Sample records for continuous insect cell

  1. A continuous-exchange cell-free protein synthesis system based on extracts from cultured insect cells.

    PubMed

    Stech, Marlitt; Quast, Robert B; Sachse, Rita; Schulze, Corina; Wüstenhagen, Doreen A; Kubick, Stefan

    2014-01-01

    In this study, we present a novel technique for the synthesis of complex prokaryotic and eukaryotic proteins by using a continuous-exchange cell-free (CECF) protein synthesis system based on extracts from cultured insect cells. Our approach consists of two basic elements: First, protein synthesis is performed in insect cell lysates which harbor endogenous microsomal vesicles, enabling a translocation of de novo synthesized target proteins into the lumen of the insect vesicles or, in the case of membrane proteins, their embedding into a natural membrane scaffold. Second, cell-free reactions are performed in a two chamber dialysis device for 48 h. The combination of the eukaryotic cell-free translation system based on insect cell extracts and the CECF translation system results in significantly prolonged reaction life times and increased protein yields compared to conventional batch reactions. In this context, we demonstrate the synthesis of various representative model proteins, among them cytosolic proteins, pharmacological relevant membrane proteins and glycosylated proteins in an endotoxin-free environment. Furthermore, the cell-free system used in this study is well-suited for the synthesis of biologically active tissue-type-plasminogen activator, a complex eukaryotic protein harboring multiple disulfide bonds.

  2. A Continuous-Exchange Cell-Free Protein Synthesis System Based on Extracts from Cultured Insect Cells

    PubMed Central

    Stech, Marlitt; Quast, Robert B.; Sachse, Rita; Schulze, Corina; Wüstenhagen, Doreen A.; Kubick, Stefan

    2014-01-01

    In this study, we present a novel technique for the synthesis of complex prokaryotic and eukaryotic proteins by using a continuous-exchange cell-free (CECF) protein synthesis system based on extracts from cultured insect cells. Our approach consists of two basic elements: First, protein synthesis is performed in insect cell lysates which harbor endogenous microsomal vesicles, enabling a translocation of de novo synthesized target proteins into the lumen of the insect vesicles or, in the case of membrane proteins, their embedding into a natural membrane scaffold. Second, cell-free reactions are performed in a two chamber dialysis device for 48 h. The combination of the eukaryotic cell-free translation system based on insect cell extracts and the CECF translation system results in significantly prolonged reaction life times and increased protein yields compared to conventional batch reactions. In this context, we demonstrate the synthesis of various representative model proteins, among them cytosolic proteins, pharmacological relevant membrane proteins and glycosylated proteins in an endotoxin-free environment. Furthermore, the cell-free system used in this study is well-suited for the synthesis of biologically active tissue-type-plasminogen activator, a complex eukaryotic protein harboring multiple disulfide bonds. PMID:24804975

  3. Continuous evolution of B. thuringiensis toxins overcomes insect resistance

    PubMed Central

    Badran, Ahmed H.; Guzov, Victor M.; Huai, Qing; Kemp, Melissa M.; Vishwanath, Prashanth; Kain, Wendy; Nance, Autumn M.; Evdokimov, Artem; Moshiri, Farhad; Turner, Keith H.; Wang, Ping; Malvar, Thomas; Liu, David R.

    2016-01-01

    The Bacillus thuringiensis δ-endotoxins (Bt toxins) are widely used insecticidal proteins in engineered crops that provide agricultural, economic, and environmental benefits. The development of insect resistance to Bt toxins endangers their long-term effectiveness. We developed a phage-assisted continuous evolution (PACE) selection that rapidly evolves high-affinity protein-protein interactions, and applied this system to evolve variants of the Bt toxin Cry1Ac that bind a cadherin-like receptor from the insect pest Trichoplusia ni (TnCAD) that is not natively targeted by wild-type Cry1Ac. The resulting evolved Cry1Ac variants bind TnCAD with high affinity (Kd = 11–41 nM), kill TnCAD-expressing insect cells that are not susceptible to wild-type Cry1Ac, and kill Cry1Ac-resistant T. ni insects up to 335-fold more potently than wild-type Cry1Ac. Our findings establish that the evolution of Bt toxins with novel insect cell receptor affinity can overcome Bt toxin resistance in insects and confer lethality approaching that of the wild-type Bt toxin against non-resistant insects. PMID:27120167

  4. Innate immune system still works at diapause, a physiological state of dormancy in insects

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

    Nakamura, Akihiro; Miyado, Kenji, E-mail: kmiyado@nch.go.jp; Takezawa, Youki

    Highlights: {yields} Two major types of cells are present in the body fluid isolated from the thoracic region of a diapausing pupa. {yields} Phagocytosis and encapsulation by these cells were observed when latex beads as foreign targets were microinjected into a pupa. {yields} Such behavior by these cells was still observed even when pupae were continuously chilled at 4 {sup o}C. {yields} Innate cellular reactions can work in diapausing insects in a dormant state. -- Abstract: Diapause is most often observed in insects and is a physiologically dormant state different from other types of dormancy, such as hibernation. It allowsmore » insects to survive in harsh environments or extend longevity. In general, larval, pupal, or adult non-diapausing insects possess an innate immune system preventing the invasion of microorganisms into their bodies; however, it is unclear whether this system works under the dormant condition of diapause. We here report the occurrence of innate cellular reactions during diapause using pupae of a giant silkmoth, Samia cynthia pryeri. Scanning electron microscopic analysis demonstrated the presence of two major types of cells in the body fluid isolated from the thoracic region of a pupa. Phagocytosis and encapsulation, characteristics of innate cellular reactions, by these cells were observed when latex beads as foreign targets were microinjected into the internal portion of a pupa. Such behavior by these cells was still observed even when pupae were continuously chilled at 4 {sup o}C. Our results indicate that innate cellular reactions can work in diapausing insects in a dormant state.« less

  5. Characterization of cell lines developed from the glassy-winged sharpshooter, Homalodisca coagulata (Hemiptera: Cicadellidae).

    PubMed

    Kamita, Shizuo G; Do, Zung N; Samra, Aman I; Hagler, James R; Hammock, Bruce D

    2005-01-01

    Four continuous cell lines were established from the embryos of the glassy-winged sharpshooter, Homalodisca coagulata (Say), an economically important insect vector of bacterial pathogens of grape, almond, citrus, oleander, and other agricultural and ornamental plantings. The cell lines were designated GWSS-Z10, GWSS-Z15, GWSS-G3, and GWSS-LH. The GWSS-Z10, GWSS-Z15, and GWSS-G3 lines were cultured in Ex-Cell 401 medium supplemented with 10% fetal bovine serum (FBS), whereas the GWSS-LH line was cultured in LH medium supplemented with 20% FBS. The cell lines were characterized in terms of their morphology, growth, protein composition, and polymerase chain reaction- amplification patterns of their chromosomal deoxyribonucleic acid. The population doubling times of GWSS-Z10, GWSS-Z15, GWSS-G3, and GWSS-LH were 46.2, 90.9, 100.3, and 60.2 h, respectively. These lines should be useful for the study of insect-pathogenic viruses of leafhoppers, aphids, treehoppers, and other related insects as well as plant-pathogenic viruses that are transmitted by these insects.

  6. Insect anaphylaxis: addressing clinical challenges.

    PubMed

    Tracy, James M; Lewis, Elena J; Demain, Jeffrey G

    2011-08-01

    Few allergic reactions are as potentially life-threatening, or frightening to the patient, as anaphylaxis. Food, medications, and insect stings are the three most common triggers of anaphylaxis, but insect allergy provides the best opportunity to understand the biology of anaphylaxis. If the physician can establish a diagnosis of insect allergy, treatment with nearly 98% effectiveness can be initiated. However, sometimes patients have a compelling history of insect sting anaphylaxis, but negative skin and blood tests. This situation presents us with a fascinating opportunity to understand the biology of insect anaphylaxis. Recent and ongoing work shows that occult mast cell disease may be critical in insect anaphylaxis. Mastocytosis, serum tryptase and basophil biology are key elements; genetic markers may potentially help us diagnose at-risk individuals and determine proper treatment. Understanding basophil activation may play an additional role both in diagnosis and knowing when therapy might be terminated. Mast cell disease, serum tryptase and basophil biology are providing an opportunity to better understand and manage insect allergy. This evolving understanding should improve long-term management of insect anaphylaxis and help us to better understand the clinical dilemma of appropriate management of the history-positive patient in which testing is unable to detect venom-specific IgE. Furthermore, omalizumab's immunomodulatory effects may play a role in difficult-to-treat insect allergy and mastocytosis. Finally, unrelated to these, but still important as an ongoing risk factor, is the continued underutilization of epinephrine for both acute and long-term management of insect anaphylaxis.

  7. FluBlok, a next generation influenza vaccine manufactured in insect cells.

    PubMed

    Cox, Manon M J; Hollister, Jason R

    2009-06-01

    FluBlok, a recombinant trivalent hemagglutinin (rHA) vaccine produced in insect cell culture using the baculovirus expression system, provides an attractive alternative to the current egg-based trivalent inactivated influenza vaccine (TIV). Its manufacturing process presents the possibility for safe and expeditious vaccine production. FluBlok contains three times more HA than TIV and does not contain egg-protein or preservatives. The high purity of the antigen enables administration at higher doses without a significant increase in side-effects in human subjects. The insect cell-baculovirus production technology is particularly suitable for influenza where annual adjustment of the vaccine is required. The baculovirus-insect expression system is generally considered a safe production system, with limited growth potential for adventitious agents. Still regulators question and challenge the safety of this novel cell substrate as FluBlok continues to advance toward product approval. This review provides an overview of cell substrate characterization for expresSF cell line used for the manufacturing of FluBlok. In addition, this review includes an update on the clinical development of FluBlok. The highly purified protein vaccine, administered at three times higher antigen content than TIV, is well tolerated and results in stronger immunogenicity, a long lasting immune response and provides cross-protection against drift influenza viruses.

  8. Successful propagation of shrimp yellow head virus in immortal mosquito cells.

    PubMed

    Gangnonngiw, Warachin; Kanthong, Nipaporn; Flegel, Timothy W

    2010-05-18

    Research on crustacean viruses is hampered by the lack of continuous cell lines susceptible to them. To overcome this problem, we previously challenged immortal mosquito and lepidopteran cell lines with shrimp yellow head virus (YHV), followed by serial, split-passage of whole cells, and showed that this produced cells that persistently expressed YHV antigens. To determine whether such insect cultures positive for YHV antigens could be used to infect shrimp Penaeus monodon with YHV, culture supernatants and whole-cell homogenates were used to challenge shrimp by injection. Shrimp injected with culture supernatants could not be infected. However, shrimp injection-challenged with whole-cell homogenates from Passage 5 (early-passage) of such cultures died with histological and clinical signs typical for yellow head disease (YHD), while homogenates of mock-passaged, YHV-challenged cells did not. By contrast, shrimp challenged with cell homogenates of late-passage cultures became infected with YHV, but survived, suggesting that YHV attenuation had occurred during its long-term serial passage in insect cells. Thus, YHV could be propagated successfully in C6/36 mosquito cells and used at low passage numbers as a source of inoculum to initiate lethal infections in shrimp. This partially solves the problem of lack of continuous shrimp cell lines for cultivation of YHV.

  9. Toxicity and mode of action of insecticidal Cry1A proteins from Bacillus thuringiensis in an insect cell line, CF-1.

    PubMed

    Portugal, Leivi; Gringorten, J Lawrence; Caputo, Guido F; Soberón, Mario; Muñoz-Garay, Carlos; Bravo, Alejandra

    2014-03-01

    Bacillus thuringiensis Cry toxins are insecticidal proteins used to control insect pests. The interaction of Cry toxins with the midgut of susceptible insects is a dynamic process involving activation of the toxin, binding to midgut receptors in the apical epithelium and conformational changes in the toxin molecule, leading to pore formation and cell lysis. An understanding of the molecular events underlying toxin mode of action is essential for the continued use of Cry toxins. In this work, we examined the mechanism of action of Cry1A toxins in the lepidopteran cell line CF-1, using native Cry1Ab and mutant forms of this protein that interfer with different steps in the mechanism of action, specifically, receptor binding, oligomerization or pore formation. These mutants lost activity against both Manduca sexta larvae and CF-1 cells. We also analyzed a mutation created in domain I of Cry1Ab, in which helix α-1 and part of helix α-2 were deleted (Cry1AbMod). Cry1AbMod is able to oligomerize in the absence of toxin receptors, and although it shows reduced activity against some susceptible insects, it kills insect pests that have developed resistance to native Cry1Ab. Cry1AbMod showed enhanced toxicity to CF-1, suggesting that oligomerization of native Cry1Ab may be a limiting step in its activity against CF-1 cells. The toxicity of Cry1Ac and Cry1AcMod were also analyzed. Our results suggest that some of the steps in the mode of action of Cry1A toxins are conserved in vivo in insect midgut cells and in vitro in an established cell line, CF-1. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. Comparative ultrastructural characterization of African horse sickness virus-infected mammalian and insect cells reveals a novel potential virus release mechanism from insect cells.

    PubMed

    Venter, E; van der Merwe, C F; Buys, A V; Huismans, H; van Staden, V

    2014-03-01

    African horse sickness virus (AHSV) is an arbovirus capable of successfully replicating in both its mammalian host and insect vector. Where mammalian cells show a severe cytopathic effect (CPE) following AHSV infection, insect cells display no CPE. These differences in cell death could be linked to the method of viral release, i.e. lytic or non-lytic, that predominates in a specific cell type. Active release of AHSV, or any related orbivirus, has, however, not yet been documented from insect cells. We applied an integrated microscopy approach to compare the nanomechanical and morphological response of mammalian and insect cells to AHSV infection. Atomic force microscopy revealed plasma membrane destabilization, integrity loss and structural deformation of the entire surface of infected mammalian cells. Infected insect cells, in contrast, showed no morphological differences from mock-infected cells other than an increased incidence of circular cavities present on the cell surface. Transmission electron microscopy imaging identified a novel large vesicle-like compartment within infected insect cells, not present in mammalian cells, containing viral proteins and virus particles. Extracellular clusters of aggregated virus particles were visualized adjacent to infected insect cells with intact plasma membranes. We propose that foreign material is accumulated within these vesicles and that their subsequent fusion with the cell membrane releases entrapped viruses, thereby facilitating a non-lytic virus release mechanism different from the budding previously observed in mammalian cells. This insect cell-specific defence mechanism contributes to the lack of cell damage observed in AHSV-infected insect cells.

  11. 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

  12. Suitability and perspectives on using recombinant insect cells for the production of virus-like particles.

    PubMed

    Yamaji, Hideki

    2014-03-01

    Virus-like particles (VLPs) can be produced in recombinant protein production systems by expressing viral surface proteins that spontaneously assemble into particulate structures similar to authentic viral or subviral particles. VLPs serve as excellent platforms for the development of safe and effective vaccines and diagnostic antigens. Among various recombinant protein production systems, the baculovirus-insect cell system has been used extensively for the production of a wide variety of VLPs. This system is already employed for the manufacture of a licensed human papillomavirus-like particle vaccine. However, the baculovirus-insect cell system has several inherent limitations including contamination of VLPs with progeny baculovirus particles. Stably transformed insect cells have emerged as attractive alternatives to the baculovirus-insect cell system. Different types of VLPs, with or without an envelope and composed of either single or multiple structural proteins, have been produced in stably transformed insect cells. VLPs produced by stably transformed insect cells have successfully elicited immune responses in vivo. In some cases, the yield of VLPs attained with recombinant insect cells was comparable to, or higher than, that obtained by baculovirus-infected insect cells. Recombinant insect cells offer a promising approach to the development and production of VLPs.

  13. Development of leafhopper cell culture to trace the early infection process of a nucleorhabdovirus, rice yellow stunt virus, in insect vector cells.

    PubMed

    Wang, Haitao; Wang, Juan; Xie, Yunjie; Fu, Zhijun; Wei, Taiyun; Zhang, Xiao-Feng

    2018-04-20

    In China, the rice pathogen Rice yellow stunt virus (RYSV), a member of the genus Nucleorhabdovirus in the family Rhabdoviridae, was a severe threat to rice production during the1960s and1970s. Fundamental aspects of the biology of this virus such as protein localization and formation of the RYSV viroplasm during infection of insect vector cells are largely unexplored. The specific role(s) of the structural proteins nucleoprotein (N) and phosphoprotein (P) in the assembly of the viroplasm during RYSV infection in insect vector is also unclear. In present study, we used continuous leafhopper cell culture, immunocytochemical techniques, and transmission electron microscopy to investigate the subcellular distributions of N and P during RYSV infection. Both GST pull-down assay and yeast two-hybrid assay were used to assess the in vitro interaction of N and P. The dsRNA interference assay was performed to study the functional roles of N and P in the assembly of RYSV viroplasm. Here we demonstrated that N and P colocalized in the nucleus of RYSV-infected Nephotettix cincticeps cell and formed viroplasm-like structures (VpLSs). The transiently expressed N and P are sufficient to form VpLSs in the Sf9 cells. In addition, the interactions of N/P, N/N and P/P were confirmed in vitro. More interestingly, the accumulation of RYSV was significantly reduced when the transcription of N gene or P gene was knocked down by dsRNA treatment. In summary, our results suggest that N and P are the main viral factors responsible for the formation of viroplasm in RYSV-infected insect cells. Early during RYSV infection in the insect vector, N and P interacted with each other in the nucleus to form viroplasm-like structures, which are essential for the infection of RYSV.

  14. Combining stable insect cell lines with baculovirus-mediated expression for multi-HA influenza VLP production.

    PubMed

    Sequeira, Daniela P; Correia, Ricardo; Carrondo, Manuel J T; Roldão, António; Teixeira, Ana P; Alves, Paula M

    2018-05-24

    Safer and broadly protective vaccines are needed to cope with the continuous evolution of circulating influenza virus strains and promising approaches based on the expression of multiple hemagglutinins (HA) in a virus-like particle (VLP) have been proposed. However, expression of multiple genes in the same vector can lead to its instability due to tandem repetition of similar sequences. By combining stable with transient expression systems we can rationally distribute the number of genes to be expressed per platform and thus mitigate this risk. In this work, we developed a modular system comprising stable and baculovirus-mediated expression in insect cells for production of multi-HA influenza enveloped VLPs. First, a stable insect High Five cell population expressing two different HA proteins from subtype H3 was established. Infection of this cell population with a baculovirus vector encoding three other HA proteins from H3 subtype proved to be as competitive as traditional co-infection approaches in producing a pentavalent H3 VLP. Aiming at increasing HA expression, the stable insect cell population was infected at increasingly higher cell concentrations (CCI). However, cultures infected at CCI of 3×10 6 cells/mL showed lower HA titers per cell in comparison to standard CCI of 2×10 6 cells/mL, a phenomenon named "cell density effect". To lessen the negative impact of this phenomenon, a tailor-made refeed strategy was designed based on the exhaustion of key nutrients during cell growth. Noteworthy, cultures supplemented and infected at a CCI of 4×10 6 cells/mL showed comparable HA titers per cell to those of CCI of 2×10 6 cells/mL, thus leading to an increase of up to 4-fold in HA titers per mL. Scalability of the modular strategy herein proposed was successfully demonstrated in 2L stirred tank bioreactors with comparable HA protein levels observed between bioreactor and shake flasks cultures. Overall, this work demonstrates the suitability of combining stable with baculovirus-mediated expression in insect cells as an efficient platform for production of multi-HA influenza VLPs, surpassing the drawbacks of traditional co-infection strategies and/or the use of larger, unstable vectors. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Recombinant protein production and insect cell culture and process

    NASA Technical Reports Server (NTRS)

    Spaulding, Glenn (Inventor); Prewett, Tacey (Inventor); Goodwin, Thomas (Inventor); Francis, Karen (Inventor); Andrews, Angela (Inventor); Oconnor, Kim (Inventor)

    1993-01-01

    A process has been developed for recombinant production of selected polypeptides using transformed insect cells cultured in a horizontally rotating culture vessel modulated to create low shear conditions. A metabolically transformed insect cell line is produced using the culture procedure regardless of genetic transformation. The recombinant polypeptide can be produced by an alternative process using the cultured insect cells as host for a virus encoding the described polypeptide such as baculovirus. The insect cells can also be a host for viral production.

  16. Recombinant Protein Production and Insect Cell Culture and Process

    NASA Technical Reports Server (NTRS)

    Spaulding, Glenn F. (Inventor); Goodwin, Thomas J. (Inventor); OConnor, Kim C. (Inventor); Francis, Karen M. (Inventor); Andrews, Angela D. (Inventor); Prewett, Tracey L. (Inventor)

    1997-01-01

    A process has been developed for recombinant production of selected polypeptides using transformed insect cells cultured in a horizontally rotating culture vessel modulated to create low shear conditions. A metabolically transformed insect cell line is produced using the culture procedure regardless of genetic transformation. The recombinant polypeptide can be produced by an alternative process using virtually infected or stably transformed insect cells containing a gene encoding the described polypeptide. The insect cells can also be a host for viral production.

  17. Methods for Maintaining Insect Cell Cultures

    PubMed Central

    Lynn, Dwight E.

    2002-01-01

    Insect cell cultures are now commonly used in insect physiology, developmental biology, pathology, and molecular biology. As the field has advanced from methods development to a standard procedure, so has the diversity of scientists using the technique. This paper describes methods that are effective for maintaining various insect cell lines. The procedures are differentiated between loosely or non-attached cell strains, attached cell strains, and strongly adherent cell strains. PMID:15455043

  18. Adventitious viruses in insect cell lines used for recombinant protein expression.

    PubMed

    Geisler, Christoph; Jarvis, Donald L

    2018-04-01

    Insect cells are widely used for recombinant protein expression, typically as hosts for recombinant baculovirus vectors, but also for plasmid-mediated transient transfection or stable genetic transformation. Insect cells are used to express proteins for research, as well as to manufacture biologicals for human and veterinary medicine. Recently, several insect cell lines used for recombinant protein expression were found to be persistently infected with adventitious viruses. This has raised questions about how these infections might affect research performed using those cell lines. Furthermore, these findings raised serious concerns about the safety of biologicals produced using those cell lines. In response, new insect cell lines lacking adventitious viruses have been isolated for use as improved research tools and safer biological manufacturing platforms. Here, we review the scientific and patent literature on adventitious viruses found in insect cell lines, affected cell lines, and new virus-free cell lines. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. In vitro anticancer effects of insect tea in TCA8113 cells.

    PubMed

    Qian, Yu; Li, Gui-Jie; Wang, Rui; Zhou, Ya-Lin; Sun, Peng; Zhao, Xin

    2014-01-01

    Insect tea is widely used a traditional drink or traditional Chinese medicine in China. This study was conducted with an aim to determine the in vitro anticancer effect of Insect tea in cancer cells. The anticancer effects of Insect tea were evaluated in human tongue carcinoma TCA8113 cells using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry analysis, nuclear staining with 4,6-diamidino-2-phenylindole (DAPI), reverse transcription-polymerase chain reaction (RT-PCR) analysis, and western bolt assay. At 200 μg/mL, Insect tea inhibited the growth of TCA8113 cells by 80.7%, which was higher than the inhibition caused by 100 μg/mL Insect tea but lower than that of 200 μg/mL green tea. Compared to the control cancer cells, Insect tea significantly (P<0.05) induced apoptosis as determined by DAPI staining and flow cytometry analysis results. Insect tea significantly induced apoptosis in cancer cells by upregulating BAX, CASP3, CASP9 and downregulating BCL2. Genes encoding nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) were significantly downregulated by Insect tea, demonstrating its anti-inflammatory properties. Insect tea also exerted a great anti-metastasis effect on cancer cells as demonstrated by decreased expression of matrix metalloproteinase (MMP) genes and increased expression of tissue inhibitors of metalloproteinases (TIMPs). The results showed that Insect tea has good in vitro anticancer effects in TCA8113 cells, like green tea.

  20. Chapter 15. transforming lepidopteran insect cells for continuous recombinant protein expression

    USDA-ARS?s Scientific Manuscript database

    The baculovirus expression vector system (BEVS) is widely used to produce large quantities of recombinant proteins. However, yields of extracellular and membrane-bound proteins obtained with this system often are very low, possibly due to the adverse effects of baculovirus infection on the host ins...

  1. Adaptation of neuronal signaling and cell stress response pathways in a multigenic response of Drosophila melanogaster to DDT selection

    USDA-ARS?s Scientific Manuscript database

    The adaptation of insect populations to insecticidal control is a continual threat human health and sustainable agriculture practices, but many complex genomic mechanisms involved remain poorly understood. A systems approach was applied to investigate the interconnections between structural and func...

  2. Sugar beet cell wall protein confers fungal and pest resistance in genetically engineered plants

    USDA-ARS?s Scientific Manuscript database

    Sugar beet biomass and sugar yield are reduced by diseases caused by microbial pathogens and insect pest infestations. Since disease and pest control measures continue to rely on harmful chemical fungicides and insecticides, biotechnological approaches offer an alternate approach for disease and pe...

  3. Spatial and Temporal Analysis of Alphavirus Replication and Assembly in Mammalian and Mosquito Cells.

    PubMed

    Jose, Joyce; Taylor, Aaron B; Kuhn, Richard J

    2017-02-14

    Sindbis virus (SINV [genus Alphavirus , family Togaviridae ]) is an enveloped, mosquito-borne virus. Alphaviruses cause cytolytic infections in mammalian cells while establishing noncytopathic, persistent infections in mosquito cells. Mosquito vector adaptation of alphaviruses is a major factor in the transmission of epidemic strains of alphaviruses. Though extensive studies have been performed on infected mammalian cells, the morphological and structural elements of alphavirus replication and assembly remain poorly understood in mosquito cells. Here we used high-resolution live-cell imaging coupled with single-particle tracking and electron microscopy analyses to delineate steps in the alphavirus life cycle in both the mammalian host cell and insect vector cells. Use of dually labeled SINV in conjunction with cellular stains enabled us to simultaneously determine the spatial and temporal differences of alphavirus replication complexes (RCs) in mammalian and insect cells. We found that the nonstructural viral proteins and viral RNA in RCs exhibit distinct spatial organization in mosquito cytopathic vacuoles compared to replication organelles from mammalian cells. We show that SINV exploits filopodial extensions for virus dissemination in both cell types. Additionally, we propose a novel mechanism for replication complex formation around glycoprotein-containing vesicles in mosquito cells that produced internally released particles that were seen budding from the vesicles by live imaging. Finally, by characterizing mosquito cell lines that were persistently infected with fluorescent virus, we show that the replication and assembly machinery are highly modified, and this allows continuous production of alphaviruses at reduced levels. IMPORTANCE Reemerging mosquito-borne alphaviruses cause serious human epidemics worldwide. Several structural and imaging studies have helped to define the life cycle of alphaviruses in mammalian cells, but the mode of virus replication and assembly in the invertebrate vector and mechanisms producing two disease outcomes in two types of cells are yet to be identified. Using transmission electron microscopy and live-cell imaging with dual fluorescent protein-tagged SINV, we show that while insect and mammalian cells display similarities in entry and exit, they present distinct spatial and temporal organizations in virus replication and assembly. By characterizing acutely and persistently infected cells, we provide new insights into alphavirus replication and assembly in two distinct hosts, resulting in high-titer virus production in mammalian cells and continuous virus production at reduced levels in mosquito cells-presumably a prerequisite for alphavirus maintenance in nature. Copyright © 2017 Jose et al.

  4. Production of Japanese encephalitis virus-like particles in insect cells.

    PubMed

    Yamaji, Hideki; Konishi, Eiji

    2013-01-01

    Virus-like particles (VLPs) are composed of one or several recombinant viral surface proteins that spontaneously assemble into particulate structures without the incorporation of virus DNA or RNA. The baculovirus-insect cell system has been used extensively for the production of recombinant virus proteins including VLPs. While the baculovirus-insect cell system directs the transient expression of recombinant proteins in a batch culture, stably transformed insect cells allow constitutive production. In our recent study, a secretory form of Japanese encephalitis (JE) VLPs was successfully produced by Trichoplusia ni BTI-TN-5B1-4 (High Five) cells engineered to coexpress the JE virus (JEV) premembrane (prM) and envelope (E) proteins. A higher yield of E protein was attained with recombinant High Five cells than with the baculovirus-insect cell system. This study demonstrated that recombinant insect cells offer a promising approach to the high-level production of VLPs for use as vaccines and diagnostic antigens.

  5. Stingless Bee Larvae Require Fungal Steroid to Pupate.

    PubMed

    Paludo, Camila R; Menezes, Cristiano; Silva-Junior, Eduardo A; Vollet-Neto, Ayrton; Andrade-Dominguez, Andres; Pishchany, Gleb; Khadempour, Lily; do Nascimento, Fabio S; Currie, Cameron R; Kolter, Roberto; Clardy, Jon; Pupo, Mônica T

    2018-01-18

    The larval stage of the stingless bee Scaptotrigona depilis must consume a specific brood cell fungus in order to continue development. Here we show that this fungus is a member of the genus Zygosaccharomyces and provides essential steroid precursors to the developing bee. Insect pupation requires ecdysteroid hormones, and as insects cannot synthesize sterols de novo, they must obtain steroids in their diet. Larval in vitro culturing assays demonstrated that consuming ergosterol recapitulates the developmental effects on S. depilis as ingestion of Zygosaccharomyces sp. cells. Thus, we determined the molecular underpinning of this intimate mutualistic symbiosis. Phylogenetic analyses showed that similar cases of bee-Zygosaccharomyces symbiosis may exist. This unprecedented case of bee-fungus symbiosis driven by steroid requirement brings new perspectives regarding pollinator-microbiota interaction and preservation.

  6. Isotope labeling of proteins in insect cells.

    PubMed

    Skora, Lukasz; Shrestha, Binesh; Gossert, Alvar D

    2015-01-01

    Protein targets of contemporary research are often membrane proteins, multiprotein complexes, secreted proteins, or other proteins of human origin. These are difficult to express in the standard expression host used for most nuclear magnetic resonance (NMR) studies, Escherichia coli. Insect cells represent an attractive alternative, since they have become a well-established expression system and simple solutions have been developed for generation of viruses to efficiently introduce the target protein DNA into cells. Insect cells enable production of a larger fraction of the human proteome in a properly folded way than bacteria, as insect cells have a very similar set of cytosolic chaperones and a closely related secretory pathway. Here, the limited and defined glycosylation pattern that insect cells produce is an advantage for structural biology studies. For these reasons, insect cells have been established as the most widely used eukaryotic expression host for crystallographic studies. In the past decade, significant advancements have enabled amino acid type-specific as well as uniform isotope labeling of proteins in insect cells, turning them into an attractive expression host for NMR studies. © 2015 Elsevier Inc. All rights reserved.

  7. Optimization of insect cell based protein production processes - online monitoring, expression systems, scale up.

    PubMed

    Druzinec, Damir; Salzig, Denise; Brix, Alexander; Kraume, Matthias; Vilcinskas, Andreas; Kollewe, Christian; Czermak, Peter

    2013-01-01

    Due to the increasing use of insect cell based expression systems in research and industrial recombinant protein production, the development of efficient and reproducible production processes remains a challenging task. In this context, the application of online monitoring techniques is intended to ensure high and reproducible product qualities already during the early phases of process development. In the following chapter, the most common transient and stable insect cell based expression systems are briefly introduced. Novel applications of insect cell based expression systems for the production of insect derived antimicrobial peptides/proteins (AMPs) are discussed using the example of G. mellonella derived gloverin. Suitable in situ sensor techniques for insect cell culture monitoring in disposable and common bioreactor systems are outlined with respect to optical and capacitive sensor concepts. Since scale up of production processes is one of the most critical steps in process development, a conclusive overview is given about scale up aspects for industrial insect cell culture processes.

  8. CRISPR-Cas9 vectors for genome editing and host engineering in the baculovirus-insect cell system.

    PubMed

    Mabashi-Asazuma, Hideaki; Jarvis, Donald L

    2017-08-22

    The baculovirus-insect cell system (BICS) has been widely used to produce many different recombinant proteins for basic research and is being used to produce several biologics approved for use in human or veterinary medicine. Early BICS were technically complex and constrained by the relatively primordial nature of insect cell protein glycosylation pathways. Since then, recombination has been used to modify baculovirus vectors-which has simplified the system-and transform insect cells, which has enhanced its protein glycosylation capabilities. Now, CRISPR-Cas9 tools for site-specific genome editing are needed to facilitate further improvements in the BICS. Thus, in this study, we used various insect U6 promoters to construct CRISPR-Cas9 vectors and assessed their utility for site-specific genome editing in two insect cell lines commonly used as hosts in the BICS. We demonstrate the use of CRISPR-Cas9 to edit an endogenous insect cell gene and alter protein glycosylation in the BICS.

  9. Multiplication of VHS virus in insect cells.

    PubMed

    Lorenzen, N; Olesen, N J

    1995-01-01

    Viral haemorrhagic septicaemia virus (VHSV) belongs to the rhabdovirus family and is a major pathogen in farmed rainbow trout. An insect cell culture traditionally used for production of recombinant proteins was found to be susceptible to VHS virus. At pH 6.2, VHSV multiplication induced formation of large syncytia similar to those obtained by baculovirus-induced expression of recombinant VHSV glycoprotein. The VHSV G protein produced in insect cells was smaller than G protein derived from fish cells. VHS virus produced in insect cells was still pathogenic to rainbow trout after 2 cell culture passages.

  10. Insect cells as factories for biomanufacturing.

    PubMed

    Drugmand, Jean-Christophe; Schneider, Yves-Jacques; Agathos, Spiros N

    2012-01-01

    Insect cells (IC) and particularly lepidopteran cells are an attractive alternative to mammalian cells for biomanufacturing. Insect cell culture, coupled with the lytic expression capacity of baculovirus expression vector systems (BEVS), constitutes a powerful platform, IC-BEVS, for the abundant and versatile formation of heterologous gene products, including proteins, vaccines and vectors for gene therapy. Such products can be manufactured on a large scale thanks to the development of efficient and scaleable production processes involving the integration of a cell growth stage and a stage of cell infection with the recombinant baculovirus vector. Insect cells can produce multimeric proteins functionally equivalent to the natural ones and engineered vectors can be used for efficient expression. Insect cells can be cultivated easily in serum- and protein-free media. A growing number of companies are currently developing an interest in producing therapeutics using IC-BEVS, and many products are today in clinical trials and on the market for veterinary and human applications. This review summarizes current knowledge on insect cell metabolism, culture conditions and applications. Copyright © 2011 Elsevier Inc. All rights reserved.

  11. Influence of simulated microgravity on the longevity of insect-cell culture

    NASA Technical Reports Server (NTRS)

    Cowger, N. L.; O'Connor, K. C.; Bivins, J. E.

    1997-01-01

    Simulated microgravity within the NASA High Aspect Rotating-Wall Vessel (HARV) provides a quiescent environment to culture fragile insect cells. In this vessel, the duration of stationary and death phase for cultures of Spodoptera frugiperda cells was greatly extended over that achieved in shaker-flask controls. For both HARV and control cultures, S. frugiperda cells grew to concentrations in excess of 1 x 10(7) viable cells ml-1 with viabilities greater than 90%. In the HARV, stationary phase was maintained 9-15 days in contrast to 4-5 days in the shaker flask. Furthermore, the rate of cell death was reduced in the HARV by a factor of 20-90 relative to the control culture and was characterized with a death rate constant of 0.01-0.02 day-1. Beginning in the stationary phase and continuing in the death phase, there was a significant decrease in population size in the HARV versus an increase in the shaker flask. This phenomenon could represent cell adaptation to simulated microgravity and/or a change in the ratio of apoptotic to necrotic cells. Differences observed in this research between the HARV and its control were attributed to a reduction in hydrodynamic forces in the microgravity vessel.

  12. An Overview and History of Glyco-Engineering in Insect Expression Systems.

    PubMed

    Geisler, Christoph; Mabashi-Asazuma, Hideaki; Jarvis, Donald L

    2015-01-01

    Insect systems, including the baculovirus-insect cell and Drosophila S2 cell systems are widely used as recombinant protein production platforms. Historically, however, no insect-based system has been able to produce glycoproteins with human-type glycans, which often influence the clinical efficacy of therapeutic glycoproteins and the overall structures and functions of other recombinant glycoprotein products. In addition, some insect cell systems produce N-glycans with immunogenic epitopes. Over the past 20 years, these problems have been addressed by efforts to glyco-engineer insect-based expression systems. These efforts have focused on introducing the capacity to produce complex-type, terminally sialylated N-glycans and eliminating the capacity to produce immunogenic N-glycans. Various glyco-engineering approaches have included genetically engineering insect cells, baculoviral vectors, and/or insects with heterologous genes encoding the enzymes required to produce various glycosyltransferases, sugars, nucleotide sugars, and nucleotide sugar transporters, as well as an enzyme that can deplete GDP-fucose. In this chapter, we present an overview and history of glyco-engineering in insect expression systems as a prelude to subsequent chapters, which will highlight various methods used for this purpose.

  13. Tracing the evolutionary origins of insect renal function.

    PubMed

    Halberg, Kenneth A; Terhzaz, Selim; Cabrero, Pablo; Davies, Shireen A; Dow, Julian A T

    2015-04-21

    Knowledge on neuropeptide receptor systems is integral to understanding animal physiology. Yet, obtaining general insight into neuropeptide signalling in a clade as biodiverse as the insects is problematic. Here we apply fluorescent analogues of three key insect neuropeptides to map renal tissue architecture across systematically chosen representatives of the major insect Orders, to provide an unprecedented overview of insect renal function and control. In endopterygote insects, such as Drosophila, two distinct transporting cell types receive separate neuropeptide signals, whereas in the ancestral exopterygotes, a single, general cell type mediates all signals. Intriguingly, the largest insect Order Coleoptera (beetles) has evolved a unique approach, in which only a small fraction of cells are targets for neuropeptide action. In addition to demonstrating a universal utility of this technology, our results reveal not only a generality of signalling by the evolutionarily ancient neuropeptide families but also a clear functional separation of the types of cells that mediate the signal.

  14. Insect biofuel cells using trehalose included in insect hemolymph leading to an insect-mountable biofuel cell.

    PubMed

    Shoji, Kan; Akiyama, Yoshitake; Suzuki, Masato; Hoshino, Takayuki; Nakamura, Nobuhumi; Ohno, Hiroyuki; Morishima, Keisuke

    2012-12-01

    In this paper, an insect biofuel cell (BFC) using trehalose included in insect hemolymph was developed. The insect BFC is based on trehalase and glucose oxidase (GOD) reaction systems which oxidize β-glucose obtained by hydrolyzing trehalose. First, we confirmed by LC-MS that a sufficient amount of trehalose was present in the cockroach hemolymph (CHL). The maximum power density obtained using the insect BFC was 6.07 μW/cm(2). The power output was kept more than 10 % for 2.5 h by protecting the electrodes with a dialysis membrane. Furthermore, the maximum power density was increased to 10.5 μW/cm(2) by using an air diffusion cathode. Finally, we succeeded in driving a melody integrated circuit (IC) and a piezo speaker by connecting five insect BFCs in series. The results indicate that the insect BFC is a promising insect-mountable battery to power environmental monitoring micro-tools.

  15. Iridovirus infection of cell cultures from the Diaprepes root weevil, Diaprepes abbreviatus

    PubMed Central

    Hunter, W. B.; Lapointe, S. L.

