mean temperatures ranged from 25 to 28°C. Total coliform counts varied from 80% above 100,000 colony-forming units (CFU)/100 ml at heavily polluted sites to 100% below 100 CFU/100 ml at unpolluted sites. Total yeast counts above 100 CFU/100 ml were typical of heavily and moderately polluted water but atypical of lightly polluted and unpolluted water. Mean total yeast counts were 2,880 CFU/100 ml for heavily polluted sites, 202 CFU/100 ml for moderately polluted sites, and 3 CFU/100 ml for lightly polluted and unpolluted sites. Total yeast counts had a positive response to increased pollution levels, and Candida krusei and phenotypically similar yeasts as a group were prevalent in polluted estuarine water but rare in unpolluted seawater. The 549 strains of yeasts and yeast-like organisms isolated were grouped into 67 species, of which the 21 most prevalent made up 86% of the total yeast population. The prevalent genera in the polluted estuary were Candida, Rhodotorula, Torulopsis, Hanseniaspora, Debaryomyces, and Trichosporon. PMID:16345683

Point counts of landbirds in bottomland hardwood forests of the Mississippi Alluvial Valley: How long and how many?

USGS Publications Warehouse

Smith, W.P.; Wiedenfeld, D.A.; Hanel, P.B.; Twedt, D.J.; Ford, R.P.; Cooper, R.J.; Smith, Winston Paul

1993-01-01

To quantify efficacy of point count sampling in bottomland hardwood forests, we examined the influence of point count duration on corresponding estimates of number of individuals and species recorded. To accomplish this we conducted a totalof 82 point counts 7 May-16 May 1992distributed among three habitats (Wet, Mesic, Dry) in each of three regions within the lower Mississippi Alluvial Valley (MAV). Each point count consisted of recording the number of individual birds (all species) seen or heard during the initial three minutes and per each minute thereafter for a period totaling ten minutes. In addition, we included 384 point counts recorded during an 8-week period in each of 3 years (1985-1987) among 56 randomly-selected forest patches within the bottomlands of western Tennessee. Each point count consisted of recording the number of individuals (excluding migrating species) during each of four, 5 minute intervals for a period totaling 20 minutes. To estimate minimum sample size, we determined sampling variation at each level (region, habitat, and locality) with the 82 point counts from the lower (MAV) and applied the procedures of Neter and Wasserman (1974:493; Applied linear statistical models). Neither the cumulative number of individuals nor number of species per sampling interval attained an asymptote after 10 or 20 minutes of sampling. For western Tennessee bottomlands, total individual and species counts relative to point count duration were similar among years and comparable to the pattern observed throughout the lower MAV. Across the MAV, we recorded a total of 1,62 1 birds distributed among 52 species with the majority (8721/1621) representing 8 species. More birds were recorded within 25-50 m than in either of the other distance categories. There was significant variation in numbers of individuals and species among point counts. For both, significant differences between region and patch (nested within region) occurred; neither habitat nor interaction between habitat and region was significant. For = 0.05 and L3 = 0.10, minimum sample size estimates (per factor level) varied by orders of magnitude depending upon the observed or specified range of desired detectable difference. For observed regional variation, 20 and 40 point counts were required to accommodate variability in total birds (MSE = 9.28) and species (MSE = 3.79), respectively; 25 percent of the mean could be achieved with 5 counts per factor level. Corresponding sample sizes required to detect differences of rarer species (e.g., Wood Thrush) were 500; for common species (e.g., Northern Cardinal) this same level of precision could be achieved with 100 counts.

Somatic cell counts in bulk milk and their importance for milk processing

NASA Astrophysics Data System (ADS)

Savić, N. R.; Mikulec, D. P.; Radovanović, R. S.

2017-09-01

Bulk tank milk somatic cell counts are the indicator of the mammary gland health in the dairy herds and may be regarded as an indirect measure of milk quality. Elevated somatic cell counts are correlated with changes in milk composition The aim of this study was to assess the somatic cell counts that significantly affect the quality of milk and dairy products. We examined the somatic cell counts in bulk tank milk samples from 38 farms during the period of 6 months, from December to the May of the next year. The flow cytometry, Fossomatic was used for determination of somatic cell counts. In the same samples content of total proteins and lactose was determined by Milcoscan. Our results showed that average values for bulk tank milk samples were 273,605/ml from morning milking and 292,895/ml from evening milking. The average values for total proteins content from morning and evening milking are 3,31 and 3,34%, respectively. The average values for lactose content from morning and evening milking are 4,56 and 4,63%, respectively. The highest somatic cell count (516,000/ml) was detected in bulk tank milk sample from evening milk in the Winter and the lowest content of lactose was 4,46%. Our results showed that obtained values for bulk tank milk somatic cell counts did not significantly affected the content of total proteins and lactose.

Short communication: Repeatability of differential goat bulk milk culture and associations with somatic cell count, total bacterial count, and standard plate count.

PubMed

Koop, G; Dik, N; Nielen, M; Lipman, L J A

2010-06-01

The aims of this study were to assess how different bacterial groups in bulk milk are related to bulk milk somatic cell count (SCC), bulk milk total bacterial count (TBC), and bulk milk standard plate count (SPC) and to measure the repeatability of bulk milk culturing. On 53 Dutch dairy goat farms, 3 bulk milk samples were collected at intervals of 2 wk. The samples were cultured for SPC, coliform count, and staphylococcal count and for the presence of Staphylococcus aureus. Furthermore, SCC (Fossomatic 5000, Foss, Hillerød, Denmark) and TBC (BactoScan FC 150, Foss) were measured. Staphylococcal count was correlated to SCC (r=0.40), TBC (r=0.51), and SPC (r=0.53). Coliform count was correlated to TBC (r=0.33), but not to any of the other variables. Staphylococcus aureus did not correlate to SCC. The contribution of the staphylococcal count to the SPC was 31%, whereas the coliform count comprised only 1% of the SPC. The agreement of the repeated measurements was low. This study indicates that staphylococci in goat bulk milk are related to SCC and make a significant contribution to SPC. Because of the high variation in bacterial counts, repeated sampling is necessary to draw valid conclusions from bulk milk culturing. 2010 American Dairy Science Association. Published by Elsevier Inc. 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.

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.

Preimpoundment Water Quality of the Wild Rice River, Norman County, Minnesota.

DTIC Science & Technology

1980-06-01

cell counts at Twin Valley for 1977 water year 25 14. Plot of total phosphorus related to phytoplankton cell counts at Twin Valley ; 30 15. Plot...of total nitrogen related to phytoplankton cell counts at Twin Valley 31 16. Bar graph of diversity indices of phytoplankton genera, 1976, 1977...statistically signifi- cant beyond the 0.02 level. There is no apparent relation of BOD to stream- flow or to suspended-sediment, phytoplankton , and bacteria

A comparison of legionella and other bacteria concentrations in cooling tower water

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

Cappabianca, R.M.; Jurinski, N.B.; Jurinski, J.B.