    2003-01-01

    We here report the development and viral infection of a Diaprepes root weevil cell culture. Embryonic tissues of the root weevil were used to establish cell cultures for use in screening viral pathogens as potential biological control agents. Tissues were seeded into a prepared solution of insect medium and kept at a temperature of 24°C. The cell culture had primarily fibroblast-like morphology with some epithelial monolayers. Root weevil cells were successfully infected in vitro with a known insect virus, Invertebrate Iridescent Virus 6. Potential uses of insect cell cultures and insect viruses are discussed. Abbreviation: IIV-6 Invertebrate Iridescent Virus 6 PMID:15841252

  16. Enhancing the Breadth of Efficacy of Therapeutic Vaccines for Breast Cancer

    DTIC Science & Technology

    2015-10-01

    from recombinant baculovirus- infected insect cells . *Currently expressing antigens in C1R cells since these cells can also be used as antigen...co-transfect SF9 insect cells . Co-transfection of SF9 cells is initially assesed by survival of the cells compared to un-infected controls. The...Clones with higher TCR production are amplified again and used to infect Hi5 insect cells for protein production. 17 Figure 10. ELISA used to

  17. Modifying an Insect Cell N-Glycan Processing Pathway Using CRISPR-Cas Technology.

    PubMed

    Mabashi-Asazuma, Hideaki; Kuo, Chu-Wei; Khoo, Kay-Hooi; Jarvis, Donald L

    2015-10-16

    Fused lobes (FDL) is an enzyme that simultaneously catalyzes a key trimming reaction and antagonizes elongation reactions in the insect N-glycan processing pathway. Accordingly, FDL function accounts, at least in part, for major differences in the N-glycosylation patterns of glycoproteins produced by insect and mammalian cells. In this study, we used the CRISPR-Cas9 system to edit the fdl gene in Drosophila melanogaster S2 cells. CRISPR-Cas9 editing produced a high frequency of site-specific nucleotide insertions and deletions, reduced the production of insect-type, paucimannosidic products (Man3GlcNAc2), and led to the production of partially elongated, mammalian-type complex N-glycans (GlcNAc2Man3GlcNAc2) in S2 cells. As CRISPR-Cas9 has not been widely used to analyze or modify protein glycosylation pathways or edit insect cell genes, these results underscore its broad utility as a tool for these purposes. Our results also confirm the key role of FDL at the major branch point distinguishing insect and mammalian N-glycan processing pathways. Finally, the new FDL-deficient S2 cell derivative produced in this study will enable future bottom-up glycoengineering efforts designed to isolate insect cell lines that can efficiently produce recombinant glycoproteins with chemically predefined oligosaccharide side-chain structures.

  18. Purification of proteins from baculovirus-infected insect cells.

    PubMed

    O'Shaughnessy, Luke; Doyle, Sean

    2011-01-01

    Expression of recombinant proteins in the baculovirus/insect cell expression system is employed because it enables post-translational protein modification and high yields of recombinant protein. The system is capable of facilitating the functional expression of many proteins - either secreted or intracellularly located within infected insect cells. Strategies for the isolation and extraction of soluble proteins are presented in this chapter and involve selective cell lysis, precipitation and chromatography. Protein insolubility, following recombinant expression in insect cells, can occur. However, using the methods described herein, it is possible to extract and purify insoluble protein using affinity, ion-exchange and gel filtration chromatography. Indeed, protein insolubility often aids protein purification.

  19. Transmission of Ehrlichia risticii, the agent of Potomac horse fever, using naturally infected aquatic insects and helminth vectors: preliminary report.

    PubMed

    Madigan, J E; Pusterla, N; Johnson, E; Chae, J S; Pusterla, J B; Derock, E; Lawler, S P

    2000-07-01

    Ehrlichia risticii, the agent of Potomac horse fever (PHF), has been recently detected in trematode stages found in snail secretions and in aquatic insects. Based on these findings, horses could conceivably be exposed to E. risticii by skin penetration with infected cercariae, by ingestion of infected cercariae in water or via metacercariae in a second intermediate host, such as an aquatic insect. In order to test this hypothesis, horses were challenged with infectious snail secretions and aquatic insects collected from a PHF endemic region in northern California. Two horses stood with their front feet in water harbouring E. risticii-infected cercariae, 2 horses drank water harbouring E. risticii-infected cercariae, and 6 horses were fed pools of different aquatic insects harbouring E. risticii-infected metacercariae. In this preliminary study, only the one horse infected orally with mature caddisflies (Dicosmoecus gilvipes) developed the clinical and haematological disease syndrome of PHF. The agent was isolated from the blood of the infected horse in a continuous cell line and identified as E. risticii by characterisation of the 16S rRNA gene. Therefore, E. risticii is maintained in nature in a complex aquatic ecosystem and transmission to horses can occur through accidental ingestion of insects such as caddisflies containing infected metacercariae. At present, the small number of horses used in this study does not exclude other insects and free trematode stages as potential sources of infection.

  20. Prostaglandins modify phosphorylation of specific proteins in the insect cell line BCIRL-HzAM1

    USDA-ARS?s Scientific Manuscript database

    Prostaglandins (PGs) play crucial roles in vertebrate biology, particularly in immune functions. Because PGs also mediate specific cell functions in insect immunity, we are investigating how these signaling molecules affect insect cells. We reported that PGs, notably PGA1, PGA2, and PGE1, up and/or ...

  1. Insect cell transformation vectors that support high level expression and promoter assessment in insect cell culture

    USDA-ARS?s Scientific Manuscript database

    A somatic transformation vector, pDP9, was constructed that provides a simplified means of producing permanently transformed cultured insect cells that support high levels of protein expression of foreign genes. The pDP9 plasmid vector incorporates DNA sequences from the Junonia coenia densovirus th...

  2. 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.

  3. Evaluation of the Pichia pastoris expression system for the production of GPCRs for structural analysis

    PubMed Central

    2011-01-01

    Background Various protein expression systems, such as Escherichia coli (E. coli), Saccharomyces cerevisiae (S. cerevisiae), Pichia pastoris (P. pastoris), insect cells and mammalian cell lines, have been developed for the synthesis of G protein-coupled receptors (GPCRs) for structural studies. Recently, the crystal structures of four recombinant human GPCRs, namely β2 adrenergic receptor, adenosine A2a receptor, CXCR4 and dopamine D3 receptor, were successfully determined using an insect cell expression system. GPCRs expressed in insect cells are believed to undergo mammalian-like posttranscriptional modifications and have similar functional properties than in mammals. Crystal structures of GPCRs have not yet been solved using yeast expression systems. In the present study, P. pastoris and insect cell expression systems for the human muscarinic acetylcholine receptor M2 subtype (CHRM2) were developed and the quantity and quality of CHRM2 synthesized by both expression systems were compared for the application in structural studies. Results The ideal conditions for the expression of CHRM2 in P. pastoris were 60 hr at 20°C in a buffer of pH 7.0. The specific activity of the expressed CHRM2 was 28.9 pmol/mg of membrane protein as determined by binding assays using [3H]-quinuclidinyl benzilate (QNB). Although the specific activity of the protein produced by P. pastoris was lower than that of Sf9 insect cells, CHRM2 yield in P. pastoris was 2-fold higher than in Sf9 insect cells because P. pastoris was cultured at high cell density. The dissociation constant (Kd) for QNB in P. pastoris was 101.14 ± 15.07 pM, which was similar to that in Sf9 insect cells (86.23 ± 8.57 pM). There were no differences in the binding affinity of CHRM2 for QNB between P. pastoris and Sf9 insect cells. Conclusion Compared to insect cells, P. pastoris is easier to handle, can be grown at lower cost, and can be expressed quicker at a large scale. Yeast, P. pastoris, and insect cells are all effective expression systems for GPCRs. The results of the present study strongly suggested that protein expression in P. pastoris can be applied to the structural and biochemical studies of GPCRs. PMID:21513509

  4. Small Interfering RNA Pathway Modulates Initial Viral Infection in Midgut Epithelium of Insect after Ingestion of Virus.

    PubMed

    Lan, Hanhong; Chen, Hongyan; Liu, Yuyan; Jiang, Chaoyang; Mao, Qianzhuo; Jia, Dongsheng; Chen, Qian; Wei, Taiyun

    2016-01-15

    Numerous viruses are transmitted in a persistent manner by insect vectors. Persistent viruses establish their initial infection in the midgut epithelium, from where they disseminate to the midgut visceral muscles. Although propagation of viruses in insect vectors can be controlled by the small interfering RNA (siRNA) antiviral pathway, whether the siRNA pathway can control viral dissemination from the midgut epithelium is unknown. Infection by a rice virus (Southern rice black streaked dwarf virus [SRBSDV]) of its incompetent vector (the small brown planthopper [SBPH]) is restricted to the midgut epithelium. Here, we show that the siRNA pathway is triggered by SRBSDV infection in continuously cultured cells derived from the SBPH and in the midgut of the intact insect. Knockdown of the expression of the core component Dicer-2 of the siRNA pathway by RNA interference strongly increased the ability of SRBSDV to propagate in continuously cultured SBPH cells and in the midgut epithelium, allowing viral titers in the midgut epithelium to reach the threshold (1.99 × 10(9) copies of the SRBSDV P10 gene/μg of midgut RNA) needed for viral dissemination into the SBPH midgut muscles. Our results thus represent the first elucidation of the threshold for viral dissemination from the insect midgut epithelium. Silencing of Dicer-2 further facilitated the transmission of SRBSDV into rice plants by SBPHs. Taken together, our results reveal the new finding that the siRNA pathway can control the initial infection of the insect midgut epithelium by a virus, which finally affects the competence of the virus's vector. Many viral pathogens that cause significant global health and agricultural problems are transmitted via insect vectors. The first bottleneck in viral infection, the midgut epithelium, is a principal determinant of the ability of an insect species to transmit a virus. Southern rice black streaked dwarf virus (SRBSDV) is restricted exclusively to the midgut epithelium of an incompetent vector, the small brown planthopper (SBPH). Here, we show that silencing of the core component Dicer-2 of the small interfering RNA (siRNA) pathway increases viral titers in the midgut epithelium past the threshold (1.99 × 10(9) copies of the SRBSDV P10 gene/μg of midgut RNA) for viral dissemination into the midgut muscles and then into the salivary glands, allowing the SBPH to become a competent vector of SRBSDV. This result is the first evidence that the siRNA antiviral pathway has a direct role in the control of viral dissemination from the midgut epithelium and that it affects the competence of the virus's vector. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  5. Small Interfering RNA Pathway Modulates Initial Viral Infection in Midgut Epithelium of Insect after Ingestion of Virus

    PubMed Central

    Lan, Hanhong; Chen, Hongyan; Liu, Yuyan; Jiang, Chaoyang; Mao, Qianzhuo; Jia, Dongsheng; Chen, Qian

    2015-01-01

    ABSTRACT Numerous viruses are transmitted in a persistent manner by insect vectors. Persistent viruses establish their initial infection in the midgut epithelium, from where they disseminate to the midgut visceral muscles. Although propagation of viruses in insect vectors can be controlled by the small interfering RNA (siRNA) antiviral pathway, whether the siRNA pathway can control viral dissemination from the midgut epithelium is unknown. Infection by a rice virus (Southern rice black streaked dwarf virus [SRBSDV]) of its incompetent vector (the small brown planthopper [SBPH]) is restricted to the midgut epithelium. Here, we show that the siRNA pathway is triggered by SRBSDV infection in continuously cultured cells derived from the SBPH and in the midgut of the intact insect. Knockdown of the expression of the core component Dicer-2 of the siRNA pathway by RNA interference strongly increased the ability of SRBSDV to propagate in continuously cultured SBPH cells and in the midgut epithelium, allowing viral titers in the midgut epithelium to reach the threshold (1.99 × 109 copies of the SRBSDV P10 gene/μg of midgut RNA) needed for viral dissemination into the SBPH midgut muscles. Our results thus represent the first elucidation of the threshold for viral dissemination from the insect midgut epithelium. Silencing of Dicer-2 further facilitated the transmission of SRBSDV into rice plants by SBPHs. Taken together, our results reveal the new finding that the siRNA pathway can control the initial infection of the insect midgut epithelium by a virus, which finally affects the competence of the virus's vector. IMPORTANCE Many viral pathogens that cause significant global health and agricultural problems are transmitted via insect vectors. The first bottleneck in viral infection, the midgut epithelium, is a principal determinant of the ability of an insect species to transmit a virus. Southern rice black streaked dwarf virus (SRBSDV) is restricted exclusively to the midgut epithelium of an incompetent vector, the small brown planthopper (SBPH). Here, we show that silencing of the core component Dicer-2 of the small interfering RNA (siRNA) pathway increases viral titers in the midgut epithelium past the threshold (1.99 × 109 copies of the SRBSDV P10 gene/μg of midgut RNA) for viral dissemination into the midgut muscles and then into the salivary glands, allowing the SBPH to become a competent vector of SRBSDV. This result is the first evidence that the siRNA antiviral pathway has a direct role in the control of viral dissemination from the midgut epithelium and that it affects the competence of the virus's vector. PMID:26537672

  6. Increasing cell-device adherence using cultured insect cells for receptor-based biosensors

    NASA Astrophysics Data System (ADS)

    Terutsuki, Daigo; Mitsuno, Hidefumi; Sakurai, Takeshi; Okamoto, Yuki; Tixier-Mita, Agnès; Toshiyoshi, Hiroshi; Mita, Yoshio; Kanzaki, Ryohei

    2018-03-01

    Field-effect transistor (FET)-based biosensors have a wide range of applications, and a bio-FET odorant sensor, based on insect (Sf21) cells expressing insect odorant receptors (ORs) with sensitivity and selectivity, has emerged. To fully realize the practical application of bio-FET odorant sensors, knowledge of the cell-device interface for efficient signal transfer, and a reliable and low-cost measurement system using the commercial complementary metal-oxide semiconductor (CMOS) foundry process, will be indispensable. However, the interfaces between Sf21 cells and sensor devices are largely unknown, and electrode materials used in the commercial CMOS foundry process are generally limited to aluminium, which is reportedly toxic to cells. In this study, we investigated Sf21 cell-device interfaces by developing cross-sectional specimens. Calcium imaging of Sf21 cells expressing insect ORs was used to verify the functions of Sf21 cells as odorant sensor elements on the electrode materials. We found that the cell-device interface was approximately 10 nm wide on average, suggesting that the adhesion mechanism of Sf21 cells may differ from that of other cells. These results will help to construct accurate signal detection from expressed insect ORs using FETs.

  7. Analysis of the genome of the sexually transmitted insect virus Hz-2V

    USDA-ARS?s Scientific Manuscript database

    Hz-2V is an insect DNA virus closely related to the baculoviruses that grow to high titers in insect cells and produces high yields of virus progeny. The capacity of this virus to replicate to high titers in insect cells may allow the use of this virus for production of large amount of proteins. Th...

  8. Recent developments in the remote radio control of insect flight.

    PubMed

    Sato, Hirotaka; Maharbiz, Michel M

    2010-01-01

    The continuing miniaturization of digital circuits and the development of low power radio systems coupled with continuing studies into the neurophysiology and dynamics of insect flight are enabling a new class of implantable interfaces capable of controlling insects in free flight for extended periods. We provide context for these developments, review the state-of-the-art and discuss future directions in this field.

  9. Recent Developments in the Remote Radio Control of Insect Flight

    PubMed Central

    Sato, Hirotaka; Maharbiz, Michel M.

    2010-01-01

    The continuing miniaturization of digital circuits and the development of low power radio systems coupled with continuing studies into the neurophysiology and dynamics of insect flight are enabling a new class of implantable interfaces capable of controlling insects in free flight for extended periods. We provide context for these developments, review the state-of-the-art and discuss future directions in this field. PMID:21629761

  10. Bridging conventional and molecular genetics of sorghum insect resistance

    USDA-ARS?s Scientific Manuscript database

    Sustainable production of sorghum, Sorghum bicolor (L.) Moench, depends on effective control of insect pests as they continue to compete with humans for the sorghum crop. Insect pests are major constraint in sorghum production, and nearly 150 insect species are serious pests of this crop worldwide,...

  11. Replication of Heliothis virescens ascovirus in insect cell lines.

    PubMed

    Asgari, S

    2006-09-01

    Ascoviruses (AVs) infect larvae of various insect pests belonging to the family Noctuidae. The result of AV infection in the hosts is cleavage of infected cells into vesicles, a unique feature of AV infection. Since insect cell lines facilitate the study of virus life cycles, attempts were made to analyze Heliothis virescens AV (HvAV3e) infection in several cell lines and compare cell pathology to larval infection. In this study, replication and cytopathological effects of HvAV3e on four different cell lines were investigated. HvAV3e replication was confirmed in three noctuid cell lines from Spodoptera frugiperda (Sf9) and Helicoverpa zea (BCIRL-Hz-AM1 and FB33). However, the virus did not replicate in the non-noctuid insect cell line from Pieris rapae (Pieridae). Despite replication of the virus in the three permissive cell lines, the cytopathological effects of the virus were significantly different from that of larval infection.

  12. Reassessing ecdysteroidogenic cells from the cell membrane receptors' perspective.

    PubMed

    Alexandratos, Alexandros; Moulos, Panagiotis; Nellas, Ioannis; Mavridis, Konstantinos; Dedos, Skarlatos G

    2016-02-05

    Ecdysteroids secreted by the prothoracic gland (PG) cells of insects control the developmental timing of their immature life stages. These cells have been historically considered as carrying out a single function in insects, namely the biochemical conversion of cholesterol to ecdysteroids and their secretion. A growing body of evidence shows that PG cells receive multiple cues during insect development so we tested the hypothesis that they carry out more than just one function in insects. We characterised the molecular nature and developmental profiles of cell membrane receptors in PG cells of Bombyx mori during the final larval stage and determined what receptors decode nutritional, developmental and physiological signals. Through iterative approaches we identified a complex repertoire of cell membrane receptors that are expressed in intricate patterns and activate previously unidentified signal transduction cascades in PG cells. The expression patterns of some of these receptors explain precisely the mechanisms that are known to control ecdysteroidogenesis. However, the presence of receptors for the notch, hedgehog and wingless signalling pathways and the expression of innate immunity-related receptors such as phagocytosis receptors, receptors for microbial ligands and Toll-like receptors call for a re-evaluation of the role these cells play in insects.

  13. Novel cell-based odorant sensor elements based on insect odorant receptors.

    PubMed

    Mitsuno, Hidefumi; Sakurai, Takeshi; Namiki, Shigehiro; Mitsuhashi, Hiroyuki; Kanzaki, Ryohei

    2015-03-15

    Development of cell-based odorant sensor elements combined not only high degree of sensitivity and selectivity but also long-term stability is crucial for their practical applications. Here we report the development of a novel cell-based odorant sensor element that sensitively and selectively detects odorants and displays increased fluorescent intensities over a long period of time. Our odorant sensor elements, based on Sf21 cell lines expressing insect odorant receptors, are sensitive to the level of several tens of parts per billion in solution, can selectively distinguish between different types of odorants based on the odorant selectivity intrinsic to the expressed receptors, and have response times of approximately 13s. Specifically, with the use of Sf21 cells and insect odorant receptors, we demonstrated that the established cell lines stably expressing insect odorant receptors are able to detect odorants with consistent responsiveness for at least 2 months, thus exceeding the short life-span normally associated with cell-based sensors. We also demonstrated the development of a compact odorant sensor chip by integrating the established insect cell lines into a microfluidic chip. The methodology we established in this study, in conjunction with the large repertoire of insect odorant receptors, will aid in the development of practical cell-based odorant sensors for various applications, including food administration and health management. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Preparation of ubiquitin-conjugated proteins using an insect cell-free protein synthesis system.

    PubMed

    Suzuki, Takashi; Ezure, Toru; Ando, Eiji; Nishimura, Osamu; Utsumi, Toshihiko; Tsunasawa, Susumu

    2010-01-01

    Ubiquitination is one of the most significant posttranslational modifications (PTMs). To evaluate the ability of an insect cell-free protein synthesis system to carry out ubiquitin (Ub) conjugation to in vitro translated proteins, poly-Ub chain formation was studied in an insect cell-free protein synthesis system. Poly-Ub was generated in the presence of Ub aldehyde (UA), a de-ubiquitinating enzyme inhibitor. In vitro ubiquitination of the p53 tumor suppressor protein was also analyzed, and p53 was poly-ubiquitinated when Ub, UA, and Mdm2, an E3 Ub ligase (E3) for p53, were added to the in vitro reaction mixture. These results suggest that the insect cell-free protein synthesis system contains enzymatic activities capable of carrying out ubiquitination. CBB-detectable ubiquitinated p53 was easily purified from the insect cell-free protein synthesis system, allowing analysis of the Ub-conjugated proteins by mass spectrometry (MS). Lys 305 of p53 was identified as one of the Ub acceptor sites using this strategy. Thus, we conclude that the insect cell-free protein synthesis system is a powerful tool for studying various PTMs of eukaryotic proteins including ubiqutination presented here.

  15. Live imaging of symbiosis: spatiotemporal infection dynamics of a GFP-labelled Burkholderia symbiont in the bean bug Riptortus pedestris

    PubMed Central

    Kikuchi, Yoshitomo; Fukatsu, Takema

    2014-01-01

    Many insects possess endosymbiotic bacteria inside their body, wherein intimate interactions occur between the partners. While recent technological advancements have deepened our understanding of metabolic and evolutionary features of the symbiont genomes, molecular mechanisms underpinning the intimate interactions remain difficult to approach because the insect symbionts are generally uncultivable. The bean bug Riptortus pedestris is associated with the betaproteobacterial Burkholderia symbiont in a posterior region of the midgut, which develops numerous crypts harbouring the symbiont extracellularly. Distinct from other insect symbiotic systems, R. pedestris acquires the Burkholderia symbiont not by vertical transmission but from the environment every generation. By making use of the cultivability and the genetic tractability of the symbiont, we constructed a transgenic Burkholderia strain labelled with green fluorescent protein (GFP), which enabled detailed observation of spatiotemporal dynamics and the colonization process of the symbiont in freshly prepared specimens. The symbiont live imaging revealed that, at the second instar, colonization of the symbiotic midgut M4 region started around 6 h after inoculation (hai). By 24 hai, the symbiont cells appeared in the main tract and also in several crypts of the M4. By 48 hai, most of the crypts were colonized by the symbiont cells. By 72 hai, all the crypts were filled up with the symbiont cells and the symbiont localization pattern continued during the subsequent nymphal development. Quantitative PCR of the symbiont confirmed the infection dynamics quantitatively. These results highlight the stinkbug-Burkholderia gut symbiosis as an unprecedented model for comprehensive understanding of molecular mechanisms underpinning insect symbiosis. PMID:24103110

  16. Role of the phosphatidylinositol-3-kinase/Akt/target of rapamycin pathway during ambidensovirus infection of insect cells.

    PubMed

    Salasc, F; Mutuel, D; Debaisieux, S; Perrin, A; Dupressoir, T; Grenet, A-S Gosselin; Ogliastro, M

    2016-01-01

    The phosphatidylinositol-3-kinase (PI3K)/Akt/target of rapamycin (TOR) signalling pathway controls cell growth and survival, and is targeted by a number of viruses at different phases of their infection cycle to control translation. Whether and how insect viruses interact with this pathway remain poorly addressed. Here, we investigated the role of PI3K/Akt/TOR signalling during lethal infection of insect cells with an insect parvovirus. Using Junonia coenia densovirus (JcDV; lepidopteran ambidensovirus 1) and susceptible insect cells as experimental models, we first described JcDV cytopathology, and showed that viral infection affects cell size, cell proliferation and survival. We deciphered the role of PI3K/Akt/TOR signalling in the course of infection and found that non-structural (NS) protein expression correlates with the inhibition of TOR and the shutdown of cellular synthesis, concomitant with the burst of viral protein expression. Together, these results suggest that NS proteins control the cellular translational machinery to favour the translation of viral mRNAs at the expense of cellular mRNAs. As a consequence of TOR inhibition, cell autophagy is activated. These results highlight new functions for NS proteins in the course of multiplication of an insect parvovirus.

  17. Comparison of two eukaryotic systems for the expression of VP6 protein of rotavirus specie A: transient gene expression in HEK293-T cells and insect cell-baculovirus system.

    PubMed

    da Silva Junior, Haroldo Cid; da Silva E Mouta Junior, Sérgio; de Mendonça, Marcos César Lima; de Souza Pereira, Mirian Claudia; da Rocha Nogueira, Alanderson; de Azevedo, Maria Luiza Borges; Leite, José Paulo Gagliardi; de Moraes, Márcia Terezinha Baroni

    2012-09-01

    The VP6 protein of rotavirus A (RVA) is a target antigen used for diagnostic assays and also for the development of new RVA vaccines. We have compared the expression of VP6 protein in human embryonic kidney (HEK293-T) cells with results obtained using a well-established insect cell-baculovirus system. The recombinant VP6 (rVP6) expressed in HEK293-T cells did not present degradation and also retained the ability to form trimers. In the insect cell-baculovirus system, rVP6 was expressed at higher levels and with protein degradation as well as partial loss of ability to form trimers was observed. Therefore, HEK293-T cells represent a less laborious alternative system than insect cells for expression of rVP6 from human RVA.

  18. Engineering N-Glycosylation Pathway in Insect Cells: Suppression of β-N-Acetylglucosaminidase and Expression of β-1,4-Galactosyltransferase.

    PubMed

    Kim, Yeon Kyu; Cha, Hyung Joon

    2015-01-01

    Most insect cells have a simple N-glycosylation process and consequently paucimannosidic or simple core glycans predominate. It has been proposed that β-N-acetylglucosaminidase (GlcNAcase), a hexosaminidase in the Golgi membrane which removes a terminal N-acetylglucosamine (GlcNAc), might contribute to simple N-glycosylation profile in several insect cells including Drosophila S2. Here, we describe GlcNAcase suppression strategy using RNA interference (RNAi) to avoid the formation of paucimannosidic glycans in insect S2 cells. In addition, we describe coexpression of β(1,4)-galactosyltransferase (GalT) as a strategy to improve N-glycosylation pattern and enable recombinant therapeutic proteins to be produced in S2 cells with more complex N-glycans.

  19. BioComponent Robots

    DTIC Science & Technology

    2014-02-13

    sterile water. Culture medium was prepared with 70% Leibovitz’s L15 medium, 18% Grace’s Insect Medium, 12% fetal bovine serum (FBS), 3.4 mg/mL yeast...SECURITY CLASSIFICATION OF: The project goal was to exploit insect cell culture and tissue engineering approaches to generate biological actuators...utilizing the unique hardiness and longevity of insect cell sources for device applications for robotics. In contrast to mammalian cells and tissues

  20. 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.

  1. Host Range Restriction of Insect-Specific Flaviviruses Occurs at Several Levels of the Viral Life Cycle.

    PubMed

    Junglen, Sandra; Korries, Marvin; Grasse, Wolfgang; Wieseler, Janett; Kopp, Anne; Hermanns, Kyra; León-Juárez, Moises; Drosten, Christian; Kümmerer, Beate Mareike

    2017-01-01

    The genus Flavivirus contains emerging arthropod-borne viruses (arboviruses) infecting vertebrates, as well as insect-specific viruses (ISVs) (i.e., viruses whose host range is restricted to insects). ISVs are evolutionary precursors to arboviruses. Knowledge of the nature of the ISV infection block in vertebrates could identify functions necessary for the expansion of the host range toward vertebrates. Mapping of host restrictions by complementation of ISV and arbovirus genome functions could generate knowledge critical to predicting arbovirus emergence. Here we isolated a novel flavivirus, termed Niénokoué virus (NIEV), from mosquitoes sampled in Côte d'Ivoire. NIEV groups with insect-specific flaviviruses (ISFs) in phylogeny and grows in insect cells but not in vertebrate cells. We generated an infectious NIEV cDNA clone and a NIEV reporter replicon to study growth restrictions of NIEV in comparison to yellow fever virus (YFV), for which the same tools are available. Efficient RNA replication of the NIEV reporter replicon was observed in insect cells but not in vertebrate cells. Initial translation of the input replicon RNA in vertebrate cells was functional, but RNA replication did not occur. Chimeric YFV carrying the envelope proteins of NIEV was recovered via electroporation in C6/36 insect cells but did not infect vertebrate cells, indicating a block at the level of entry. Since the YF/NIEV chimera readily produced infectious particles in insect cells but not in vertebrate cells despite efficient RNA replication, restriction is also determined at the level of assembly/release. Taking the results together, the ability of ISF to infect vertebrates is blocked at several levels, including attachment/entry and RNA replication as well as assembly/release. IMPORTANCE Most viruses of the genus Flavivirus , e.g., YFV and dengue virus, are mosquito borne and transmitted to vertebrates during blood feeding of mosquitoes. Within the last decade, an increasing number of viruses with a host range exclusively restricted to insects in close relationship to the vertebrate-pathogenic flaviviruses were discovered in mosquitoes. To identify barriers that could block the arboviral vertebrate tropism, we set out to identify the steps at which the ISF replication cycle fails in vertebrates. Our studies revealed blocks at several levels, suggesting that flavivirus host range expansion from insects to vertebrates was a complex process that involved overcoming multiple barriers.

  2. Host Range Restriction of Insect-Specific Flaviviruses Occurs at Several Levels of the Viral Life Cycle

    PubMed Central

    Junglen, Sandra; Korries, Marvin; Grasse, Wolfgang; Wieseler, Janett; Kopp, Anne; Hermanns, Kyra; León-Juárez, Moises; Drosten, Christian

    2017-01-01

    ABSTRACT The genus Flavivirus contains emerging arthropod-borne viruses (arboviruses) infecting vertebrates, as well as insect-specific viruses (ISVs) (i.e., viruses whose host range is restricted to insects). ISVs are evolutionary precursors to arboviruses. Knowledge of the nature of the ISV infection block in vertebrates could identify functions necessary for the expansion of the host range toward vertebrates. Mapping of host restrictions by complementation of ISV and arbovirus genome functions could generate knowledge critical to predicting arbovirus emergence. Here we isolated a novel flavivirus, termed Niénokoué virus (NIEV), from mosquitoes sampled in Côte d’Ivoire. NIEV groups with insect-specific flaviviruses (ISFs) in phylogeny and grows in insect cells but not in vertebrate cells. We generated an infectious NIEV cDNA clone and a NIEV reporter replicon to study growth restrictions of NIEV in comparison to yellow fever virus (YFV), for which the same tools are available. Efficient RNA replication of the NIEV reporter replicon was observed in insect cells but not in vertebrate cells. Initial translation of the input replicon RNA in vertebrate cells was functional, but RNA replication did not occur. Chimeric YFV carrying the envelope proteins of NIEV was recovered via electroporation in C6/36 insect cells but did not infect vertebrate cells, indicating a block at the level of entry. Since the YF/NIEV chimera readily produced infectious particles in insect cells but not in vertebrate cells despite efficient RNA replication, restriction is also determined at the level of assembly/release. Taking the results together, the ability of ISF to infect vertebrates is blocked at several levels, including attachment/entry and RNA replication as well as assembly/release. IMPORTANCE Most viruses of the genus Flavivirus, e.g., YFV and dengue virus, are mosquito borne and transmitted to vertebrates during blood feeding of mosquitoes. Within the last decade, an increasing number of viruses with a host range exclusively restricted to insects in close relationship to the vertebrate-pathogenic flaviviruses were discovered in mosquitoes. To identify barriers that could block the arboviral vertebrate tropism, we set out to identify the steps at which the ISF replication cycle fails in vertebrates. Our studies revealed blocks at several levels, suggesting that flavivirus host range expansion from insects to vertebrates was a complex process that involved overcoming multiple barriers. PMID:28101536

  3. Increasing cell–device adherence using cultured insect cells for receptor-based biosensors

    PubMed Central

    Mitsuno, Hidefumi; Sakurai, Takeshi; Okamoto, Yuki; Tixier-Mita, Agnès; Toshiyoshi, Hiroshi; Mita, Yoshio; Kanzaki, Ryohei

    2018-01-01

    Field-effect transistor (FET)-based biosensors have a wide range of applications, and a bio-FET odorant sensor, based on insect (Sf21) cells expressing insect odorant receptors (ORs) with sensitivity and selectivity, has emerged. To fully realize the practical application of bio-FET odorant sensors, knowledge of the cell–device interface for efficient signal transfer, and a reliable and low-cost measurement system using the commercial complementary metal-oxide semiconductor (CMOS) foundry process, will be indispensable. However, the interfaces between Sf21 cells and sensor devices are largely unknown, and electrode materials used in the commercial CMOS foundry process are generally limited to aluminium, which is reportedly toxic to cells. In this study, we investigated Sf21 cell–device interfaces by developing cross-sectional specimens. Calcium imaging of Sf21 cells expressing insect ORs was used to verify the functions of Sf21 cells as odorant sensor elements on the electrode materials. We found that the cell–device interface was approximately 10 nm wide on average, suggesting that the adhesion mechanism of Sf21 cells may differ from that of other cells. These results will help to construct accurate signal detection from expressed insect ORs using FETs. PMID:29657822

  4. Bacterial cell motility of Burkholderia gut symbiont is required to colonize the insect gut.

    PubMed

    Lee, Jun Beom; Byeon, Jin Hee; Jang, Ho Am; Kim, Jiyeun Kate; Yoo, Jin Wook; Kikuchi, Yoshitomo; Lee, Bok Luel

    2015-09-14

    We generated a Burkholderia mutant, which is deficient of an N-acetylmuramyl-l-alanine amidase, AmiC, involved in peptidoglycan degradation. When non-motile ΔamiC mutant Burkholderia cells harboring chain form were orally administered to Riptortus insects, ΔamiC mutant cells were unable to establish symbiotic association. But, ΔamiC mutant complemented with amiC gene restored in vivo symbiotic association. ΔamiC mutant cultured in minimal medium restored their motility with single-celled morphology. When ΔamiC mutant cells harboring single-celled morphology were administered to the host insect, this mutant established normal symbiotic association, suggesting that bacterial motility is essential for the successful symbiosis between host insect and Burkholderia symbiont. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  5. Shifting behaviour: epigenetic reprogramming in eusocial insects.

    PubMed

    Patalano, Solenn; Hore, Timothy A; Reik, Wolf; Sumner, Seirian

    2012-06-01

    Epigenetic modifications are ancient and widely utilised mechanisms that have been recruited across fungi, plants and animals for diverse but fundamental biological functions, such as cell differentiation. Recently, a functional DNA methylation system was identified in the honeybee, where it appears to underlie queen and worker caste differentiation. This discovery, along with other insights into the epigenetics of social insects, allows provocative analogies to be drawn between insect caste differentiation and cellular differentiation, particularly in mammals. Developing larvae in social insect colonies are totipotent: they retain the ability to specialise as queens or workers, in a similar way to the totipotent cells of early embryos before they differentiate into specific cell lineages. Further, both differentiating cells and insect castes lose phenotypic plasticity by committing to their lineage, losing the ability to be readily reprogrammed. Hence, a comparison of the epigenetic mechanisms underlying lineage differentiation (and reprogramming) between cells and social insects is worthwhile. Here we develop a conceptual model of how loss and regain of phenotypic plasticity might be conserved for individual specialisation in both cells and societies. This framework forges a novel link between two fields of biological research, providing predictions for a unified approach to understanding the molecular mechanisms underlying biological complexity. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. Natural aquatic insect carriers of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV).

    PubMed

    Sudhakaran, R; Haribabu, P; Kumar, S Rajesh; Sarathi, M; Ahmed, V P Ishaq; Babu, V Sarath; Venkatesan, C; Hameedl, A S Sahul

    2008-04-01

    Five different species of aquatic insects were collected from nursery ponds containing the freshwater prawn Macrobrachium rosenbergii infected with Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV). The insects were screened as potential natural carriers of MrNV and XSV. RT-PCR (reverse transcription polymerase chain reaction) analysis gave positive results for MrNV and XSV in Belostoma sp., Aesohna sp., Cybister sp. and Notonecta sp., and negative results for Nepa sp. An Aedes albopictus mosquito cell line (C6/36) was used for infectivity assays, with viral inoculum prepared from the aquatic insects, since C6/36 cells have recently been shown to be susceptible to infection with MrNV and XSV. The C6/36 cells were harvested 4 d post-challenge for examination by electron microscopy. This revealed aggregation of viral particles throughout the cytoplasm for cells challenged with inocula from all the insect species except Nepa sp. Our results indicate that several aquatic insect species may present a risk for MrNV and XSV transmission to M. rosenbergii.

  7. Utility of temporally distinct baculovirus promoters for constitutive and baculovirus-inducible transgene expression in transformed insect cells.

    PubMed

    Lin, Chi-Hung; Jarvis, Donald L

    2013-05-10

    Genetically transformed lepidopteran insect cell lines have biotechnological applications as constitutive recombinant protein production platforms and improved hosts for baculovirus-mediated recombinant protein production. Insect cell transformation is often accomplished with a DNA construct(s) encoding a foreign protein(s) under the transcriptional control of a baculovirus immediate early promoter, such as the ie1 promoter. However, the potential utility of increasingly stronger promoters from later baculovirus gene classes, such as delayed early (39K), late (p6.9), and very late (polh), has not been systematically assessed. Hence, we produced DNA constructs encoding secreted alkaline phosphatase (SEAP) under the transcriptional control of each of the four temporally distinct classes of baculovirus promoters, used them to transform insect cells, and compared the levels of SEAP RNA and protein production obtained before and after baculovirus infection. The ie1 construct was the only one that supported SEAP protein production by transformed insect cells prior to baculovirus infection, confirming that only immediate early promoters can be used to isolate transformed insect cells for constitutive recombinant protein production. However, baculovirus infection activated transgene expression by all four classes of baculovirus promoters. After infection, cells transformed with the very late (polh) and late (p6.9) promoter constructs produced the highest levels of SEAP RNA, but only low levels of SEAP protein. Conversely, cells transformed with the immediate early (ie1) and delayed early (39K) promoter constructs produced lower levels of RNA, but equal or higher levels of SEAP protein. Unexpectedly, the 39K promoter construct provided tightly regulated, baculovirus-inducible protein production at higher levels than the later promoter constructs. Thus, this study demonstrated the utility of the 39K promoter for insect cell engineering, particularly when one requires higher levels of effector protein production than obtained with ie1 and/or when constitutive transgene expression adversely impacts host cell fitness and/or genetic stability. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. Anaphylaxis and insect allergy.

    PubMed

    Demain, Jeffrey G; Minaei, Ashley A; Tracy, James M

    2010-08-01

    Anaphylaxis is an acute-onset and potentially life-threatening allergic reaction that can be caused by numerous allergic triggers including stinging insects. This review focuses on recent advances, natural history, risk factors and therapeutic considerations. Recent work suggests that concerns over insect allergy diagnosis continue to exist. This is especially true with individuals who have a convincing history of a serious life-threatening anaphylactic event, but lack the necessary diagnostic criteria of venom-specific IgE by skin test or in-vitro diagnostic methods to confirm the diagnosis. The role of occult mastocytosis or increased basophile reactivity may play a role in this subset population. Additionally, epinephrine continues to be underutilized as the primary acute intervention for an anaphylactic reaction in the emergent setting. The incidence of anaphylaxis continues to rise across all demographic groups, especially those less than 20 years of age. Fortunately, the fatalities related to anaphylaxis appear to have decreased over the past decades. Our understanding of various triggers, associated risk factors, as well as an improved understanding and utilization of biological markers such as serum tryptase have improved. Our ability to treat insect anaphylaxis by venom immunotherapy is highly effective. Unfortunately, anaphylaxis continues to be underappreciated and undertreated especially in regard to insect sting anaphylaxis. This includes the appropriate use of injectable epinephrine as the primary acute management tool. These findings suggest that continued education of the general population, primary care healthcare providers and emergency departments is required.

  9. Characterization of Nonjunctional Hemichannels in Caterpillar Cells

    PubMed Central

    Luo, Kaijun; Turnbull, Matthew W.

    2011-01-01

    Recent studies have demonstrated that hemichannels, which form gap junctions when paired from apposing cells, may serve additional roles when unpaired including cell adhesion and paracrine communication. Hemichannels in mammals are formed by connexins or pannexins, while in insects they are formed by pannexin homologues termed innexins. The formation of functional gap junctions by insect innexins has been established, although their ability to form functional nonjunctional hemichannels has not been reported. Here the characteristics of nonjunctional hemichannels were examined in three lepidopteran cell types, two cell lines (High Five and Sf9) and explanted hemocytes from Heliothis virescens (Fabricius) (Lepidoptera: Noctuidae). Selective fluorescent dye uptake by hemichannels was observed in a significant minority of cells, using fluorescence microscopy and flow cytometry. Carbenoxelone, an inhibitor of mammalian junctions, disrupted dye uptake, while flufenamic acid and mefloquine did not. The presence of Ca2+ and Mg2+ in the media increased hemichannel activity. Additionally, lipopolysaccharide, a stimulator of immune activity in lepidopterans, decreased dye uptake. These results demonstrate for the first time the activity of nonjunctional hemichannels in insect cells, as well as pharmacological tools to manipulate them. These results will facilitate the further examination of the role of innexins and nonjunctional hemichannels in insect cell biology, including paracrine signaling, and comparative studies of mammalian pannexins and insect innexins. PMID:21521140

  10. Impact of a human CMP-sialic acid transporter on recombinant glycoprotein sialylation in glycoengineered insect cells.