1994-05-01

A field study was conducted in which water samples collected from air conditioning cooling water reservoirs of high-rise buildings throughout an urban area were assayed for Legionella and for total bacteria. Buildings included within the study had ongoing biocidal treatment programs for the cooling towers. Separate sample analyses were performed to measure the viable colony concentrations of total bacteria and of Legionella in the process waters. The occurrence and viable counts of Legionella in 304 environmental water samples were determined by inoculating them onto plates of buffered charcoal yeast extract (BCYE) agar medium (a presumptive screening method). The samples weremore » collected during summer months between July and September. BCYE plate cultures of 50 (16.4%) of the samples yielded Legionella with viable counts ranging from 2 to 608 colony forming units per milliliter. In the water samples, 281 (92.4%) yielded viable counts of bacteria that ranged from 9 to 1.2 x 10{sup 6} per milliliter. This study demonstrates that Legionella are commonly present in the water of air conditioning cooling towers and that there is no significant correlation between concurrently sampled culture plate counts of Legionella and total bacteria plate counts. Correspondingly, there is no demonstrated validity for use of total bacterial counts as an inferential surrogate for the concentration of Legionella in the water. 19 refs., 3 figs., 1 tab.« less

Lower white blood cell counts in elite athletes training for highly aerobic sports.

PubMed

Horn, P L; Pyne, D B; Hopkins, W G; Barnes, C J

2010-11-01

White cell counts at rest might be lower in athletes participating in selected endurance-type sports. Here, we analysed blood tests of elite athletes collected over a 10-year period. Reference ranges were established for 14 female and 14 male sports involving 3,679 samples from 937 females and 4,654 samples from 1,310 males. Total white blood cell counts and counts of neutrophils, lymphocytes and monocytes were quantified. Each sport was scaled (1-5) for its perceived metabolic stress (aerobic-anaerobic) and mechanical stress (concentric-eccentric) by 13 sports physiologists. Substantially lower total white cell and neutrophil counts were observed in aerobic sports of cycling and triathlon (~16% of test results below the normal reference range) compared with team or skill-based sports such as water polo, cricket and volleyball. Mechanical stress of sports had less effect on the distribution of cell counts. The lower white cell counts in athletes in aerobic sports probably represent an adaptive response, not underlying pathology.

Associations Between Nutritional Parameters and Clinicopathologic Factors in Patients with Gastric Cancer: A Comprehensive Study.

PubMed

Brewczyński, Adam; Jabłońska, Beata; Pawlicki, Krzysztof

2017-07-01

The aim of this study was to assess and analyze the nutritional status of gastric cancer (GC) patients. The analysis included 207 patients with GC treated in a large center of oncology. Patients were divided into two groups according to the cutoff value of the mean prognostic nutritional index (PNI): those with a PNI < 52.78 and those with a PNI ≥ 52.78. The higher PNI was associated with lower age and higher total protein and hemoglobin levels (P < 0.01). The total lymphocyte count (P = 0.02), albumin, total protein and PNI (P < 0.01) were significantly higher in stable-weight patients and lower in the group with weight loss > 10% (P = 0.000031). Body mass index (BMI) after disease recognition, albumin and total protein (0.003) levels, total lymphocyte count, and PNI were significantly lower in patients with nutritional risk. Significantly lower BMI before disease and BMI after disease recognition were noted in smoking patients. Significantly higher total lymphocyte count was observed in smoking patients (P < 0.01). Significantly lower PNI was noted in tumors with lymph node metastasis (N+). G3 tumors were associated with the lowest total lymphocyte count (P = 0.01). Assessment of nutritional status using PNI calculation should be the standard management of patients with GC before treatment.

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.

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.

STEFFY-software to calculate nuclide-specific total counting efficiency in well-type γ-ray detectors.

PubMed

Pommé, S

2012-09-01

A software package is presented to calculate the total counting efficiency for the decay of radionuclides in a well-type γ-ray detector. It is specifically applied to primary standardisation of activity by means of 4πγ-counting with a NaI(Tl) well-type scintillation detector. As an alternative to Monte Carlo simulations, the software combines good accuracy with superior speed and ease-of-use. It is also well suited to investigate uncertainties associated with the 4πγ-counting method for a variety of radionuclides and detector dimensions. In this paper, the underlying analytical models for the radioactive decay and subsequent counting efficiency of the emitted radiation in the detector are summarised. Copyright © 2012 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.

Comparison of fluorescence microscopy and solid-phase cytometry methods for counting bacteria in water

USGS Publications Warehouse

Lisle, John T.; Hamilton, Martin A.; Willse, Alan R.; McFeters, Gordon A.

2004-01-01

Total direct counts of bacterial abundance are central in assessing the biomass and bacteriological quality of water in ecological and industrial applications. Several factors have been identified that contribute to the variability in bacterial abundance counts when using fluorescent microscopy, the most significant of which is retaining an adequate number of cells per filter to ensure an acceptable level of statistical confidence in the resulting data. Previous studies that have assessed the components of total-direct-count methods that contribute to this variance have attempted to maintain a bacterial cell abundance value per filter of approximately 106 cells filter-1. In this study we have established the lower limit for the number of bacterial cells per filter at which the statistical reliability of the abundance estimate is no longer acceptable. Our results indicate that when the numbers of bacterial cells per filter were progressively reduced below 105, the microscopic methods increasingly overestimated the true bacterial abundance (range, 15.0 to 99.3%). The solid-phase cytometer only slightly overestimated the true bacterial abundances and was more consistent over the same range of bacterial abundances per filter (range, 8.9 to 12.5%). The solid-phase cytometer method for conducting total direct counts of bacteria was less biased and performed significantly better than any of the microscope methods. It was also found that microscopic count data from counting 5 fields on three separate filters were statistically equivalent to data from counting 20 fields on a single filter.

Evaluation of the impact of banking umbilical cord blood units with high cell dose for ethnically diverse patients.

PubMed

Stritesky, Gretta; Wadsworth, Kimberly; Duffy, Merry; Buck, Kelly; Dehn, Jason

2018-02-01

Umbilical cord blood units provide an important stem cell source for transplantation, particularly for patients of ethnic diversity who may not have suitably matched available, adult-unrelated donors. However, with the cost of cord blood unit acquisition from public banks significantly higher than that for adult-unrelated donors, attention is focused on decreasing cost yet still providing cord blood units to patients in need. Historical practices of banking units with low total nucleated cell counts, including units with approximately 90 × 10 7 total nucleated cells, indicates that most banked cord blood units have much lower total nucleated cell counts than are required for transplant. The objective of this study was to determine the impact on the ability to identify suitable cord blood units for transplantation if the minimum total nucleated cell count for banking were increased from 90 × 10 7 to 124 or 149 × 10 7 . We analyzed ethnically diverse patients (median age, 3 years) who underwent transplantation of a single cord blood unit in 2005 to 2016. A cord blood unit search was evaluated to identify units with equal or greater human leukocyte antigen matching and a greater total nucleated cell count than that of the transplanted cord blood unit (the replacement cord blood unit). If the minimum total nucleated cell count for banking increased to 124 or 149 × 10 7 , then from 75 to 80% of patients would still have at least 1 replacement cord blood unit in the current (2016) cord blood unit inventory. The best replacement cord blood units were often found among cords with the same ethnic background as the patient. The current data suggest that, if the minimum total nucleated cell count were increased for banking, then it would likely lead to an inventory of more desirable cord blood units while having minimal impact on the identification of suitable cord blood units for transplantation. © 2017 AABB.

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.

Transcriptome Analysis Revealed Changes of Multiple Genes Involved in Haliotis discus hannai Innate Immunity during Vibrio parahemolyticus Infection.

PubMed

Nam, Bo-Hye; Jung, Myunghee; Subramaniyam, Sathiyamoorthy; Yoo, Seung-il; Markkandan, Kesavan; Moon, Ji-Young; Kim, Young-Ok; Kim, Dong-Gyun; An, Cheul Min; Shin, Younhee; Jung, Ho-jin; Park, Jun-hyung

2016-01-01

Abalone (Haliotis discus hannai) is one of the most valuable marine aquatic species in Korea, Japan and China. Tremendous exposure to bacterial infection is common in aquaculture environment, especially by Vibrio sp. infections. It's therefore necessary and urgent to understand the mechanism of H. discus hannai host defense against Vibrio parahemolyticus infection. However studies on its immune system are hindered by the lack of genomic resources. In the present study, we sequenced the transcriptome of control and bacterial challenged H. discus hannai tissues. Totally, 138 MB of reference transcriptome were obtained from de novo assembly of 34 GB clean bases from ten different libraries and annotated with the biological terms (GO and KEGG). A total of 10,575 transcripts exhibiting the differentially expression at least one pair of comparison and the functional annotations highlight genes related to immune response, cell adhesion, immune regulators, redox molecules and mitochondrial coding genes. Mostly, these groups of genes were dominated in hemocytes compared to other tissues. This work is a prerequisite for the identification of those physiological traits controlling H. discus hannai ability to survive against Vibrio infection.

Transcriptome Analysis Revealed Changes of Multiple Genes Involved in Haliotis discus hannai Innate Immunity during Vibrio parahemolyticus Infection

PubMed Central

Nam, Bo-Hye; Jung, Myunghee; Subramaniyam, Sathiyamoorthy; Yoo, Seung-il; Markkandan, Kesavan; Moon, Ji-Young; Kim, Young-Ok; Kim, Dong-Gyun; An, Cheul Min; Shin, Younhee; Jung, Ho-jin; Park, Jun-hyung

2016-01-01

Abalone (Haliotis discus hannai) is one of the most valuable marine aquatic species in Korea, Japan and China. Tremendous exposure to bacterial infection is common in aquaculture environment, especially by Vibrio sp. infections. It’s therefore necessary and urgent to understand the mechanism of H. discus hannai host defense against Vibrio parahemolyticus infection. However studies on its immune system are hindered by the lack of genomic resources. In the present study, we sequenced the transcriptome of control and bacterial challenged H. discus hannai tissues. Totally, 138 MB of reference transcriptome were obtained from de novo assembly of 34 GB clean bases from ten different libraries and annotated with the biological terms (GO and KEGG). A total of 10,575 transcripts exhibiting the differentially expression at least one pair of comparison and the functional annotations highlight genes related to immune response, cell adhesion, immune regulators, redox molecules and mitochondrial coding genes. Mostly, these groups of genes were dominated in hemocytes compared to other tissues. This work is a prerequisite for the identification of those physiological traits controlling H. discus hannai ability to survive against Vibrio infection. PMID:27088873

Short communication: bulk tank total bacterial count in dairy sheep: factors of variation and relationship with somatic cell count.

PubMed

Gonzalo, C; Carriedo, J A; Beneitez, E; Juárez, M T; De La Fuente, L F; San Primitivo, F

2006-02-01

A total of 9,353 records for bulk tank total bacterial count (TBC) were obtained over 1 yr from 315 dairy ewe flocks belonging to the Sheep Improvement Consortium (CPO) in Castilla-León (Spain). Analysis of variance showed significant effects of flock, breed, month within flock, dry therapy, milking type and installation, and logSCC on logTBC. Flock and month within flock were important variation factors as they accounted for 22.0 and 22.1% of the variance, respectively. Considerable repeatability values were obtained for both random factors. Hand milking and bucket-milking machines elicited highest logTBC (5.31), whereas parlor systems with looped milkline (5.01) elicited the lowest logTBC. The implementation of dry therapy practice (5.12) showed significantly lower logTBC than when not used (5.25). Variability in logTBC among breeds ranged from 5.24 (Awassi) to 5.07 (Churra). However, clinical outbreaks of contagious agalactia did not increase TBC significantly. A statistically significant relationship was found between logTBC and logSCC, the correlation coefficient between the variables being r = 0.23. Programs for improving milk hygiene should be implemented for both total bacterial count and somatic cell count variables at the same time.

Comparison of m-Endo LES, MacConkey, and Teepol media for membrane filtration counting of total coliform bacteria in water.

PubMed Central

Grabow, W O; du Preez, M

1979-01-01

Total coliform counts obtained by means of standard membrane filtration techniques, using MacConkey agar, m-Endo LES agar, Teepol agar, and pads saturated with Teepol broth as growth media, were compared. Various combinations of these media were used in tests on 490 samples of river water and city wastewater after different stages of conventional purification and reclamation processes including lime treatment, and filtration, active carbon treatment, ozonation, and chlorination. Endo agar yielded the highest average counts for all these samples. Teepol agar generally had higher counts then Teepol broth, whereas MacConkey agar had the lowest average counts. Identification of 871 positive isolates showed that Aeromonas hydrophila was the species most commonly detected. Species of Escherichia, Citrobacter, Klebsiella, and Enterobacter represented 55% of isolates which conformed to the definition of total coliforms on Endo agar, 54% on Teepol agar, and 45% on MacConkey agar. Selection for species on the media differed considerably. Evaluation of these data and literature on alternative tests, including most probable number methods, indicated that the technique of choice for routine analysis of total coliform bacteria in drinking water is membrane filtration using m-Endo LES agar as growth medium without enrichment procedures or a cytochrome oxidase restriction. PMID:394678

Guidebooks for estimating total transit usage through extrapolating incomplete counts : final report.