    PubMed

    Mabashi-Asazuma, Hideaki; Shi, Xianzong; Geisler, Christoph; Kuo, Chu-Wei; Khoo, Kay-Hooi; Jarvis, Donald L

    2013-02-01

    Insect cells are widely used for recombinant glycoprotein production, but they cannot provide the glycosylation patterns required for some biotechnological applications. This problem has been addressed by genetically engineering insect cells to express mammalian genes encoding various glycoprotein glycan processing functions. However, for various reasons, the impact of a mammalian cytosine-5'-monophospho (CMP)-sialic acid transporter has not yet been examined. Thus, we transformed Spodoptera frugiperda (Sf9) cells with six mammalian genes to generate a new cell line, SfSWT-4, that can produce sialylated glycoproteins when cultured with the sialic acid precursor, N-acetylmannosamine. We then super-transformed SfSWT-4 with a human CMP-sialic acid transporter (hCSAT) gene to isolate a daughter cell line, SfSWT-6, which expressed the hCSAT gene in addition to the other mammalian glycogenes. SfSWT-6 cells had higher levels of cell surface sialylation and also supported higher levels of recombinant glycoprotein sialylation, particularly when cultured with low concentrations of N-acetylmannosamine. Thus, hCSAT expression has an impact on glycoprotein sialylation, can reduce the cost of recombinant glycoprotein production and therefore should be included in ongoing efforts to glycoengineer the baculovirus-insect cell system. The results of this study also contributed new insights into the endogenous mechanism and potential mechanisms of CMP-sialic acid accumulation in the Golgi apparatus of lepidopteran insect cells.

  11. Glycobiotechnology of the Insect Cell-Baculovirus Expression System Technology.

    PubMed

    Palomares, Laura A; Srivastava, Indresh K; Ramírez, Octavio T; Cox, Manon M J

    2018-06-10

    The insect cell-baculovirus expression system technology (BEST) has a prominent role in producing recombinant proteins to be used as research and diagnostic reagents and vaccines. The glycosylation profile of proteins produced by the BEST is composed predominantly of terminal mannose glycans, and, in Trichoplusia ni cell lines, core α3 fucosylation, a profile different to that in mammals. Insects contain all the enzymatic activities needed for complex N- and O-glycosylation and sialylation, although few reports of complex glycosylation and sialylation by the BEST exist. The insect cell line and culture conditions determine the glycosylation profile of proteins produced by the BEST. The promoter used, dissolved oxygen tension, presence of sugar precursors, bovine serum or hemolymph, temperature, and the time of harvest all influence glycosylation, although more research is needed. The lack of activity of glycosylation enzymes possibly results from the transcription regulation and stress imposed by baculovirus infection. To solve this limitation, the glycosylation pathway of insect cells has been engineered to produce complex sialylated glycans and to eliminate α3 fucosylation, either by generating transgenic cell lines or by using baculovirus vectors. These strategies have been successful. Complex glycosylation, sialylation, and inhibition of α3 fucosylation have been achieved, although the majority of glycans still have terminal mannose residues. The implication of insect glycosylation in the proteins produced by the BEST is discussed. Graphical Abstract.

  12. Genomic Analysis and Isolation of RNA Polymerase II Dependent Promoters from Spodoptera frugiperda.

    PubMed

    Bleckmann, Maren; Fritz, Markus H-Y; Bhuju, Sabin; Jarek, Michael; Schürig, Margitta; Geffers, Robert; Benes, Vladimir; Besir, Hüseyin; van den Heuvel, Joop

    2015-01-01

    The Baculoviral Expression Vector System (BEVS) is the most commonly used method for high expression of recombinant protein in insect cells. Nevertheless, expression of some target proteins--especially those entering the secretory pathway--provides a severe challenge for the baculovirus infected insect cells, due to the reorganisation of intracellular compounds upon viral infection. Therefore, alternative strategies for recombinant protein production in insect cells like transient plasmid-based expression or stable expression cell lines are becoming more popular. However, the major bottleneck of these systems is the lack of strong endogenous polymerase II dependent promoters, as the strong baculoviral p10 and polH promoters used in BEVS are only functional in presence of the viral transcription machinery during the late phase of infection. In this work we present a draft genome and a transcriptome analysis of Sf21 cells for the identification of the first known endogenous Spodoptera frugiperda promoters. Therefore, putative promoter sequences were identified and selected because of high mRNA level or in analogy to other strong promoters in other eukaryotic organism. The chosen endogenous Sf21 promoters were compared to early viral promoters for their efficiency to trigger eGFP expression using transient plasmid based transfection in a BioLector Microfermentation system. Furthermore, promoter activity was not only shown in Sf21 cells but also in Hi5 cells. The novel endogenous Sf21 promoters were ranked according to their activity and expand the small pool of available promoters for stable insect cell line development and transient plasmid expression in insect cells. The best promoter was used to improve plasmid based transient transfection in insect cells substantially.

  13. Analysis and functional annotation of expressed sequence tags from the fall armyworm Spodoptera frugiperda

    PubMed Central

    Deng, Youping; Dong, Yinghua; Thodima, Venkata; Clem, Rollie J; Passarelli, A Lorena

    2006-01-01

    Background Little is known about the genome sequences of lepidopteran insects, although this group of insects has been studied extensively in the fields of endocrinology, development, immunity, and pathogen-host interactions. In addition, cell lines derived from Spodoptera frugiperda and other lepidopteran insects are routinely used for baculovirus foreign gene expression. This study reports the results of an expressed sequence tag (EST) sequencing project in cells from the lepidopteran insect S. frugiperda, the fall armyworm. Results We have constructed an EST database using two cDNA libraries from the S. frugiperda-derived cell line, SF-21. The database consists of 2,367 ESTs which were assembled into 244 contigs and 951 singlets for a total of 1,195 unique sequences. Conclusion S. frugiperda is an agriculturally important pest insect and genomic information will be instrumental for establishing initial transcriptional profiling and gene function studies, and for obtaining information about genes manipulated during infections by insect pathogens such as baculoviruses. PMID:17052344

  14. The silencing suppressor (NSs) protein of the plant virus Tomato spotted wilt virus enhances heterologous protein expression and baculovirus pathogenicity in cells and lepidopteran insects.

    PubMed

    de Oliveira, Virgínia Carla; da Silva Morgado, Fabricio; Ardisson-Araújo, Daniel Mendes Pereira; Resende, Renato Oliveira; Ribeiro, Bergmann Morais

    2015-11-01

    In this work, we showed that cell death induced by a recombinant (vAcNSs) Autographa californica multiple nucleopolyhedrovirus (AcMNPV) expressing the silencing suppressor (NSs) protein of Tomato spotted wilt virus (TSWV) was enhanced on permissive and semipermissive cell lines. The expression of a heterologous gene (firefly luciferase) during co-infection of insect cells with vAcNSs and a second recombinant baculovirus (vAgppolhfluc) was shown to increase when compared to single vAgppolhfluc infections. Furthermore, the vAcNSs mean time-to-death values were significantly lower than those for wild-type AcMNPV on larvae of Spodoptera frugiperda and Anticarsia gemmatalis. These results showed that the TSWV-NSs protein could efficiently increase heterologous protein expression in insect cells as well as baculovirus pathogenicity and virulence, probably by suppressing the gene-silencing machinery in insects.

  15. Characterization of an Sf-rhabdovirus-negative Spodoptera frugiperda cell line as an alternative host for recombinant protein production in the baculovirus-insect cell system.

    PubMed

    Maghodia, Ajay B; Geisler, Christoph; Jarvis, Donald L

    2016-06-01

    Cell lines derived from the fall armyworm, Spodoptera frugiperda (Sf), are widely used as hosts for recombinant protein production in the baculovirus-insect cell system (BICS). However, it was recently discovered that these cell lines are contaminated with a virus, now known as Sf-rhabdovirus [1]. The detection of this adventitious agent raised a potential safety issue that could adversely impact the BICS as a commercial recombinant protein production platform. Thus, we examined the properties of Sf-RVN, an Sf-rhabdovirus-negative Sf cell line, as a potential alternative host. Nested RT-PCR assays showed Sf-RVN cells had no detectable Sf-rhabdovirus over the course of 60 passages in continuous culture. The general properties of Sf-RVN cells, including their average growth rates, diameters, morphologies, and viabilities after baculovirus infection, were virtually identical to those of Sf9 cells. Baculovirus-infected Sf-RVN and Sf9 cells produced equivalent levels of three recombinant proteins, including an intracellular prokaryotic protein and two secreted eukaryotic glycoproteins, and provided similar N-glycosylation patterns. In fact, except for the absence of Sf-rhabdovirus, the only difference between Sf-RVN and Sf9 cells was SF-RVN produced higher levels of infectious baculovirus progeny. These results show Sf-RVN cells can be used as improved, alternative hosts to circumvent the potential safety hazard associated with the use of Sf-rhabdovirus-contaminated Sf cells for recombinant protein manufacturing with the BICS. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Establishment of a highly efficient virus-inducible CRISPR/Cas9 system in insect cells.

    PubMed

    Dong, Zhan-Qi; Chen, Ting-Ting; Zhang, Jun; Hu, Nan; Cao, Ming-Ya; Dong, Fei-Fan; Jiang, Ya-Ming; Chen, Peng; Lu, Cheng; Pan, Min-Hui

    2016-06-01

    Although current antiviral strategies can inhibit baculovirus infection and decrease viral DNA replication to a certain extent, novel tools are required for specific and accurate elimination of baculovirus genomes from infected insects. Using the newly developed clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease (CRISPR/Cas9) technology, we disrupted a viral genome in infected insect cells in vitro as a defense against viral infection. We optimized the CRISPR/Cas9 system to edit foreign and viral genome in insect cells. Using Bombyx mori nucleopolyhedrovirus (BmNPV) as a model, we found that the CRISPR/Cas9 system was capable of cleaving the replication key factor ie-1 in BmNPV thus effectively inhibiting virus proliferation. Furthermore, we constructed a virus-inducible CRISPR/Cas9 editing system, which minimized the probability of off-target effects and was rapidly activated after viral infection. This is the first report describing the application of the CRISPR/Cas9 system in insect antiviral research. Establishment of a highly efficient virus-inducible CRISPR/Cas9 system in insect cells provides insights to produce virus-resistant transgenic strains for future. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Prostaglandin Actions in Established Insect Cell Lines

    USDA-ARS?s Scientific Manuscript database

    Prostaglandins (PGs) are oxygenated metabolites of arachidonic acid (AA) and two other C20 polyunsaturated fatty acids that serve as biochemical signals that mediate a wide range of physiological functions in animal cells. For example, PGs influence protein expression in establish insect cell lines ...

  18. Transforming Lepidopteran Insect Cells for Improved Protein Processing and Expression

    USDA-ARS?s Scientific Manuscript database

    The lepidopteran insect cells used with the baculovirus expression vector system (BEVS) are capable of synthesizing and accurately processing foreign proteins. However, proteins expressed in baculovirus-infected cells often fail to be completely processed, or are not processed in a manner that meet...

  19. Ebola virus-like particles produced in insect cells exhibit dendritic cell stimulating activity and induce neutralizing antibodies

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

    Ye Ling; Lin Jianguo; Sun Yuliang

    2006-08-01

    Recombinant baculoviruses (rBV) expressing Ebola virus VP40 (rBV-VP40) or GP (rBV-GP) proteins were generated. Infection of Sf9 insect cells by rBV-VP40 led to assembly and budding of filamentous particles from the cell surface as shown by electron microscopy. Ebola virus-like particles (VLPs) were produced by coinfection of Sf9 cells with rBV-VP40 and rBV-GP, and incorporation of Ebola GP into VLPs was demonstrated by SDS-PAGE and Western blot analysis. Recombinant baculovirus infection of insect cells yielded high levels of VLPs, which were shown to stimulate cytokine secretion from human dendritic cells similar to VLPs produced in mammalian cells. The immunogenicity ofmore » Ebola VLPs produced in insect cells was evaluated by immunization of mice. Analysis of antibody responses showed that most of the GP-specific antibodies were of the IgG2a subtype, while no significant level of IgG1 subtype antibodies specific for GP was induced, indicating the induction of a Th1-biased immune response. Furthermore, sera from Ebola VLP immunized mice were able to block infection by Ebola GP pseudotyped HIV virus in a single round infection assay, indicating that a neutralizing antibody against the Ebola GP protein was induced. These results show that production of Ebola VLPs in insect cells using recombinant baculoviruses represents a promising approach for vaccine development against Ebola virus infection.« less

  20. A New theraphosid Spider Toxin Causes Early Insect Cell Death by Necrosis When Expressed In Vitro during Recombinant Baculovirus Infection

    PubMed Central

    Ardisson-Araújo, Daniel Mendes Pereira; Morgado, Fabrício Da Silva; Schwartz, Elisabeth Ferroni; Corzo, Gerardo; Ribeiro, Bergmann Morais

    2013-01-01

    Baculoviruses are the most studied insect viruses in the world and are used for biological control of agricultural and forest insect pests. They are also used as versatile vectors for expression of heterologous proteins. One of the major problems of their use as biopesticides is their slow speed to kill insects. Thus, to address this shortcoming, insect-specific neurotoxins from arachnids have been introduced into the baculovirus genome solely aiming to improve its virulence. In this work, an insecticide-like toxin gene was obtained from a cDNA derived from the venom glands of the theraphosid spider Brachypelma albiceps. The mature form of the peptide toxin (called Ba3) has a high content of basic amino acid residues, potential for three possible disulfide bonds, and a predicted three-stranded β-sheetDifferent constructions of the gene were engineered for recombinant baculovirus Autographa californica multiple nuclepolyhedrovirus (AcMNPV) expression. Five different forms of Ba3 were assessed; (1) the full-length sequence, (2) the pro-peptide and mature region, (3) only the mature region, and the mature region fused to an (4) insect or a (5) virus-derived signal peptide were inserted separately into the genome of the baculovirus. All the recombinant viruses induced cell death by necrosis earlier in infection relative to a control virus lacking the toxin gene. However, the recombinant virus containing the mature portion of the toxin gene induced a faster cell death than the other recombinants. We found that the toxin construct with the signal peptide and/or pro-peptide regions delayed the necrosis phenotype. When infected cells were subjected to ultrastructural analysis, the cells showed loss of plasma membrane integrity and structural changes in mitochondria before death. Our results suggest this use of baculovirus is a potential tool to help understand or to identify the effect of insect-specific toxic peptides when produced during infection of insect cells. PMID:24349574

  1. Intracellular localization of adeno-associated viral proteins expressed in insect cells.

    PubMed

    Gallo-Ramírez, Lilí E; Ramírez, Octavio T; Palomares, Laura A

    2011-01-01

    Production of vectors derived from adeno-associated virus (AAVv) in insect cells represents a feasible option for large-scale applications. However, transducing particles yields obtained in this system are low compared with total capsid yields, suggesting the presence of genome encapsidation bottlenecks. Three components are required for AAVv production: viral capsid proteins (VP), the recombinant AAV genome, and Rep proteins for AAV genome replication and encapsidation. Little is known about the interaction between the three components in insect cells, which have intracellular conditions different to those in mammalian cells. In this work, the localization of AAV proteins in insect cells was assessed for the first time with the purpose of finding potential limiting factors. Unassembled VP were located either in the cytoplasm or in the nucleus. Their transport into the nucleus was dependent on protein concentration. Empty capsids were located in defined subnuclear compartments. Rep proteins expressed individually were efficiently translocated into the nucleus. Their intranuclear distribution was not uniform and differed from VP distribution. While Rep52 distribution and expression levels were not affected by AAV genomes or VP, Rep78 distribution and stability changed during coexpression. Expression of all AAV components modified capsid intranuclear distribution, and assembled VP were found in vesicles located in the nuclear periphery. Such vesicles were related to baculovirus infection, highlighting its role in AAVv production in insect cells. The results obtained in this work suggest that the intracellular distribution of AAV proteins allows their interaction and does not limit vector production in insect cells. Copyright © 2011 American Institute of Chemical Engineers (AIChE).

  2. Interference of a synthetic C18 juvenile hormone with mammalian cells in vitro, I. Effects on growth and morphology.

    PubMed

    Zielińska, Z M; Laskowska-Bozek, H; Jastreboff, P

    1978-01-01

    Some of structural and functional analogs of juvenile hormones are now under field examinations as growth inhibitors of some pest-insect populations. So far however very little is known about the possible interference of these compounds with mammalian cells or organisms. In this research the interference of a synthetic preparation of the insect C18 juvenile hormone with mouse embryo fibroblasts (ME-cells) and mouse cells of an established line (L-cells) was studied. Aliquots of juvenile hormone solution or those of the solvent (DMSO plus ethanol, 9:1) were included into the culture medium and after defined times of contact the cells were tested for their morphology, pattern of growth, proliferation rate and viability. The data for the parameters under examination were evaluated by means of the analysis of variance and checked by the Tuckey test. The sensitivity of ME-cells and L-cells to the agent tested was compared by means of the analysis of variance of the data for mitotic indices of these cells and by evaluation of the number of dead cells in cultures under the particular conditions of the experiments. The main findings can be summarized as follows: 1. Cells of both types are evidently more sensitive to juvenile hormone than to the solvent. 2. ME-cells are more sensitive to both agents than are L-cells. 3. The concentrations of the hormone in the medium required to evoked the cytocidal effect on the mouse cells similarly as those affecting some insect non-target cells were far above concentrations found in insect blood, but they were of the same order of magnitude as those used in physiological experiments with insect organs in vitro.

  3. Modeling emerald ash borer spread in Ohio and Michigan

    Treesearch

    Anantha Prasad; Louis Iverson; Matthew Peters; Jonathan Bossenbroek; Davis Sydnor; Mark Schwartz

    2008-01-01

    Our group has been modelling the spread of emerald ash borer (EAB) in Ohio using a spatially explicit cell-based model that takes into account the insect's flight characteristics (Insect Flight Model) as well as external factors that enable the insects to travel passively (Insect Ride Model).

  4. Manipulation of host plant cells and tissues by gall-inducing insects and adaptive strategies used by different feeding guilds.

    PubMed

    Oliveira, D C; Isaias, R M S; Fernandes, G W; Ferreira, B G; Carneiro, R G S; Fuzaro, L

    2016-01-01

    Biologists who study insect-induced plant galls are faced with the overwhelming diversity of plant forms and insect species. A challenge is to find common themes amidst this diversity. We discuss common themes that have emerged from our cytological and histochemical studies of diverse neotropical insect-induced galls. Gall initiation begins with recognition of reactive plant tissues by gall inducers, with subsequent feeding and/or oviposition triggering a cascade of events. Besides, to induce the gall structure insects have to synchronize their life cycle with plant host phenology. We predict that reactive oxygen species (ROS) play a role in gall induction, development and histochemical gradient formation. Controlled levels of ROS mediate the accumulation of (poly)phenols, and phytohormones (such as auxin) at gall sites, which contributes to the new cell developmental pathways and biochemical alterations that lead to gall formation. The classical idea of an insect-induced gall is a chamber lined with a nutritive tissue that is occupied by an insect that directly harvests nutrients from nutritive cells via its mouthparts, which function mechanically and/or as a delivery system for salivary secretions. By studying diverse gall-inducing insects we have discovered that insects with needle-like sucking mouthparts may also induce a nutritive tissue, whose nutrients are indirectly harvested as the gall-inducing insects feeds on adjacent vascular tissues. Activity of carbohydrate-related enzymes across diverse galls corroborates this hypothesis. Our research points to the importance of cytological and histochemical studies for elucidating mechanisms of induced susceptibility and induced resistance. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Rift valley fever virus infection of human cells and insect hosts is promoted by protein kinase C epsilon.

    PubMed

    Filone, Claire Marie; Hanna, Sheri L; Caino, M Cecilia; Bambina, Shelly; Doms, Robert W; Cherry, Sara

    2010-11-24

    As an arthropod-borne human pathogen, Rift Valley fever virus (RVFV) cycles between an insect vector and mammalian hosts. Little is known about the cellular requirements for infection in either host. Here we developed a tissue culture model for RVFV infection of human and insect cells that is amenable to high-throughput screening. Using this approach we screened a library of 1280 small molecules with pharmacologically defined activities and identified 59 drugs that inhibited RVFV infection with 15 inhibiting RVFV replication in both human and insect cells. Amongst the 15 inhibitors that blocked infection in both hosts was a subset that inhibits protein kinase C. Further studies found that infection is dependent upon the novel protein kinase C isozyme epsilon (PKCε) in both human and insect cells as well as in adult flies. Altogether, these data show that inhibition of cellular factors required for early steps in the infection cycle including PKCε can block RVFV infection, and may represent a starting point for the development of anti-RVFV therapeutics.

  6. Investigating Engineered Ribonucleoprotein Particles to Improve Oral RNAi Delivery in Crop Insect Pests

    PubMed Central

    Gillet, François-Xavier; Garcia, Rayssa A.; Macedo, Leonardo L. P.; Albuquerque, Erika V. S.; Silva, Maria C. M.; Grossi-de-Sa, Maria F.

    2017-01-01

    Genetically modified (GM) crops producing double-stranded RNAs (dsRNAs) are being investigated largely as an RNA interference (RNAi)-based resistance strategy against crop insect pests. However, limitations of this strategy include the sensitivity of dsRNA to insect gut nucleases and its poor insect cell membrane penetration. Working with the insect pest cotton boll weevil (Anthonomus grandis), we showed that the chimeric protein PTD-DRBD (peptide transduction domain—dsRNA binding domain) combined with dsRNA forms a ribonucleoprotein particle (RNP) that improves the effectiveness of the RNAi mechanism in the insect. The RNP slows down nuclease activity, probably by masking the dsRNA. Furthermore, PTD-mediated internalization in insect gut cells is achieved within minutes after plasma membrane contact, limiting the exposure time of the RNPs to gut nucleases. Therefore, the RNP provides an approximately 2-fold increase in the efficiency of insect gene silencing upon oral delivery when compared to naked dsRNA. Taken together, these data demonstrate the role of engineered RNPs in improving dsRNA stability and cellular entry, representing a path toward the design of enhanced RNAi strategies in GM plants against crop insect pests. PMID:28503153

  7. Investigating Engineered Ribonucleoprotein Particles to Improve Oral RNAi Delivery in Crop Insect Pests.

    PubMed

    Gillet, François-Xavier; Garcia, Rayssa A; Macedo, Leonardo L P; Albuquerque, Erika V S; Silva, Maria C M; Grossi-de-Sa, Maria F

    2017-01-01

    Genetically modified (GM) crops producing double-stranded RNAs (dsRNAs) are being investigated largely as an RNA interference (RNAi)-based resistance strategy against crop insect pests. However, limitations of this strategy include the sensitivity of dsRNA to insect gut nucleases and its poor insect cell membrane penetration. Working with the insect pest cotton boll weevil ( Anthonomus grandis ), we showed that the chimeric protein PTD-DRBD (peptide transduction domain-dsRNA binding domain) combined with dsRNA forms a ribonucleoprotein particle (RNP) that improves the effectiveness of the RNAi mechanism in the insect. The RNP slows down nuclease activity, probably by masking the dsRNA. Furthermore, PTD-mediated internalization in insect gut cells is achieved within minutes after plasma membrane contact, limiting the exposure time of the RNPs to gut nucleases. Therefore, the RNP provides an approximately 2-fold increase in the efficiency of insect gene silencing upon oral delivery when compared to naked dsRNA. Taken together, these data demonstrate the role of engineered RNPs in improving dsRNA stability and cellular entry, representing a path toward the design of enhanced RNAi strategies in GM plants against crop insect pests.

  8. Insect Pollinated Crops, Insect Pollinators and US Agriculture: Trend Analysis of Aggregate Data for the Period 1992–2009

    PubMed Central

    Calderone, Nicholas W.

    2012-01-01

    In the US, the cultivated area (hectares) and production (tonnes) of crops that require or benefit from insect pollination (directly dependent crops: apples, almonds, blueberries, cucurbits, etc.) increased from 1992, the first year in this study, through 1999 and continued near those levels through 2009; aggregate yield (tonnes/hectare) remained unchanged. The value of directly dependent crops attributed to all insect pollination (2009 USD) decreased from $14.29 billion in 1996, the first year for value data in this study, to $10.69 billion in 2001, but increased thereafter, reaching $15.12 billion by 2009. The values attributed to honey bees and non-Apis pollinators followed similar patterns, reaching $11.68 billion and $3.44 billion, respectively, by 2009. The cultivated area of crops grown from seeds resulting from insect pollination (indirectly dependent crops: legume hays, carrots, onions, etc.) was stable from 1992 through 1999, but has since declined. Production of those crops also declined, albeit not as rapidly as the decline in cultivated area; this asymmetry was due to increases in aggregate yield. The value of indirectly dependent crops attributed to insect pollination declined from $15.45 billion in 1996 to $12.00 billion in 2004, but has since trended upward. The value of indirectly dependent crops attributed to honey bees and non-Apis pollinators, exclusive of alfalfa leafcutter bees, has declined since 1996 to $5.39 billion and $1.15 billion, respectively in 2009. The value of alfalfa hay attributed to alfalfa leafcutter bees ranged between $4.99 and $7.04 billion. Trend analysis demonstrates that US producers have a continued and significant need for insect pollinators and that a diminution in managed or wild pollinator populations could seriously threaten the continued production of insect pollinated crops and crops grown from seeds resulting from insect pollination. PMID:22629374

  9. Soluble forms of the cell adhesion molecule L1 produced by insect and baculovirus-transduced mammalian cells enhance Schwann cell motility.

    PubMed

    Lavdas, Alexandros A; Efrose, Rodica; Douris, Vassilis; Gaitanou, Maria; Papastefanaki, Florentia; Swevers, Luc; Thomaidou, Dimitra; Iatrou, Kostas; Matsas, Rebecca

    2010-12-01

    For biotechnological applications, insect cell lines are primarily known as hosts for the baculovirus expression system that is capable to direct synthesis of high levels of recombinant proteins through use of powerful viral promoters. Here, we demonstrate the implementation of two alternative approaches based on the baculovirus system for production of a mammalian recombinant glycoprotein, comprising the extracellular part of the cell adhesion molecule L1, with potential important therapeutic applications in nervous system repair. In the first approach, the extracellular part of L1 bearing a myc tag is produced in permanently transformed insect cell lines and purified by affinity chromatography. In the second approach, recombinant baculoviruses that express L1-Fc chimeric protein, derived from fusion of the extracellular part of L1 with the Fc part of human IgG1, under the control of a mammalian promoter are used to infect mammalian HEK293 and primary Schwann cells. Both the extracellular part of L1 bearing a myc tag accumulating in the supernatants of insect cultures as well as L1-Fc secreted by transduced HEK293 or Schwann cells are capable of increasing the motility of Schwann cells with similar efficiency in a gap bridging bioassay. In addition, baculovirus-transduced Schwann cells show enhanced motility when grafted on organotypic cultures of neonatal brain slices while they retain their ability to myelinate CNS axons. This proof-of-concept that the migratory properties of myelin-forming cells can be modulated by recombinant protein produced in insect culture as well as by means of baculovirus-mediated adhesion molecule expression in mammalian cells may have beneficial applications in the field of CNS therapies. ©2010 The Authors. Journal of Neurochemistry © 2010 International Society for Neurochemistry.

  10. Damage signals in the insect immune response

    PubMed Central

    Krautz, Robert; Arefin, Badrul; Theopold, Ulrich

    2014-01-01

    Insects and mammals share an ancient innate immune system comprising both humoral and cellular responses. The insect immune system consists of the fat body, which secretes effector molecules into the hemolymph and several classes of hemocytes, which reside in the hemolymph and of protective border epithelia. Key features of wound- and immune responses are shared between insect and mammalian immune systems including the mode of activation by commonly shared microbial (non-self) patterns and the recognition of these patterns by dedicated receptors. It is unclear how metazoan parasites in insects, which lack these shared motifs, are recognized. Research in recent years has demonstrated that during entry into the insect host, many eukaryotic pathogens leave traces that alert potential hosts of the damage they have afflicted. In accordance with terminology used in the mammalian immune systems, these signals have been dubbed danger- or damage-associated signals. Damage signals are necessary byproducts generated during entering hosts either by mechanical or proteolytic damage. Here, we briefly review the current stage of knowledge on how wound closure and wound healing during mechanical damage is regulated and how damage-related signals contribute to these processes. We also discuss how sensors of proteolytic activity induce insect innate immune responses. Strikingly damage-associated signals are also released from cells that have aberrant growth, including tumor cells. These signals may induce apoptosis in the damaged cells, the recruitment of immune cells to the aberrant tissue and even activate humoral responses. Thus, this ensures the removal of aberrant cells and compensatory proliferation to replace lost tissue. Several of these pathways may have been co-opted from wound healing and developmental processes. PMID:25071815

  11. A baculovirus (Bombyx mori nuclear polyhedrosis virus) repeat element functions as a powerful constitutive enhancer in transfected insect cells.

    PubMed

    Lu, M; Farrell, P J; Johnson, R; Iatrou, K

    1997-12-05

    It has been previously reported that baculovirus homologous regions, the regions of baculovirus genomes that contain the origins of DNA replication, can augment the expression of a small number of baculovirus genes in vitro. We are now reporting that a region of the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) containing the homologous region 3 (HR3) acts as an enhancer for the promoter of a nonviral gene, the cytoplasmic actin gene of the silkmoth B. mori. Incorporation of the HR3 sequences of BmNPV into an actin promoter-based expression cassette results in an augmentation of transgene expression in transfected cells by two orders of magnitude relative to the control recombinant expression cassette. This increase is due to a corresponding increase in the rate of transcription from the actin promoter and not to replication of the expression cassette and occurs only when the HR3 element is linked to the expression cassette in cis. A comparable degree of enhancement in the activity of the silkworm actin promoter occurs also in heterologous lepidopteran cells. Concomitant supplementation of transfected cells with the BmIE1 trans-activator, which was previously shown to be capable of functioning in vitro as a transcriptional co-activator of the cytoplasmic actin gene promoter, results in more than a 1,000-fold increase in the level of expression of recombinant proteins placed under the control of the actin gene promoter. These findings provide the foundation for the development of a nonlytic insect cell expression system for continuous high-level expression of recombinant proteins. Such a system should provide levels of expression of recombinant proteins comparable to those obtained from baculovirus expression systems and should also have the additional advantage of continuous production in a cellular environment that, in contrast to that generated by a baculovirus infection, supports continuously proper posttranslational modifications of recombinant proteins and the capability of expression of proteins from genomic as well as cDNA sequences.

  12. Adenylate kinase 2 (AK2) promotes cell proliferation in insect development

    PubMed Central

    2012-01-01

    Background Adenylate kinase 2 (AK2) is a phosphotransferase that catalyzes the reversible reaction 2ADP(GDP) ↔ ATP(GTP) + AMP and influences cellular energy homeostasis. However, the role of AK2 in regulating cell proliferation remains unclear because AK2 has been reported to be involved in either cell proliferation or cell apoptosis in different cell types of various organisms. Results This study reports AK2 promotion of cell proliferation using the lepidopteran insect Helicoverpa armigera and its epidermal cell line HaEpi as models. Western blot analysis indicates that AK2 constitutively expresses in various tissues during larval development. Immunocytochemistry analysis indicates that AK2 localizes in the mitochondria. The recombinant expressed AK2 in E. coli promotes cell growth and viability of HaEpi cell line by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. AK2 knockdown in larvae by RNA interference causes larval growth defects, including body weight decrease and development delay. AK2 knockdown in larvae also decreases the number of circulating haemocytes. The mechanism for such effects might be the suppression of gene transcription involved in insect development caused by AK2 knockdown. Conclusion These results show that AK2 regulates cell growth, viability, and proliferation in insect growth and development. PMID:23020757

  13. Cytorhabdovirus phosphoprotein shows RNA silencing suppressor activity in plants, but not in insect cells.

    PubMed

    Mann, Krin S; Johnson, Karyn N; Dietzgen, Ralf G

    2015-02-01

    RNA silencing in plants and insects provides an antiviral defense and as a countermeasure most viruses encode RNA silencing suppressors (RSS). For the family Rhabdoviridae, no detailed functional RSS studies have been reported in plant hosts and insect vectors. In agroinfiltrated Nicotiana benthamiana leaves we show for the first time for a cytorhabdovirus, lettuce necrotic yellows virus (LNYV), that one of the nucleocapsid core proteins, phosphoprotein (P) has relatively weak local RSS activity and delays systemic silencing of a GFP reporter. Analysis of GFP small RNAs indicated that the P protein did not prevent siRNA accumulation. To explore RSS activity in insects, we used a Flock House virus replicon system in Drosophila S2 cells. In contrast to the plant host, LNYV P protein did not exhibit RSS activity in the insect cells. Taken together our results suggest that P protein may target plant-specific components of RNA silencing post siRNA biogenesis. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Transcriptional Profiling of Midgut Immunity Response and Degeneration in the Wandering Silkworm, Bombyx mori

    PubMed Central

    Xiao, Guohua; Yang, Bing; Zhang, Jie; Li, Xuquan; Guan, Jingmin; Shao, Qimiao; Beerntsen, Brenda T.; Zhang, Peng; Wang, Chengshu; Ling, Erjun

    2012-01-01

    Background Lepidoptera insects have a novel development process comprising several metamorphic stages during their life cycle compared with vertebrate animals. Unlike most Lepidoptera insects that live on nectar during the adult stage, the Bombyx mori silkworm adults do not eat anything and die after egg-laying. In addition, the midguts of Lepidoptera insects produce antimicrobial proteins during the wandering stage when the larval tissues undergo numerous changes. The exact mechanisms responsible for these phenomena remain unclear. Principal Findings We used the silkworm as a model and performed genome-wide transcriptional profiling of the midgut between the feeding stage and the wandering stage. Many genes concerned with metabolism, digestion, and ion and small molecule transportation were down-regulated during the wandering stage, indicating that the wandering stage midgut loses its normal functions. Microarray profiling, qRT-PCR and western blot proved the production of antimicrobial proteins (peptides) in the midgut during the wandering stage. Different genes of the immune deficiency (Imd) pathway were up-regulated during the wandering stage. However, some key genes belonging to the Toll pathway showed no change in their transcription levels. Unlike butterfly (Pachliopta aristolochiae), the midgut of silkworm moth has a layer of cells, indicating that the development of midgut since the wandering stage is not usual. Cell division in the midgut was observed only for a short time during the wandering stage. However, there was extensive cell apoptosis before pupation. The imbalance of cell division and apoptosis probably drives the continuous degeneration of the midgut in the silkworm since the wandering stage. Conclusions This study provided an insight into the mechanism of the degeneration of the silkworm midgut and the production of innate immunity-related proteins during the wandering stage. The imbalance of cell division and apoptosis induces irreversible degeneration of the midgut. The Imd pathway probably regulates the production of antimicrobial peptides in the midgut during the wandering stage. PMID:22937093

  15. Transcriptional profiling of midgut immunity response and degeneration in the wandering silkworm, Bombyx mori.

    PubMed

    Xu, Qiuyun; Lu, Anrui; Xiao, Guohua; Yang, Bing; Zhang, Jie; Li, Xuquan; Guan, Jingmin; Shao, Qimiao; Beerntsen, Brenda T; Zhang, Peng; Wang, Chengshu; Ling, Erjun

    2012-01-01

    Lepidoptera insects have a novel development process comprising several metamorphic stages during their life cycle compared with vertebrate animals. Unlike most Lepidoptera insects that live on nectar during the adult stage, the Bombyx mori silkworm adults do not eat anything and die after egg-laying. In addition, the midguts of Lepidoptera insects produce antimicrobial proteins during the wandering stage when the larval tissues undergo numerous changes. The exact mechanisms responsible for these phenomena remain unclear. We used the silkworm as a model and performed genome-wide transcriptional profiling of the midgut between the feeding stage and the wandering stage. Many genes concerned with metabolism, digestion, and ion and small molecule transportation were down-regulated during the wandering stage, indicating that the wandering stage midgut loses its normal functions. Microarray profiling, qRT-PCR and western blot proved the production of antimicrobial proteins (peptides) in the midgut during the wandering stage. Different genes of the immune deficiency (Imd) pathway were up-regulated during the wandering stage. However, some key genes belonging to the Toll pathway showed no change in their transcription levels. Unlike butterfly (Pachliopta aristolochiae), the midgut of silkworm moth has a layer of cells, indicating that the development of midgut since the wandering stage is not usual. Cell division in the midgut was observed only for a short time during the wandering stage. However, there was extensive cell apoptosis before pupation. The imbalance of cell division and apoptosis probably drives the continuous degeneration of the midgut in the silkworm since the wandering stage. This study provided an insight into the mechanism of the degeneration of the silkworm midgut and the production of innate immunity-related proteins during the wandering stage. The imbalance of cell division and apoptosis induces irreversible degeneration of the midgut. The Imd pathway probably regulates the production of antimicrobial peptides in the midgut during the wandering stage.

  16. Forest insect conditions in the Northeast - 1954

    Treesearch

    W. E. Waters

    1955-01-01

    Forest insects continued to be a major cause of timber loss in the Northeast in 1954. A diversity of destructive pests caused these losses in different ways: by outright killing trees; by reducing growth; by reducing merchantable volume; and by reducing the quality of forest products. Some of the insects caused serious damage to woodlands that have high recreational...

  17. High entomotoxicity and mechanism of the fungal GalNAc/Gal-specific Rhizoctonia solani lectin in pest insects.

    PubMed

    Hamshou, Mohamad; Van Damme, Els J M; Caccia, Silvia; Cappelle, Kaat; Vandenborre, Gianni; Ghesquière, Bart; Gevaert, Kris; Smagghe, Guy

    2013-03-01

    Whole insect assays where Rhizoctonia solani agglutinin (RSA) was fed to larval stages of the cotton leaf-worm Spodoptera littoralis and the pea aphid Acyrthosiphon pisum demonstrated a high concentration-dependent entomotoxicity, suggesting that this GalNAc/Gal-specific fungal lectin might be a good control agent for different pest insects. RSA at 10 mg/g in the solid diet of 2nd-instar caterpillars caused 84% weight reduction after 8 days with none of the caterpillars reaching the 4th-instar stage. In sucking aphids, 50% mortality was achieved after 3 days with 9 μM of RSA in the liquid diet. Feeding of FITC-labeled RSA to both insect pest species revealed strong lectin binding at the apical/luminal side of the midgut epithelium with the brush border zone, suggesting the insect midgut as a primary insecticide target tissue for RSA. This was also confirmed with cell cultures in vitro, where there was high fluorescence binding at the microvillar zone with primary cultures of larval midgut columnar cells of S. littoralis, and also at the surface with the insect midgut CF-203 cell line without lectin uptake in the midgut cells. In vitro assays using insect midgut CF-203 cells, revealed that RSA was highly toxic with an EC50 of 0.3 μM. Preincubation with GalNAc and saponin indicated that this action of RSA was carbohydrate-binding dependent and happened at the surface of the cells. Intoxicated CF-203 cells showed symptoms of apoptosis as nuclear condensation and DNA fragmentation, and this concurred with an increase of caspase-3/7, -8 and -9 activities. Finally, RSA affinity chromatography of membrane extracts of CF-203 cells followed by LC-MS/MS allowed the identification of 5747 unique peptides, among which four putatively glycosylated membrane proteins that are associated with apoptosis induction, namely Fas-associated factor, Apoptosis-linked gene-2, Neuroglian and CG2076, as potential binding targets for RSA. These data are discussed in relation to the physiological effects of RSA. Copyright © 2012 Elsevier Ltd. All rights reserved.