DOT National Transportation Integrated Search

2016-09-01

This report provides guidance for transit agencies to estimate transit usage for reporting to the National Transit : Database (NTD) when their counting procedure that is designed to perform full counts misses some trips. Transit usage : refers to unl...

Hawaii Census 2000 Blocks

EPA Pesticide Factsheets

This data layer represents Census 2000 demographic data derived from the PL94-171 redistricting files and SF3. Census geographic entities include blocks, blockgroups and tracts. Tiger line files are the source of the geometry representing the Census blocks. Attributes include total population counts, racial/ethnic, and poverty/income information. Racial/ethnic classifications are represented in units of blocks, blockgroups and tracts. Poverty and income data are represented in units of blockgroups and tracts. Percentages of each racial/ethnic group have been calculated from the population counts. Total Minority counts and percentages were compiled from each racial/ethnic non-white category. Categories compiled to create the Total Minority count includes the following: African American, Asian, American Indian, Pacific Islander, White Hispanic, Other and all mixed race categories. The percentage poverty attribute represents the percent of the population living at or below poverty level. The per capita income attribute represents the sum of all income within the geographic entity, divided by the total population of that entity. Special fields designed to be used for EJ analysis have been derived from the PL data and include the following: Percentage difference of block, blockgroup and total minority from the state and county averages, percentile rank for each percent total minority within state and county entitie

Hawaii Census 2000 Tracts

EPA Pesticide Factsheets

This data layer represents Census 2000 demographic data derived from the PL94-171 redistricting files and SF3. Census geographic entities include blocks, blockgroups and tracts. Tiger line files are the source of the geometry representing the Census blocks. Attributes include total population counts, racial/ethnic, and poverty/income information. Racial/ethnic classifications are represented in units of blocks, blockgroups and tracts. Poverty and income data are represented in units of blockgroups and tracts. Percentages of each racial/ethnic group have been calculated from the population counts. Total Minority counts and percentages were compiled from each racial/ethnic non-white category. Categories compiled to create the Total Minority count includes the following: African American, Asian, American Indian, Pacific Islander, White Hispanic, Other and all mixed race categories. The percentage poverty attribute represents the percent of the population living at or below poverty level. The per capita income attribute represents the sum of all income within the geographic entity, divided by the total population of that entity. Special fields designed to be used for EJ analysis have been derived from the PL data and include the following: Percentage difference of block, blockgroup and total minority from the state and county averages, percentile rank for each percent total minority within state and county entitie

Hawaii Census 2000 Block Groups

EPA Pesticide Factsheets

This data layer represents Census 2000 demographic data derived from the PL94-171 redistricting files and SF3. Census geographic entities include blocks, blockgroups and tracts. Tiger line files are the source of the geometry representing the Census blocks. Attributes include total population counts, racial/ethnic, and poverty/income information. Racial/ethnic classifications are represented in units of blocks, blockgroups and tracts. Poverty and income data are represented in units of blockgroups and tracts. Percentages of each racial/ethnic group have been calculated from the population counts. Total Minority counts and percentages were compiled from each racial/ethnic non-white category. Categories compiled to create the Total Minority count includes the following: African American, Asian, American Indian, Pacific Islander, White Hispanic, Other and all mixed race categories. The percentage poverty attribute represents the percent of the population living at or below poverty level. The per capita income attribute represents the sum of all income within the geographic entity, divided by the total population of that entity. Special fields designed to be used for EJ analysis have been derived from the PL data and include the following: Percentage difference of block, blockgroup and total minority from the state and county averages, percentile rank for each percent total minority within state and county entitie

Nondestructive detection of total viable count changes of chilled pork in high oxygen storage condition based on hyperspectral technology

NASA Astrophysics Data System (ADS)

Zheng, Xiaochun; Peng, Yankun; Li, Yongyu; Chao, Kuanglin; Qin, Jianwei

2017-05-01

The plate count method is commonly used to detect the total viable count (TVC) of bacteria in pork, which is timeconsuming and destructive. It has also been used to study the changes of the TVC in pork under different storage conditions. In recent years, many scholars have explored the non-destructive methods on detecting TVC by using visible near infrared (VIS/NIR) technology and hyperspectral technology. The TVC in chilled pork was monitored under high oxygen condition in this study by using hyperspectral technology in order to evaluate the changes of total bacterial count during storage, and then evaluate advantages and disadvantages of the storage condition. The VIS/NIR hyperspectral images of samples stored in high oxygen condition was acquired by a hyperspectral system in range of 400 1100nm. The actual reference value of total bacteria was measured by standard plate count method, and the results were obtained in 48 hours. The reflection spectra of the samples are extracted and used for the establishment of prediction model for TVC. The spectral preprocessing methods of standard normal variate transformation (SNV), multiple scatter correction (MSC) and derivation was conducted to the original reflectance spectra of samples. Partial least squares regression (PLSR) of TVC was performed and optimized to be the prediction model. The results show that the near infrared hyperspectral technology based on 400-1100nm combined with PLSR model can describe the growth pattern of the total bacteria count of the chilled pork under the condition of high oxygen very vividly and rapidly. The results obtained in this study demonstrate that the nondestructive method of TVC based on NIR hyperspectral has great potential in monitoring of edible safety in processing and storage of meat.

Assessment of background hydrogen by the Monte Carlo computer code MCNP-4A during measurements of total body nitrogen.

PubMed

Ryde, S J; al-Agel, F A; Evans, C J; Hancock, D A

2000-05-01

The use of a hydrogen internal standard to enable the estimation of absolute mass during measurement of total body nitrogen by in vivo neutron activation is an established technique. Central to the technique is a determination of the H prompt gamma ray counts arising from the subject. In practice, interference counts from other sources--e.g., neutron shielding--are included. This study reports use of the Monte Carlo computer code, MCNP-4A, to investigate the interference counts arising from shielding both with and without a phantom containing a urea solution. Over a range of phantom size (depth 5 to 30 cm, width 20 to 40 cm), the counts arising from shielding increased by between 4% and 32% compared with the counts without a phantom. For any given depth, the counts increased approximately linearly with width. For any given width, there was little increase for depths exceeding 15 centimeters. The shielding counts comprised between 15% and 26% of those arising from the urea phantom. These results, although specific to the Swansea apparatus, suggest that extraneous hydrogen counts can be considerable and depend strongly on the subject's size.

Effect of gamma irradiation on microbial quality of minimally processed carrot and lettuce: A case study in Greater Accra region of Ghana

NASA Astrophysics Data System (ADS)

Frimpong, G. K.; Kottoh, I. D.; Ofosu, D. O.; Larbi, D.

2015-05-01

The effect of ionizing radiation on the microbiological quality on minimally processed carrot and lettuce was studied. The aim was to investigate the effect of irradiation as a sanitizing agent on the bacteriological quality of some raw eaten salad vegetables obtained from retailers in Accra, Ghana. Minimally processed carrot and lettuce were analysed for total viable count, total coliform count and pathogenic organisms. The samples collected were treated and analysed for a 15 day period. The total viable count for carrot ranged from 1.49 to 14.01 log10 cfu/10 g while that of lettuce was 0.70 to 8.5 7 log10 cfu/10 g. It was also observed that total coliform count for carrot was 1.46-7.53 log10 cfu/10 g and 0.14-7.35 log10 cfu/10 g for lettuce. The predominant pathogenic organisms identified were Bacillus cereus, Cronobacter sakazakii, Staphylococcus aureus, and Klebsiella spp. It was concluded that 2 kGy was most effective for medium dose treatment of minimally processed carrot and lettuce.

A statistical treatment of bioassay pour fractions

NASA Astrophysics Data System (ADS)

Barengoltz, Jack; Hughes, David

A bioassay is a method for estimating the number of bacterial spores on a spacecraft surface for the purpose of demonstrating compliance with planetary protection (PP) requirements (Ref. 1). The details of the process may be seen in the appropriate PP document (e.g., for NASA, Ref. 2). In general, the surface is mechanically sampled with a damp sterile swab or wipe. The completion of the process is colony formation in a growth medium in a plate (Petri dish); the colonies are counted. Consider a set of samples from randomly selected, known areas of one spacecraft surface, for simplicity. One may calculate the mean and standard deviation of the bioburden density, which is the ratio of counts to area sampled. The standard deviation represents an estimate of the variation from place to place of the true bioburden density commingled with the precision of the individual sample counts. The accuracy of individual sample results depends on the equipment used, the collection method, and the culturing method. One aspect that greatly influences the result is the pour fraction, which is the quantity of fluid added to the plates divided by the total fluid used in extracting spores from the sampling equipment. In an analysis of a single sample’s counts due to the pour fraction, one seeks to answer the question: What is the probability that if a certain number of spores are counted with a known pour fraction, that there are an additional number of spores in the part of the rinse not poured. This is given for specific values by the binomial distribution density, where detection (of culturable spores) is success and the probability of success is the pour fraction. A special summation over the binomial distribution, equivalent to adding for all possible values of the true total number of spores, is performed. This distribution when normalized will almost yield the desired quantity. It is the probability that the additional number of spores does not exceed a certain value. Of course, for a desired value of uncertainty, one must invert the calculation. However, this probability of finding exactly the number of spores in the poured part is correct only in the case where all values of the true number of spores greater than or equal to the adjusted count are equally probable. This is not realistic, of course, but the result can only overestimate the uncertainty. So it is useful. In probability speak, one has the conditional probability given any true total number of spores. Therefore one must multiply it by the probability of each possible true count, before the summation. If the counts for a sample set (of which this is one sample) are available, one may use the calculated variance and the normal probability distribution. In this approach, one assumes a normal distribution and neglects the contribution from spatial variation. The former is a common assumption. The latter can only add to the conservatism (over estimate the number of spores at some level of confidence). A more straightforward approach is to assume a Poisson probability distribution for the measured total sample set counts, and use the product of the number of samples and the mean number of counts per sample as the mean of the Poisson distribution. It is necessary to set the total count to 1 in the Poisson distribution when actual total count is zero. Finally, even when the planetary protection requirements for spore burden refer only to the mean values, they require an adjustment for pour fraction and method efficiency (a PP specification based on independent data). The adjusted mean values are a 50/50 proposition (e.g., the probability of the true total counts in the sample set exceeding the estimate is 0.50). However, this is highly unconservative when the total counts are zero. No adjustment to the mean values occurs for either pour fraction or efficiency. The recommended approach is once again to set the total counts to 1, but now applied to the mean values. Then one may apply the corrections to the revised counts. It can be shown by the methods developed in this work that this change is usually conservative enough to increase the level of confidence in the estimate to 0.5. 1. NASA. (2005) Planetary protection provisions for robotic extraterrestrial missions. NPR 8020.12C, April 2005, National Aeronautics and Space Administration, Washington, DC. 2. NASA. (2010) Handbook for the Microbiological Examination of Space Hardware, NASA-HDBK-6022, National Aeronautics and Space Administration, Washington, DC.

Mold and aflatoxin reduction by gamma radiation of packed hot peppers and their evolution during storage.

PubMed

Iqbal, Qumer; Amjad, Muhammad; Asi, Muhammad Rafique; Ariño, Agustin

2012-08-01

The effect of gamma radiation on moisture content, total mold counts, Aspergillus counts, and aflatoxins of three hot pepper hybrids (Sky Red, Maha, and Wonder King) was investigated. Whole dried peppers packed in polyethylene bags were gamma irradiated at 0 (control), 2, 4, and 6 kGy and stored at 25°C for 90 days. Gamma radiation proved to be effective in reducing total mold and Aspergillus counts in a dose-dependent relationship. Total mold counts in irradiated peppers immediately after treatments were significantly lowered compared with those in nonirradiated samples, achieving 90 and 99% reduction at 2- and 4-kGy doses, respectively. Aspergillus counts were significantly reduced, by 93 and 97%, immediately after irradiation at doses of 2 and 4 kGy, respectively. A radiation dose of 6 kGy completely eliminated the population of total molds and Aspergillus fungi. The evolution of total molds in control and irradiated samples indicated no further fungal proliferation during 3 months of storage at 25°C. Aflatoxin levels were slightly affected by radiation doses of 2 and 4 kGy and showed a nonsignificant reduction of 6% at the highest radiation dose of 6 kGy. The distinct effectiveness of gamma radiation in molds and aflatoxins can be explained by the target theory of food irradiation, which states that the likelihood of a microorganism or a molecule being inactivated by gamma rays increases as its size increases.

A rapid and universal bacteria-counting approach using CdSe/ZnS/SiO2 composite nanoparticles as fluorescence probe.

PubMed

Fu, Xin; Huang, Kelong; Liu, Suqin

2010-02-01

In this paper, a rapid, simple, and sensitive method was described for detection of the total bacterial count using SiO(2)-coated CdSe/ZnS quantum dots (QDs) as a fluorescence marker that covalently coupled with bacteria using glutaraldehyde as the crosslinker. Highly luminescent CdSe/ZnS were prepared by applying cadmium oxide and zinc stearate as precursors instead of pyrophoric organometallic precursors. A reverse-microemulsion technique was used to synthesize CdSe/ZnS/SiO(2) composite nanoparticles with a SiO(2) surface coating. Our results showed that CdSe/ZnS/SiO(2) composite nanoparticles prepared with this method possessed highly luminescent, biologically functional, and monodispersive characteristics, and could successfully be covalently conjugated with the bacteria. As a demonstration, it was found that the method had higher sensitivity and could count bacteria in 3 x 10(2) CFU/mL, lower than the conventional plate counting and organic dye-based method. A linear relationship of the fluorescence peak intensity (Y) and the total bacterial count (X) was established in the range of 3 x 10(2)-10(7) CFU/mL using the equation Y = 374.82X-938.27 (R = 0.99574). The results of the determination for the total count of bacteria in seven real samples were identical with the conventional plate count method, and the standard deviation was satisfactory.