  18. Multiorganismal insects: diversity and function of resident microorganisms.

    PubMed

    Douglas, Angela E

    2015-01-07

    All insects are colonized by microorganisms on the insect exoskeleton, in the gut and hemocoel, and within insect cells. The insect microbiota is generally different from microorganisms in the external environment, including ingested food. Specifically, certain microbial taxa are favored by the conditions and resources in the insect habitat, by their tolerance of insect immunity, and by specific mechanisms for their transmission. The resident microorganisms can promote insect fitness by contributing to nutrition, especially by providing essential amino acids, B vitamins, and, for fungal partners, sterols. Some microorganisms protect their insect hosts against pathogens, parasitoids, and other parasites by synthesizing specific toxins or modifying the insect immune system. Priorities for future research include elucidation of microbial contributions to detoxification, especially of plant allelochemicals in phytophagous insects, and resistance to pathogens; as well as their role in among-insect communication; and the potential value of manipulation of the microbiota to control insect pests.

  19. Multiorganismal Insects: Diversity and Function of Resident Microorganisms

    PubMed Central

    Douglas, Angela E.

    2015-01-01

    All insects are colonized by microorganisms on the insect exoskeleton, in the gut and hemocoel, and within insect cells. The insect microbiota is generally different from microorganisms in the external environment, including ingested food. Specifically, certain microbial taxa are favored by the conditions and resources in the insect habitat, by their tolerance of insect immunity, and by specific mechanisms for their transmission. The resident microorganisms can promote insect fitness by contributing to nutrition, especially by providing essential amino acids, B vitamins, and, for fungal partners, sterols. Some microorganisms protect their insect hosts against pathogens, parasitoids, and other parasites by synthesizing specific toxins or modifying the insect immune system. Priorities for future research include elucidation of microbial contributions to detoxification, especially of plant allelochemicals in phytophagous insects, and resistance to pathogens; as well as their role in among-insect communication; and the potential value of manipulation of the microbiota to control insect pests. PMID:25341109

  20. Cultivating Insect Cells To Produce Recombinant Proteins

    NASA Technical Reports Server (NTRS)

    Spaulding, Glenn; Goodwin, Thomas; Prewett, Tacey; Andrews, Angela; Francis, Karen; O'Connor, Kim

    1996-01-01

    Method of producing recombinant proteins involves growth of insect cells in nutrient solution in cylindrical bioreactor rotating about cylindrical axis, oriented horizontally and infecting cells with viruses into which genes of selected type cloned. Genes in question those encoding production of desired proteins. Horizontal rotating bioreactor preferred for use in method, denoted by acronym "HARV", described in "High-Aspect-Ratio Rotating Cell-Culture Vessel" (MSC-21662).

  1. Engineering Sialic Acid Synthesis Ability in Insect Cells.

    PubMed

    Viswanathan, Karthik; Narang, Someet; Betenbaugh, Michael J

    2015-01-01

    Insect cells lack the ability to synthesize the sialic acid donor molecule CMP-sialic acid or its precursor, sialic acid. In this chapter, we describe a method to engineer CMP-sialic acid synthesis capability into Spodoptera frugiperda (Sf9) cells, a prototypical insect cell line, by recombinant expression of sialic acid synthesis pathway genes using baculovirus technology. Co-expression of a sialuria mutant UDP-GlcNAc-2-epimerase/ManNAc kinase (EKR263L), wild-type sialic acid 9-phosphate synthase (SAS), and wild-type CMP-sialic acid synthetase (CSAS) in the presence of GlcNAc leads to synthesis of CMP-sialic acids synthesis to support sialylation of N-glycans on glycoproteins.

  2. Intracellular ice formation in insects: unresolved after 50 years?

    PubMed

    Sinclair, Brent J; Renault, David

    2010-01-01

    Many insects survive internal ice formation. The general model of freeze tolerance is of extracellular ice formation (EIF) whereby ice formation in the haemocoel leads to osmotic dehydration of the cells, whose contents remain unfrozen. However, survivable intracellular ice formation (IIF) has been reported in fat body and certain other cells of some insects. Although the cellular location of ice has been determined only in vitro, several lines of evidence suggest that IIF occurs in vivo. Both cell-to-cell propagation of intracellular ice and inoculation from the haemocoel may be important, although the route of ice into the cell is unclear. It is unclear why some cells survive IIF and others do not, but it is suggested that the shape, size, and low water content of fat body cells may predispose them towards surviving ice formation. We speculate that IIF may reduce water loss in some freeze tolerant species, but there are too few data to build a strong conceptual model of the advantages of IIF. We suggest that new developments in microscopy and other forms of imaging may allow investigation of the cellular location of ice in freeze tolerant insects in vivo.

  3. Potent insect gut binding lectin from Sclerotium rolfsii impart resistance to sucking and chewing type insects in cotton.

    PubMed

    Vanti, Gulamnabi L; Katageri, Ishwarappa S; Inamdar, Shashikala R; Hiremathada, Vamadevaiah; Swamy, Bale M

    2018-04-30

    Cotton is an important crop that is continuously cultivated around the world. However, its production has decreased in recent times due to wide ranging insects and also current practices of using synthetic insecticides that are not precise and their residues impairing the biodiversity. Hence, the search for newer classes of efficient entomotoxic proteins continues. Genetically modified cotton crops with cry genes from Bacillus thuringiensis, have been cultivated across the world, which overcome the chewing type insect menace. In the present study, we assess the development of transgenic cotton plants by Agrobacterium, wherein the confirmed kanamycin resistant T 0 plants were advanced to T 1 generation and the gene integration was studied by molecular analysis. Western blot and ELISA assays demonstrated the expression of 0.46% lectin of the total soluble leaf proteins. In planta bioassay showed 69% of aphid, Aphis gossypii population reduction with T 1 generation plants. Whereas 100% insect mortality is occurred in Spodoptera litura larvae by 96 h. Present findings shows the potent insecticidal effect of Sclerotium rolfsii lectin on sucking (homopteran) and chewing (lepidopteron) insects, underlining its significance and strengthening genetic resources in cotton breeding against different order insect pests. Copyright © 2018 Elsevier B.V. All rights reserved.

  4. Expression, purification and characterization of the secreted luciferase of the copepod Metridia longa from Sf9 insect cells

    PubMed Central

    Stepanyuk, Galina A.; Xu, Hao; Wu, Chia-Kuei; Markova, Svetlana V.; Lee, John; Vysotski, Eugene S.; Wang, Bi-Cheng

    2008-01-01

    Metridia luciferase is a secreted luciferase from a marine copepod and uses coelenterazine as a substrate to produce a blue bioluminescence (λmax = 480 nm). This luciferase has been successfully applied as a bioluminescent reporter in mammalian cells. The main advantage of secreted luciferase as a reporter is the capability of measuring intracellular events without destroying the cells or tissues and this property is well suited for development of high throughput screening technologies. However because Metridia luciferase is a Cys-rich protein, E. coli expression systems produce an incorrectly folded protein, hindering its biochemical characterization and application for development of in vitro bioluminescent assays. Here we report the successful expression of Metridia luciferase with its signal peptide for secretion, in insect (Sf9) cells using the baculovirus expression system. Functionally active luciferase secreted by insect cells into the culture media has been efficiently purified with a yield of high purity protein of 2–3 mg/L. This Metridia luciferase expressed in the insect cell system is a monomeric protein showing 3.5-fold greater bioluminescence activity than luciferase expressed and purified from E. coli. The near coincidence of the experimental mass of Metridia luciferase purified from insect cells with that calculated from amino acid sequence, indicates that luciferase does not undergo posttranslational modifications such as phosphorylation or glycosylation and also, the cleavage site of the signal peptide for secretion is at VQA-KS, as predicted from sequence analysis. PMID:18595733

  5. A comparative assessment of cytotoxicity of commonly used agricultural insecticides to human and insect cells.

    PubMed

    Yun, Xinming; Huang, Qingchun; Rao, Wenbing; Xiao, Ciying; Zhang, Tao; Mao, Zhifan; Wan, Ziyi

    2017-03-01

    The cytotoxic potential of 13 commonly used agricultural insecticides was examined using cell-based systems with three human HepG2, Hek293, HeLa cells and three insect Tn5B1-4, Sf-21, and Drosophila S2 cells. Data showed that (1) an enhancement of some insecticides (e.g. pyrethroids) on cells proliferation; (2) an inhibition of some insecticides on cells viability; (3) various levels of susceptibility of different cells to the same insecticide; and (4) the cell type dependent sensitivity to different insecticides. The degree of cytotoxicity of insecticides on human cells was significantly lower than that on insect cells (P<0.05). Methomyl, even 20μg/ml, showed little cytotoxicity at 24h exposure whereas emamectin benzoate possessed the strongest cytotoxic potential in a dose-dependent fashion. The results revealed comparable cytotoxic property of agricultural insecticides against intact cells. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Comparative toxicity and micronuclei formation in Tribolium castaneum, Callosobruchus maculatus and Sitophilus oryzae exposed to high doses of gamma radiation.

    PubMed

    Ahmadi, Mehrdad; Mozdarani, Hossein; Abd-Alla, Adly M M

    2015-07-01

    The effects of gamma radiation on mortality and micronucleus formation in Tribolium castaneum Herbst, Callosobruchus maculatus (F.) and Sitophilus oryzae (L.) genital cells were evaluated. Two groups of healthy and active adult insects 1-3 and 8-10 days old were irradiated with various doses (50-200 Gy) gamma ray. Seven days post-irradiation; mortality rates and micronucleus formation were assessed in genital cells of the irradiated insects. The results show that with increasing gamma doses, the mortality rate of each species increased and T. castaneum and S. oryzae showed the low and high sensitivity respectively. It was shown that the micronucleus appearance in the tested insects had correlation with amount and intensity of radiation doses. Moreover our results indicate different levels in the genotoxicity of gamma radiation among the insects' genital cells under study. The frequency of micronuclei in genital cells of 1-3 days old insects exposed to 50 and 200 Gy were 12.6 and 38.8 Mn/1000 cells in T. castaneum, 20.8 and 46.8 Mn/1000 cells in C. maculatus and 16.8 and 57.2 Mn/1000 cells in S. oryzae respectively. A high sensitivity of the genital cells to irradiation exposure was seen in S. oryzae correlated with its high mortality rate compared with the other two species. These results might be indicative of inflicting chromosomal damage expressed as micronucleus in high mortality rates observed in the pest population; an indication of genotoxic effects of radiation on the studied species. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

    PubMed Central

    Vincent, Thomas R.; Canham, James; Toyota, Masatsugu; Avramova, Marieta; Mugford, Sam T.; Gilroy, Simon; Miller, Anthony J.; Hogenhout, Saskia; Sanders, Dale

    2017-01-01

    Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant defense response to microbial elicitors and to wounding caused by chewing insects, eliciting systemic calcium signals in plants. However, the role of calcium in vivo during biotic stress is still unclear. This protocol describes the use of a genetically-encoded calcium sensor to detect calcium signals in plants during feeding by a hemipteran pest. Hemipterans such as aphids pierce a small number of cells with specialized, elongated sucking mouthparts, making them the ideal tool to study calcium dynamics when a plant is faced with a biotic stress, which is distinct from a wounding response. In addition, fluorescent biosensors are revolutionizing the measurement of signaling molecules in vivo in both animals and plants. Expressing a GFP-based calcium biosensor, GCaMP3, in the model plant Arabidopsis thaliana allows for the real-time imaging of plant calcium dynamics during insect feeding, with a high spatial and temporal resolution. A repeatable and robust assay has been developed using the fluorescence microscopy of detached GCaMP3 leaves, allowing for the continuous measurement of cytosolic calcium dynamics before, during, and after insect feeding. This reveals a highly-localized rapid calcium elevation around the aphid feeding site that occurs within a few minutes. The protocol can be adapted to other biotic stresses, such as additional insect species, while the use of Arabidopsis thaliana allows for the rapid generation of mutants to facilitate the molecular analysis of the phenomenon. PMID:28829425

  8. CRISPR/Cas9 in insects: Applications, best practices and biosafety concerns.

    PubMed

    Taning, Clauvis Nji Tizi; Van Eynde, Benigna; Yu, Na; Ma, Sanyuan; Smagghe, Guy

    2017-04-01

    Discovered as a bacterial adaptive immune system, CRISPR/Cas9 (clustered, regularly interspaced, short palindromic repeat/CRISPR associated) is being developed as an attractive tool in genome editing. Due to its high specificity and applicability, CRISPR/Cas9-mediated gene editing has been employed in a multitude of organisms and cells, including insects, for not only fundamental research such as gene function studies, but also applied research such as modification of organisms of economic importance. Despite the rapid increase in the use of CRISPR in insect genome editing, results still differ from each study, principally due to existing differences in experimental parameters, such as the Cas9 and guide RNA form, the delivery method, the target gene and off-target effects. Here, we review current reports on the successes of CRISPR/Cas9 applications in diverse insects and insect cells. We furthermore summarize several best practices to give a useful checklist of CRISPR/Cas9 experimental setup in insects for beginners. Lastly, we discuss the biosafety concerns related to the release of CRISPR/Cas9-edited insects into the environment. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Both cell-autonomous mechanisms and hormones contribute to sexual development in vertebrates and insects.

    PubMed

    Bear, Ashley; Monteiro, Antónia

    2013-08-01

    The differentiation of male and female characteristics in vertebrates and insects has long been thought to proceed via different mechanisms. Traditionally, vertebrate sexual development was thought to occur in two phases: a primary and a secondary phase, the primary phase involving the differentiation of the gonads, and the secondary phase involving the differentiation of other sexual traits via the influence of sex hormones secreted by the gonads. In contrast, insect sexual development was thought to depend exclusively on cell-autonomous expression of sex-specific genes. Recently, however, new evidence indicates that both vertebrates and insects rely on sex hormones as well as cell-autonomous mechanisms to develop sexual traits. Collectively, these new data challenge the traditional vertebrate definitions of primary and secondary sexual development, call for a redefinition of these terms, and indicate the need for research aimed at explaining the relative dependence on cell-autonomous versus hormonally guided sexual development in animals. © 2013 The Authors. BioEssays published by WILEY Periodicals, Inc.

  10. Active Solute Transport across Frog Skin and Epithelial Cell Systems According to the Association-Induction Hypothesis,

    DTIC Science & Technology

    1980-01-01

    indicated in insect Malpighian tubules, insect midgut , choroid plexus and gastric mucosa. 9.2.5 Conciliation of the Conflict Between Models of "Homocellular...SeApeinaebility is therefore under close I Ling (P;. 14) control by hormones or drugs that react with receptor sites on these protelns and so...bears remembering that in active transport across bifacial cells the key cation is not always No% Thus in MalPighian tubules of insects the key Lon

  11. Production of G protein-coupled receptors in an insect-based cell-free system.

    PubMed

    Sonnabend, Andrei; Spahn, Viola; Stech, Marlitt; Zemella, Anne; Stein, Christoph; Kubick, Stefan

    2017-10-01

    The biochemical analysis of human cell membrane proteins remains a challenging task due to the difficulties in producing sufficient quantities of functional protein. G protein-coupled receptors (GPCRs) represent a main class of membrane proteins and drug targets, which are responsible for a huge number of signaling processes regulating various physiological functions in living cells. To circumvent the current bottlenecks in GPCR studies, we propose the synthesis of GPCRs in eukaryotic cell-free systems based on extracts generated from insect (Sf21) cells. Insect cell lysates harbor the fully active translational and translocational machinery allowing posttranslational modifications, such as glycosylation and phosphorylation of de novo synthesized proteins. Here, we demonstrate the production of several GPCRs in a eukaryotic cell-free system, performed within a short time and in a cost-effective manner. We were able to synthesize a variety of GPCRs ranging from 40 to 133 kDa in an insect-based cell-free system. Moreover, we have chosen the μ opioid receptor (MOR) as a model protein to analyze the ligand binding affinities of cell-free synthesized MOR in comparison to MOR expressed in a human cell line by "one-point" radioligand binding experiments. Biotechnol. Bioeng. 2017;114: 2328-2338. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

  12. Antigenic characterization of bovine ephemeral fever rhabdovirus G and GNS glycoproteins expressed from recombinant baculoviruses.

    PubMed

    Johal, Jasjit; Gresty, Karryn; Kongsuwan, Kritaya; Walker, Peter J

    2008-01-01

    Recombinant baculoviruses expressing the BEFV envelope glycoprotein G and non-structural glycoprotein G(NS) were constructed. The G protein expressed in insect cells was located on the cell surface and induced spontaneous cell fusion at mildly acidic pH. The expressed G protein reacted with MAbs to continuous and conformational neutralization sites (G1, G2, G3b and G4), but not to conformational site G3a. The expressed G(NS) protein was also located on the cell surface but did not exhibit fusogenic activity. The G(NS) protein reacted with polyclonal antiserum produced from vaccinia-virus-expressed recombinant G(NS) but did not react with G protein antibodies. A His(6)-tagged, soluble form of the G protein was expressed and purified by Ni(2+)-NTA chromatography. The purified G protein reacted with BEFV-neutralizing MAbs to all continuous and conformational antigenic sites. The highly protective characteristics of the native BEFV G protein suggest that the secreted, baculovirus-expressed product may be a useful vaccine antigen.

  13. High altitude solar power platform. [aircraft design analysis

    NASA Technical Reports Server (NTRS)

    Bailey, M. D.; Bower, M. V.

    1992-01-01

    Solar power is a preeminent alternative to conventional aircraft propulsion. With the continued advances in solar cells, fuel cells, and composite materials technology, the solar powered airplane is no longer a simple curiosity constrained to flights of several feet in altitude or minutes of duration. A high altitude solar powered platform (HASPP) has several potential missions, including communications and agriculture. In remote areas, a HASPP could be used as a communication link. In large farming areas, a HASPP could perform remote sensing of crops. The impact of HASPP in continuous flight for one year on agricultural monitoring mission is presented. This mission provides farmers with near real-time data twice daily from an altitude which allows excellant resolution on water conditions, crop diseases, and insect infestation. Accurate, timely data will enable farmers to increase their yield and efficiency. A design for HASPP for the foregoing mission is presented. In the design power derived from solar cells covering the wings is used for propulsion, avionics, and sensors. Excess power produced midday will be stored in fuel cells for use at night to maintain altitude and course.

  14. Hearing in Insects.

    PubMed

    Göpfert, Martin C; Hennig, R Matthias

    2016-01-01

    Insect hearing has independently evolved multiple times in the context of intraspecific communication and predator detection by transforming proprioceptive organs into ears. Research over the past decade, ranging from the biophysics of sound reception to molecular aspects of auditory transduction to the neuronal mechanisms of auditory signal processing, has greatly advanced our understanding of how insects hear. Apart from evolutionary innovations that seem unique to insect hearing, parallels between insect and vertebrate auditory systems have been uncovered, and the auditory sensory cells of insects and vertebrates turned out to be evolutionarily related. This review summarizes our current understanding of insect hearing. It also discusses recent advances in insect auditory research, which have put forward insect auditory systems for studying biological aspects that extend beyond hearing, such as cilium function, neuronal signal computation, and sensory system evolution.

  15. 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.

  16. Comparison between the Amount of Environmental Change and the Amount of Transcriptome Change

    PubMed Central

    Ogata, Norichika; Kozaki, Toshinori; Yokoyama, Takeshi; Hata, Tamako; Iwabuchi, Kikuo

    2015-01-01

    Cells must coordinate adjustments in genome expression to accommodate changes in their environment. We hypothesized that the amount of transcriptome change is proportional to the amount of environmental change. To capture the effects of environmental changes on the transcriptome, we compared transcriptome diversities (defined as the Shannon entropy of frequency distribution) of silkworm fat-body tissues cultured with several concentrations of phenobarbital. Although there was no proportional relationship, we did identify a drug concentration “tipping point” between 0.25 and 1.0 mM. Cells cultured in media containing lower drug concentrations than the tipping point showed uniformly high transcriptome diversities, while those cultured at higher drug concentrations than the tipping point showed uniformly low transcriptome diversities. The plasticity of transcriptome diversity was corroborated by cultivations of fat bodies in MGM-450 insect medium without phenobarbital and in 0.25 mM phenobarbital-supplemented MGM-450 insect medium after previous cultivation (cultivation for 80 hours in MGM-450 insect medium without phenobarbital, followed by cultivation for 10 hours in 1.0 mM phenobarbital-supplemented MGM-450 insect medium). Interestingly, the transcriptome diversities of cells cultured in media containing 0.25 mM phenobarbital after previous cultivation (cultivation for 80 hours in MGM-450 insect medium without phenobarbital, followed by cultivation for 10 hours in 1.0 mM phenobarbital-supplemented MGM-450 insect medium) were different from cells cultured in media containing 0.25 mM phenobarbital after previous cultivation (cultivation for 80 hours in MGM-450 insect medium without phenobarbital). This hysteretic phenomenon of transcriptome diversities indicates multi-stability of the genome expression system. Cellular memories were recorded in genome expression networks as in DNA/histone modifications. PMID:26657512

  17. 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.

  18. Comparative proteomics analysis of Spodoptera frugiperda cells during Autographa californica multiple nucleopolyhedrovirus infection.

    PubMed

    Yu, Qian; Xiong, Youhua; Gao, Hang; Liu, Jianliang; Chen, Zhiqiang; Wang, Qin; Wen, Dongling

    2015-08-04

    Increasing evidence sugggest that in addition of balculovirus controling insect host, host cells also responds to balculovirus infection. However, compared to existing knowledge on virus gene, host cell responses are relatively poorly understood. In this study, Spodoptera frugiperda (Sf9) cells were infected with Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The protein composition and protein changes of Spodoptera frugiperda (Sf9) cells of different infection stages were analysed by isobaric tag for relative and absolute quantification (iTRAQ) techniques. A total of 4004 Sf9 proteins were identified by iTRAQ and 413 proteins were found as more than 1.5-fold changes in abundance. The 413 proteins were categorised according to GO classification for insects and were categorised into: biological process, molecular function and cellular component. The determination of the protein changes in infected Sf9 cells would help to better understanding of host cell responses and facilitate better design of this virus-host cell interaction in pest insect control and other related fields.

  19. Cell-cell signaling controls Xylella fastidiosa interactions with both insects and plants

    PubMed Central

    Newman, Karyn L.; Almeida, Rodrigo P. P.; Purcell, Alexander H.; Lindow, Steven E.

    2004-01-01

    Xylella fastidiosa, which causes Pierce's disease of grapevine and other important plant diseases, is a xylem-limited bacterium that depends on insect vectors for transmission. Although many studies have addressed disease symptom development and transmission of the pathogen by vectors, little is known about the bacterial mechanisms driving these processes. Recently available X. fastidiosa genomic sequences and molecular tools have provided new routes for investigation. Here, we show that a diffusible signal molecule is required for biofilm formation in the vector and for vector transmission to plants. We constructed strains of X. fastidiosa mutated in the rpfF gene and determined that they are unable to produce the signal activity. In addition, rpfF mutants are more virulent than the wild type when mechanically inoculated into plants. This signal therefore directs interaction of X. fastidiosa with both its insect vector and plant host. Interestingly, rpfF mutants can still form in planta biofilms, which differ architecturally from biofilms in insects, suggesting that biofilm architecture, rather than a passive response to the environment, is actively determined by X. fastidiosa gene expression. This article reports a cell-cell signaling requirement for vector transmission. Identification of the genes regulated by rpfF should elucidate bacterial factors involved in transmission and biofilm formation in the insect. PMID:14755059

  20. Baculovirus IE2 Stimulates the Expression of Heat Shock Proteins in Insect and Mammalian Cells to Facilitate Its Proper Functioning.

    PubMed

    Tung, Hsuan; Wei, Sung-Chan; Lo, Huei-Ru; Chao, Yu-Chan

    2016-01-01

    Baculoviruses have gained popularity as pest control agents and for protein production in insect systems. These viruses are also becoming popular for gene expression, tissue engineering and gene therapy in mammalian systems. Baculovirus infection triggers a heat shock response, and this response is crucial for its successful infection of host insect cells. However, the viral protein(s) or factor(s) that trigger this response are not yet clear. Previously, we revealed that IE2-an early gene product of the baculovirus-could form unique nuclear bodies for the strong trans-activation of various promoters in mammalian cells. Here, we purified IE2 nuclear bodies from Vero E6 cells and investigated the associated proteins by using mass spectrometry. Heat shock proteins (HSPs) were found to be one of the major IE2-associated proteins. Our experiments show that HSPs are greatly induced by IE2 and are crucial for the trans-activation function of IE2. Interestingly, blocking both heat shock protein expression and the proteasome pathway preserved the IE2 protein and its nuclear body structure, and revived its function. These observations reveal that HSPs do not function directly to assist the formation of the nuclear body structure, but may rather protect IE2 from proteasome degradation. Aside from functional studies in mammalian cells, we also show that HSPs were stimulated and required to determine IE2 protein levels, in insect cells infected with baculovirus. Upon inhibiting the expression of heat shock proteins, baculovirus IE2 was substantially suppressed, resulting in a significantly suppressed viral titer. Thus, we demonstrate a unique feature in that IE2 can function in both insect and non-host mammalian cells to stimulate HSPs, which may be associated with IE2 stabilization and lead to the protection of the its strong gene activation function in mammalian cells. On the other hand, during viral infection in insect cells, IE2 could also strongly stimulate HSPs and ultimately affect viral replication.

  1. Bioreactor concepts for cell culture-based viral vaccine production.

    PubMed

    Gallo-Ramírez, Lilí Esmeralda; Nikolay, Alexander; Genzel, Yvonne; Reichl, Udo

    2015-01-01

    Vaccine manufacturing processes are designed to meet present and upcoming challenges associated with a growing vaccine market and to include multi-use facilities offering a broad portfolio and faster reaction times in case of pandemics and emerging diseases. The final products, from whole viruses to recombinant viral proteins, are very diverse, making standard process strategies hardly universally applicable. Numerous factors such as cell substrate, virus strain or expression system, medium, cultivation system, cultivation method, and scale need consideration. Reviewing options for efficient and economical production of human vaccines, this paper discusses basic factors relevant for viral antigen production in mammalian cells, avian cells and insect cells. In addition, bioreactor concepts, including static systems, single-use systems, stirred tanks and packed-beds are addressed. On this basis, methods towards process intensification, in particular operational strategies, the use of perfusion systems for high product yields, and steps to establish continuous processes are introduced.

  2. Characterization of a novel insect-specific flavivirus from Brazil: Potential for inhibition of infection of arthropod cells with medically important flaviviruses.

    DOE PAGES

    Kenney, Joan L.; Solberg, Owen D.; Langevin, Stanley A.; ...

    2014-01-12

    In the past decade, there has been an upsurge in the number of newly described insect-specific flaviviruses isolated pan-globally. We recently described the isolation of a novel flavivirus (tentatively designated ‘Nhumirim virus’; NHUV) that represents an example of a unique subset of apparently insect-specific viruses that phylogenetically affiliate with dual-host mosquito-borne flaviviruses despite appearing to be limited to replication in mosquito cells. We characterized the in vitro growth potential and 3' untranslated region (UTR) sequence homology with alternative flaviviruses, and evaluated the virus’s capacity to suppress replication of representative Culex spp.-vectored pathogenic flaviviruses in mosquito cells. Only mosquito cell linesmore » were found to support NHUV replication, further reinforcing the insect-specific phenotype of this virus. Analysis of the sequence and predicted RNA secondary structures of the 3' UTR indicated NHUV to be most similar to viruses within the yellow fever serogroup and Japanese encephalitis serogroup, and viruses in the tick-borne flavivirus clade. NHUV was found to share the fewest conserved sequence elements when compared with traditional insect-specific flaviviruses. This suggests that, despite apparently being insect specific, this virus probably diverged from an ancestral mosquito-borne flavivirus. Co-infection experiments indicated that prior or concurrent infection of mosquito cells with NHUV resulted in a significant reduction in virus production of West Nile virus (WNV), St Louis encephalitis virus (SLEV) and Japanese encephalitis virus. As a result, the inhibitory effect was most effective against WNV and SLEV with over a 106-fold and 104-fold reduction in peak titres, respectively.« less

  3. Characterization of a novel insect-specific flavivirus from Brazil: Potential for inhibition of infection of arthropod cells with medically important flaviviruses.

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

    Kenney, Joan L.; Solberg, Owen D.; Langevin, Stanley A.

    In the past decade, there has been an upsurge in the number of newly described insect-specific flaviviruses isolated pan-globally. We recently described the isolation of a novel flavivirus (tentatively designated ‘Nhumirim virus’; NHUV) that represents an example of a unique subset of apparently insect-specific viruses that phylogenetically affiliate with dual-host mosquito-borne flaviviruses despite appearing to be limited to replication in mosquito cells. We characterized the in vitro growth potential and 3' untranslated region (UTR) sequence homology with alternative flaviviruses, and evaluated the virus’s capacity to suppress replication of representative Culex spp.-vectored pathogenic flaviviruses in mosquito cells. Only mosquito cell linesmore » were found to support NHUV replication, further reinforcing the insect-specific phenotype of this virus. Analysis of the sequence and predicted RNA secondary structures of the 3' UTR indicated NHUV to be most similar to viruses within the yellow fever serogroup and Japanese encephalitis serogroup, and viruses in the tick-borne flavivirus clade. NHUV was found to share the fewest conserved sequence elements when compared with traditional insect-specific flaviviruses. This suggests that, despite apparently being insect specific, this virus probably diverged from an ancestral mosquito-borne flavivirus. Co-infection experiments indicated that prior or concurrent infection of mosquito cells with NHUV resulted in a significant reduction in virus production of West Nile virus (WNV), St Louis encephalitis virus (SLEV) and Japanese encephalitis virus. As a result, the inhibitory effect was most effective against WNV and SLEV with over a 106-fold and 104-fold reduction in peak titres, respectively.« less

  4. Characterization of a novel insect-specific flavivirus from Brazil: potential for inhibition of infection of arthropod cells with medically important flaviviruses

    PubMed Central

    Kenney, Joan L.; Solberg, Owen D.; Langevin, Stanley A.; Brault, Aaron C.

    2015-01-01

    In the past decade there has been an upsurge in the number of newly described insect-specific flaviviruses isolated pan-globally. We recently described the isolation of a novel flavivirus (tentatively designated “Nhumirim virus”; NHUV) (Pauvolid-Correa et al., in review) that represents an example of a unique subset of apparently insect-specific viruses that phylogenetically affiliate with dual-host mosquito-borne flaviviruses despite appearing to be limited to replication in mosquito cells. We characterized the in vitro growth potential, 3’ untranslated region (UTR) sequence homology with alternative flaviviruses, and evaluated the virus’s capacity to suppress replication of representative Culex spp. vectored pathogenic flaviviruses in mosquito cells. Only mosquito cell lines were found to support NHUV replication, further reinforcing the insect-specific phenotype of this virus. Analysis of the sequence and predicted RNA secondary structures of the 3’ UTR indicate NHUV to be most similar to viruses within the yellow fever serogroup, Japanese encephalitis serogroup, and viruses in the tick-borne flavivirus clade. NHUV was found to share the fewest conserved sequence elements when compared to traditional insect-specific flaviviruses. This suggests that, despite being apparently insect-specific, this virus likely diverged from an ancestral mosquito-borne flavivirus. Co-infection experiments indicated that prior or concurrent infection of mosquito cells with NHUV resulted in significant reduction in viral production of West Nile virus (WNV), St. Louis encephalitis virus (SLEV) and Japanese encephalitis virus. The inhibitory effect was most effective against WNV and SLEV with over a million-fold and 10,000-fold reduction in peak titers, respectively. PMID:25146007

  5. Insect Cell-Derived Cofactors Become Fully Functional after Proteinase K and Heat Treatment for High-Fidelity Amplification of Glycosylphosphatidylinositol-Anchored Recombinant Scrapie and BSE Prion Proteins

    PubMed Central

    Imamura, Morikazu; Kato, Nobuko; Okada, Hiroyuki; Yoshioka, Miyako; Iwamaru, Yoshifumi; Shimizu, Yoshihisa; Mohri, Shirou; Yokoyama, Takashi; Murayama, Yuichi

    2013-01-01

    The central event in prion infection is the conformational conversion of host-encoded cellular prion protein (PrPC) into the pathogenic isoform (PrPSc). Diverse mammalian species possess the cofactors required for in vitro replication of PrPSc by protein-misfolding cyclic amplification (PMCA), but lower organisms, such as bacteria, yeasts, and insects, reportedly lack the essential cofactors. Various cellular components, such as RNA, lipids, and other identified cofactor molecules, are commonly distributed in both eukaryotes and prokaryotes, but the reasons for the absence of cofactor activity in lower organisms remain to be elucidated. Previously, we reported that brain-derived factors were necessary for the in vitro replication of glycosylphosphatidylinositol-anchored baculovirus-derived recombinant PrP (Bac-PrP). Here, we demonstrate that following protease digestion and heat treatment, insect cell lysates had the functional cofactor activity required for Bac-PrP replication by PMCA. Mammalian PrPSc seeds and Bac-PrPSc generated by PMCA using Bac-PrP and insect cell-derived cofactors showed similar pathogenicity and produced very similar lesions in the brains of inoculated mice. These results suggested that the essential cofactors required for the high-fidelity replication of mammalian PrPSc were present in the insect cells but that the cofactor activity was masked or inhibited in the native state. We suggest that not only RNA, but also DNA, are the key components of PMCA, although other cellular factors were necessary for the expression of the cofactor activity of nucleic acids. PMCA using only insect cell-derived substances (iPMCA) was highly useful for the ultrasensitive detection of PrPSc of some prion strains. PMID:24367521

  6. Baculovirus: an Insect-derived Vector for Diverse Gene Transfer Applications

    PubMed Central

    Airenne, Kari J; Hu, Yu-Chen; Kost, Thomas A; Smith, Richard H; Kotin, Robert M; Ono, Chikako; Matsuura, Yoshiharu; Wang, Shu; Ylä-Herttuala, Seppo

    2013-01-01

    Insect-derived baculoviruses have emerged as versatile and safe workhorses of biotechnology. Baculovirus expression vectors (BEVs) have been applied widely for crop and forest protection, as well as safe tools for recombinant protein production in insect cells. However, BEVs ability to efficiently transduce noninsect cells is still relatively poorly recognized despite the fact that efficient baculovirus-mediated in vitro and ex vivo gene delivery into dormant and dividing vertebrate cells of diverse origin has been described convincingly by many authors. Preliminary proof of therapeutic potential has also been established in preclinical studies. This review summarizes the advantages and current status of baculovirus-mediated gene delivery. Stem cell transduction, preclinical animal studies, tissue engineering, vaccination, cancer gene therapy, viral vector production, and drug discovery are covered. PMID:23439502

  7. Evolutionary conservation and changes in insect TRP channels.

    PubMed

    Matsuura, Hironori; Sokabe, Takaaki; Kohno, Keigo; Tominaga, Makoto; Kadowaki, Tatsuhiko

    2009-09-10

    TRP (Transient Receptor Potential) channels respond to diverse stimuli and thus function as the primary integrators of varied sensory information. They are also activated by various compounds and secondary messengers to mediate cell-cell interactions as well as to detect changes in the local environment. Their physiological roles have been primarily characterized only in mice and fruit flies, and evolutionary studies are limited. To understand the evolution of insect TRP channels and the mechanisms of integrating sensory inputs in insects, we have identified and compared TRP channel genes in Drosophila melanogaster, Bombyx mori, Tribolium castaneum, Apis mellifera, Nasonia vitripennis, and Pediculus humanus genomes as part of genome sequencing efforts. All the insects examined have 2 TRPV, 1 TRPN, 1 TRPM, 3 TRPC, and 1 TRPML subfamily members, demonstrating that these channels have the ancient origins in insects. The common pattern also suggests that the mechanisms for detecting mechanical and visual stimuli and maintaining lysosomal functions may be evolutionarily well conserved in insects. However, a TRPP channel, the most ancient TRP channel, is missing in B. mori, A. mellifera, and N. vitripennis. Although P. humanus and D. melanogaster contain 4 TRPA subfamily members, the other insects have 5 TRPA subfamily members. T. castaneum, A. mellifera, and N. vitripennis contain TRPA5 channels, which have been specifically retained or gained in Coleoptera and Hymenoptera. Furthermore, TRPA1, which functions for thermotaxis in Drosophila, is missing in A. mellifera and N. vitripennis; however, they have other Hymenoptera-specific TRPA channels (AmHsTRPA and NvHsTRPA). NvHsTRPA expressed in HEK293 cells is activated by temperature increase, demonstrating that HsTRPAs function as novel thermal sensors in Hymenoptera. The total number of insect TRP family members is 13-14, approximately half that of mammalian TRP family members. As shown for mammalian TRP channels, this may suggest that single TRP channels are responsible for integrating diverse sensory inputs to maintain the insect sensory systems. The above results demonstrate that there are both evolutionary conservation and changes in insect TRP channels. In particular, the evolutionary processes have been accelerated in the TRPA subfamily, indicating divergence in the mechanisms that insects use to detect environmental temperatures.

  8. Molecular dissection of nutrient exchange at the insect-microbial interface.

    PubMed

    Douglas, Angela E

    2014-10-01

    Genome research is transforming our understanding of nutrient exchange between insects and intracellular bacteria. A key characteristic of these bacteria is their small genome size and gene content. Their fastidious and inflexible nutritional requirements are met by multiple metabolites from the insect host cell. Although the bacteria have generally retained genes coding the synthesis of nutrients required by the insect, some apparently critical genes have been lost, and compensated for by shared metabolic pathways with the insect host or supplementary bacteria with complementary metabolic capabilities. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Conserved Genetic Pathways Controlling the Development of the Diffuse Endocrine System in Vertebrates and Drosophila

    PubMed Central

    Hartenstein, Volker; Takashima, Shigeo; Adams, Katrina

    2014-01-01

    The midgut epithelium is formed by absorptive enterocytes, secretory cells and endocrine cells. Each of these lineages is derived from the pluripotent progenitors that constitute the embryonic endoderm; the mature midgut retains pools of self-renewing stem cells that continue to produce all lineages. Recent findings in vertebrates and Drosophila shed light on the genetic mechanism that specifies the fate of the different lineages. A pivotal role is played by the Notch signaling pathway that, in a manner that appears to be very similar to the way in which Notch signaling selects neural progenitors within the neurectoderm, distinguishes the fate of secretory/endocrine cells and enterocytes. Proneural genes encoding bHLH transcription factors are expressed and required in prospective endocrine cells; activation of the Notch pathways restricts the number of these cells and promotes enterocyte development. In this review we compare the development of the intestinal endocrine cells in vertebrates and insects and summarize recent findings dealing with genetic pathways controlling this cell type. PMID:20005229

  10. Prostaglandin A2 influences gene expression in an established insect cell line (BCIRL-HzAm1) cells

    USDA-ARS?s Scientific Manuscript database

    Prostaglandins (PGs) and other eicosanoids are oxygenated metabolites of arachidonic acid and two other C20 polyunsaturated fatty acids. While most well studied in mammals, PGs exert important actions in insects and virtually all other invertebrates. We have been researching the mechanisms of PG a...

  11. INSECT FAT BODY: ENERGY, METABOLISM, AND REGULATION

    PubMed Central

    Arrese, Estela L.; Soulages, Jose L.