The Effects of Gamma and Proton Radiation Exposure on Hematopoietic Cell Counts in the Ferret Model

PubMed Central

Sanzari, Jenine K.; Wan, X. Steven; Krigsfeld, Gabriel S.; Wroe, Andrew J.; Gridley, Daila S.; Kennedy, Ann R.

2014-01-01

Exposure to total-body radiation induces hematological changes, which can detriment one's immune response to wounds and infection. Here, the decreases in blood cell counts after acute radiation doses of γ-ray or proton radiation exposure, at the doses and dose-rates expected during a solar particle event (SPE), are reported in the ferret model system. Following the exposure to γ-ray or proton radiation, the ferret peripheral total white blood cell (WBC) and lymphocyte counts decreased whereas neutrophil count increased within 3 hours. At 48 hours after irradiation, the WBC, neutrophil, and lymphocyte counts decreased in a dose-dependent manner but were not significantly affected by the radiation type (γ-rays verses protons) or dose rate (0.5 Gy/minute verses 0.5 Gy/hour). The loss of these blood cells could accompany and contribute to the physiological symptoms of the acute radiation syndrome (ARS). PMID:25356435

The role of hybridization in improving the immune response and thermal tolerance of abalone.

PubMed

Liang, Shuang; Luo, Xuan; You, Weiwei; Luo, Lianzhong; Ke, Caihuan

2014-07-01

Recently, frequent death of cultured abalone drew our attention to the stress tolerance of abalone. Hybridization is an effective way of genetic improvement in aquaculture, which can introduce improved traits to the hybrids. In this study, we challenged the hybrids between Haliotis discus hannai and Haliotis gigantea, and their parents with bacteria (vibrio harveyi, vibrio alginolyticus and vibrio parahemolyticus), then held them at 20 °C and 28 °C, survival rates of the parental populations and hybrid populations were recorded. Then we tested the immune responses and thermal-induced responses of the four populations at different temperatures. Total hemocyte count (THC), respiratory burst, superoxide dismutase activity (SOD), acid phosphatase activity (ACP), alkaline phosphatase activity (AKP), myeloperoxidase activity (MPO), and HSP70 expression were determined on day 1 and day 7 of the temperature exposure. Results showed higher survival rates of the hybrids than their parents against bacteria challenge. For immune parameters, THCs were evaluated at 28 °C, while increased THC was also observed in H. discus hannai ♀ × H. gigantea ♂ (DG) and H. discus hannai ♀ × H. discus hannai ♂ (DD) at 12 °C (day 7); at 28 °C, respiratory burst was activated (day 1 and 7), while SOD activity first rose then fell over 7-days exposure; AKP activity was elevated at 12 °C and 28 °C (day 1), most notably in DG, and an increased level of ACP was observed in DG at 28 °C (day 7); MPO activity was suppressed at 12 °C and 28 °C on day 1, but recovered on day 7. For HSP70, increased HSP70 levels were observed in all populations at 28 °C (day 1), and DD got the lowest HSP70 level after 7-days exposure at 28 °C. Overall, the results suggest that temperature changes could significantly affect the physiological status of abalone, and hybrids may be more resistant to disease and thermal stresses than their parents. Copyright © 2014 Elsevier Ltd. All rights reserved.

Immunostimulation and yellow head virus (YHV) disease resistance induced by a lignin-based pulping by-product in black tiger shrimp (Penaeus monodon Linn.).

PubMed

Srisapoome, Prapansak; Hamano, Kaoru; Tsutsui, Isao; Iiyama, Kenji

2018-01-01

Yellow head virus (YHV) is classified as one of the most serious pathogens causing a harmful disease in many penaeids, especially black tiger shrimp (Penaeus monodon), with high economic loss. To determine a potent and practical prophylactic strategy for controlling this disease, the toxicity of the by-product kraft lignin and its ability to control severe YHV infection were investigated in juvenile black tiger shrimp (15.9 ± 1.2 g body weight). The median lethal dosage at 96 h (96-hrs LD 50 ) of lignin in shrimp was 297 mg/L. Lignin was further added to shrimp diets via top-dressing to assess its ability to elicit immune stimulation activity. At 14 days after feeding, shrimp fed 1, 3, 5 and 10 g of lignin/kg of diet exhibited significantly higher levels of phagocytic activity (PA) than the control group (P < 0.05). However, differences in total hemocyte count among treatments were not significant during the experimental period (P > 0.05). Additionally, lignin supplementation at 1-10 g/kg for 14 days failed to protect experimental shrimp against YHV infection. The antiviral activity of lignin against YHV in black tiger shrimp was notable in vitro because compared to control shrimp (96.7 ± 5.8%; P < 0.05), shrimp injected with a pre-incubated solution of YHV and lignin at 1, 5, 10 and 20 mg/L exhibited significantly lower mortality rates, 23.3 ± 5.8, 16.7 ± 5.8, 23.3 ± 5.8, and 20.0 ± 0.0%, respectively, after a lethal dose of YHV at 14-20 days after injection. These potent effects were clearly supported and confirmed by histopathological and RT-PCR analyses. Based on these results, the pulping by-product kraft lignin efficiently inhibits YHV infection in black tiger shrimp. This information will facilitate the development of practical methods to control yellow head disease in the marine shrimp culture industry. Copyright © 2017 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

Digital computing cardiotachometer

NASA Technical Reports Server (NTRS)

Smith, H. E.; Rasquin, J. R.; Taylor, R. A. (Inventor)

1973-01-01

A tachometer is described which instantaneously measures heart rate. During the two intervals between three succeeding heart beats, the electronic system: (1) measures the interval by counting cycles from a fixed frequency source occurring between the two beats; and (2) computes heat rate during the interval between the next two beats by counting the number of times that the interval count must be counted to zero in order to equal a total count of sixty times (to convert to beats per minute) the frequency of the fixed frequency source.

42 CFR Appendix E to Part 5 - Criteria for Designation of Areas Having Shortages of Podiatric Professional(s)

Code of Federal Regulations, 2010 CFR

2010-10-01

... their time to foot care, the total available foot care practitioners will be computed as follows: Number... for the delivery of podiatric services. 2. The area's ratio of population to foot care practitioners... Count. The population count used will be the total permanent resident civilian population of the area...

Effects of lint cleaning on lint trash particle size distribution

USDA-ARS?s Scientific Manuscript database

Cotton quality trash measurements used today typically yield a single value for trash parameters for a lint sample (i.e. High Volume Instrument – percent area; Advanced Fiber Information System – total count, trash size, dust count, trash count, and visible foreign matter). A Cotton Trash Identifica...

Fluorometric determination of the DNA concentration in municipal drinking water.

PubMed Central

McCoy, W F; Olson, B H

1985-01-01

DNA concentrations in municipal drinking water samples were measured by fluorometry, using Hoechst 33258 fluorochrome. The concentration, extraction, and detection methods used were adapted from existing techniques. The method is reproducible, fast, accurate, and simple. The amounts of DNA per cell for five different bacterial isolates obtained from drinking water samples were determined by measuring DNA concentration and total cell concentration (acridine orange epifluorescence direct cell counting) in stationary pure cultures. The relationship between DNA concentration and epifluorescence total direct cell concentration in 11 different drinking water samples was linear and positive; the amounts of DNA per cell in these samples did not differ significantly from the amounts in pure culture isolates. We found significant linear correlations between DNA concentration and colony-forming unit concentration, as well as between epifluorescence direct cell counts and colony-forming unit concentration. DNA concentration measurements of municipal drinking water samples appear to monitor changes in bacteriological quality at least as well as total heterotrophic plate counting and epifluorescence direct cell counting. PMID:3890737

Hematological and plasma biochemical reference ranges of Alaskan seabirds: Their ecological significance and clinical importance

USGS Publications Warehouse

Newman, S.H.; Piatt, John F.; White, J.

1997-01-01

Blood was analyzed from 151 pelagic marine birds to establish reference ranges for hematological and plasma biochemical parameters from healthy, wild populations of Pacific seabirds. Of the 13 species examined, 9 were from the Family Alcidae (N = 122 individuals) and the remainder (N = 29) from the Families Phalacrocoracidae, Laridae, and Procellariidae. Three of 8 hematological parameters (total white blood cell count, lymphocyte count and eosinophil count) differed significantly among species, as did 9 of 13 plasma biochemical parameters (alkaline phosphatase, aspartate aminotransferase, creatine kinase, cholesterol, glucose, lactate dehydrogenase, total bilirubin, total protein and field total protein). There were no differences among species for packed cell volume, buffy coat, cell counts of heterophils, monocytes and basophils, or for concentrations of alanine aminotransferase, triglycerides, uric acid and calcium. Plasma calcium concentration, triglyceride levels and field total protein varied significantly between sexes, with females having higher mean concentrations of all 3 parameters. However, no significant relationships between measures of breeding condition (brood patch size, subcutaneous and mesenteric fat deposits, or ovarian follicle size and ovary weight) and calcium or alkaline phosphatase concentrations in female birds could be identified. Alanine aminotransferase and uric acid were the only analytes which did not differ significantly between species or sexes.

Enumeration of total aerobic microorganisms in foods by SimPlate Total Plate Count-Color Indicator methods and conventional culture methods: collaborative study.

PubMed

Feldsine, Philip T; Leung, Stephanie C; Lienau, Andrew H; Mui, Linda A; Townsend, David E

2003-01-01

The relative efficacy of the SimPlate Total Plate Count-Color Indicator (TPC-CI) method (SimPlate 35 degrees C) was compared with the AOAC Official Method 966.23 (AOAC 35 degrees C) for enumeration of total aerobic microorganisms in foods. The SimPlate TPC-CI method, incubated at 30 degrees C (SimPlate 30 degrees C), was also compared with the International Organization for Standardization (ISO) 4833 method (ISO 30 degrees C). Six food types were analyzed: ground black pepper, flour, nut meats, frozen hamburger patties, frozen fruits, and fresh vegetables. All foods tested were naturally contaminated. Nineteen laboratories throughout North America and Europe participated in the study. Three method comparisons were conducted. In general, there was <0.3 mean log count difference in recovery among the SimPlate methods and their corresponding reference methods. Mean log counts between the 2 reference methods were also very similar. Repeatability (Sr) and reproducibility (SR) standard deviations were similar among the 3 method comparisons. The SimPlate method (35 degrees C) and the AOAC method were comparable for enumerating total aerobic microorganisms in foods. Similarly, the SimPlate method (30 degrees C) was comparable to the ISO method when samples were prepared and incubated according to the ISO method.

Tower counts

USGS Publications Warehouse

Woody, Carol Ann; Johnson, D.H.; Shrier, Brianna M.; O'Neal, Jennifer S.; Knutzen, John A.; Augerot, Xanthippe; O'Neal, Thomas A.; Pearsons, Todd N.

2007-01-01

Counting towers provide an accurate, low-cost, low-maintenance, low-technology, and easily mobilized escapement estimation program compared to other methods (e.g., weirs, hydroacoustics, mark-recapture, and aerial surveys) (Thompson 1962; Siebel 1967; Cousens et al. 1982; Symons and Waldichuk 1984; Anderson 2000; Alaska Department of Fish and Game 2003). Counting tower data has been found to be consistent with that of digital video counts (Edwards 2005). Counting towers do not interfere with natural fish migration patterns, nor are fish handled or stressed; however, their use is generally limited to clear rivers that meet specific site selection criteria. The data provided by counting tower sampling allow fishery managers to determine reproductive population size, estimate total return (escapement + catch) and its uncertainty, evaluate population productivity and trends, set harvest rates, determine spawning escapement goals, and forecast future returns (Alaska Department of Fish and Game 1974-2000 and 1975-2004). The number of spawning fish is determined by subtracting subsistence, sport-caught fish, and prespawn mortality from the total estimated escapement. The methods outlined in this protocol for tower counts can be used to provide reasonable estimates ( plus or minus 6%-10%) of reproductive salmon population size and run timing in clear rivers. 

Comparison of line transects and point counts for monitoring spring migration in forested wetlands

USGS Publications Warehouse

Wilson, R.R.; Twedt, D.J.; Elliott, A.B.

2000-01-01

We compared the efficacy of 400-m line transects and sets of three point counts at detecting avian richness and abundance in bottomland hardwood forests and intensively managed cottonwood (Populus deltoides) plantations within the Mississippi Alluvial Valley. We detected more species and more individuals on line transects than on three point counts during 218 paired surveys conducted between 24 March and 3 June, 1996 and 1997. Line transects also yielded more birds per unit of time, even though point counts yielded higher estimates of relative bird density. In structurally more-complex bottomland hardwood forests, we detected more species and individuals on line transects, but in more-open cottonwood plantations, transects surpassed point counts only at detecting species within 50 m of the observer. Species richness and total abundance of Nearctic-Neotropical migrants and temperate migrants were greater on line transects within bottomland hardwood forests. Within cottonwood plantations, however, only species richness of Nearctic-Neotropical migrants and total abundance of temperate migrants were greater on line transects. Because we compared survey techniques using the same observer, within the same forest stand on a given day, we assumed that the technique yielding greater estimates of avian species richness and total abundance per unit of effort is superior. Thus, for monitoring migration within hardwood forests of the Mississippi Alluvial Valley, we recommend using line transects instead of point counts.