    2010-01-01

    The fat body plays major roles in the life of insects. It is a dynamic tissue involved in multiple metabolic functions. One of these functions is to store and release energy in response to the energy demands of the insect. Insects store energy reserves in the form of glycogen and triglycerides in the adipocytes, the main fat body cell. Insect adipocytes can store a great amount of lipid reserves as cytoplasmic lipid droplets. Lipid metabolism is essential for growth and reproduction and provides energy needed during extended nonfeeding periods. This review focuses on energy storage and release and summarizes current understanding of the mechanisms underlying these processes in insects. PMID:19725772

  12. Stem cell autotomy and niche interaction in different systems

    PubMed Central

    Dorn, David C; Dorn, August

    2015-01-01

    The best known cases of cell autotomy are the formation of erythrocytes and thrombocytes (platelets) from progenitor cells that reside in special niches. Recently, autotomy of stem cells and its enigmatic interaction with the niche has been reported from male germline stem cells (GSCs) in several insect species. First described in lepidopterans, the silkmoth, followed by the gipsy moth and consecutively in hemipterans, foremost the milkweed bug. In both, moths and the milkweed bug, GSCs form finger-like projections toward the niche, the apical cells (homologs of the hub cells in Drosophila). Whereas in the milkweed bug the projection terminals remain at the surface of the niche cells, in the gipsy moth they protrude deeply into the singular niche cell. In both cases, the projections undergo serial retrograde fragmentation with progressing signs of autophagy. In the gipsy moth, the autotomized vesicles are phagocytized and digested by the niche cell. In the milkweed bug the autotomized vesicles accumulate at the niche surface and disintegrate. Autotomy and sprouting of new projections appears to occur continuously. The significance of the GSC-niche interactions, however, remains enigmatic. Our concept on the signaling relationship between stem cell-niche in general and GSC and niche (hub cells and cyst stem cells) in particular has been greatly shaped by Drosophila melanogaster. In comparing the interactions of GSCs with their niche in Drosophila with those in species exhibiting GSC autotomy it is obvious that additional or alternative modes of stem cell-niche communication exist. Thus, essential signaling pathways, including niche-stem cell adhesion (E-cadherin) and the direction of asymmetrical GSC division - as they were found in Drosophila - can hardly be translated into the systems where GSC autotomy was reported. It is shown here that the serial autotomy of GSC projections shows remarkable similarities with Wallerian axonal destruction, developmental axon pruning and dying-back degeneration in neurodegenerative diseases. Especially the hypothesis of an existing evolutionary conserved “autodestruction program” in axons that might also be active in GSC projections appears attractive. Investigations on the underlying signaling pathways have to be carried out. There are two other well known cases of programmed cell autotomy: the enucleation of erythroblasts in the process of erythrocyte maturation and the segregation of thousands of thrombocytes (platelets) from one megakaryocyte. Both progenitor cell types - erythroblasts and megakaryocytes - are associated with a niche in the bone marrow, erythroblasts with a macrophage, which they surround, and the megakaryocytes with the endothelial cells of sinusoids and their extracellular matrix. Although the regulatory mechanisms may be specific in each case, there is one aspect that connects all described processes of programmed cell autotomy and neuronal autodestruction: apoptotic pathways play always a prominent role. Studies on the role of male GSC autotomy in stem cell-niche interaction have just started but are expected to reveal hitherto unknown ways of signal exchange. Spermatogenesis in mammals advance our understanding of insect spermatogenesis. Mammal and insect spermatogenesis share some broad principles, but a comparison of the signaling pathways is difficult. We have intimate knowledge from Drosophila, but of almost no other insect, and we have only limited knowledge from mammals. The discovery of stem cell autotomy as part of the interaction with the niche promises new general insights into the complicated stem cell-niche interdependence. PMID:26240680

  13. The components of shear stress affecting insect cells used with the baculovirus expression vector system.

    PubMed

    Weidner, Tobias; Druzinec, Damir; Mühlmann, Martina; Buchholz, Rainer; Czermak, Peter

    2017-09-26

    Insect-based expression platforms such as the baculovirus expression vector system (BEVS) are widely used for the laboratory- and industrial-scale production of recombinant proteins. Thereby, major drawbacks to gain high-quality proteins are the lytic infection cycle and the shear sensitivity of infected insect cells due to turbulence and aeration. Smaller bubbles were formerly assumed to be more harmful than larger ones, but we found that cell damage is also dependent on the concentration of protective agents such as Pluronic®. At the appropriate concentration, Pluronic forms a layer around air bubbles and hinders the attachment of cells, thus limiting the damage. In this context, we used microaeration to vary bubble sizes and confirmed that size is not the most important factor, but the total gas surface area in the reactor is. If the surface area exceeds a certain threshold, the concentration of Pluronic is no longer sufficient for cell protection. To investigate the significance of shear forces, a second study was carried out in which infected insect cells were cultivated in a hollow fiber module to protect them from shear forces. Both model studies revealed important aspects of the design and scale-up of BEVS processes for the production of recombinant proteins.

  14. Toxicological characteristics of edible insects in China: A historical review.

    PubMed

    Gao, Yu; Wang, Di; Xu, Meng-Lei; Shi, Shu-Sen; Xiong, Jin-Feng

    2018-04-10

    Edible insects are ideal food sources, which contain important nutrients and health-promoting compounds. With a rapid development of industrial insect farming, insect-derived food is a novel and emerging food industry. Edible insects have been traditionally consumed in various communities, while continuously gaining relevance in today's society; however, they currently remain underutilized. Although there are a large number of literature on edible insects, these literature primarily focus on the nutritional value edible insects. The toxicity assessment data of edible insects remain incomprehensive, especially for the new national standard that is currently in effect; and many data and conclusions are not accurately specified/reported. Therefore, we performed a literature review and summarized the data on the toxicological assessment of edible insects in China. The review first describes the research progress on safety toxicological assessment, and then offers references regarding the development of 34 edible insect species in China. These data can be a platform for the development of future toxicological assessment strategies, which can be carried out by a multidisciplinary team, possibly consisting of food engineers, agronomists, farmers, and so on, to improve the acceptability of edible insects. Copyright © 2018 Elsevier Ltd. All rights reserved.

  15. Roles for insulin and ecdysteroids in differentiation of an insect cell line of epidermal origin.

    PubMed

    Hatt, P J; Moriniere, M; Oberlander, H; Porcheron, P

    1994-10-01

    During postembryonic development of insects, molting cycles affect epidermal cells with alternate periods of proliferation and differentiation. Cells of the cell line established from imaginal discs of the Indian meal moth (IAL-PID2) differentiate under the action of the molting hormone, 20-hydroxyecdysone, in a manner that is meaningful in terms of the development of the tissue from which they were derived. In particular, the hormone caused an accumulation of the cells in the G2 phase of their cycle and induced the formation of epithelial-like aggregates and the synthesis of specific proteoglycans. Recent discovery of members of the insulin superfamily in insects and the role of growth factors played by this family of molecules in vertebrates led us to check for their potential effects on IAL-PID2 cell cycle regulation. On the one hand, our results showed that insulin was involved in partial resumption of the cell cycle after an arrest caused by serum deprivation, but that other growth factors present in fetal calf serum were needed for full completion of mitosis. On the other hand, the cytostatic effect of 20-hydroxyecdysone was reversible, and, prior exposure of the cells to the hormone allowed the cells to complete one cell cycle in serum-free medium. These results suggest that the production of autocrine growth factors induced by ecdysteroids could circumvent the absence of serum. This cell culture model provides potential for further study of interactions between ecdysteroids and growth factor homologs during differentiation of insect epidermal cells.

  16. Yellow fever virus envelope protein expressed in insect cells is capable of syncytium formation in lepidopteran cells and could be used for immunodetection of YFV in human sera.

    PubMed

    Barros, Maria C E S; Galasso, Tatiane G C M; Chaib, Antônio J M; Degallier, Nicolas; Nagata, Tatsuya; Ribeiro, Bergmann M

    2011-05-27

    Yellow fever is an haemorrhagic disease caused by a virus that belongs to the genus Flavivirus (Flaviviridae family) and is transmitted by mosquitoes. Among the viral proteins, the envelope protein (E) is the most studied one, due to its high antigenic potencial. Baculovirus are one of the most popular and efficient eukaryotic expression system. In this study a recombinant baculovirus (vSynYFE) containing the envelope gene (env) of the 17D vaccine strain of yellow fever virus was constructed and the recombinant protein antigenicity was tested. Insect cells infected with vSynYFE showed syncytium formation, which is a cytopathic effect characteristic of flavivirus infection and expressed a polypeptide of around 54 kDa, which corresponds to the expected size of the recombinant E protein. Furthermore, the recombinant E protein expression was also confirmed by fluorescence microscopy of vSynYFE-infected insect cells. Total vSynYFE-infected insect extracts used as antigens detected the presence of antibodies for yellow fever virus in human sera derived from yellow fever-infected patients in an immunoassay and did not cross react with sera from dengue virus-infected patients. The E protein expressed by the recombinant baculovirus in insect cells is antigenically similar to the wild protein and it may be useful for different medical applications, from improved diagnosis of the disease to source of antigens for the development of a subunit vaccine.

  17. A gene delivery system for insect cells mediated by arginine-rich cell-penetrating peptides.

    PubMed

    Chen, Yung-Jen; Liu, Betty Revon; Dai, Yun-Hao; Lee, Cheng-Yi; Chan, Ming-Huan; Chen, Hwei-Hsien; Chiang, Huey-Jenn; Lee, Han-Jung

    2012-02-10

    Most bioactive macromolecules, such as protein, DNA and RNA, basically cannot permeate into cells freely from outside the plasma membrane. Cell-penetrating peptides (CPPs) are a group of short peptides that possess the ability to traverse the cell membrane and have been considered as candidates for mediating gene and drug delivery into living cells. In this study, we demonstrate that three arginine-rich CPPs (SR9, HR9 and PR9) are able to form stable complexes with plasmid DNA and deliver DNA into insect Sf9 cells in a noncovalent manner. The transferred plasmid DNA containing enhanced green fluorescent protein (EGFP) and red fluorescent protein (RFP) coding regions could be expressed in cells functionally assayed at both the protein and RNA levels. Furthermore, treatment of cells with CPPs and CPP/DNA complexes resulted in a viability of 84-93% indicating these CPPs are not cytotoxic. These results suggest that arginine-rich CPPs appear to be a promising tool for insect transgenesis. Copyright © 2011 Elsevier B.V. All rights reserved.

  18. Insect response to plant defensive protease inhibitors.

    PubMed

    Zhu-Salzman, Keyan; Zeng, Rensen

    2015-01-07

    Plant protease inhibitors (PIs) are natural plant defense proteins that inhibit proteases of invading insect herbivores. However, their anti-insect efficacy is determined not only by their potency toward a vulnerable insect system but also by the response of the insect to such a challenge. Through the long history of coevolution with their host plants, insects have developed sophisticated mechanisms to circumvent antinutritional effects of dietary challenges. Their response takes the form of changes in gene expression and the protein repertoire in cells lining the alimentary tract, the first line of defense. Research in insect digestive proteases has revealed the crucial roles they play in insect adaptation to plant PIs and has brought about a new appreciation of how phytophagous insects employ this group of molecules in both protein digestion and counterdefense. This review provides researchers in related fields an up-to-date summary of recent advances.

  19. Plant Defense Inhibitors Affect the Structures of Midgut Cells in Drosophila melanogaster and Callosobruchus maculatus

    PubMed Central

    Li-Byarlay, Hongmei; Pittendrigh, Barry R.; Murdock, Larry L.

    2016-01-01

    Plants produce proteins such as protease inhibitors and lectins as defenses against herbivorous insects and pathogens. However, no systematic studies have explored the structural responses in the midguts of insects when challenged with plant defensive proteins and lectins across different species. In this study, we fed two kinds of protease inhibitors and lectins to the fruit fly Drosophila melanogaster and alpha-amylase inhibitors and lectins to the cowpea bruchid Callosobruchus maculatus. We assessed the changes in midgut cell structures by comparing them with such structures in insects receiving normal diets or subjected to food deprivation. Using light and transmission electron microscopy in both species, we observed structural changes in the midgut peritrophic matrix as well as shortened microvilli on the surfaces of midgut epithelial cells in D. melanogaster. Dietary inhibitors and lectins caused similar lesions in the epithelial cells but not much change in the peritrophic matrix in both species. We also noted structural damages in the Drosophila midgut after six hours of starvation and changes were still present after 12 hours. Our study provided the first evidence of key structural changes of midguts using a comparative approach between a dipteran and a coleopteran. Our particular observation and discussion on plant–insect interaction and dietary stress are relevant for future mode of action studies of plant defensive protein in insect physiology. PMID:27594789

  20. Plant Defense Inhibitors Affect the Structures of Midgut Cells in Drosophila melanogaster and Callosobruchus maculatus.

    PubMed

    Li-Byarlay, Hongmei; Pittendrigh, Barry R; Murdock, Larry L

    2016-01-01

    Plants produce proteins such as protease inhibitors and lectins as defenses against herbivorous insects and pathogens. However, no systematic studies have explored the structural responses in the midguts of insects when challenged with plant defensive proteins and lectins across different species. In this study, we fed two kinds of protease inhibitors and lectins to the fruit fly Drosophila melanogaster and alpha-amylase inhibitors and lectins to the cowpea bruchid Callosobruchus maculatus. We assessed the changes in midgut cell structures by comparing them with such structures in insects receiving normal diets or subjected to food deprivation. Using light and transmission electron microscopy in both species, we observed structural changes in the midgut peritrophic matrix as well as shortened microvilli on the surfaces of midgut epithelial cells in D. melanogaster. Dietary inhibitors and lectins caused similar lesions in the epithelial cells but not much change in the peritrophic matrix in both species. We also noted structural damages in the Drosophila midgut after six hours of starvation and changes were still present after 12 hours. Our study provided the first evidence of key structural changes of midguts using a comparative approach between a dipteran and a coleopteran. Our particular observation and discussion on plant-insect interaction and dietary stress are relevant for future mode of action studies of plant defensive protein in insect physiology.

  1. Spinosad induces programmed cell death involves mitochondrial dysfunction and cytochrome C release in Spodoptera frugiperda Sf9 cells.

    PubMed

    Yang, Mingjun; Wang, Bo; Gao, Jufang; Zhang, Yang; Xu, Wenping; Tao, Liming

    2017-02-01

    Spinosad, a reduced-risk insecticide, acts on the nicotinic acetylcholine receptors and the gamma-aminobutyric acid receptor in the nervous system of target insects. However, its mechanism of action in non-neural insect cells is unclear. This study aimed to evaluate mitochondrial functional changes associated with spinosad in Spodoptera frugiperda (Sf9) insect cells. Our results indicate that in Sf9 cells, spinosad induces programmed cell death and mitochondrial dysfunction through enhanced reactive oxygen species production, mitochondrial permeability transition pore (mPTP) opening, and mitochondrial membrane potential collapse, eventually leading to cytochrome C release and apoptosis. The cytochrome C release induced by spinosad treatment was partly inhibited by the mPTP inhibitors cyclosporin A and bongkrekic acid. Subsequently, we found that spinosad downregulated Bcl-2 expression and upregulated p53 and Bax expressions, activated caspase-9 and caspase-3, and triggered PARP cleavage in Sf9 cells. These findings suggested that spinosad-induced programmed cell death was modulated by mitochondrial dysfunction and cytochrome C release. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Rhythmic autocrine activity in cultured insect epidermal cells.

    PubMed

    Mesnier, M; Partiaoglou, N; Oberlander, H; Porcheron, P

    2000-05-01

    It is now well established that ecdysteroids can be produced in insects in the absence of prothoracic glands. In this respect, it has been shown that cells in culture can produce ecdysteroids. Our aims were: (1) to determine whether ecdysteroid target cells of epidermal origin could also be the source of ecdysteroids; (2) to monitor more accurately the kinetics of ecdysteroid production; and (3) to check for possible relationships between this synthetic activity and dynamics of cell division. An insect cell line (IAL-PID2) established from imaginal discs of the Indian meal moth, Plodia interpunctella, with wild-type sensitivity to ecdysteroids was used in our study. Our results showed that the Plodia cell line exhibited autocrine activity. When division of IAL-PID2 cells was synchronized, a rhythmic production of ecdysteroids was observed. However, further experiments indicated that this rhythmicity could be cell autonomous. This led us to anticipate the existence of two cell subpopulations that would be able to produce ecdysteroids rhythmically, a minor one that would be cell cycle serum-independent population, and a major population that would need serum growth factors to proliferate and produce ecdysteroids. Qualitative study of the ecdysteroid content of the media clearly showed that ecdysone was the major immunoreactive product. Taken together, our findings clearly show that an insect cell line of epidermal origin is capable of rhythmic autocrine production of ecdysteroids. These results support the hypothesis that alternate sites for ecdysteroid production in vivo may exist and could play a role in local regulation of development. We now plan to determine the cellular basis of this rhythmic autocrine activity and to confirm the existence of growth factor-autonomous cells in the culture as well as the potent role played by ecdysteroids in the cross-talk between various cell subpopulations. Copyright 2000 Wiley-Liss, Inc.

  3. Toxicity of the mycotoxin fumonisin B1 on the insect Sf9 cell line.

    PubMed

    Zhang, He; Zhang, Liyang; Diao, Xue; Li, Na; Liu, Chenglan

    2017-04-01

    Fumonisins are a type of mycotoxin produced by Fusarium spp., mainly F. proliferatum and F. vertieilliodes, and represent a potential hazard to the health of animals and human beings. The toxicity and mechanism of action of fumonisins is ambiguous, and it is unclear whether fumonisins are toxic to insect cells. This study examines the toxicity of fumonisin B 1 (FB 1 ) and its mechanism of action in the Spodoptera frugiperda Sf9 cell line. We found that FB 1 inhibited Sf9 cellular proliferation and arrested cell growth at the G 2 /M phase. Morphological observation showed that FB 1 induced swelling, vacuole formation, and loss of adhesion in Sf9 cells. Flow cytometry analysis showed that FB 1 caused depolarization of the cell membrane potential and hyperpolarization of the mitochondrial membrane potential. To uncover potential genes associated with the molecular mechanisms of FB 1 , 41 differentially expressed genes were identified by transcriptome analyses after FB 1 treatment. These genes are putatively involved in detoxification metabolism, insect hormone regulation, cell apoptosis, and other related processes. Finally, six differentially expressed genes were chosen and validated by quantitative real-time PCR (QRT-PCR). Our test could provide a reference for other kinds of insect cells studies on FB 1 stress. At the same time, our studies try to provide a possible for FB 1 as a precursor compounds of biological insecticide. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. The avian cell line AGE1.CR.pIX characterized by metabolic flux analysis

    PubMed Central

    2014-01-01

    Background In human vaccine manufacturing some pathogens such as Modified Vaccinia Virus Ankara, measles, mumps virus as well as influenza viruses are still produced on primary material derived from embryonated chicken eggs. Processes depending on primary cell culture, however, are difficult to adapt to modern vaccine production. Therefore, we derived previously a continuous suspension cell line, AGE1.CR.pIX, from muscovy duck and established chemically-defined media for virus propagation. Results To better understand vaccine production processes, we developed a stoichiometric model of the central metabolism of AGE1.CR.pIX cells and applied flux variability and metabolic flux analysis. Results were compared to literature dealing with mammalian and insect cell culture metabolism focusing on the question whether cultured avian cells differ in metabolism. Qualitatively, the observed flux distribution of this avian cell line was similar to distributions found for mammalian cell lines (e.g. CHO, MDCK cells). In particular, glucose was catabolized inefficiently and glycolysis and TCA cycle seem to be only weakly connected. Conclusions A distinguishing feature of the avian cell line is that glutaminolysis plays only a minor role in energy generation and production of precursors, resulting in low extracellular ammonia concentrations. This metabolic flux study is the first for a continuous avian cell line. It provides a basis for further metabolic analyses to exploit the biotechnological potential of avian and vertebrate cell lines and to develop specific optimized cell culture processes, e.g. vaccine production processes. PMID:25077436

  5. The avian cell line AGE1.CR.pIX characterized by metabolic flux analysis.

    PubMed

    Lohr, Verena; Hädicke, Oliver; Genzel, Yvonne; Jordan, Ingo; Büntemeyer, Heino; Klamt, Steffen; Reichl, Udo

    2014-07-30

    In human vaccine manufacturing some pathogens such as Modified Vaccinia Virus Ankara, measles, mumps virus as well as influenza viruses are still produced on primary material derived from embryonated chicken eggs. Processes depending on primary cell culture, however, are difficult to adapt to modern vaccine production. Therefore, we derived previously a continuous suspension cell line, AGE1.CR.pIX, from muscovy duck and established chemically-defined media for virus propagation. To better understand vaccine production processes, we developed a stoichiometric model of the central metabolism of AGE1.CR.pIX cells and applied flux variability and metabolic flux analysis. Results were compared to literature dealing with mammalian and insect cell culture metabolism focusing on the question whether cultured avian cells differ in metabolism. Qualitatively, the observed flux distribution of this avian cell line was similar to distributions found for mammalian cell lines (e.g. CHO, MDCK cells). In particular, glucose was catabolized inefficiently and glycolysis and TCA cycle seem to be only weakly connected. A distinguishing feature of the avian cell line is that glutaminolysis plays only a minor role in energy generation and production of precursors, resulting in low extracellular ammonia concentrations. This metabolic flux study is the first for a continuous avian cell line. It provides a basis for further metabolic analyses to exploit the biotechnological potential of avian and vertebrate cell lines and to develop specific optimized cell culture processes, e.g. vaccine production processes.

  6. Insect remote sensing using a polarization sensitive cw lidar system in chinese rice fields

    NASA Astrophysics Data System (ADS)

    Zhu, Shiming; Malmqvist, Elin; Li, Yiyun; Jansson, Samuel; Li, Wansha; Duan, Zheng; Fu, Wei; Svanberg, Katarina; Bood, Joakim; Feng, Hongqiang; Åkesson, Susanne; Song, Ziwei; Zhang, Baoxin; Zhao, Guangyu; Li, Dunsong; Brydegaard, Mikkel; Svanberg, Sune

    2018-04-01

    A joint Chinese-Swedish field campaign of Scheimpflug continuous-wave lidar monitoring of rice-field flying pest insects was pursued in very hot July weather conditions close to Guangzhou, China. The occurrence of insects, birds and bats with almost 200 hours of round-the-clock polarization-sensitive recordings was studied. Wing-beat frequency recordings and depolarization properties were used for target classification. Influence of weather conditions on the flying fauna was also investigated.

  7. Yellow fever virus envelope protein expressed in insect cells is capable of syncytium formation in lepidopteran cells and could be used for immunodetection of YFV in human sera

    PubMed Central

    2011-01-01

    Background Yellow fever is an haemorrhagic disease caused by a virus that belongs to the genus Flavivirus (Flaviviridae family) and is transmitted by mosquitoes. Among the viral proteins, the envelope protein (E) is the most studied one, due to its high antigenic potencial. Baculovirus are one of the most popular and efficient eukaryotic expression system. In this study a recombinant baculovirus (vSynYFE) containing the envelope gene (env) of the 17D vaccine strain of yellow fever virus was constructed and the recombinant protein antigenicity was tested. Results Insect cells infected with vSynYFE showed syncytium formation, which is a cytopathic effect characteristic of flavivirus infection and expressed a polypeptide of around 54 kDa, which corresponds to the expected size of the recombinant E protein. Furthermore, the recombinant E protein expression was also confirmed by fluorescence microscopy of vSynYFE-infected insect cells. Total vSynYFE-infected insect extracts used as antigens detected the presence of antibodies for yellow fever virus in human sera derived from yellow fever-infected patients in an immunoassay and did not cross react with sera from dengue virus-infected patients. Conclusions The E protein expressed by the recombinant baculovirus in insect cells is antigenically similar to the wild protein and it may be useful for different medical applications, from improved diagnosis of the disease to source of antigens for the development of a subunit vaccine. PMID:21619598

  8. Development of serum-free media for lepidopteran insect cell lines.

    PubMed

    Agathos, Spiros N

    2007-01-01

    Lepidopteran insect cell culture technology has progressed to the point of becoming an essential part of one of the most successful eukaryotic expression systems and is increasingly used industrially on a large scale. Therefore, there is a constant need for convenient and low-cost culture media capable of supporting good insect cell growth and ensuring high yield of baculovirus as well as the strong expression of recombinant proteins. Vertebrate sera or invertebrate hemolymph were essential supplements in first-generation insect cell media. These supplements, however, are cumbersome and expensive for routine large-scale culture; thus, their use is now circumvented by substituting the essential growth factors present in these supplements with serum-free substances. Such non-serum supplements are typically of non-animal origin and include protein hydrolysates, lipid emulsions, and specialized substances (e.g., surfactants and shear damage protecting chemicals). These supplements need to complement the defined, synthetic basal medium to ensure that the fundamental nutritional needs of the cells are satisfied. Although there is a significant number of proprietary serum-free and low-protein or protein-free media on the market, the lack of information concerning their detailed composition is a drawback in their adoption for different applications, including their adaptation to the metabolic and kinetic analysis and monitoring of a given insect cell based bioprocess. Hence, there is wide appeal for formulating serum-free media based on a rational assessment of the metabolic requirements of the lepidopteran cells during both the growth and the production phases. Techniques such as statistical experimental design and genetic algorithms adapted to the cellular behavior and the bioreactor operation mode (batch, fed-batch, or perfusion) permit the formulation of versatile serum- and protein-free media. These techniques are illustrated with recent developments of serum-free media for the cultivation of commercially important Spodoptera frugiperda and Trichoplusia ni cell lines.

  9. Distribution of the Primary Endosymbiont (Candidatus Uzinura Diaspidicola) Within Host Insects from the Scale Insect Family Diaspididae

    PubMed Central

    Gruwell, Matthew E.; Flarhety, Meghan; Dittmar, Katharina

    2012-01-01

    It has long been known that armored scale insects harbor endosymbiotic bacteria inside specialized cells called bacteriocytes. Originally, these endosymbionts were thought to be fungal symbionts but they are now known to be bacterial and have been named Uzinura diaspidicola. Bacteriocyte and endosymbiont distribution patterns within host insects were visualized using in situ hybridization via 16S rRNA specific probes. Images of scale insect embryos, eggs and adult scale insects show patterns of localized bacteriocytes in embryos and randomly distributed bacteriocytes in adults. The symbiont pocket was not found in the armored scale insect eggs that were tested. The pattern of dispersed bacteriocytes in adult scale insects suggest that Uzinura and Blattabacteria may share some homologous traits that coincide with similar life style requirements, such as dispersal in fat bodies and uric acid recycling. PMID:26467959

  10. T Cell Gene Therapy to Eradicate Disseminated Breast Cancers

    DTIC Science & Technology

    2012-05-01

    examined in a mouse engraftment model. 50x106 mouse T cells transduced with anti-CEA IgTCR and Tandem CARs were injected i.v. into 350 rads γ- irradiated ...proteins in insect cell expression system for testing their effectiveness in inhibiting tick feeding by using them as vaccines to immunize the host...genes essential for sperm development in the male tick Amblyomma hebraeum Koch (Acari: Ixodidae). Insect Biochem Mol Biol. 2008 Jul; 38 (7): 721-729

  11. Gradients of metabolite accumulation and redifferentiation of nutritive cells associated with vascular tissues in galls induced by sucking insects

    PubMed Central

    Carneiro, Renê Gonçalves da Silva; Isaias, Rosy Mary dos Santos

    2015-01-01

    Plant cells respond to abiotic and biotic stimuli, which generate adaptive phenotypes in plant organs. In the case of plant galls, cell phenotypes are adaptive for the gall inducer and assume characteristics mainly linked to its protection and nutrition. Herein, the cytological development and histochemical profile of Nothotrioza cattleiani galls, a sucking insect, on the leaves of Psidium cattleianum are compared with those of other galls, especially N. myrtoidis galls, searching for conserved and divergent alterations in cell fates and cycles. Leaf cell fates are completely changed within galls, except for epidermal cells, but the comparison between Nothotrioza spp. galls shows conserved fates. Nevertheless, cytological development of N. cattleiani galls is different from the standby-redifferentiation of N. myrtoidis galls. Starch and lignins, and reducing sugars form centrifugal and centripetal gradients of accumulation, respectively. Proteins, total phenolics, terpenoids, proanthocyanidins and reactive oxygen species are detected in bidirectional gradients, i.e. weak or undetectable reaction in the median cortical cells that is gradually more intense in the cell layers towards the inner and outer surfaces of the gall. True nutritive cells associated with vascular tissues, together with the bidirectional gradients of metabolite accumulation, are herein reported for the first time in insect galls. The globoid galls of N. cattleiani, though macro-morphologically similar to the galls of N. myrtoidis, are distinct and unique among insect galls, as far as the cellular, subcellular and histochemical traits are concerned. Thus, the traits of the galls on P. cattleianum studied herein represent the extended phenotypes of their inducers. PMID:26209687

  12. Studies of the Biology of Phleboviruses in Sandflies.

    DTIC Science & Technology

    1983-02-01

    8217Ahiebotomus fever, sand fly fever, arbovirus, medical entomology, Phiebotomus, Lutzomyia, vector-borne diseases, insect cell cultures, _)_Laboratory...parenteral administration. Most of the viruses replicated in sand flies after intrathorazic inoculation; however, the insects were quite refractory to oral...cells was also established and tested for its ability to support the growth of a number of different arbaviruses. Most of the rhabdoviruses

  13. Localization of Bacillus thuringiensis Cry1A toxin-binding molecules in gypsy moth larval gut sections using fluorescence microscopy

    Treesearch

    Algimantas P. Valaitis

    2011-01-01

    The microbial insecticide Bacillus thuringiensis (Bt) produces Cry toxins, proteins that bind to the brush border membranes of gut epithelial cells of insects that ingest it, disrupting the integrity of the membranes, and leading to cell lysis and insect death. In gypsy moth, Lymantria dispar, two toxin-binding molecules for the...

  14. Genome scale transcriptomics of baculovirus-insect interactions.

    PubMed

    Nguyen, Quan; Nielsen, Lars K; Reid, Steven

    2013-11-12

    Baculovirus-insect cell technologies are applied in the production of complex proteins, veterinary and human vaccines, gene delivery vectors' and biopesticides. Better understanding of how baculoviruses and insect cells interact would facilitate baculovirus-based production. While complete genomic sequences are available for over 58 baculovirus species, little insect genomic information is known. The release of the Bombyx mori and Plutella xylostella genomes, the accumulation of EST sequences for several Lepidopteran species, and especially the availability of two genome-scale analysis tools, namely oligonucleotide microarrays and next generation sequencing (NGS), have facilitated expression studies to generate a rich picture of insect gene responses to baculovirus infections. This review presents current knowledge on the interaction dynamics of the baculovirus-insect system' which is relatively well studied in relation to nucleocapsid transportation, apoptosis, and heat shock responses, but is still poorly understood regarding responses involved in pro-survival pathways, DNA damage pathways, protein degradation, translation, signaling pathways, RNAi pathways, and importantly metabolic pathways for energy, nucleotide and amino acid production. We discuss how the two genome-scale transcriptomic tools can be applied for studying such pathways and suggest that proteomics and metabolomics can produce complementary findings to transcriptomic studies.

  15. Sterile-Insect Methods for Control of Mosquito-Borne Diseases: An Analysis

    PubMed Central

    Benedict, Mark; Bellini, Romeo; Clark, Gary G.; Dame, David A.; Service, Mike W.; Dobson, Stephen L.

    2010-01-01

    Abstract Effective vector control, and more specifically mosquito control, is a complex and difficult problem, as illustrated by the continuing prevalence (and spread) of mosquito-transmitted diseases. The sterile insect technique and similar methods control certain agricultural insect pest populations in a species-specific, environmentally sound, and effective manner; there is increased interest in applying this approach to vector control. Such an approach, like all others in use and development, is not a one-size-fits-all solution, and will be more appropriate in some situations than others. In addition, the proposed release of pest insects, and more so genetically modified pest insects, is bound to raise questions in the general public and the scientific community as to such a method's efficacy, safety, and sustainability. This article attempts to address these concerns and indicate where sterile-insect methods are likely to be useful for vector control. PMID:19725763

  16. Insect cells-baculovirus system for the production of difficult to express proteins.

    PubMed

    Osz-Papai, Judit; Radu, Laura; Abdulrahman, Wassim; Kolb-Cheynel, Isabelle; Troffer-Charlier, Nathalie; Birck, Catherine; Poterszman, Arnaud

    2015-01-01

    The production of sufficient quantities of homogenous protein not only is an essential prelude for structural investigations but also represents a rate-limiting step for many human functional studies. Although technologies for expression of recombinant proteins and complexes have been improved tremendously, in many cases, protein production remains a challenge and can be associated with considerable investment. This chapter describes simple and efficient protocols for expression screening and optimization of protein production in insect cells using the baculovirus expression system. We describe the procedure, starting from the cloning of a gene of interest into an expression transfer baculovirus vector, followed by generation of the recombinant virus by homologous recombination, evaluation of protein expression, and scale-up. Handling of insect cell cultures and preparation of bacmid for co-transfection are also detailed.

  17. Air-dried cells from the anhydrobiotic insect, Polypedilum vanderplanki, can survive long term preservation at room temperature and retain proliferation potential after rehydration.

    PubMed

    Watanabe, Kazuyo; Imanishi, Shigeo; Akiduki, Gaku; Cornette, Richard; Okuda, Takashi

    2016-08-01

    Pv11, a cell line derived from the anhydrobiotic insect, Polypedilum vanderplanki, was preserved in a dry form (only 6% residual moisture) at room temperature for up to 251 days and restarted proliferating after rehydration. A previous study already reported survival of Pv11 cells after desiccation, but without subsequent proliferation. Here, the protocol was improved to increase survival and achieve proliferation of Pv11 cells after dry storage. The method basically included preincubation, desiccation and rehydration processes and each step was investigated. So far, preincubation in a 600 mM trehalose solution for 48 h before dehydration was the most favourable preconditioning to achieve successful dry preservation of Pv11 cells, allowing about 16% of survival after rehydration and subsequent cell proliferation. Although the simple air-dry method established for Pv11 cells here was not applicable for successful dry-preservation of other insect cell lines, Pv11 is the first dry-preservable animal cell line and will surely contribute not only to basic but also applied sciences. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  18. The significance of multiple mating in the social wasp Vespula maculifrons.

    PubMed

    Goodisman, Michael A D; Kovacs, Jennifer L; Hoffman, Eric A

    2007-09-01

    The evolution of the complex societies displayed by social insects depended partly on high relatedness among interacting group members. Therefore, behaviors that depress group relatedness, such as multiple mating by reproductive females (polyandry), are unexpected in social insects. Nevertheless, the queens of several social insect species mate multiply, suggesting that polyandry provides some benefits that counteract the costs. However, few studies have obtained evidence for links between rates of polyandry and fitness in naturally occurring social insect populations. We investigated if polyandry was beneficial in the social wasp Vespula maculifrons. We used genetic markers to estimate queen mate number in V. maculifrons colonies and assessed colony fitness by counting the number of cells that colonies produced. Our results indicated that queen mate number was directly, strongly, and significantly correlated with the number of queen cells produced by colonies. Because V. maculifrons queens are necessarily reared in queen cells, our results demonstrate that high levels of polyandry are associated with colonies capable of producing many new queens. These data are consistent with the explanation that polyandry is adaptive in V. maculifrons because it provides a fitness advantage to queens. Our research may provide a rare example of an association between polyandry and fitness in a natural social insect population and help explain why queens in this taxon mate multiply.

  19. Siberian forest insects: ready for export

    Treesearch

    Yuri N. Baranchikov

    1998-01-01

    Existing publications on Palaearctic insect invaders to North America forests are devoted exclusively to Europe-U.S. comparisons. This is understandable from both geographical and historical points of view. But as the history of mankind continues, new economical relations are established which, unfortunately, make new possibilities for the introductions of pests. The...

  20. Insects and Diseases of Cottonwood

    Treesearch

    R.C. Morris; T.H. Filer; J.D. Solomon; Francis I. McCracken; N.A. Overgaard; M.J. Weiss

    1975-01-01

    Insects and disease organisms are a continuing threat to cottonwood (Populus deltoides Bartr.), especially during the tree's first 5 years. The danger is intensified in large plantings of a single species and age because rapid buildup of damaging agents can occur. This booklet, will help forest nurserymen and plantation managers identify and...

  1. Ionotropic Receptor-dependent moist and dry cells control hygrosensation in Drosophila.

    PubMed

    Knecht, Zachary A; Silbering, Ana F; Cruz, Joyner; Yang, Ludi; Croset, Vincent; Benton, Richard; Garrity, Paul A

    2017-06-16

    Insects use hygrosensation (humidity sensing) to avoid desiccation and, in vectors such as mosquitoes, to locate vertebrate hosts. Sensory neurons activated by either dry or moist air ('dry cells' and 'moist cells') have been described in many insects, but their behavioral roles and the molecular basis of their hygrosensitivity remain unclear. We recently reported that Drosophila hygrosensation relies on three Ionotropic Receptors (IRs) required for dry cell function: IR25a, IR93a and IR40a (Knecht et al., 2016). Here, we discover Drosophila moist cells and show that they require IR25a and IR93a together with IR68a, a conserved, but orphan IR. Both IR68a- and IR40a-dependent pathways drive hygrosensory behavior: each is important for dry-seeking by hydrated flies and together they underlie moist-seeking by dehydrated flies. These studies reveal that humidity sensing in Drosophila , and likely other insects, involves the combined activity of two molecularly related but neuronally distinct hygrosensing systems.

  2. Autophagy pathway induced by a plant virus facilitates viral spread and transmission by its insect vector.

    PubMed

    Chen, Yong; Chen, Qian; Li, Manman; Mao, Qianzhuo; Chen, Hongyan; Wu, Wei; Jia, Dongsheng; Wei, Taiyun

    2017-11-01

    Many viral pathogens are persistently transmitted by insect vectors and cause agricultural or health problems. Generally, an insect vector can use autophagy as an intrinsic antiviral defense mechanism against viral infection. Whether viruses can evolve to exploit autophagy to promote their transmission by insect vectors is still unknown. Here, we show that the autophagic process is triggered by the persistent replication of a plant reovirus, rice gall dwarf virus (RGDV) in cultured leafhopper vector cells and in intact insects, as demonstrated by the appearance of obvious virus-containing double-membrane autophagosomes, conversion of ATG8-I to ATG8-II and increased level of autophagic flux. Such virus-containing autophagosomes seem able to mediate nonlytic viral release from cultured cells or facilitate viral spread in the leafhopper intestine. Applying the autophagy inhibitor 3-methyladenine or silencing the expression of Atg5 significantly decrease viral spread in vitro and in vivo, whereas applying the autophagy inducer rapamycin or silencing the expression of Torc1 facilitate such viral spread. Furthermore, we find that activation of autophagy facilitates efficient viral transmission, whereas inhibiting autophagy blocks viral transmission by its insect vector. Together, these results indicate a plant virus can induce the formation of autophagosomes for carrying virions, thus facilitating viral spread and transmission by its insect vector. We believe that such a role for virus-induced autophagy is common for vector-borne persistent viruses during their transmission by insect vectors.

  3. Autophagy pathway induced by a plant virus facilitates viral spread and transmission by its insect vector

    PubMed Central

    Mao, Qianzhuo; Chen, Hongyan; Wu, Wei

    2017-01-01

    Many viral pathogens are persistently transmitted by insect vectors and cause agricultural or health problems. Generally, an insect vector can use autophagy as an intrinsic antiviral defense mechanism against viral infection. Whether viruses can evolve to exploit autophagy to promote their transmission by insect vectors is still unknown. Here, we show that the autophagic process is triggered by the persistent replication of a plant reovirus, rice gall dwarf virus (RGDV) in cultured leafhopper vector cells and in intact insects, as demonstrated by the appearance of obvious virus-containing double-membrane autophagosomes, conversion of ATG8-I to ATG8-II and increased level of autophagic flux. Such virus-containing autophagosomes seem able to mediate nonlytic viral release from cultured cells or facilitate viral spread in the leafhopper intestine. Applying the autophagy inhibitor 3-methyladenine or silencing the expression of Atg5 significantly decrease viral spread in vitro and in vivo, whereas applying the autophagy inducer rapamycin or silencing the expression of Torc1 facilitate such viral spread. Furthermore, we find that activation of autophagy facilitates efficient viral transmission, whereas inhibiting autophagy blocks viral transmission by its insect vector. Together, these results indicate a plant virus can induce the formation of autophagosomes for carrying virions, thus facilitating viral spread and transmission by its insect vector. We believe that such a role for virus-induced autophagy is common for vector-borne persistent viruses during their transmission by insect vectors. PMID:29125860

  4. Standardization of a Continuous Assay for Glycosidases and Its Use for Screening Insect Gut Samples at Individual and Populational Levels

    PubMed Central

    Profeta, Gerson S.; Pereira, Jessica A. S.; Costa, Samara G.; Azambuja, Patricia; Garcia, Eloi S.; Moraes, Caroline da Silva; Genta, Fernando A.

    2017-01-01

    Glycoside Hydrolases (GHs) are enzymes able to recognize and cleave glycosidic bonds. Insect GHs play decisive roles in digestion, in plant-herbivore, and host-pathogen interactions. GH activity is normally measured by the detection of a release from the substrate of products as sugars units, colored, or fluorescent groups. In most cases, the conditions for product release and detection differ, resulting in discontinuous assays. The current protocols result in using large amounts of reaction mixtures for the obtainment of time points in each experimental replica. These procedures restrain the analysis of biological materials with limited amounts of protein and, in the case of studies regarding small insects, implies in the pooling of samples from several individuals. In this respect, most studies do not assess the variability of GH activities across the population of individuals from the same species. The aim of this work is to approach this technical problem and have a deeper understanding of the variation of GH activities in insect populations, using as models the disease vectors Rhodnius prolixus (Hemiptera: Triatominae) and Lutzomyia longipalpis (Diptera: Phlebotominae). Here we standardized continuous assays using 4-methylumbelliferyl derived substrates for the detection of α-Glucosidase, β-Glucosidase, α-Mannosidase, N-acetyl-hexosaminidase, β-Galactosidase, and α-Fucosidase in the midgut of R. prolixus and L. longipalpis with results similar to the traditional discontinuous protocol. The continuous assays allowed us to measure GH activities using minimal sample amounts with a higher number of measurements, resulting in data that are more reliable and less time and reagent consumption. The continuous assay also allows the high-throughput screening of GH activities in small insect samples, which would be not applicable to the previous discontinuous protocol. We applied continuous GH measurements to 90 individual samples of R. prolixus anterior midgut homogenates using a high-throughput protocol. α-Glucosidase and α-Mannosidase activities showed the normal distribution in the population. β-Glucosidase, β-Galactosidase, N-acetyl-hexosaminidase, and α-Fucosidase activities showed non-normal distributions. These results indicate that GHs fluorescent-based high-throughput assays apply to insect samples and that the frequency distribution of digestive activities should be considered in data analysis, especially if a small number of samples is used. PMID:28553236

  5. Topical Hazard Evaluation Program of Candidate Insect Repellent AI3-20816-a, US Department of Agriculture Proprietary Compound, September 1978-November 1980.

    DTIC Science & Technology

    1981-02-11

    GROUND, MD 21010 TOPICAL HAZARD EVALUATION PROGRAM OF CANDIDATE INSECT REPELLENT AI 3- 20816 -aUS DEPARTMENT OF AGRICULTURE PROPRIETARY COMPOUND STUDY NO...irritation A13- 20816 -a Photochemical irritation Topical Hazard Evaluation Sensitization Candidate repellent ALD Skin irritation 20. ABSTRACT (Continue an...reverse e e if necessary and identify by block number) A hazard evaluation of candidate insect repellent A13- 20816 -a was performed by means of laboratory

  6. Chandipura virus growth kinetics in vertebrate cell lines, insect cell lines & embryonated eggs.

    PubMed

    Jadi, R S; Sudeep, A B; Kumar, Satyendra; Arankalle, V A; Mishra, A C

    2010-08-01

    Since not much information on Chandipura virus is available, an attempt was made to study the growth kinetics of the virus in certain vertebrate, invertebrate cell lines and embryonated chicken eggs. Comparative study of Chandipura virus (CHPV) growth kinetics in three vertebrate cell lines [Vero E6, Rhabdo myosarcoma (RD), Porcine stable kidney (PS) cell lines], two insect cell lines [Aedes aegypti (AA) and Phlebotomus papatasi (PP-9) cell lines] and embryonated pathogen free chicken eggs was conducted, by tissue culture infective dose 50 per cent (TCID(50)) and indirect immunofluorescence assay (IFA). All the cell lines and embryonated egg supported the growth of CHPV and yielded high virus titre. The vertebrate cell lines showed distinct cytopathic effect (CPE) within 4-6 h post infection (PI), while no CPE was observed in insect cell lines. PP-9 cell line was the most sensitive system to CHPV as viral antigen could be detected at 1 h PI by IFA. Our results demonstrated that all the systems were susceptible to CHPV and achieved high yield of virus. However, the PP-9 cell line had an edge over the others due to its high sensitivity to the virus which might be useful for detection and isolation of the virus during epidemics.

  7. Histology and cell wall biochemistry of stone cells in the physical defence of conifers against insects.

    PubMed

    Whitehill, Justin G A; Henderson, Hannah; Schuetz, Mathias; Skyba, Oleksandr; Yuen, Macaire Man Saint; King, John; Samuels, A Lacey; Mansfield, Shawn D; Bohlmann, Jörg

    2016-08-01

    Conifers possess an array of physical and chemical defences against stem-boring insects. Stone cells provide a physical defence associated with resistance against bark beetles and weevils. In Sitka spruce (Picea sitchensis), abundance of stone cells in the cortex of apical shoots is positively correlated with resistance to white pine weevil (Pissodes strobi). We identified histological, biochemical and molecular differences in the stone cell phenotype of weevil resistant (R) or susceptible (S) Sitka spruce genotypes. R trees displayed significantly higher quantities of cortical stone cells near the apical shoot node, the primary site for weevil feeding. Lignin, cellulose, xylan and mannan were the most abundant components of stone cell secondary walls, respectively. Lignin composition of stone cells isolated from R trees contained a higher percentage of G-lignin compared with S trees. Transcript profiling revealed higher transcript abundance in the R genotype of coumarate 3-hydroxylase, a key monolignol biosynthetic gene. Developing stone cells in current year apical shoots incorporated fluorescent-tagged monolignol into the secondary cell wall, while mature stone cells of previous year apical shoots did not. Stone cell development is an ephemeral process, and fortification of shoot tips in R trees is an effective strategy against insect feeding. © 2015 John Wiley & Sons Ltd.

  8. Protein Expression in Insect and Mammalian Cells Using Baculoviruses in Wave Bioreactors.

    PubMed

    Kadwell, Sue H; Overton, Laurie K

    2016-01-01

    Many types of disposable bioreactors for protein expression in insect and mammalian cells are now available. They differ in design, capacity, and sensor options, with many selections available for either rocking platform, orbitally shaken, pneumatically mixed, or stirred-tank bioreactors lined with an integral disposable bag (Shukla and Gottschalk, Trends Biotechnol 31(3):147-154, 2013). WAVE Bioreactors™ were among the first disposable systems to be developed (Singh, Cytotechnology 30:149-158, 1999). Since their commercialization in 1999, Wave Bioreactors have become routinely used in many laboratories due to their ease of operation, limited utility requirements, and protein expression levels comparability to traditional stirred-tank bioreactors. Wave Bioreactors are designed to use a presterilized Cellbag™, which is attached to a rocking platform and inflated with filtered air provided by the bioreactor unit. The Cellbag can be filled with medium and cells and maintained at a set temperature. The rocking motion, which is adjusted through angle and rock speed settings, provides mixing of oxygen (and CO2, which is used to control pH in mammalian cell cultures) from the headspace created in the inflated Cellbag with the cell culture medium and cells. This rocking motion can be adjusted to prevent cell shear damage. Dissolved oxygen and pH can be monitored during scale-up, and samples can be easily removed to monitor other parameters. Insect and mammalian cells grow very well in Wave Bioreactors (Shukla and Gottschalk, Trends Biotechnol 31(3):147-154, 2013). Combining Wave Bioreactor cell growth capabilities with recombinant baculoviruses engineered for insect or mammalian cell expression has proven to be a powerful tool for rapid production of a wide range of proteins.

  9. Development of insect resistant maize plants expressing a chitinase gene from the cotton leaf worm, Spodoptera littoralis

    PubMed Central

    Osman, Gamal H.; Assem, Shireen K.; Alreedy, Rasha M.; El-Ghareeb, Doaa K.; Basry, Mahmoud A.; Rastogi, Anshu; Kalaji, Hazem M.

    2015-01-01

    Due to the importance of chitinolytic enzymes for insect, nematode and fungal growth, they are receiving attention concerning their development as biopesticides or chemical defense proteins in transgenic plants and as microbial biocontrol agents. Targeting chitin associated with the extracellular matrices or cell wall by insect chitinases may be an effective approach for controlling pest insects and pathogenic fungi. The ability of chitinases to attack and digest chitin in the peritrophic matrix or exoskeleton raises the possibility to use them as insect control method. In this study, an insect chitinase cDNA from cotton leaf worm (Spodoptera littoralis) has been synthesized. Transgenic maize plant system was used to improve its tolerance against insects. Insect chitinase transcripts and proteins were expressed in transgenic maize plants. The functional integrity and expression of chitinase in progenies of the transgenic plants were confirmed by insect bioassays. The bioassays using transgenic corn plants against corn borer (Sesamia cretica) revealed that ~50% of the insects reared on transgenic corn plants died, suggesting that transgenic maize plants have enhanced resistance against S. cretica. PMID:26658494

  10. The evolutionary development of plant-feeding insects and their nutritional endosymbionts.

    PubMed

    Skidmore, Isabel H; Hansen, Allison K

    2017-12-01

    Herbivorous insects have evolved diverse mechanisms enabling them to feed on plants with suboptimal nutrient availability. Low nutrient availability negatively impacts insect herbivore development and fitness. To overcome this obstacle numerous insect lineages have evolved intimate associations with nutritional endosymbionts. This is especially true for insects that specialize on nitrogen-poor substrates, as these insects are highly dependent on intracellular symbionts to provide nitrogen lacking in their insect host's diet. Emerging evidence in these systems suggest that the symbiont's and/or the insect's biosynthetic pathways are dynamically regulated throughout the insect's development to potentially cope with the insect's changing nutritional demands. In this review, we evaluate the evolutionary development of symbiotic insect cells (bacteriocytes) by comparing and contrasting genes and mechanisms involved in maintaining and regulating the nutritional symbiosis throughout insect development in a diversity of insect herbivore-endosymbiont associations. With new advances in genome sequencing and functional genomics, we evaluate to what extent nutritional symbioses are shaped by (i) the regulation of symbiont titer, (ii) the regulation of insect symbiosis genes, and (iii) the regulation of symbiont genes. We discuss how important these mechanisms are for the biosynthesis of essential amino acids and vitamins across insect life stages in divergent insect-symbiont systems. We conclude by suggesting future directions of research to further elucidate the evolutionary development of bacteriocytes and the impact of these nutritional symbioses on insect-plant interactions. © 2017 Institute of Zoology, Chinese Academy of Sciences.

  11. Single inverted terminal repeats of the Junonia coenia Densovirus promotes somatic chromosomal integration of vector plasmids in insect cells and supports high efficiency expression

    USDA-ARS?s Scientific Manuscript database

    Plasmids that contain a disrupted genome of the Junonia coenia densovirus (JcDNV) integrate into the chromosomes of the somatic cells of insects. When subcloned individually, both the P9 inverted terminal repeat (P9-ITR) and the P93-ITR promote the chromosomal integration of vector plasmids in insec...

  12. Interaction of compass sensing and object-motion detection in the locust central complex.

    PubMed

    Bockhorst, Tobias; Homberg, Uwe

    2017-07-01

    Goal-directed behavior is often complicated by unpredictable events, such as the appearance of a predator during directed locomotion. This situation requires adaptive responses like evasive maneuvers followed by subsequent reorientation and course correction. Here we study the possible neural underpinnings of such a situation in an insect, the desert locust. As in other insects, its sense of spatial orientation strongly relies on the central complex, a group of midline brain neuropils. The central complex houses sky compass cells that signal the polarization plane of skylight and thus indicate the animal's steering direction relative to the sun. Most of these cells additionally respond to small moving objects that drive fast sensory-motor circuits for escape. Here we investigate how the presentation of a moving object influences activity of the neurons during compass signaling. Cells responded in one of two ways: in some neurons, responses to the moving object were simply added to the compass response that had adapted during continuous stimulation by stationary polarized light. By contrast, other neurons disadapted, i.e., regained their full compass response to polarized light, when a moving object was presented. We propose that the latter case could help to prepare for reorientation of the animal after escape. A neuronal network based on central-complex architecture can explain both responses by slight changes in the dynamics and amplitudes of adaptation to polarized light in CL columnar input neurons of the system. NEW & NOTEWORTHY Neurons of the central complex in several insects signal compass directions through sensitivity to the sky polarization pattern. In locusts, these neurons also respond to moving objects. We show here that during polarized-light presentation, responses to moving objects override their compass signaling or restore adapted inhibitory as well as excitatory compass responses. A network model is presented to explain the variations of these responses that likely serve to redirect flight or walking following evasive maneuvers. Copyright © 2017 the American Physiological Society.

  13. Combinatory annotation of cell membrane receptors and signalling pathways of Bombyx mori prothoracic glands

    PubMed Central

    Moulos, Panagiotis; Samiotaki, Martina; Panayotou, George; Dedos, Skarlatos G.

    2016-01-01

    The cells of prothoracic glands (PG) are the main site of synthesis and secretion of ecdysteroids, the biochemical products of cholesterol conversion to steroids that shape the morphogenic development of insects. Despite the availability of genome sequences from several insect species and the extensive knowledge of certain signalling pathways that underpin ecdysteroidogenesis, the spectrum of signalling molecules and ecdysteroidogenic cascades is still not fully comprehensive. To fill this gap and obtain the complete list of cell membrane receptors expressed in PG cells, we used combinatory bioinformatic, proteomic and transcriptomic analysis and quantitative PCR to annotate and determine the expression profiles of genes identified as putative cell membrane receptors of the model insect species, Bombyx mori, and subsequently enrich the repertoire of signalling pathways that are present in its PG cells. The genome annotation dataset we report here highlights modules and pathways that may be directly involved in ecdysteroidogenesis and aims to disseminate data and assist other researchers in the discovery of the role of such receptors and their ligands. PMID:27576083

  14. Using a cellular model to explore human-facilitated spread of risk of EAB in Minnesota

    Treesearch

    Anantha Prasad; Louis Iverson; Matthew Peters; Steve Matthews

    2011-01-01

    The Emerald Ash Borer has made inroads to Minnesota in the past two years, killing ash trees. We use our spatially explicit cell based model called EAB-SHIFT to calculate the risk of infestation owing to flight characteristics and short distance movement of the insect (insect flight model, IFM), and the human facilitated agents like roads, campgrounds etc. (insect ride...

  15. Insect anaphylaxis: where are we? The stinging facts 2012.

    PubMed

    Tracy, James M; Khan, Fatima S; Demain, Jeffrey G

    2012-08-01

    Insect allergy remains an important cause of morbidity and mortality in the United States. In 2011, the third iteration of the stinging insect hypersensitivity practice parameter was published, the first being published in 1999 and the second in 2004. Since the 2004 edition, our understanding of insect hypersensitivity has continued to expand and has been incorporated into the 2011 edition. This work will review the relevant changes in the management of insect hypersensitivity occurring since 2004 and present our current understanding of the insect hypersensitivity diagnosis and management. Since the 2004 commissioning by the Joint Task Force (JTF) on Practice Parameters of 'Stinging insect hypersensitivity: a practice parameter update', there have been important contributions to our understanding of insect allergy. These contributions were incorporated into the 2011 iteration. Similar efforts were made by the European Allergy Asthma and Clinical Immunology Interest Group in 2005 and most recently in 2011 by the British Society of Allergy and Clinical Immunology. Our understanding of insect allergy, including the natural history, epidemiology, diagnostic testing, and risk factors, has greatly expanded. This evolution of knowledge should provide improved long-term management of stinging insect hypersensitivity. This review will focus primarily on the changes between the 2004 and 2011 stinging insect practice parameter commissioned by the JTF on Practice Parameters, but will, where appropriate, highlight the differences between working groups.

  16. Three-way interaction among plants, bacteria, and coleopteran insects.

    PubMed

    Wielkopolan, Beata; Obrępalska-Stęplowska, Aleksandra

    2016-08-01

    Coleoptera, the largest and the most diverse Insecta order, is characterized by multiple adaptations to plant feeding. Insect-associated microorganisms can be important mediators and modulators of interactions between insects and plants. Interactions between plants and insects are highly complex and involve multiple factors. There are various defense mechanisms initiated by plants upon attack by herbivorous insects, including the development of morphological structures and the synthesis of toxic secondary metabolites and volatiles. In turn, herbivores have adapted to feeding on plants and further sophisticated adaptations to overcome plant responses may continue to evolve. Herbivorous insects may detoxify toxic phytocompounds, sequester poisonous plant factors, and alter their own overall gene expression pattern. Moreover, insects are associated with microbes, which not only considerably affect insects, but can also modify plant defense responses to the benefit of their host. Plants are also frequently associated with endophytes, which may act as bioinsecticides. Therefore, it is very important to consider the factors influencing the interaction between plants and insects. Herbivorous insects cause considerable damage to global crop production. Coleoptera is the largest and the most diverse order in the class Insecta. In this review, various aspects of the interactions among insects, microbes, and plants are described with a focus on coleopteran species, their bacterial symbionts, and their plant hosts to demonstrate that many factors contribute to the success of coleopteran herbivory.

  17. Insects as model systems in cell biology.

    PubMed

    Keil, Thomas A; Steinbrecht, R Alexander

    2010-01-01

    For almost 100 years, insects have been favorable "model systems" in biology. Just to mention a few examples: fruit flies in genetics and developmental biology; bugs and caterpillars in hormone research; houseflies, blowflies, and locusts in neurobiology; silk moths in pheromone research; honeybees and crickets in neuroethology. For more than 50 years the electron microscope (EM) has been a valuable tool in analyzing the structure of cells and organs of these creatures. However, progress in specimen preparation was relatively slow compared with mammalian material and, in 1970, it was taken for granted that insects were much more difficult to fix than mammals. Since then, methods have dramatically improved, and satisfactory results can now be obtained routinely with chemical as well as cryofixation. In this chapter we briefly demonstrate what can be achieved with insect material, and help the researcher to find the most appropriate method for her/his systems and scientific questions. Copyright © 2010 Elsevier Inc. All rights reserved.

  18. Emergence of mammalian cell-adapted vesicular stomatitis virus from persistent infections of insect vector cells.

    PubMed

    Novella, Isabel S; Ebendick-Corpus, Bonnie E; Zárate, Selene; Miller, Eric L

    2007-06-01

    Arboviruses (arthropod-borne viruses) represent quintessential generalists, with the ability to infect and perform well in multiple hosts. However, antagonistic pleiotropy imposed a cost during the adaptation to persistent replication of vesicular stomatitis virus in sand fly cells and resulted in strains that initially replicated poorly in hamster cells, even when the virus was allowed to replicate periodically in the latter. Once a debilitated strain started replicating continuously in mammalian cells, fitness increased significantly. Fitness recovery did not entail back mutations or compensatory mutations, but instead, we observed the replacement of persistence-adapted genomes by mammalian cell-adapted strains with a full set of new, unrelated sequence changes. These mammalian cell-adapted genomes were present at low frequencies in the populations with a history of persistence for up to a year and quickly became dominant during mammalian infection, but coexistence was not stable in the long term. Periodic acute replication in mammalian cells likely contributed to extending the survival of minority genomes, but these genomes were also found in strictly persistent populations.

  19. Conserved genetic pathways controlling the development of the diffuse endocrine system in vertebrates and Drosophila.

    PubMed

    Hartenstein, Volker; Takashima, Shigeo; Adams, Katrina L

    2010-05-01

    The midgut epithelium is formed by absorptive enterocytes, secretory cells and endocrine cells. Each of these lineages is derived from the pluripotent progenitors that constitute the embryonic endoderm; the mature midgut retains pools of self-renewing stem cells that continue to produce all lineages. Recent findings in vertebrates and Drosophila shed light on the genetic mechanism that specifies the fate of the different lineages. A pivotal role is played by the Notch signaling pathway that, in a manner that appears to be very similar to the way in which Notch signaling selects neural progenitors within the neurectoderm, distinguishes the fate of secretory/endocrine cells and enterocytes. Proneural genes encoding bHLH transcription factors are expressed and required in prospective endocrine cells; activation of the Notch pathways restricts the number of these cells and promotes enterocyte development. In this review we compare the development of the intestinal endocrine cells in vertebrates and insects and summarize recent findings dealing with genetic pathways controlling this cell type. Copyright 2009. Published by Elsevier Inc.

  20. Correlation of cell surface proteins of distinct Beauveria bassiana cell types and adaption to varied environment and interaction with the host insect.

    PubMed

    Yang, Zhi; Jiang, Hongyan; Zhao, Xin; Lu, Zhuoyue; Luo, Zhibing; Li, Xuebing; Zhao, Jing; Zhang, Yongjun

    2017-02-01

    The insect fungal pathogen Beauveria bassiana produces a number of distinct cell types that include aerial conidia, blastospores and haemolymph-derived cells, termed hyphal bodies, to adapt varied environment niches and within the host insect. These cells display distinct biochemical properties and surface structures, and a highly ordered outermost brush-like structure uniquely present on hyphal bodies, but not on any in vitro cells. Here, we found that the outermost structure on the hyphal bodies mainly consisted of proteins associated to structural wall components in that most of it could be removed by dithiothreitol (DTT) or proteinase K. DTT-treatment also caused delayed germination, decreased tolerance to ultraviolet irradiation and virulence of conidia or blastospores, with decreased adherence and alternated carbohydrate epitopes, suggesting involvement in fungal development, stress responses and virulence. To characterize these cell surface molecules, proteins were released from the living cells using DTT, and identified and quantitated using label-free quantitative mass spectrometry. Thereafter, a series of bioinformatics programs were used to predict cell surface-associated proteins (CSAPs), and 96, 166 and 54 CSAPs were predicted from the identified protein pools of conidia, blastospores and hyphal bodies, respectively, which were involved in utilization of carbohydrate, nitrogen, and lipid, detoxification, pathogen-host interaction, and likely other cellular processes. Thirteen, sixty-nine and six CSAPs were exclusive in conidia, blastospores and hyphal bodies, respectively, which were verified by eGFP-tagged proteins at their N-terminus. Our data provide a crucial cue to understand mechanism of B. bassiana to adapt to varied environment and interaction with insect host. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Curcumin induces autophagic cell death in Spodoptera frugiperda cells.

    PubMed

    Veeran, Sethuraman; Shu, Benshui; Cui, Gaofeng; Fu, Shengjiao; Zhong, Guohua

    2017-06-01

    The increasing interest in the role of autophagy (type II cell death) in the regulation of insect toxicology has propelled study of investigating autophagic cell death pathways. Turmeric, the rhizome of the herb Curcuma longa (Mañjaḷ in Tamil, India and Jiānghuáng in Chinese) have been traditionally used for the pest control either alone or combination with other botanical pesticides. However, the mechanisms by which Curcuma longa or curcumin exerts cytotoxicity in pests are not well understood. In this study, we investigated the potency of Curcuma longa (curcumin) as a natural pesticide employing Sf9 insect line. Autophagy induction effect of curcumin on Spodoptera frugiperda (Sf9) cells was investigated using various techniques including cell proliferation assay, morphology analysis with inverted phase contrast microscope and Transmission Electron Microscope (TEM) analysis. Autophagy was evaluated using the fluorescent dye monodansylcadaverine (MDC). Cell death measurement was examined using 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) within the concentrations of 5-15μg/mL. Curcumin inhibited the growth of the Sf9 cells and induced autophagic cell death in a time and dose dependent manner. Staining the cells with MDC showed the presence of autophagic vacuoles while increased in a dose and time dependent manner. At the ultrastructural level transmission electron microscopy, cells revealed massive autophagy vacuole accumulation and absence of chromatin condensation. Protein expression levels of ATG8-I and ATG8-II, well-established markers of autophagy related protein were elevated in a time dependent manner after curcumin treatment. The present study proves that curcumin induces autophagic cell death in Sf9 insect cell line and this is the first report of cytotoxic effect of curcumin in insect cells and that will be utilized as natural pesticides in future. Copyright © 2017. Published by Elsevier Inc.

  2. The Yersinia pseudotuberculosis and Yersinia pestis toxin complex is active against cultured mammalian cells.

    PubMed

    Hares, Michelle C; Hinchliffe, Stewart J; Strong, Philippa C R; Eleftherianos, Ioannis; Dowling, Andrea J; ffrench-Constant, Richard H; Waterfield, Nick

    2008-11-01

    The toxin complex (Tc) genes were first identified in the insect pathogen Photorhabdus luminescens and encode approximately 1 MDa protein complexes which are toxic to insect pests. Subsequent genome sequencing projects have revealed the presence of tc orthologues in a range of bacterial pathogens known to be associated with insects. Interestingly, members of the mammalian-pathogenic yersiniae have also been shown to encode Tc orthologues. Studies in Yersinia enterocolitica have shown that divergent tc loci either encode insect-active toxins or play a role in colonization of the gut in gastroenteritis models of rats. So far little is known about the activity of the Tc proteins in the other mammalian-pathogenic yersiniae. Here we present work to suggest that Tc proteins in Yersinia pseudotuberculosis and Yersinia pestis are not insecticidal toxins but have evolved for mammalian pathogenicity. We show that Tc is secreted by Y. pseudotuberculosis strain IP32953 during growth in media at 28 degrees C and 37 degrees C. We also demonstrate that oral toxicity of strain IP32953 to Manduca sexta larvae is not due to Tc expression and that lysates of Escherichia coli BL21 expressing the Yersinia Tc proteins are not toxic to Sf9 insect cells but are toxic to cultured mammalian cell lines. Cell lysates of E. coli BL21 expressing the Y. pseudotuberculosis Tc proteins caused actin ruffles, vacuoles and multi-nucleation in cultured human gut cells (Caco-2); similar morphology was observed after application of a lysate of E. coli BL21 expressing the Y. pestis Tc proteins to mouse fibroblast NIH3T3 cells, but not Caco-2 cells. Finally, transient expression of the individual Tc proteins in Caco-2 and NIH3T3 cell lines reproduced the actin and nuclear rearrangement observed with the topical applications. Together these results add weight to the growing hypothesis that the Tc proteins in Y. pseudotuberculosis and Y. pestis have been adapted for mammalian pathogenicity. We further conclude that Tc proteins from Y. pseudotuberculosis and Y. pestis display differential mammalian cell specificity in their toxicity.

  3. Duck hepatitis A virus structural proteins expressed in insect cells self-assemble into virus-like particles with strong immunogenicity in ducklings.

    PubMed

    Wang, Anping; Gu, Lingling; Wu, Shuang; Zhu, Shanyuan

    2018-02-01

    Duck hepatitis A virus (DHAV), a non-enveloped ssRNA virus, can cause a highly contagious disease in young ducklings. The three capsid proteins of VP0, VP1 and VP3 are translated within a single large open reading frame (ORF) and hydrolyzed by protease 3CD. However, little is known on whether the recombinant viral structural proteins (VPs) expressed in insect cells could spontaneously assemble into virus-like particles (VLPs) and whether these VLPs could induce protective immunity in young ducklings. To address these issues, the structural polyprotein precursor gene P1 and the protease gene 3CD were amplified by PCR, and the recombinant proteins were expressed in insect cells using a baculovirus expression system for the characterization of their structures and immunogenicity. The recombinant proteins expressed in Sf9 cells were detected by indirect immunofluorescence assay and Western blot analysis. Electron microscopy showed that the recombinant proteins spontaneously assembled into VLPs in insect cells. Western blot analysis of the purified VLPs revealed that the VLPs were composed with the three structural proteins. In addition, vaccination with the VLPs induced high humoral immune response and provided strong protection. Therefore, our findings may provide a framework for development of new vaccines for the prevention of duck viral hepatitis. Copyright © 2018 Elsevier B.V. All rights reserved.

  4. Genome-Wide RNAi Screen Identifies Broadly-Acting Host Factors That Inhibit Arbovirus Infection

    PubMed Central

    Yasunaga, Ari; Hanna, Sheri L.; Li, Jianqing; Cho, Hyelim; Rose, Patrick P.; Spiridigliozzi, Anna; Gold, Beth; Diamond, Michael S.; Cherry, Sara

    2014-01-01

    Vector-borne viruses are an important class of emerging and re-emerging pathogens; thus, an improved understanding of the cellular factors that modulate infection in their respective vertebrate and insect hosts may aid control efforts. In particular, cell-intrinsic antiviral pathways restrict vector-borne viruses including the type I interferon response in vertebrates and the RNA interference (RNAi) pathway in insects. However, it is likely that additional cell-intrinsic mechanisms exist to limit these viruses. Since insects rely on innate immune mechanisms to inhibit virus infections, we used Drosophila as a model insect to identify cellular factors that restrict West Nile virus (WNV), a flavivirus with a broad and expanding geographical host range. Our genome-wide RNAi screen identified 50 genes that inhibited WNV infection. Further screening revealed that 17 of these genes were antiviral against additional flaviviruses, and seven of these were antiviral against other vector-borne viruses, expanding our knowledge of invertebrate cell-intrinsic immunity. Investigation of two newly identified factors that restrict diverse viruses, dXPO1 and dRUVBL1, in the Tip60 complex, demonstrated they contributed to antiviral defense at the organismal level in adult flies, in mosquito cells, and in mammalian cells. These data suggest the existence of broadly acting and functionally conserved antiviral genes and pathways that restrict virus infections in evolutionarily divergent hosts. PMID:24550726

  5. Insects of the Idaho National Laboratory: A compilation and review

    Treesearch

    Nancy Hampton

    2005-01-01

    Large tracts of important sagebrush (Artemisia L.) habitat in southeastern Idaho, including thousands of acres at the Idaho National Laboratory (INL), continue to be lost and degraded through wildland fire and other disturbances. The roles of most insects in sagebrush ecosystems are not well understood, and the effects of habitat loss and alteration...

  6. Climate analyses to assess risks from invasive forest insects: Simple matching to advanced models

    Treesearch

    Robert C. Venette

    2017-01-01

    Purpose of Review. The number of invasive alien insects that adversely affect trees and forests continues to increase as do associated ecological, economic, and sociological impacts. Prevention strategies remain the most cost-effective approach to address the issue, but risk management decisions, particularly those affecting international trade,...

  7. Quality control tests of lab-reared Cydia pomonella and Cactoblastis cactorum field performance: Comparison of laboratory and field bioassays.

    USDA-ARS?s Scientific Manuscript database

    Research, operational, and commercial programs which rely on mass-reared insects of high quality and performance, need accurate methods for monitoring quality degradation during each step of production, handling and release. With continued interest in the use of the sterile insect technique (SIT) a...

  8. Efficient production of antibody Fab fragment by transient gene expression in insect cells.

    PubMed

    Mori, Keita; Hamada, Hirotsugu; Ogawa, Takafumi; Ohmuro-Matsuyama, Yuki; Katsuda, Tomohisa; Yamaji, Hideki

    2017-08-01

    Transient gene expression allows a rapid production of diverse recombinant proteins in early-stage preclinical and clinical developments of biologics. Insect cells have proven to be an excellent platform for the production of functional recombinant proteins. In the present study, the production of an antibody Fab fragment by transient gene expression in lepidopteran insect cells was investigated. The DNA fragments encoding heavy-chain (Hc; Fd fragment) and light-chain (Lc) genes of an Fab fragment were individually cloned into the plasmid vector pIHAneo, which contained the Bombyx mori actin promoter downstream of the B. mori nucleopolyhedrovirus (BmNPV) IE-1 transactivator and the BmNPV HR3 enhancer for high-level expression. Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were co-transfected with the resultant plasmid vectors using linear polyethyleneimine. When the transfection efficiency was evaluated, a plasmid vector encoding an enhanced green fluorescent protein (EGFP) gene was also co-transfected. Transfection and culture conditions were optimized based on both the flow cytometry of the EGFP expression in transfected cells and the yield of the secreted Fab fragments determined by enzyme-linked immunosorbent assay (ELISA). Under optimal conditions, a yield of approximately 120 mg/L of Fab fragments was achieved in 5 days in a shake-flask culture. Transient gene expression in insect cells may offer a promising approach to the high-throughput production of recombinant proteins. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  9. Effect of microwave exposure on the ovarian development of Drosophila melanogaster.

    PubMed

    Panagopoulos, Dimitris J

    2012-06-01

    In the present experiments the effect of GSM radiation on ovarian development of virgin Drosophila melanogaster female insects was studied. Newly emerged adult female flies were collected and divided into separate identical groups. After the a lapse of certain number of hours-different for each group-the insects (exposed and sham-exposed) were dissected and their intact ovaries were collected and photographed under an optical microscope with the same magnification. The size of the ovaries was compared between exposed and sham-exposed virgin female insects, during the time needed for the completion of oogenesis and maturation of the first eggs in the ovarioles. Immediately after the intact ovaries were photographed, they were further dissected into individual ovarioles and treated for TUNEL and acridine-orange assays to determine the degree of DNA damage in the egg chamber cells. The study showed that the ovarian size of the exposed insects is significantly smaller than that of the corresponding sham-exposed insects, due to destruction of egg chambers by the GSM radiation, after DNA damage and consequent cell death induction in the egg chamber cells of the virgin females as shown in previous experiments on inseminated females. The difference in ovarian size between sham-exposed and exposed virgin female flies becomes most evident 39-45 h after eclosion when the first eggs within the ovaries are at the late vitellogenic and post-vitellogenic stages (mid-late oogenesis). More than 45 h after eclosion, the difference in ovarian size decreases, as the first mature eggs of the sham-exposed insects are leaving the ovaries and are laid.

  10. Genomic Instability and Breast Cancer

    DTIC Science & Technology

    2011-01-01

    interaction between CCDC98 and BRCA1 (Kim et al., 2007; Liu et al., 2007; Wang et al., 2007). BRCC36 expressed and purified from insect cells was...Figure 7. (A) An in vitro DUB assay was conducted using K63 ubiquitin chains as substrate and insect cell-expressed BRCC36, the BRCC36/KIAA0157...Tandom Affinity Purification (TAP), Irradiation , Immuno- staining, and Immunoprecipitation—All of these procedures were performed as described

  11. Regulation of the Inflammasome, a Modulator of Caspase-Mediated Cytokine Production

    DTIC Science & Technology

    2008-07-01

    and amplified. For protein expression, insect cells were cultured in suspension at 27C using Erlenmeyer culture flasks or Cytostir bioreactors ...in bacterial or insect cell expression systems, exhibiting poor expression levels and solubility. The NALP pyrin domain has been successfully produced... mammalian codon supplementation. Protein overexpression was induced by 0.5 mM IPTG (isopropyl β-D-1- thiogalactopyranoside) in standard LB (Luria-Bertani

  12. Stiffness distribution in insect cuticle: a continuous or a discontinuous profile?

    PubMed

    Rajabi, H; Jafarpour, M; Darvizeh, A; Dirks, J-H; Gorb, S N

    2017-07-01

    Insect cuticle is a biological composite with a high degree of complexity in terms of both architecture and material composition. Given the complex morphology of many insect body parts, finite-element (FE) models play an important role in the analysis and interpretation of biomechanical measurements, taken by either macroscopic or nanoscopic techniques. Many previous studies show that the interpretation of nanoindentation measurements of this layered composite material is very challenging. To develop accurate FE models, it is of particular interest to understand more about the variations in the stiffness through the thickness of the cuticle. Considering the difficulties of making direct measurements, in this study, we use the FE method to analyse previously published data and address this issue numerically. For this purpose, sets of continuous or discontinuous stiffness profiles through the thickness of the cuticle were mathematically described. The obtained profiles were assigned to models developed based on the cuticle of three insect species with different geometries and layer configurations. The models were then used to simulate the mechanical behaviour of insect cuticles subjected to nanoindentation experiments. Our results show that FE models with discontinuous exponential stiffness gradients along their thickness were able to predict the stress and deformation states in insect cuticle very well. Our results further suggest that, for more accurate measurements and interpretation of nanoindentation test data, the ratio of the indentation depth to cuticle thickness should be limited to 7% rather than the traditional '10% rule'. The results of this study thus might be useful to provide a deeper insight into the biomechanical consequences of the distinct material distribution in insect cuticle and also to form a basis for more realistic modelling of this complex natural composite. © 2017 The Author(s).

  13. The role of cytochrome c on apoptosis induced by Anagrapha falcifera multiple nuclear polyhedrosis virus in insect Spodoptera litura cells.

    PubMed

    Liu, Kaiyu; Shu, Duanyang; Song, Na; Gai, Zhongchao; Yuan, Yuan; Li, Juan; Li, Min; Guo, Shuying; Peng, Jianxin; Hong, Huazhu

    2012-01-01

    There are conflicting reports on the role of cytochrome c during insect apoptosis. Our previous studies have showed that cytochrome c released from the mitochondria was an early event by western blot analysis and caspase-3 activation was closely related to cytochrome c release during apoptosis induced by baculovirus in Spodoptera litura cells (Sl-1 cell line). In the present study, alteration in mitochondrial morphology was observed by transmission electron microscopy, and cytochrome c release from mitochondria in apoptotic Sl-1 cells induced with Anagrapha falcifera multiple nuclear polyhedrosis virus (AfMNPV) has further been confirmed by immunofluoresence staining protocol, suggesting that structural disruption of mitochondria and the release of cytochrome c are important events during Lepidoptera insect cell apoptosis. We also used Sl-1 cell-free extract system and the technique of RNA interference to further investigate the role of cytochrome c in apoptotic Sl-1 cells induced by AfMNPV. Caspase-3 activity in cell-free extracts supplemented with exogenous cytochrome c was determined and showed an increase with the extension of incubation time. DsRNA-mediated silencing of cytochrome c resulted in the inhibition of apoptosis and protected the cells from AfMNPV-induced cell death. Silencing of expression of cytochrome c had a remarkable effect on pro-caspase-3 and pro-caspase-9 activation and resulted in the reduction of caspase-3 and caspase-9 activity in Sl-1 cells undergoing apoptosis. Caspase-9 inhibitor could inhibit activation of pro-caspase-3, and the inhibition of the function of Apaf-1 with FSBA blocked apoptosis, hinting that Apaf-1 could be involved in Sl-1 cell apoptosis induced by AfMNPV. Taken together, these results strongly demonstrate that cytochrome c plays an important role in apoptotic signaling pathways in Lepidopteran insect cells.

  14. 7 CFR 457.139 - Fresh market tomato (dollar plan) crop insurance provisions.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... the crop. Freeze. The formation of ice in the cells of the plant or its fruit, caused by low air... insect infestation, unless no effective control measure exists for such disease or insect infestation; or...

  15. 7 CFR 457.139 - Fresh market tomato (dollar plan) crop insurance provisions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... the crop. Freeze. The formation of ice in the cells of the plant or its fruit, caused by low air... insect infestation, unless no effective control measure exists for such disease or insect infestation; or...

  16. Plant Virus–Insect Vector Interactions: Current and Potential Future Research Directions

    PubMed Central

    Dietzgen, Ralf G.; Mann, Krin S.; Johnson, Karyn N.