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

Measurement of total-body cobalt-57 vitamin B12 absorption with a gamma camera.

PubMed

Cardarelli, J A; Slingerland, D W; Burrows, B A; Miller, A

1985-08-01

Previously described techniques for the measurement of the absorption of [57Co]vitamin B12 by total-body counting have required an iron room equipped with scanning or multiple detectors. The present study uses simplifying modifications which make the technique more available and include the use of static geometry, the measurement of body thickness to correct for attenuation, a simple formula to convert the capsule-in-air count to a 100% absorption count, and finally the use of an adequately shielded gamma camera obviating the need of an iron room.

Microplastics in the Southern Ocean.

PubMed

Isobe, Atsuhiko; Uchiyama-Matsumoto, Kaori; Uchida, Keiichi; Tokai, Tadashi

2017-01-15

A field survey to collect microplastics with sizes <5mm was conducted in the Southern Ocean in 2016. We performed five net-tows and collected 44 pieces of plastic. Total particle counts of the entire water column, which is free of vertical mixing, were computed using the surface concentration (particle count per unit seawater volume) of microplastics, wind speed, and significant wave height during the observation period. Total particle counts at two stations near Antarctica were estimated to be in the order of 100,000pieceskm -2 . Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Clorate Metabolism in Pure Cultures of E.Coli 0157:H7 Pretreated with Either Nitrate or Chlorate

USDA-ARS?s Scientific Manuscript database

Experiments were conducted to determine the effects of 5, 7.5, and 10 mM nitrate, and 5, 10, or 20 mM chlorate on total E. coli counts, chlorate metabolism, and volatile fatty acid (VFA) concentrations in anaerobic ruminal fluid cultures. Nitrate did not affect total E. coli counts (P = 0.05), chlor...

Rapid staining and enumeration of small numbers of total bacteria in water by solid-phase laser cytometry

NASA Technical Reports Server (NTRS)

Broadaway, Susan C.; Barton, Stephanie A.; Pyle, Barry H.

2003-01-01

The nucleic acid stain SYBR Green I was evaluated for use with solid-phase laser cytometry to obtain total bacterial cell counts from several water sources with small bacterial numbers. Results were obtained within 30 min and exceeded or equaled counts on R2A agar plates incubated for 14 days at room temperature.

Effect of vacuum and modified atmosphere packaging on microbiological properties of cold-smoked trout

NASA Astrophysics Data System (ADS)

Đorđević, J.; Pavlićević, N.; Bošković, M.; Janjić, J.; Glišić, M.; Starčević, M.; Baltić, M. Ž.

2017-09-01

Because of the importance of different packaging methods for the extension of fish shelf life, as a highly perishable food, the aim of the present study was to examine the effect of vacuum and modified atmosphere packaging on the total Enterobacteriaceae and lactic acid bacteria counts of cold-smoked Salmon trout (Oncorhynchus mykiss) stored at 3°C during six weeks. Trout fillets were vacuumed packaged (VP) or packaged in one of two different modified atmospheres, with gas ratio of 50%CO2/50%N2 (MAP1) and 90%CO2/10%N2 (MAP2) and analysed on days 0, 7, 14, 21, 28, 35 and 42. Both the total Enterobacteriaceae and total lactic acid bacteria counts increased in the trout fillets in all packaging types during storage. A significantly lower total Enterobacteriaceae count was determined in the MAP fish compared to the VP fish, with the weakest growth rate and lowest numbers attained in MAP2 fillets. The lactic acid bacteria count was higher in trout packaged in MAP compared to VP, with the highest number in the MAP with 90% CO2 (MAP2).

Fingerprint Ridge Count: A Polygenic Trait Useful in Classroom Instruction.

ERIC Educational Resources Information Center

Mendenhall, Gordon; And Others

1989-01-01

Describes the use of the polygenic trait of total fingerprint ridge count in the classroom as a laboratory investigation. Presents information on background of topic, fingerprint patterns which are classified into three major groups, ridge count, the inheritance model, and activities. Includes an example data sheet format for fingerprints. (RT)

Evaluation of three portable samplers for monitoring airborne fungi

NASA Technical Reports Server (NTRS)

Mehta, S. K.; Mishra, S. K.; Pierson, D. L.

1996-01-01

Airborne fungi were monitored at five sample sites with the Burkard portable, the RCS Plus, and the SAS Super 90 air samplers; the Andersen 2-stage impactor was used for comparison. All samplers were calibrated before being used simultaneously to collect 100-liter samples at each site. The Andersen and Burkard samplers retrieved equivalent volumes of airborne fungi; the SAS Super 90 and RCS Plus measurements did not differ from each other but were significantly lower than those obtained with the Andersen or Burkard samplers. Total fungal counts correlated linearly with Cladosporium and Penicillium counts. Alternaria species, although present at all sites, did not correlate with total count or with amounts of any other fungal genera. Sampler and location significantly influenced fungal counts, but no interactions between samplers and locations were found.

Comparison of cell counting methods in rodent pulmonary toxicity studies: automated and manual protocols and considerations for experimental design

PubMed Central

Zeidler-Erdely, Patti C.; Antonini, James M.; Meighan, Terence G.; Young, Shih-Houng; Eye, Tracy J.; Hammer, Mary Ann; Erdely, Aaron

2016-01-01

Pulmonary toxicity studies often use bronchoalveolar lavage (BAL) to investigate potential adverse lung responses to a particulate exposure. The BAL cellular fraction is counted, using automated (i.e. Coulter Counter®), flow cytometry or manual (i.e. hemocytometer) methods, to determine inflammatory cell influx. The goal of the study was to compare the different counting methods to determine which is optimal for examining BAL cell influx after exposure by inhalation or intratracheal instillation (ITI) to different particles with varying inherent pulmonary toxicities in both rat and mouse models. General findings indicate that total BAL cell counts using the automated and manual methods tended to agree after inhalation or ITI exposure to particle samples that are relatively nontoxic or at later time points after exposure to a pneumotoxic particle when the response resolves. However, when the initial lung inflammation and cytotoxicity was high after exposure to a pneumotoxic particle, significant differences were observed when comparing cell counts from the automated, flow cytometry and manual methods. When using total BAL cell count for differential calculations from the automated method, depending on the cell diameter size range cutoff, the data suggest that the number of lung polymorphonuclear leukocytes (PMN) varies. Importantly, the automated counts, regardless of the size cutoff, still indicated a greater number of total lung PMN when compared with the manual method, which agreed more closely with flow cytometry. The results suggest that either the manual method or flow cytometry would be better suited for BAL studies where cytotoxicity is an unknown variable. PMID:27251196