    2016-01-01

    Acquisition and transmission by an insect vector is central to the infection cycle of the majority of plant pathogenic viruses. Plant viruses can interact with their insect host in a variety of ways including both non-persistent and circulative transmission; in some cases, the latter involves virus replication in cells of the insect host. Replicating viruses can also elicit both innate and specific defense responses in the insect host. A consistent feature is that the interaction of the virus with its insect host/vector requires specific molecular interactions between virus and host, commonly via proteins. Understanding the interactions between plant viruses and their insect host can underpin approaches to protect plants from infection by interfering with virus uptake and transmission. Here, we provide a perspective focused on identifying novel approaches and research directions to facilitate control of plant viruses by better understanding and targeting virus–insect molecular interactions. We also draw parallels with molecular interactions in insect vectors of animal viruses, and consider technical advances for their control that may be more broadly applicable to plant virus vectors. PMID:27834855

  17. Plant Virus-Insect Vector Interactions: Current and Potential Future Research Directions.

    PubMed

    Dietzgen, Ralf G; Mann, Krin S; Johnson, Karyn N

    2016-11-09

    Acquisition and transmission by an insect vector is central to the infection cycle of the majority of plant pathogenic viruses. Plant viruses can interact with their insect host in a variety of ways including both non-persistent and circulative transmission; in some cases, the latter involves virus replication in cells of the insect host. Replicating viruses can also elicit both innate and specific defense responses in the insect host. A consistent feature is that the interaction of the virus with its insect host/vector requires specific molecular interactions between virus and host, commonly via proteins. Understanding the interactions between plant viruses and their insect host can underpin approaches to protect plants from infection by interfering with virus uptake and transmission. Here, we provide a perspective focused on identifying novel approaches and research directions to facilitate control of plant viruses by better understanding and targeting virus-insect molecular interactions. We also draw parallels with molecular interactions in insect vectors of animal viruses, and consider technical advances for their control that may be more broadly applicable to plant virus vectors.

  18. Activity of insulin growth factors and shrimp neurosecretory organ extracts on a lepidopteran cell line.

    PubMed

    Hatt, P J; Liebon, C; Morinière, M; Oberlander, H; Porcheron, P

    1997-01-01

    Ecdysteroids, or molting hormones, have been proven to be key differentiation regulators for epidermal cells in the postembryonic development of arthropods. Regulators of cell proliferation, however, remain largely unknown. To date, no diffusible insect peptidic growth factors have been characterized. Molecules structurally related to insulin have been discovered in insects, as in other eucaryotes. We developed in vitro tests for the preliminary characterization of potential growth factors in arthropods by adapting the procedures designed to detect such factors in vertebrates to an insect cell line (IAL-PID2) established from imaginal discs of the Indian meal moth. We verified the ability of these tests to measure the proliferation of IAL-PID2 cells. We tested mammalian insulin and insulin-like growth factors (IGF-I, IGF-II). Following an arrest of cell proliferation by serum deprivation, IGF-I and IGF-II caused partial resumption of the cell cycle, evidenced by DNA synthesis. In contrast, the addition of 20-hydroxyecdysone arrested the proliferation of the IAL-PID2 cells. The cell line was then used in a test for functional characterization of potential growth factors originating from the penaeid shrimp, Penaeus vannamei. Crude extracts of neurosecretory and nervous tissues, eyestalks, and ventral neural chain compensated for serum deprivation and stimulated completion of mitosis. Arch.

  19. The Phylogenetic Origin of oskar Coincided with the Origin of Maternally Provisioned Germ Plasm and Pole Cells at the Base of the Holometabola

    PubMed Central

    Lynch, Jeremy A.; Özüak, Orhan; Khila, Abderrahman; Abouheif, Ehab; Desplan, Claude; Roth, Siegfried

    2011-01-01

    The establishment of the germline is a critical, yet surprisingly evolutionarily labile, event in the development of sexually reproducing animals. In the fly Drosophila, germ cells acquire their fate early during development through the inheritance of the germ plasm, a specialized maternal cytoplasm localized at the posterior pole of the oocyte. The gene oskar (osk) is both necessary and sufficient for assembling this substance. Both maternal germ plasm and oskar are evolutionary novelties within the insects, as the germline is specified by zygotic induction in basally branching insects, and osk has until now only been detected in dipterans. In order to understand the origin of these evolutionary novelties, we used comparative genomics, parental RNAi, and gene expression analyses in multiple insect species. We have found that the origin of osk and its role in specifying the germline coincided with the innovation of maternal germ plasm and pole cells at the base of the holometabolous insects and that losses of osk are correlated with changes in germline determination strategies within the Holometabola. Our results indicate that the invention of the novel gene osk was a key innovation that allowed the transition from the ancestral late zygotic mode of germline induction to a maternally controlled establishment of the germline found in many holometabolous insect species. We propose that the ancestral role of osk was to connect an upstream network ancestrally involved in mRNA localization and translational control to a downstream regulatory network ancestrally involved in executing the germ cell program. PMID:21552321

  20. Insect abundance over Chinese rice fields in relation to environmental parameters, studied with a polarization-sensitive CW near-IR lidar system

    NASA Astrophysics Data System (ADS)

    Zhu, Shiming; Malmqvist, Elin; Li, Wansha; Jansson, Samuel; Li, Yiyun; Duan, Zheng; Svanberg, Katarina; Feng, Hongqiang; Song, Ziwei; Zhao, Guangyu; Brydegaard, Mikkel; Svanberg, Sune

    2017-07-01

    Effective monitoring of flying insects is of major societal importance in view of the role of insects as indispensable pollinators, destructive disease vectors and economically devastating agricultural pests. The present paper reports on monitoring of flying agricultural pests using a continuous-wave lidar system in a rice-field location in Southern China. Using a Scheimpflug arrangement, range resolution over several 100 m long observational paths was achieved. The system operates with two perpendicularly polarized near-infrared lasers, which are activated intermittently, and back-scattered radiation from insects was recorded by a linear array detector placed after a linear polarizer. Our polarization sensitive system was used to monitor the flying insect diurnal activity and also the influence of changes in weather conditions, e.g., the occurrence of rain. Activity strongly peaked at dusk and rose again, although to a lower extent, just before dawn. At the onset of rainfall, a strong increase in insect counts occurred which was interpreted as the rain-induced bringing down of high-altitude migrant insects.

  1. Insect Ferritins: typical or atypical?

    PubMed Central

    Pham, Daphne Q. D.; Winzerling, Joy J.

    2010-01-01

    Insects transmit millions of cases of disease each year, and cost millions of dollars in agricultural losses. The control of insect-borne diseases is vital for numerous developing countries, and the management of agricultural insect pests is a very serious business for developed countries. Control methods should target insect-specific traits in order to avoid non-target effects, especially in mammals. Since insect cells have had a billion years of evolutionary divergence from those of vertebrates, they differ in many ways that might be promising for the insect control field—especially, in iron metabolism because current studies have indicated that significant differences exist between insect and mammalian systems. Insect iron metabolism differs from that of vertebrates in the following respects. Insect ferritins have a heavier mass than mammalian ferritins. Unlike their mammalian counterparts, the insect ferritin subunits are often glycosylated and are synthesized with a signal peptide. The crystal structure of insect ferritin also shows a tetrahedral symmetry consisting of 12 heavy chain and 12 light chain subunits in contrast to that of mammalian ferritin that exhibits an octahedral symmetry made of 24 heavy chain and 24 light chain subunits. Insect ferritins associate primarily with the vacuolar system and serve as iron transporters—quite the opposite of the mammalian ferritins, which are mainly cytoplasmic and serve as iron storage proteins. This review will discuss these differences. PMID:20230873

  2. Baculovirus-mediated expression of GPCRs in insect cells.

    PubMed

    Saarenpää, Tuulia; Jaakola, Veli-Pekka; Goldman, Adrian

    2015-01-01

    G-protein-coupled receptors (GPCRs) are a large family of seven transmembrane proteins that influence a considerable number of cellular events. For this reason, they are one of the most studied receptor types for their pharmacological and structural properties. Solving the structure of several GPCR receptor types has been possible using almost all expression systems, including Escherichia coli, yeast, mammalian, and insect cells. So far, however, most of the GPCR structures solved have been done using the baculovirus insect cell expression system. The reason for this is mainly due to cost-effectiveness, posttranslational modification efficiency, and overall effortless maintenance. The system has evolved so much that variables starting from vector type, purification tags, cell line, and growth conditions can be varied and optimized countless ways to suit the needs of new constructs. Here, we present the array of techniques that enable the rapid and efficient optimization of expression steps for maximal protein quality and quantity, including our emendations. © 2015 Elsevier Inc. All rights reserved.

  3. Insects as weapons of war, terror, and torture.

    PubMed

    Lockwood, Jeffrey A

    2012-01-01

    For thousands of years insects have been incorporated into human conflict, with the goals of inflicting pain, destroying food, and transmitting pathogens. Early methods used insects as "found" weapons, functioning as tactical arms (e.g., hurled nests) or in strategic habitats (e.g., mosquito-infested swamps). In the twentieth century the relationship between insects and disease was exploited; vectors were mass-produced to efficiently deliver pathogens to an enemy. The two most sophisticated programs were those of the Japanese in World War II with plague-infected fleas and cholera-coated flies and of the Americans during the Cold War with yellow fever-infected mosquitoes. With continued advances, defenses in the form of insecticides and vaccines meant that insects were no longer considered as battlefield weapons. However, in recent times sociopolitical changes have put insects back into the realm of human conflict through asymmetrical conflicts pitting combatants from nonindustrialized regions against forces from militarily and economically superior nations. Copyright © 2012 by Annual Reviews. All rights reserved.

  4. Laboratory evaluation for a potential birth control diet for fruit fly sterilization insect technique (SIT)

    USDA-ARS?s Scientific Manuscript database

    A potential fruit fly steilizing diet was evaluated on fertility, mating, survival, and protein anaylsis for fruit fly species in Hawaii. Insects were continuously fed an agar diet with lufenuron(LFN) for an initial 7d after emergence and then switched to a control diet to simulate the actual field ...

  5. Exotic pests: major threats to forest health

    Treesearch

    J. Robert Bridges

    1995-01-01

    Over 360 exotic forest insects and about 20 exotic diseases have become established in the U.S. Many of these organisms have become serious pests, causing great economic impacts and irreversible ecological harm. Despite efforts to exclude exotic species, forest insects and disease organisms continue to be introduced at a rather rapid rate. In the last few years, one...

  6. Proteomic Basis of the Antibody Response to Monkeypox Virus Infection Examined in Cynomolgus Macaques and a Comparison to Human Smallpox Vaccination

    DTIC Science & Technology

    2010-12-30

    collected after challenges were gamma- irradiated (6 Mrad) to destroy any infectious virus. Previous results indicated minimal damage to serum immuno...in Sf9 insect cells using Gateway baculovirus expression (Invitrogen). All ORF clones were fully sequenced. Recombinant proteins carried GST-tags and... insect cell expression, increased the likelihood that all products were correctly folded and functional. Successfully cloned, expressed and size

  7. [Use of a novel baculovirus vector to express nucleoprotein gene of Crimean-Congo hemorrhagic fever virus in both insect and mammalian cells].

    PubMed

    Ma, Benjiang; Hang, Changshou; Zhao, Yun; Wang, Shiwen; Xie, Yanxiang

    2002-09-01

    To construct a novel baculovirus vector which is capable of promoting the high-yield expression of foreign gene in mammalian cells and to express by this vector the nucleoprotein (NP) gene of Crimean-Congo hemorrhagic fever virus (CCHFV) Chinese isolate (Xinjiang hemorrhagic fever virus, XHFV) BA88166 in insect and Vero cells. Human cytomegalovirus (CMV) immediate early (IE) promoter was ligated to the baculovirus vector pFastBac1 downstream of the polyhedrin promoter to give rise to the novel vector pCB1. XHFV NP gene was cloned to this vector and was well expressed in COS-7 cells and Vero cells by means of recombinant plasmid transfection and baculovirus infection. The XHFV NP gene in vector pCB1 could be well expressed in mammalian cells. Vero cells infected with recombinant baculovirus harboring NP gene could be employed as antigens to detect XHF serum specimens whose results were in good correlation with those of ELISA and in parallel with clinical diagnoses. This novel baculovirus vector is able to express the foreign gene efficiently in both insect and mammalian cells, which provides not only the convenient diagnostic antigens but also the potential for developing recombinant virus vaccines and gene therapies.

  8. Edible insects in China: Utilization and prospects.

    PubMed

    Feng, Ying; Chen, Xiao-Ming; Zhao, Min; He, Zhao; Sun, Long; Wang, Cheng-Ye; Ding, Wei-Feng

    2018-04-01

    The use of edible insects has a long history in China, where they have been consumed for more than 2000 years. In general, the level of acceptance is high for the consumption of insects in China. Many studies on edible insects have been conducted in the last 20 years, and the scope of the research includes the culture of entomophagy and the identification, nutritional value, farming and breeding of edible insects, in addition to food production and safety. Currently, 324 species of insects from 11 orders are documented that are either edible or associated with entomophagy in China, which include the common edible species, some less commonly consumed species and some medicinal insects. However, only approximately 10 to 20 types of insects are regularly consumed. The nutritional values for 174 species are available in China, including edible, feed and medicinal species. Although the nutritional values vary among species, all the insects examined contain protein, fat, vitamins and minerals at levels that meet human nutritional requirements. Edible insects were, and continue to be, consumed by different ethnic groups in many parts of China. People directly consume insects or food products made from insects. The processing of products from insect protein powder, oil and chitin, and the development of healthcare foods has been studied in China. People also consume insects indirectly by eating livestock that were fed insects, which may be a more acceptable pathway to use insects in human diets. Although limited, the data on the food safety of insects indicate that insects are safe for food or feed. Incidences of allergic reactions after consuming silkworm pupae, cicadas and crickets have been reported in China. Insect farming is a unique breeding industry in rural China and is a source of income for local people. Insects are reared and bred for human food, medicine and animal feed using two approaches in China: the insects are either fully domesticated and reared completely in captivity or are partially raised in captivity, and the insect habitat is manipulated to increase production. Depending on the type of relationship the insect has with humans, plants and the environment, different farming strategies are used. The social and scientific communities must work together to promote the use of insects as food and feed. © 2017 Institute of Zoology, Chinese Academy of Sciences.

  9. Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells.

    PubMed

    Fabrick, Jeffrey A; Hull, J Joe

    2017-04-20

    Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

  10. A cell-free translocation system using extracts of cultured insect cells to yield functional membrane proteins.

    PubMed

    Ezure, Toru; Nanatani, Kei; Sato, Yoko; Suzuki, Satomi; Aizawa, Keishi; Souma, Satoshi; Ito, Masaaki; Hohsaka, Takahiro; von Heijine, Gunnar; Utsumi, Toshihiko; Abe, Keietsu; Ando, Eiji; Uozumi, Nobuyuki

    2014-01-01

    Cell-free protein synthesis is a powerful method to explore the structure and function of membrane proteins and to analyze the targeting and translocation of proteins across the ER membrane. Developing a cell-free system based on cultured cells for the synthesis of membrane proteins could provide a highly reproducible alternative to the use of tissues from living animals. We isolated Sf21 microsomes from cultured insect cells by a simplified isolation procedure and evaluated the performance of the translocation system in combination with a cell-free translation system originating from the same source. The isolated microsomes contained the basic translocation machinery for polytopic membrane proteins including SRP-dependent targeting components, translocation channel (translocon)-dependent translocation, and the apparatus for signal peptide cleavage and N-linked glycosylation. A transporter protein synthesized with the cell-free system could be functionally reconstituted into a lipid bilayer. In addition, single and double labeling with non-natural amino acids could be achieved at both the lumen side and the cytosolic side in this system. Moreover, tail-anchored proteins, which are post-translationally integrated by the guided entry of tail-anchored proteins (GET) machinery, were inserted correctly into the microsomes. These results showed that the newly developed cell-free translocation system derived from cultured insect cells is a practical tool for the biogenesis of properly folded polytopic membrane proteins as well as tail-anchored proteins.

  11. Topological and Functional Characterization of an Insect Gustatory Receptor

    PubMed Central

    Zhang, Hui-Jie; Anderson, Alisha R.; Trowell, Stephen C.; Luo, A-Rong; Xiang, Zhong-Huai; Xia, Qing-You

    2011-01-01

    Insect gustatory receptors are predicted to have a seven-transmembrane structure and are distantly related to insect olfactory receptors, which have an inverted topology compared with G-protein coupled receptors, including mammalian olfactory receptors. In contrast, the topology of insect gustatory receptors remains unknown. Except for a few examples from Drosophila, the specificity of individual insect gustatory receptors is also unknown. In this study, the total number of identified gustatory receptors in Bombyx mori was expanded from 65 to 69. BmGr8, a silkmoth gustatory receptor from the sugar receptor subfamily, was expressed in insect cells. Membrane topology studies on BmGr8 indicate that, like insect olfactory receptors, it has an inverted topology relative to G protein-coupled receptors. An orphan GR from the bitter receptor family, BmGr53, yielded similar results. We infer, from the finding that two distantly related BmGrs have an intracellular N-terminus and an odd number of transmembrane spans, that this is likely to be a general topology for all insect gustatory receptors. We also show that BmGr8 functions independently in Sf9 cells and responds in a concentration-dependent manner to the polyalcohols myo-inositol and epi-inositol but not to a range of mono- and di-saccharides. BmGr8 is the first chemoreceptor shown to respond specifically to inositol, an important or essential nutrient for some Lepidoptera. The selectivity of BmGr8 responses is consistent with the known responses of one of the gustatory receptor neurons in the lateral styloconic sensilla of B. mori, which responds to myo-inositol and epi-inositol but not to allo-inositol. PMID:21912618

  12. Chaperokine function of recombinant Hsp72 produced in insect cells using a baculovirus expression system is retained.

    PubMed

    Zheng, Hongying; Nagaraja, Ganachari M; Kaur, Punit; Asea, Edwina E; Asea, Alexzander

    2010-01-01

    Extracellular heat shock protein 72 (Hsp72; inducible form of the 70-kDa heat shock protein) plays a critical role in innate and adaptive immune responses and has shown promise as an ideal adjuvant for the optimization of antigen-specific anti-tumor vaccines. Recent studies suggest that to correctly elucidate the mechanisms by which Hsp72 exerts its beneficial effects in vitro, great care must be taken to ensure that endotoxin by-products do not invalidate the findings. In this study, we have taken advantage of the baculovirus expression vector system for production of endotoxin-free recombinant Hsp72. The coding sequence of human hsp72 was recombined into the baculovirus immediately downstream of the strong polyhedron gene promoter. Ninety-six h post-infection of Sf9 insect cells with recombinant baculovirus, maximal levels of Hsp72 protein were detected. The recombinant human Hsp72 was purified by affinity chromatography from insect cells, and purity was confirmed by SDS-PAGE and mass spectrometry. The purified human recombinant Hsp72(bv) (Hsp72 produced using the BEVS) was demonstrated to have no endotoxin contamination and was shown to have stimulated potent calcium flux in the human monocytic cell line. Furthermore, recombinant Hsp72(bv) enhanced the tolerance of neuroblastoma cells to heat stress-induced cell death and displayed classical chaperokine functions including augmentation of inflammatory cytokine productions in mouse splenocytes. The production of functional, endotoxin-free recombinant human Hsp72(bv) in insect cells is inexpensive and convenient and eliminates the need of special procedures for endotoxin depletion. Endotoxin-free recombinant human Hsp72(bv) can now be used to unlock the important role Hsp72 plays in modulating immune function.

  13. Chaperokine Function of Recombinant Hsp72 Produced in Insect Cells Using a Baculovirus Expression System Is Retained*

    PubMed Central

    Zheng, Hongying; Nagaraja, Ganachari M.; Kaur, Punit; Asea, Edwina E.; Asea, Alexzander

    2010-01-01

    Extracellular heat shock protein 72 (Hsp72; inducible form of the 70-kDa heat shock protein) plays a critical role in innate and adaptive immune responses and has shown promise as an ideal adjuvant for the optimization of antigen-specific anti-tumor vaccines. Recent studies suggest that to correctly elucidate the mechanisms by which Hsp72 exerts its beneficial effects in vitro, great care must be taken to ensure that endotoxin by-products do not invalidate the findings. In this study, we have taken advantage of the baculovirus expression vector system for production of endotoxin-free recombinant Hsp72. The coding sequence of human hsp72 was recombined into the baculovirus immediately downstream of the strong polyhedron gene promoter. Ninety-six h post-infection of Sf9 insect cells with recombinant baculovirus, maximal levels of Hsp72 protein were detected. The recombinant human Hsp72 was purified by affinity chromatography from insect cells, and purity was confirmed by SDS-PAGE and mass spectrometry. The purified human recombinant Hsp72bv (Hsp72 produced using the BEVS) was demonstrated to have no endotoxin contamination and was shown to have stimulated potent calcium flux in the human monocytic cell line. Furthermore, recombinant Hsp72bv enhanced the tolerance of neuroblastoma cells to heat stress-induced cell death and displayed classical chaperokine functions including augmentation of inflammatory cytokine productions in mouse splenocytes. The production of functional, endotoxin-free recombinant human Hsp72bv in insect cells is inexpensive and convenient and eliminates the need of special procedures for endotoxin depletion. Endotoxin-free recombinant human Hsp72bv can now be used to unlock the important role Hsp72 plays in modulating immune function. PMID:19861412

  14. RNA Interference in Insect Vectors for Plant Viruses.

    PubMed

    Kanakala, Surapathrudu; Ghanim, Murad

    2016-12-12

    Insects and other arthropods are the most important vectors of plant pathogens. The majority of plant pathogens are disseminated by arthropod vectors such as aphids, beetles, leafhoppers, planthoppers, thrips and whiteflies. Transmission of plant pathogens and the challenges in managing insect vectors due to insecticide resistance are factors that contribute to major food losses in agriculture. RNA interference (RNAi) was recently suggested as a promising strategy for controlling insect pests, including those that serve as important vectors for plant pathogens. The last decade has witnessed a dramatic increase in the functional analysis of insect genes, especially those whose silencing results in mortality or interference with pathogen transmission. The identification of such candidates poses a major challenge for increasing the role of RNAi in pest control. Another challenge is to understand the RNAi machinery in insect cells and whether components that were identified in other organisms are also present in insect. This review will focus on summarizing success cases in which RNAi was used for silencing genes in insect vector for plant pathogens, and will be particularly helpful for vector biologists.

  15. RNA Interference in Insect Vectors for Plant Viruses

    PubMed Central

    Kanakala, Surapathrudu; Ghanim, Murad

    2016-01-01

    Insects and other arthropods are the most important vectors of plant pathogens. The majority of plant pathogens are disseminated by arthropod vectors such as aphids, beetles, leafhoppers, planthoppers, thrips and whiteflies. Transmission of plant pathogens and the challenges in managing insect vectors due to insecticide resistance are factors that contribute to major food losses in agriculture. RNA interference (RNAi) was recently suggested as a promising strategy for controlling insect pests, including those that serve as important vectors for plant pathogens. The last decade has witnessed a dramatic increase in the functional analysis of insect genes, especially those whose silencing results in mortality or interference with pathogen transmission. The identification of such candidates poses a major challenge for increasing the role of RNAi in pest control. Another challenge is to understand the RNAi machinery in insect cells and whether components that were identified in other organisms are also present in insect. This review will focus on summarizing success cases in which RNAi was used for silencing genes in insect vector for plant pathogens, and will be particularly helpful for vector biologists. PMID:27973446

  16. Towards the elements of successful insect RNAi.

    PubMed

    Scott, Jeffrey G; Michel, Kristin; Bartholomay, Lyric C; Siegfried, Blair D; Hunter, Wayne B; Smagghe, Guy; Zhu, Kun Yan; Douglas, Angela E

    2013-12-01

    RNA interference (RNAi), the sequence-specific suppression of gene expression, offers great opportunities for insect science, especially to analyze gene function, manage pest populations, and reduce disease pathogens. The accumulating body of literature on insect RNAi has revealed that the efficiency of RNAi varies between different species, the mode of RNAi delivery, and the genes being targeted. There is also variation in the duration of transcript suppression. At present, we have a limited capacity to predict the ideal experimental strategy for RNAi of a particular gene/insect because of our incomplete understanding of whether and how the RNAi signal is amplified and spread among insect cells. Consequently, development of the optimal RNAi protocols is a highly empirical process. This limitation can be relieved by systematic analysis of the molecular physiological basis of RNAi mechanisms in insects. An enhanced conceptual understanding of RNAi function in insects will facilitate the application of RNAi for dissection of gene function, and to fast-track the application of RNAi to both control pests and develop effective methods to protect beneficial insects and non-insect arthropods, particularly the honey bee (Apis mellifera) and cultured Pacific white shrimp (Litopenaeus vannamei) from viral and parasitic diseases. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Occurrence, distribution, and possible functional roles of simple sequence repeats in phytoplasma genomes

    USDA-ARS?s Scientific Manuscript database

    Phytoplasmas are unculturable, cell wall-less bacteria that parasitize plants and insects. This transkingdom life cycle requires rapid responses to vastly different environments including transitions from plant phloem sieve elements to various insect tissues and alterations of diverse plant hosts. ...

  18. Development of a novel set of Gateway-compatible vectors for live imaging in insect cells.

    PubMed

    Maroniche, G A; Mongelli, V C; Alfonso, V; Llauger, G; Taboga, O; del Vas, Mariana

    2011-10-01

    Insect genomics is a growing area of research. To exploit fully the genomic data that are being generated, high-throughput systems for the functional characterization of insect proteins and their interactomes are required. In this work, a Gateway-compatible vector set for expression of fluorescent fusion proteins in insect cells was developed. The vector set was designed to express a protein of interest fused to any of four different fluorescent proteins [green fluorescent protein (GFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and mCherry] by either the C-terminal or the N-terminal ends. Additionally, a collection of organelle-specific fluorescent markers was assembled for colocalization with fluorescent recombinant proteins of interest. Moreover, the vector set was proven to be suitable for simultaneously detecting up to three proteins by multiple labelling. The use of the vector set was exemplified by defining the subcellular distribution of Mal de Río Cuarto virus (MRCV) outer coat protein P10 and by analysing the in vivo self-interaction of the MRCV viroplasm matrix protein P9-1 in Förster resonance energy transfer (FRET) experiments. In conclusion, we have developed a valuable tool for high-throughput studies of protein subcellular localization that will aid in the elucidation of the function of newly described insect and virus proteins. © 2011 The Authors. Insect Molecular Biology © 2011 The Royal Entomological Society.

  19. The future of our National Forests: Enhancing adaptive capacity

    Treesearch

    Linda A. Joyce

    2012-01-01

    Ecosystems are changing in response to observed changes in climate, extreme events, and disturbances such as fire and insects. We read not only that these changes are occurring but that we should expect them to continue - changes in temperature; more extreme events; and more disturbances such as fire and insects that are not like the past. Key point: these changes in...

  20. Improving mycoinsecticides for insect biological control.

    PubMed

    Ortiz-Urquiza, Almudena; Luo, Zhibing; Keyhani, Nemat O

    2015-02-01

    The desire for decreased reliance on chemical pesticides continues to fuel interest in alternative means for pest control including the use of naturally occurring microbial insect pathogens. Insects, as vectors of disease causing agents or as agricultural pests, are responsible for millions of deaths and significant economic losses worldwide, placing stresses on productivity (GDP) and human health and welfare. In addition, alterations in climate change are likely to affect insect ranges, expanding their access to previously constrained geographic areas, a potentially worrisome outcome. Metarhizium anisopliae and Beauveria bassiana, two cosmopolitan fungal pathogens of insects found in almost all ecosystems, are the most commonly applied mycoinsecticides for a variety of insect control purposes. The availability of the complete genomes for both organisms coupled to robust technologies for their transformation has led to several advances in engineering these fungi for greater efficacy and/or utility in pest control applications. Here, we will provide an overview of the fungal-insect and fungal-plant interactions that occur and highlight recent advances in the genetic engineering of these fungi. The latter work has resulted in the development of strains displaying (1) increased resistance to abiotic stress, (2) increased cuticular targeting and degradation, (3) increased virulence via expression of insecticidal protein/peptide toxins, (4) the ability to block transmission of disease causing agents, and (5) the ability to target specific insect hosts, decrease host fecundity, and/or alter insect behaviors.

  1. Food for the Future: A Study of Insects as a Protein Source

    NASA Astrophysics Data System (ADS)

    Riggs, S.

    2017-12-01

    This study is designed to gain a sustainable, organic food source containing the proper amino acids, minerals, and protein to sustain the needs of human life on Earth as the current economy and environment will not be able to equip the needs for the future. The hypotheses are if available protein is increased in an insect's diet then their nutritional value will increase to fulfill a human's daily protein requirements in one serving size or less for each species tested and if there is a higher content of protein in the insects, then food created with it will receive higher ratings. Protein supplements were added to an insect's natural diet to increase nutritional value. Protein value in the insects increased to fulfill a human's daily dietary protein requirement in a third of a serving size. Biuret and absorption spectrometry testing demonstrates this correlation. Insects increased protein in their body showing a positive correlation to the hypothesis. Week one, protein values doubled and tripled in some species, unexpectedly. After three weeks, protein still continued increasing. There was high success in increasing the protein value in the different species of insects chosen. Is there a taste difference benefit with a higher content of protein in the insects? Over 55% of participants rated the brownies with more protein higher than the control groups, and overall 88% preferred brownies with insects opposed to without, supporting the second hypothesis.

  2. Recombinant Human Lysyl Oxidase-like 2 Secreted from Human Embryonic Kidney Cells Displays Complex and Acidic Glycans at All Three N-Linked Glycosylation Sites.

    PubMed

    Go, Eden P; Moon, Hee-Jung; Mure, Minae; Desaire, Heather

    2018-05-04

    Human lysyl oxidase-like 2 (hLOXL2), a glycoprotein implicated in tumor progression and organ fibrosis, is a molecular target for anticancer and antifibrosis treatment. This glycoprotein contains three predicted N-linked glycosylation sites; one is near the protein's active site, and at least one more is known to facilitate the protein's secretion. Because the glycosylation impacts the protein's biology, we sought to characterize the native, mammalian glycosylation profile and to determine how closely this profile is recapitulated when the protein is expressed in insect cells. All three glycosylation sites on the protein, expressed in human embryonic kidney (HEK) cells, were characterized individually using a mass spectrometry-based glycopeptide analysis workflow. These data were compared to the glycosylation profile of the same protein expressed in insect cells. We found that the producer cell type imparts a substantial influence on the glycosylation of this important protein. The more-relevant version, expressed in HEK cells, contains large, acidic glycoforms; these glycans are not generated in insect cells. The glycosylation differences likely have structural and functional consequences, and these data should be considered when generating protein for functional studies or for high-throughput screening campaigns.

  3. Contributions of insects and droughts to growth decline of trembling aspen mixed boreal forest of western Canada.

    PubMed

    Chen, Lei; Huang, Jian-Guo; Dawson, Andria; Zhai, Lihong; Stadt, Kenneth J; Comeau, Philip G; Whitehouse, Caroline

    2018-02-01

    Insects, diseases, fire and drought and other disturbances associated with global climate change contribute to forest decline and mortality in many parts of the world. Forest decline and mortality related to drought or insect outbreaks have been observed in North American aspen forests. However, little research has been done to partition and estimate their relative contributions to growth declines. In this study, we combined tree-ring width and basal area increment series from 40 trembling aspen (Populus tremuloides Michx.) sites along a latitudinal gradient (from 52° to 58°N) in western Canada and attempted to investigate the effect of drought and insect outbreaks on growth decline, and simultaneously partition and quantify their relative contributions. Results indicated that the influence of drought on forest decline was stronger than insect outbreaks, although both had significant effects. Furthermore, the influence of drought and insect outbreaks showed spatiotemporal variability. In addition, our data suggest that insect outbreaks could be triggered by warmer early spring temperature instead of drought, implicating that potentially increased insect outbreaks are expected with continued warming springs, which may further exacerbate growth decline and death in North America aspen mixed forests. © 2017 John Wiley & Sons Ltd.

  4. Insect aquaplaning: Nepenthes pitcher plants capture prey with the peristome, a fully wettable water-lubricated anisotropic surface.

    PubMed

    Bohn, Holger F; Federle, Walter

    2004-09-28

    Pitcher plants of the genus Nepenthes have highly specialized leaves adapted to attract, capture, retain, and digest arthropod prey. Several mechanisms have been proposed for the capture of insects, ranging from slippery epicuticular wax crystals to downward-pointing lunate cells and alkaloid secretions that anesthetize insects. Here we report that perhaps the most important capture mechanism has thus far remained overlooked. It is based on special surface properties of the pitcher rim (peristome) and insect "aquaplaning." The peristome is characterized by a regular microstructure with radial ridges of smooth overlapping epidermal cells, which form a series of steps toward the pitcher inside. This surface is completely wettable by nectar secreted at the inner margin of the peristome and by rain water, so that homogenous liquid films cover the surface under humid weather conditions. Only when wet, the peristome surface is slippery for insects, so that most ant visitors become trapped. By measuring friction forces of weaver ants (Oecophylla smaragdina) on the peristome surface of Nepenthes bicalcarata, we demonstrate that the two factors preventing insect attachment to the peristome, i.e., water lubrication and anisotropic surface topography, are effective against different attachment structures of the insect tarsus. Peristome water films disrupt attachment only for the soft adhesive pads but not for the claws, whereas surface topography leads to anisotropic friction only for the claws but not for the adhesive pads. Experiments on Nepenthes alata show that the trapping mechanism of the peristome is also essential in Nepenthes species with waxy inner pitcher walls.

  5. Propagation of Homalodisca Coagulata Virus-01 via Homalodisca Vitripennis cell culture

    USDA-ARS?s Scientific Manuscript database

    The glassy-winged sharpshooter (Homalodisca vitripennis) is a highly vagile and polyphagous insect found throughout the southwestern United States. These insects are the predominant vectors of Xylella fastidiosa, a xylem-limited bacterium that is the causal agent of Pierce's disease (PD) of grapevin...

  6. When can immunotherapy for insect sting allergy be stopped?

    PubMed

    Müller, Ulrich R; Ring, Johannes

    2015-01-01

    Stings by Hymenoptera (honey bees, vespids, ants) can cause systemic allergic reactions (SARs). Venom immunotherapy (VIT) is highly effective and reduces an allergic patient's risk of a recurrent SAR to less than 5-20%. The risk of a recurrent SAR to a re-sting decreases the longer VIT is continued. The recommended duration of VIT is at least 3 to 5 years. Risk factors for recurrent SARs to a sting after stopping VIT have been identified and discussed: Recommendations concerning stopping VIT: For patients without any of the identified risk factors, VIT should be continued for 5 rather than 3 years. In patients with definite risk factors, a longer duration of VIT has to be discussed before stopping it. In mast cell disorders, VIT for life is recommended. Because of the residual risk of SARs after VIT, all patients are advised to carry an epinephrine autoinjector indefinitely and to continue to take measures to avoid Hymenoptera stings. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  7. Morphology and ultrastructure of the germarium in panoistic ovarioles of a basal "apterygotous" insect, Thermobia domestica.

    PubMed

    Tworzydlo, Waclaw; Kisiel, Elzbieta; Jankowska, Wladyslawa; Bilinski, Szczepan M

    2014-06-01

    It has been shown that in Drosophila the germline stem cells (GSCs), similar to the germline and non-germline stem cells of other species, develop and function in specialized microenvironments formed by somatic cells, referred to as the niches. In the fruit fly ovaries, the female GSCs divide asymmetrically to produce new GSCs and the progenitor cells, the cystoblasts (Cbs). Each Cb then divides to generate the cyst composed of 16 interconnected sibling cells, the cystocytes. After cyst formation, specific molecules are transferred to one of the cystocytes which differentiates into the oocyte, whereas the other 15 cystocytes become the nurse cells. We have studied morphology and ultrastructure of the germaria in the ovarioles (ovaries) of a basal "apterygotous" insect, the firebrat (Thermobia domestica). Our analyses have revealed that in this insect, putative GSCs are present along the anterior apex of the germarium. These cells are separated from each other and from the basement lamina covering the ovariole by characteristic somatic cells, termed the apical somatic cells (ASCs), or their elongated processes. We believe that all the ASCs of a given ovariole constitute a "dispersed" niche in which putative GSCs are anchored. Our analyses have additionally shown that in Thermobia, both the Cbs and young (meiotic) oocytes are always individual and never form syncytial cysts. These findings indicate that in certain basal insects the syncytial phase of oogenesis has been eliminated during evolution. Finally, we show that in the early meiotic oocytes of Thermobia, during the so-called bouquet stage, prominent Balbiani bodies (Bbs) are formed. Analysis of serial micrographs indicates that the Bbs invariably arise next to the segment of the nuclear envelope to which the telomeres of the bouquet chromosomes are attached. We suggest, in the light of these data, that the localization of the Bb together with the polar attachment of the bouquet chromosomes play a crucial role in the early asymmetrization of Thermobia oocytes. Copyright © 2014 Elsevier GmbH. All rights reserved.

  8. Process analytical technology (PAT) in insect and mammalian cell culture processes: dielectric spectroscopy and focused beam reflectance measurement (FBRM).

    PubMed

    Druzinec, Damir; Weiss, Katja; Elseberg, Christiane; Salzig, Denise; Kraume, Matthias; Pörtner, Ralf; Czermak, Peter

    2014-01-01

    Modern bioprocesses demand for a careful definition of the critical process parameters (CPPs) already during the early stages of process development in order to ensure high-quality products and satisfactory yields. In this context, online monitoring tools can be applied to recognize unfavorable changes of CPPs during the production processes and to allow for early interventions in order to prevent losses of production batches due to quality issues. Process analytical technologies such as the dielectric spectroscopy or focused beam reflectance measurement (FBRM) are possible online monitoring tools, which can be applied to monitor cell growth as well as morphological changes. Since the dielectric spectroscopy only captures cells with intact cell membranes, even information about dead cells with ruptured or leaking cell membranes can be derived. The following chapter describes the application of dielectric spectroscopy on various virus-infected and non-infected cell lines with respect to adherent as well as suspension cultures in common stirred tank reactors. The adherent mammalian cell lines Vero (African green monkey kidney cells) and hMSC-TERT (telomerase-immortalized human mesenchymal stem cells) are thereby cultured on microcarrier, which provide the required growth surface and allow the cultivation of these cells even in dynamic culture systems. In turn, the insect-derived cell lines S2 and Sf21 are used as examples for cells typically cultured in suspension. Moreover, the FBRM technology as a further monitoring tool for cell culture applications has been included in this chapter using the example of Drosophila S2 insect cells.

  9. Hyperspectral Imaging and Spectroscopy of Fluorescently Coupled Acyl-CoA: Cholesterol Acyltransferase in Insect Cells

    NASA Technical Reports Server (NTRS)

    Malak, H.; Mahtani, H.; Herman, P.; Vecer, J.; Lu, X.; Chang, T. Y.; Richmond, Robert C.; Whitaker, Ann F. (Technical Monitor)

    2001-01-01

    A high-performance hyperspectral imaging module with high throughput of light suitable for low-intensity fluorescence microscopic imaging and subsequent analysis, including single-pixel-defined emission spectroscopy, was tested on Sf21 insect cells expressing green fluorescence associated with recombinant green fluorescent protein linked or not with the membrane protein acyl-CoA:cholesterol acyltransferase. The imager utilized the phenomenon of optical activity as a new technique providing information over a spectral range of 220-1400 nm, and was inserted between the microscope and an 8-bit CCD video-rate camera. The resulting fluorescence image did not introduce observable image aberrations. The images provided parallel acquisition of well resolved concurrent spatial and spectral information such that fluorescence associated with green fluorescent protein alone was demonstrated to be diffuse within the Sf21 insect cell, and that green fluorescence associated with the membrane protein was shown to be specifically concentrated within regions of the cell cytoplasm. Emission spectra analyzed from different regions of the fluorescence image showed blue shift specific for the regions of concentration associated with the membrane protein.

  10. Effects of gamma irradiation on the midgut ultrastructure of Glossina palpalis subspecies

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

    Stiles, J.K.; Molyneux, D.H.; Wallbanks, K.R.

    1989-05-01

    In the sterile insect technique, insects are sterilized prior to release in areas where they are pests. The sterile males compete for and with fertile wild individuals for mates, thus reducing the population's reproductive rate. Tsetse fly (Glossina spp.) populations have been eradicated after release of laboratory-bred flies sterilized by gamma irradiation. However, no studies exist on radiation-induced damage to the midgut morphology and function of the radiation-sterilized insects. After G. palpalis palpalis and G. p. gambiensis were subjected to 130 Gy gamma radiation, their midgut damage and recovery were monitored by electron microscopy. The first sign of damage wasmore » atrophy and loss of the microvillous border from epithelial cells. The rate of cell degeneration increased, with young as well as old cells being affected and cellular debris filling the ectoperitrophic space. Muscle cells were destroyed, patches of basal lamina were left bare, intracellular virus- and rickettsia-like organisms became more frequent, and many replacement cells became unusually large. Partial recovery occurred from the 10th day postirradiation. Such changes in midgut ultrastructure and the corresponding inhibition of functions may increase the susceptibility of the fly to trypanosome infection.« less

  11. Infection Density Dynamics of the Citrus Greening Bacterium “Candidatus Liberibacter asiaticus” in Field Populations of the Psyllid Diaphorina citri and Its Relevance to the Efficiency of Pathogen Transmission to Citrus Plants

    PubMed Central

    Ukuda-Hosokawa, Rie; Sadoyama, Yasutsune; Kishaba, Misaki; Kuriwada, Takashi; Anbutsu, Hisashi

    2015-01-01

    Huanglongbing, or citrus greening, is a devastating disease of citrus plants recently spreading worldwide, which is caused by an uncultivable bacterial pathogen, “Candidatus Liberibacter asiaticus,” and vectored by a phloem-sucking insect, Diaphorina citri. We investigated the infection density dynamics of “Ca. Liberibacter asiaticus” in field populations of D. citri with experiments using field-collected insects to address how “Ca. Liberibacter asiaticus” infection density in the vector insect is relevant to pathogen transmission to citrus plants. Of 500 insects continuously collected from “Ca. Liberibacter asiaticus”-infected citrus trees with pathological symptoms in the spring and autumn of 2009, 497 (99.4%) were “Ca. Liberibacter asiaticus” positive. The infections were systemic across head-thorax and abdomen, ranging from 103 to 107 bacteria per insect. In spring, the infection densities were low in March, at ∼103 bacteria per insect, increasing up to 106 to 107 bacteria per insect in April and May, and decreasing to 105 to 106 bacteria per insect in late May, whereas the infection densities were constantly ∼106 to 107 bacteria per insect in autumn. Statistical analysis suggested that several factors, such as insect sex, host trees, and collection dates, may be correlated with “Ca. Liberibacter asiaticus” infection densities in field D. citri populations. Inoculation experiments with citrus seedlings using field-collected “Ca. Liberibacter asiaticus”-infected insects suggested that (i) “Ca. Liberibacter asiaticus”-transmitting insects tend to exhibit higher infection densities than do nontransmitting insects, (ii) a threshold level (∼106 bacteria per insect) of “Ca. Liberibacter asiaticus” density in D. citri is required for successful transmission to citrus plants, and (iii) D. citri attaining the threshold infection level transmits “Ca. Liberibacter asiaticus” to citrus plants in a stochastic manner. These findings provide valuable insights into understanding, predicting, and controlling this notorious citrus pathogen. PMID:25819961

  12. Development of a baculovirus vector carrying a small hairpin RNA for suppression of sf-caspase-1 expression and improvement of recombinant protein production.

    PubMed

    Zhang, Xiaoyue; Xu, Keyan; Ou, Yanmei; Xu, Xiaodong; Chen, Hongying

    2018-05-02

    The Baculovirus expression vector system (BEVS) is a transient expression platform for recombinant protein production in insect cells. Baculovirus infection of insect cells will shutoff host translation and induce apoptosis and lead to the termination of protein expression. Previous reports have demonstrated the enhancement of protein yield in BEVS using stable insect cell lines expressing interference RNA to suppress the expression of caspase-1. In this study, short-hairpin RNA (shRNA) expression cassettes targeting Spodoptera frugiperda caspase-1 (Sf-caspase-1) were constructed and inserted into an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) vector. Using the recombinant baculovirus vectors, we detected the suppression of Sf-caspase-1 expression and cell apoptosis. Green fluorescent protein (GFP), Discosoma sp. Red (DsRed) and firefly luciferase were then expressed as reporter proteins. The results showed that suppression of apoptosis enhanced the accumulation of exogenous proteins at 2 and 3 days post infection. After 4 days post infection, the activity of the reporter proteins remained higher in BEVS using the baculovirus carrying shRNA in comparison with the control without shRNA, but the accumulated protein levels showed no obvious difference between them, suggesting that apoptosis suppression resulted in improved protein folding rather than translation efficiency at the very late stage of baculovirus infection. The baculovirus vector developed in this study would be a useful tool for the production of active proteins suitable for structural and functional studies or pharmaceutical applications in Sf9 cells, and it also has the potential to be adapted for the improvement of protein expression in different insect cell lines that can be infected by AcMNPV.

  13. Role of cyclic di-GMP in Xylella fastidiosa biofilm formation, plant virulence, and insect transmission.

    PubMed

    Chatterjee, Subhadeep; Killiny, Nabil; Almeida, Rodrigo P P; Lindow, Steven E

    2010-10-01

    Xylella fastidiosa must coordinately regulate a variety of traits contributing to biofilm formation, host plant and vector colonization, and transmission between plants. Traits such as production of extracellular polysaccharides (EPS), adhesins, extracellular enzymes, and pili are expressed in a cell-density-dependent fashion mediated by a cell-to-cell signaling system involving a fatty acid diffusible signaling factor (DSF). The expression of gene PD0279 (which has a GGDEF domain) is downregulated in the presence of DSF and may be involved in intracellular signaling by modulating the levels of cyclic di-GMP. PD0279, designated cyclic di-GMP synthase A (cgsA), is required for biofilm formation, plant virulence, and vector transmission. cgsA mutants exhibited a hyperadhesive phenotype in vitro and overexpressed gumJ, hxfA, hxfB, xadA, and fimA, which promote attachment of cells to surfaces and, hence, biofilm formation. The mutants were greatly reduced in virulence to grape albeit still transmissible by insect vectors, although at a reduced level compared with transmission rates of the wild-type strain, despite the fact that similar numbers of cells of the cgsA mutant were acquired by the insects from infected plants. High levels of EPS were measured in cgsA mutants compared with wild-type strains, and scanning electron microscopy analysis also revealed a thicker amorphous layer surrounding the mutants. Overexpression of cgsA in a cgsA-complemented mutant conferred the opposite phenotypes in vitro. These results suggest that decreases of cyclic di-GMP result from the accumulation of DSF as cell density increases, leading to a phenotypic transition from a planktonic state capable of colonizing host plants to an adhesive state that is insect transmissible.

  14. Midgut pseudotumors and the maintenance of tissue homeostasis: studies on aging and manipulated stick insects.

    PubMed

    Holtmann, Matthias; Dorn, August

    2009-02-01

    Stick insects (Carausius morosus) develop pseudotumors in aging adults. Pseudotumor formation starts at the M2 midgut region where an accumulation of stomatogastric nerve terminals is observed. Pseudotumors arise from dying columnar cells whose basal parts form an "amorphous substance" at the basement membrane whereas the apical parts, including the nucleus, are expelled into the gut lumen. The "amorphous substance" is ensheathed by hemocytes. These nodules, which do not melanize, characterize the phenotype of the pseudotumors. With age, cell death and pseudotumor infestation increases. It is shown that the maintenance of midgut tissue homoeostasis is disturbed and becomes more serious with growing pseudotumor incidence. The increased death rate of differentiated columnar cells is no longer compensated by the proliferation of regenerative cells, i.e., intestinal stem cells, in the midgut nidi. The appearance of "holes" in the intestinal wall is evidently a causative factor of premature death. Extirpation of the hypocerebral ganglion in young adults of the stick insect (before the onset of spontaneous pseudotumor formation) provokes the apoptosis of a large number of columnar cells within 24 h and the formation of pseudotumors that are histologically identical with spontaneous ones. We conclude that the stomatogastric nervous system plays a decisive role in the regulatory mechanism maintaining midgut tissue homeostasis. The possibility of experimentally manipulating the regulatory system provides a valuable tool for the exploration of extrinsic factors involved into the feedback circuitry of tissue homeostasis. The fact that comparable pseudotumors were observed in a number of orthopteromorphan species, where they could also be induced by the interruption of the stomatogastric nervous system, indicates that its role in tissue homoeostasis may be widespread in insects and possibly represent a general principle. (c) 2008 Wiley-Liss, Inc.

  15. EFFECTS OF METHOPRENE, ITS METABOLITES, AND BREAKDOWN PRODUCTS ON RETINOID-ACTIVATED PATHWAYS IN TRANSFECTED CELL LINES

    EPA Science Inventory

    Methoprene is a terpene-based insecticide designed to act as an agonist of insect juvenile hormone, which is essential for the transition from larval to adult forms in some metamorphic insects. Recent evidence suggests that a methoprene metabolite, methoprene acid, activates a ve...

  16. A non-autonomous insect piggyBac trasposable element is mobile in tobacco

    USDA-ARS?s Scientific Manuscript database

    The piggyBac transposable element, originally isolated from a virus in an insect cell line, is a valuable molecular tool for transgenesis and mutagenesis of invertebrates. For heterologous transgenesis in a variety of mammals, transfer of the piggyBac transposable element from an ectopic plasmid onl...

  17. Deltabaculoviruses encode a functional type I budded virus envelope fusion protein

    USDA-ARS?s Scientific Manuscript database

    Envelope fusion proteins (F proteins) are major constituents of budded viruses (BVs) of alpha- and betabaculoviruses (Baculoviridae) and are essential for the systemic infection of insect larvae and insect cells in culture. An F protein homolog gene was absent in gammabaculoviruses. Here we show tha...

  18. Unlinked genetic loci control the reduced transcription of aminopeptidase N 1 and 3 in the European corn borer and determine tolerance to Bacillus thuringiensis Cry1Ab toxin

    USDA-ARS?s Scientific Manuscript database

    Crystalline (Cry) toxins from Bacillus thuringiensis (Bt) control insect feeding damage on crop plants via foliar applications or by expression within transgenic plants, but continued Bt use is threatened by the buildup of insect resistance traits. Aminopeptidase N (apn) gene family members encode m...

  19. Effects of mesh bag enclosure and termites on fine woody debris decomposition in a subtropical forest

    Treesearch

    Allison M. Stoklosa; Michael D. Ulyshen; Zhaofei Fan; Morgan Varner; Sebastian Seibold; Jorg Muller

    2016-01-01

    The role of insects in terrestrial decomposition remains poorly resolved, particularly for infrequently studied substrates like small diameter woody debris.  Uncertainty about how mesh bags used to exclude arthropods may affect decomposition rates continues to impede progress in this area.  We sought to (1) measure how insects affect the decomposition of small diameter...

  20. Approach and rationale to developing an IPM program: Examples of insect management in British Columbia reforestation nurseries

    Treesearch

    David Trotter

    2002-01-01

    Insects, weeds, and diseases are a significant part of the production process that nursery growers must consider in order to effectively grow the desired conifer seedling. For the pests and seedlings, the underlying theme is survival, which encompasses 3 major components: stimulus, recognition, and response (Shigo 1991). Thus, the continuation of any system depends on...

  1. O Antigen Modulates Insect Vector Acquisition of the Bacterial Plant Pathogen Xylella fastidiosa

    PubMed Central

    Rapicavoli, Jeannette N.; Kinsinger, Nichola; Perring, Thomas M.; Backus, Elaine A.; Shugart, Holly J.; Walker, Sharon

    2015-01-01

    Hemipteran insect vectors transmit the majority of plant pathogens. Acquisition of pathogenic bacteria by these piercing/sucking insects requires intimate associations between the bacterial cells and insect surfaces. Lipopolysaccharide (LPS) is the predominant macromolecule displayed on the cell surface of Gram-negative bacteria and thus mediates bacterial interactions with the environment and potential hosts. We hypothesized that bacterial cell surface properties mediated by LPS would be important in modulating vector-pathogen interactions required for acquisition of the bacterial plant pathogen Xylella fastidiosa, the causative agent of Pierce's disease of grapevines. Utilizing a mutant that produces truncated O antigen (the terminal portion of the LPS molecule), we present results that link this LPS structural alteration to a significant decrease in the attachment of X. fastidiosa to blue-green sharpshooter foreguts. Scanning electron microscopy confirmed that this defect in initial attachment compromised subsequent biofilm formation within vector foreguts, thus impairing pathogen acquisition. We also establish a relationship between O antigen truncation and significant changes in the physiochemical properties of the cell, which in turn affect the dynamics of X. fastidiosa adhesion to the vector foregut. Lastly, we couple measurements of the physiochemical properties of the cell with hydrodynamic fluid shear rates to produce a Comsol model that predicts primary areas of bacterial colonization within blue-green sharpshooter foreguts, and we present experimental data that support the model. These results demonstrate that, in addition to reported protein adhesin-ligand interactions, O antigen is crucial for vector-pathogen interactions, specifically in the acquisition of this destructive agricultural pathogen. PMID:26386068

  2. O antigen modulates insect vector acquisition of the bacterial plant pathogen Xylella fastidiosa.

    PubMed

    Rapicavoli, Jeannette N; Kinsinger, Nichola; Perring, Thomas M; Backus, Elaine A; Shugart, Holly J; Walker, Sharon; Roper, M Caroline

    2015-12-01

    Hemipteran insect vectors transmit the majority of plant pathogens. Acquisition of pathogenic bacteria by these piercing/sucking insects requires intimate associations between the bacterial cells and insect surfaces. Lipopolysaccharide (LPS) is the predominant macromolecule displayed on the cell surface of Gram-negative bacteria and thus mediates bacterial interactions with the environment and potential hosts. We hypothesized that bacterial cell surface properties mediated by LPS would be important in modulating vector-pathogen interactions required for acquisition of the bacterial plant pathogen Xylella fastidiosa, the causative agent of Pierce's disease of grapevines. Utilizing a mutant that produces truncated O antigen (the terminal portion of the LPS molecule), we present results that link this LPS structural alteration to a significant decrease in the attachment of X. fastidiosa to blue-green sharpshooter foreguts. Scanning electron microscopy confirmed that this defect in initial attachment compromised subsequent biofilm formation within vector foreguts, thus impairing pathogen acquisition. We also establish a relationship between O antigen truncation and significant changes in the physiochemical properties of the cell, which in turn affect the dynamics of X. fastidiosa adhesion to the vector foregut. Lastly, we couple measurements of the physiochemical properties of the cell with hydrodynamic fluid shear rates to produce a Comsol model that predicts primary areas of bacterial colonization within blue-green sharpshooter foreguts, and we present experimental data that support the model. These results demonstrate that, in addition to reported protein adhesin-ligand interactions, O antigen is crucial for vector-pathogen interactions, specifically in the acquisition of this destructive agricultural pathogen. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  3. Bacillus thuringiensis: A story of a successful bioinsecticide

    PubMed Central

    Bravo, Alejandra; Likitvivatanavong, Supaporn; Gill, Sarjeet S.; Soberón, Mario

    2013-01-01

    Bacillus thuringiensis (Bt) bacteria are insect pathogens that rely on insecticidal pore forming proteins known as Cry and Cyt toxins to kill their insect larval hosts. At least four different non-structurally related families of proteins form the Cry toxin group of toxins. The expression of certain Cry toxins in transgenic crops has contributed to an efficient control of insect pests resulting in a significant reduction in chemical insecticide use. The mode of action of the three domain Cry toxin family involves sequential interaction of these toxins with several insect midgut proteins facilitating the formation of a pre-pore oligomer structure and subsequent membrane insertion that leads to the killing of midgut insect cells by osmotic shock. In this manuscript we review recent progress in understanding the mode of action of this family of proteins in lepidopteran, dipteran and coleopteran insects. Interestingly, similar Cry-binding proteins have been identified in the three insect orders, as cadherin, aminopeptidase-N and alkaline phosphatase suggesting a conserved mode of action. Also, recent data on insect responses to Cry toxin attack is discussed. Finally, we review the different Bt based products, including transgenic crops, that are currently used in agriculture. PMID:21376122

  4. Optimisation of insect cell growth in deep-well blocks: development of a high-throughput insect cell expression screen.

    PubMed

    Bahia, Daljit; Cheung, Robert; Buchs, Mirjam; Geisse, Sabine; Hunt, Ian

    2005-01-01

    This report describes a method to culture insects cells in 24 deep-well blocks for the routine small-scale optimisation of baculovirus-mediated protein expression experiments. Miniaturisation of this process provides the necessary reduction in terms of resource allocation, reagents, and labour to allow extensive and rapid optimisation of expression conditions, with the concomitant reduction in lead-time before commencement of large-scale bioreactor experiments. This therefore greatly simplifies the optimisation process and allows the use of liquid handling robotics in much of the initial optimisation stages of the process, thereby greatly increasing the throughput of the laboratory. We present several examples of the use of deep-well block expression studies in the optimisation of therapeutically relevant protein targets. We also discuss how the enhanced throughput offered by this approach can be adapted to robotic handling systems and the implications this has on the capacity to conduct multi-parallel protein expression studies.

  5. Roles of silkworm endoplasmic reticulum chaperones in the secretion of recombinant proteins expressed by baculovirus system.

    PubMed

    Imai, Saki; Kusakabe, Takahiro; Xu, Jian; Li, Zhiqing; Shirai, Shintaro; Mon, Hiroaki; Morokuma, Daisuke; Lee, Jae Man

    2015-11-01

    Baculovirus expression vector system (BEVS) is widely used for production of recombinant eukaryotic proteins in insect larvae or cultured cells. BEVS has advantages over bacterial expression system in producing post-translationally modified secreted proteins. However, for some unknown reason, it is very difficult for insects to secrete sufficiently for certain proteins of interest. To understand the reasons why insect cells fail to secrete some kinds of recombinant proteins, we here employed three mammalian proteins as targets, EPO, HGF, and Wnt3A, with different secretion levels in BEVS and investigated their mRNA transcriptions from the viral genome, subcellular localizations, and interactions with silkworm ER chaperones. Moreover, we observed that no significantly influence on the secretion amounts of all three proteins when depleting or overexpressing most endogenous ER chaperone genes in cultured silkworm cells. However, among all detected ER chaperones, the depletion of BiP severely decreased the recombinant protein secretion in BEVS, indicating the possible central role of Bip in silkworm secretion pathway.

  6. Production of Hev b5 as a fluorescent biotin-binding tripartite fusion protein in insect cells.

    PubMed

    Nordlund, Henri R; Laitinen, Olli H; Uotila, Sanna T H; Kulmala, Minna; Kalkkinen, Nisse; Kulomaa, Markku S

    2005-10-14

    The presented green fluorescent protein and streptavidin core-based tripartite fusion system provides a simple and efficient way for the production of proteins fused to it in insect cells. This fusion protein forms a unique tag, which serves as a multipurpose device enabling easy optimization of production, one-step purification via streptavidin-biotin interaction, and visualization of the fusion protein during downstream processing and in applications. In the present study, we demonstrate the successful production, purification, and detection of a natural rubber latex allergen Hev b5 with this system. We also describe the production of another NRL allergen with the system, Hev b1, which formed large aggregates and gave small yields in purification. The aggregates were detected at early steps by microscopical inspection of the infected insect cells producing this protein. Therefore, this fusion system can also be utilized as a fast indicator of the solubility of the expressed fusion proteins and may therefore be extremely useful in high-throughput expression approaches.

  7. Understanding regulation of the host-mediated gut symbiont population and the symbiont-mediated host immunity in the Riptortus-Burkholderia symbiosis system.

    PubMed

    Kim, Jiyeun Kate; Lee, Jun Beom; Jang, Ho Am; Han, Yeon Soo; Fukatsu, Takema; Lee, Bok Luel

    2016-11-01

    Valuable insect models have tremendously contributed to our understanding of innate immunity and symbiosis. Bean bug, Riptortus pedestris, is a useful insect symbiosis model due to harboring cultivable monospecific gut symbiont, genus Burkholderia. Bean bug is a hemimetabolous insect whose immunity is not well-understood. However, we recently identified three major antimicrobial peptides of Riptortus and examined the relationship between gut symbiosis and host immunity. We found that the presence of Burkholderia gut symbiont positively affects Riptortus immunity. From studying host regulation mechanisms of symbiont population, we revealed that the symbiotic Burkholderia cells are much more susceptible to Riptortus immune responses than the cultured cells. We further elucidated that the immune-susceptibility of the Burkholderia gut symbionts is due to the drastic change of bacterial cell envelope. Finally, we show that the immune-susceptible Burkholderia symbionts are able to prosper in host owing to the suppression of immune responses of the symbiotic midgut. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. In vivo preventive effects of insect tea on buccal mucosa cancer in ICR mice.

    PubMed

    Zhao, Xin; Wang, Rui; Qian, Yu; Li, Guijie; Zhou, Yalin; Sun, Peng

    2014-01-01

    Insect tea is a particular drink or health product in China and it is also used as Chinese medicine now. Its functional effects need to be proved. The ICR mice buccal mucosa cancer model was established by injecting the mice with U14 cells and mice was treated with insect tea. Tumor volumes and lymph node metastasis rates were determined. And the buccal mucosa tissues and cancer cervical lymph node were also checked by histology test, real-time polymerase chain reaction, and western blot assays. The tumor volumes for the group treated with insect tea mice was smaller than those from the control mice. The sections of buccal mucosa cancer tissue showed that the canceration of insect tea mice was weaker than control mice. Insect tea significantly induced apoptosis in buccal mucosa tissues by upregulating Bax, caspases, and downregulating Bcl-2. Nuclear factor-κB, inducible nitric oxide synthase, and COX-2 gene, was significantly downregulated by insect tea, demonstrating its anti-inflammatory properties. Insect tea also exerted a great anti-metastasis effect on tissues as demonstrated by decreased expression of Matrix metalloproteinases genes and increased expression of tissue inhibitors of matrix metalloproteinases. The highest concentration of 1600 mg/kg oral gavage and 400 mg/mL smear insect tea showed the best anticancer effects. Based on the results, insect tea showed the strong in vivo buccal mucosa cancer preventive effects.

  9. Stinging insect allergy: state of the art 2015.

    PubMed

    Tankersley, Michael S; Ledford, Dennis K

    2015-01-01

    Stinging insect allergy is responsible for more than 10% of all cases of anaphylaxis. The potential culprit insects are diverse and vary with geography. The incidence of insect allergy is declining in some areas and increasing in others, possibly due to effects of climate change, introduction of species into new areas, outdoor recreational activities, and movement of human populations that brings insects into contact with a greater number of people. Flying Hymenoptera and imported fire ant stings are responsible for the majority of patients evaluated for insect anaphylaxis. The most efficient means of identifying allergy to insects is skin testing although falsely positive and negative results occur. The limitations of testing coupled with the natural temporal variability of allergic sensitivity complicate the interpretation of test results. The clinical history is of paramount importance to be certain that the test results are relevant; therefore, screening or testing before a history of a sting reaction is not advisable. Mast cell disorders are associated with severe anaphylaxis from insect stings and should be considered in affected subjects. Insect immunotherapy, using venoms for most insects and whole-body extracts for imported fire ants, is proven effective in reducing the likelihood of anaphylaxis due to subsequent stings from 40%-60% to less than 5%. Future clinical application of component testing or in vitro cellular tests, such as the basophil activation test, may improve optimal choices for immunotherapy. Published by Elsevier Inc.

  10. An Automated Flying-Insect-Detection System

    NASA Technical Reports Server (NTRS)

    Vann, Timi; Andrews, Jane C.; Howell, Dane; Ryan, Robert

    2005-01-01

    An automated flying-insect-detection system (AFIDS) was developed as a proof-of-concept instrument for real-time detection and identification of flying insects. This type of system has use in public health and homeland security decision support, agriculture and military pest management, and/or entomological research. Insects are first lured into the AFIDS integrated sphere by insect attractants. Once inside the sphere, the insect's wing beats cause alterations in light intensity that is detected by a photoelectric sensor. Following detection, the insects are encouraged (with the use of a small fan) to move out of the sphere and into a designated insect trap where they are held for taxonomic identification or serological testing. The acquired electronic wing beat signatures are preprocessed (Fourier transformed) in real-time to display a periodic signal. These signals are sent to the end user where they are graphically displayed. All AFIDS data are pre-processed in the field with the use of a laptop computer equipped with LABVIEW. The AFIDS software can be programmed to run continuously or at specific time intervals when insects are prevalent. A special DC-restored transimpedance amplifier reduces the contributions of low-frequency background light signals, and affords approximately two orders of magnitude greater AC gain than conventional amplifiers. This greatly increases the signal-to-noise ratio and enables the detection of small changes in light intensity. The AFIDS light source consists of high-intensity Al GaInP light-emitting diodes (LEDs). The AFIDS circuitry minimizes brightness fluctuations in the LEDs and when integrated with an integrating sphere, creates a diffuse uniform light field. The insect wing beats isotropically scatter the diffuse light in the sphere and create wing beat signatures that are detected by the sensor. This configuration minimizes variations in signal associated with insect flight orientation.

  11. Before the 'Big Chill': Patterns of plant-insect associations from the Neogene of Iceland

    NASA Astrophysics Data System (ADS)

    Wappler, Torsten; Grímsson, Friðgeir

    2016-07-01

    Iceland is the only known terrestrial place in the subarctic North Atlantic providing a fairly continuous sedimentary and plant fossil record over the past 15 million years. While the basic palaeobotanical framework of this pattern has been well established during the last decade, less attention has been paid to the abundant insect traces on fossil leaves/leaflets. Here, we assess the diversity and frequency of insect herbivory on 4349 fossil angiosperm leaves/leaflets from six plant-bearing sedimentary formations exposed at 18 localities. By combining analyses of environmental factors, species interactions, ecology, biogeography, and the geological history, our results demonstrate how patterns of herbivory have changed over time in relation to temperature fluctuations that profoundly influenced levels of insect-mediated damage diversity and frequency. In addition, higher structural complexity, particularly the establishment of species-rich herb layer communities seems to have positively influenced the structure of insect communities in early late Miocene palaeoforests of Iceland.

  12. Colour constancy in insects.

    PubMed

    Chittka, Lars; Faruq, Samia; Skorupski, Peter; Werner, Annette

    2014-06-01

    Colour constancy is the perceptual phenomenon that the colour of an object appears largely unchanged, even if the spectral composition of the illuminating light changes. Colour constancy has been found in all insect species so far tested. Especially the pollinating insects offer a remarkable opportunity to study the ecological significance of colour constancy since they spend much of their adult lives identifying and choosing between colour targets (flowers) under continuously changing ambient lighting conditions. In bees, whose colour vision is best studied among the insects, the compensation provided by colour constancy is only partial and its efficiency depends on the area of colour space. There is no evidence for complete 'discounting' of the illuminant in bees, and the spectral composition of the light can itself be used as adaptive information. In patchy illumination, bees adjust their spatial foraging to minimise transitions between variously illuminated zones. Modelling allows the quantification of the adaptive benefits of various colour constancy mechanisms in the economy of nature. We also discuss the neural mechanisms and cognitive operations that might underpin colour constancy in insects.

  13. Silkworm (Bombyx mori) hemolymph unable to substitute fetal bovine serum in insect cell culture

    NASA Astrophysics Data System (ADS)

    Suparto, Irma H.; Khalam, Chandra Nur; Praira, Willy; Sajuthi, Dondin

    2014-03-01

    Fetal Bovine Serum (FBS) in animal cell culture media is an important source of nutrients for cell growth. However, the harvest and collection of FBS cause bioethical concerns. Efforts to reduce and preferably replace FBS with synthetic or other natural alternatives are continually being explored. Hemolymph silkworm (Bombyx mori) contains many nutrients needed for the process of metamorphosis. Therefore, there is possibility as an alternative nutritional supplement for cell culture to reduce the use of FBS. The objective of this study was to evaluate the macrocomponent of hemolymph and the possibility as medium supplement for Spodoptera fugiperda (Sf9) cell culture. Proximate analyses showed that hemolymph contains 89.76% of water, 2.52 mg/mL carbohydrate, 2.35% fat and 55.61 mg/mL protein. Further protein analysis, it consists of 15 fractions containing molecular weight of 22 - 152 kDa. The use of hemolymph as FBS substitution in Sf9 cell culture with various concentrations was unable to maintain and support cell growth. Further research still needed by prior adaptation of the tissue culture to minimal nutrition media before introduction of the hemolymph as supplement.

  14. The bat-bird-bug battle: daily flight activity of insects and their predators over a rice field revealed by high-resolution Scheimpflug Lidar

    NASA Astrophysics Data System (ADS)

    Malmqvist, Elin; Jansson, Samuel; Zhu, Shiming; Li, Wansha; Svanberg, Katarina; Svanberg, Sune; Rydell, Jens; Song, Ziwei; Bood, Joakim; Brydegaard, Mikkel; Åkesson, Susanne

    2018-04-01

    We present the results of, to our knowledge, the first Lidar study applied to continuous and simultaneous monitoring of aerial insects, bats and birds. It illustrates how common patterns of flight activity, e.g. insect swarming around twilight, depend on predation risk and other constraints acting on the faunal components. Flight activity was monitored over a rice field in China during one week in July 2016, using a high-resolution Scheimpflug Lidar system. The monitored Lidar transect was about 520 m long and covered approximately 2.5 m3. The observed biomass spectrum was bimodal, and targets were separated into insects and vertebrates in a categorization supported by visual observations. Peak flight activity occurred at dusk and dawn, with a 37 min time difference between the bat and insect peaks. Hence, bats started to feed in declining insect activity after dusk and stopped before the rise in activity before dawn. A similar time difference between insects and birds may have occurred, but it was not obvious, perhaps because birds were relatively scarce. Our observations are consistent with the hypothesis that flight activity of bats is constrained by predation in bright light, and that crepuscular insects exploit this constraint by swarming near to sunset/sunrise to minimize predation from bats.

  15. Fossil evidence for key innovations in the evolution of insect diversity.

    PubMed

    Nicholson, David B; Ross, Andrew J; Mayhew, Peter J

    2014-10-22

    Explaining the taxonomic richness of the insects, comprising over half of all described species, is a major challenge in evolutionary biology. Previously, several evolutionary novelties (key innovations) have been posited to contribute to that richness, including the insect bauplan, wings, wing folding and complete metamorphosis, but evidence over their relative importance and modes of action is sparse and equivocal. Here, a new dataset on the first and last occurrences of fossil hexapod (insects and close relatives) families is used to show that basal families of winged insects (Palaeoptera, e.g. dragonflies) show higher origination and extinction rates in the fossil record than basal wingless groups (Apterygota, e.g. silverfish). Origination and extinction rates were maintained at levels similar to Palaeoptera in the more derived Polyneoptera (e.g. cockroaches) and Paraneoptera (e.g. true bugs), but extinction rates subsequently reduced in the very rich group of insects with complete metamorphosis (Holometabola, e.g. beetles). Holometabola show evidence of a recent slow-down in their high net diversification rate, whereas other winged taxa continue to diversify at constant but low rates. These data suggest that wings and complete metamorphosis have had the most effect on family-level insect macroevolution, and point to specific mechanisms by which they have influenced insect diversity through time. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

  16. Insect Analogue to the Lotus Leaf: A Planthopper Wing Membrane Incorporating a Low-Adhesion, Nonwetting, Superhydrophobic, Bactericidal, and Biocompatible Surface.

    PubMed

    Watson, Gregory S; Green, David W; Cribb, Bronwen W; Brown, Christopher L; Meritt, Christopher R; Tobin, Mark J; Vongsvivut, Jitraporn; Sun, Mingxia; Liang, Ai-Ping; Watson, Jolanta A

    2017-07-19

    Nature has produced many intriguing and spectacular surfaces at the micro- and nanoscales. These small surface decorations act for a singular or, in most cases, a range of functions. The minute landscape found on the lotus leaf is one such example, displaying antiwetting behavior and low adhesion with foreign particulate matter. Indeed the lotus leaf has often been considered the "benchmark" for such properties. One could expect that there are animal counterparts of this self-drying and self-cleaning surface system. In this study, we show that the planthopper insect wing (Desudaba danae) exhibits a remarkable architectural similarity to the lotus leaf surface. Not only does the wing demonstrate a topographical likeness, but some surface properties are also expressed, such as nonwetting behavior and low adhering forces with contaminants. In addition, the insect-wing cuticle exhibits an antibacterial property in which Gram-negative bacteria (Porphyromonas gingivalis) are killed over many consecutive waves of attacks over 7 days. In contrast, eukaryote cell associations, upon contact with the insect membrane, lead to a formation of integrated cell sheets (e.g., among human stem cells (SHED-MSC) and human dermal fibroblasts (HDF)). The multifunctional features of the insect membrane provide a potential natural template for man-made applications in which specific control of liquid, solid, and biological contacts is desired and required. Moreover, the planthopper wing cuticle provides a "new" natural surface with which numerous interfacial properties can be explored for a range of comparative studies with both natural and man-made materials.

  17. The production of multiprotein complexes in insect cells using the baculovirus expression system.

    PubMed

    Abdulrahman, Wassim; Radu, Laura; Garzoni, Frederic; Kolesnikova, Olga; Gupta, Kapil; Osz-Papai, Judit; Berger, Imre; Poterszman, Arnaud

    2015-01-01

    The production of a homogeneous protein sample in sufficient quantities is an essential prerequisite not only for structural investigations but represents also a rate-limiting step for many functional studies. In the cell, a large fraction of eukaryotic proteins exists as large multicomponent assemblies with many subunits, which act in concert to catalyze specific activities. Many of these complexes cannot be obtained from endogenous source material, so recombinant expression and reconstitution are then required to overcome this bottleneck. This chapter describes current strategies and protocols for the efficient production of multiprotein complexes in large quantities and of high quality, using the baculovirus/insect cell expression system.

  18. Toxicity of allyl esters in insect cell lines and in Spodoptera littoralis larvae.

    PubMed

    Giner, Marta; Avilla, Jesús; Balcells, Mercè; Caccia, Silvia; Smagghe, Guy

    2012-01-01

    We investigated the effects of five allyl esters, two aromatic (allyl cinnamate and allyl 2-furoate) and three aliphatic (allyl hexanoate, allyl heptanoate, and allyl octanoate) in established insect cell lines derived from different species and tissues. We studied embryonic cells of the fruit fly Drosophila melanogaster (S2) (Diptera) and the beet armyworm Spodoptera exigua (Se4) (Lepidoptera), fat body cells of the Colorado potato beetle Leptinotarsa decemlineata (CPB) (Coleoptera), ovarian cells of the silkmoth Bombyx mori (Bm5), and midgut cells of the spruce budworm Choristoneura fumiferana (CF203) (Lepidoptera). Cytotoxicity was determined with use of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] and trypan blue. In addition, we tested the entomotoxic action of allyl cinnamate against the cotton leafworm Spodoptera littoralis .The median (50%) cytotoxic concentrations (EC₅₀s) of the five allyl esters in the MTT bioassays ranged between 0.25 and 27 mM with significant differences among allyl esters (P = 0.0012), cell lines (P < 0.0001), and the allyl ester-cell line interaction (P < 0.0001). Allyl cinnamate was the most active product, and CF203 the most sensitive cell line. In the trypan blue bioassays, cytotoxicity was produced rapidly and followed the same trend observed in the MTT bioassay. In first instars of S. littoralis, allyl cinnamate killed all larvae at 0.25% in the diet after 1 day, while this happened in third instars after 5 days. The LC₅₀ in first instars was 0.08%. In addition, larval weight gain was reduced (P < 0.05) after 1 day of feeding on diet with 0.05%. In conclusion, the data provide evidence of the significant but differential cytotoxicity among allyl esters in insect cells of different species and tissues. Midgut cells show high sensitivity, indicating the insect midgut as a primary target tissue. Allyl cinnamate caused rapid toxic effects in S. littoralis larvae at low concentrations, suggesting further potential for use in pest control. © 2011 Wiley Periodicals, Inc.

  19. Nonaggressive systemic mastocytosis (SM) without skin lesions associated with insect-induced anaphylaxis shows unique features versus other indolent SM.

    PubMed

    Alvarez-Twose, Iván; Zanotti, Roberta; González-de-Olano, David; Bonadonna, Patrizia; Vega, Arantza; Matito, Almudena; Sánchez-Muñoz, Laura; Morgado, José Mário; Perbellini, Omar; García-Montero, Andrés; De Matteis, Giovanna; Teodósio, Cristina; Rossini, Maurizio; Jara-Acevedo, María; Schena, Donatella; Mayado, Andrea; Zamò, Alberto; Mollejo, Manuela; Sánchez-López, Paula; Cabañes, Nieves; Orfao, Alberto; Escribano, Luis

    2014-02-01

    Indolent systemic mastocytosis (ISM) without skin lesions (ISMs(-)) shows a higher prevalence in males, lower serum baseline tryptase levels, and KIT mutation more frequently restricted to bone marrow (BM) mast cells (MCs) than ISM with skin lesions (ISMs(+)). Interestingly, in almost one-half of ISMs(-) patients, MC-mediator release episodes are triggered exclusively by insects. We aimed to determine the clinical and laboratory features of ISMs(-) associated with insect-induced anaphylaxis (insectISMs(-)) versus other patients with ISM. A total of 335 patients presenting with MC activation syndrome, including 143 insectISMs(-), 72 ISMs(-) triggered by other factors (otherISMs(-)), 56 ISMs(+), and 64 nonclonal MC activation syndrome, were studied. Compared with otherISMs(-) and ISMs(+) patients, insectISMs(-) cases showed marked male predominance (78% vs 53% and 46%; P < .001), a distinct pattern of MC-related symptoms, and significantly lower median serum baseline tryptase levels (22.4 vs 28.7 and 45.8 μg/L; P ≤ .009). Moreover, insectISMs(-) less frequently presented BM MC aggregates (46% vs 70% and 81%; P ≤ .001), and they systematically showed MC-restricted KIT mutation. ISMs(-) patients with anaphylaxis triggered exclusively by insects display clinical and laboratory features that are significantly different from other ISM cases, including other ISMs(-) and ISMs(+) patients, suggesting that they represent a unique subgroup of ISM with a particularly low BM MC burden in the absence of adverse prognostic factors. Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  20. Reducing the maladaptive attractiveness of solar panels to polarotactic insects.

    PubMed

    Horváth, Gábor; Blahó, Miklós; Egri, Adám; Kriska, György; Seres, István; Robertson, Bruce

    2010-12-01

    Human-made objects (e.g., buildings with glass surfaces) can reflect horizontally polarized light so strongly that they appear to aquatic insects to be bodies of water. Insects that lay eggs in water are especially attracted to such structures because these insects use horizontal polarization of light off bodies of water to find egg-laying sites. Thus, these sources of polarized light can become ecological traps associated with reproductive failure and mortality in organisms that are attracted to them and by extension with rapid population declines or collapse. Solar panels are a new source of polarized light pollution. Using imaging polarimetry, we measured the reflection-polarization characteristics of different solar panels and in multiple-choice experiments in the field we tested their attractiveness to mayflies, caddis flies, dolichopodids, and tabanids. At the Brewster angle, solar panels polarized reflected light almost completely (degree of polarization d ≈ 100%) and substantially exceeded typical polarization values for water (d ≈ 30-70%). Mayflies (Ephemeroptera), stoneflies (Trichoptera), dolichopodid dipterans, and tabanid flies (Tabanidae) were the most attracted to solar panels and exhibited oviposition behavior above solar panels more often than above surfaces with lower degrees of polarization (including water), but in general they avoided solar cells with nonpolarizing white borders and white grates. The highly and horizontally polarizing surfaces that had nonpolarizing, white cell borders were 10- to 26-fold less attractive to insects than the same panels without white partitions. Although solar panels can act as ecological traps, fragmenting their solar-active area does lessen their attractiveness to polarotactic insects. The design of solar panels and collectors and their placement relative to aquatic habitats will likely affect populations of aquatic insects that use polarized light as a behavioral cue. © 2010 Society for Conservation Biology.

  1. Dual Insect specific virus infection limits Arbovirus replication in Aedes mosquito cells.

    PubMed

    Schultz, Michaela J; Frydman, Horacio M; Connor, John H

    2018-05-01

    Aedes mosquitoes are vectors for many pathogenic viruses. Cell culture systems facilitate the investigation of virus growth in the mosquito vector. We found Zika virus (ZIKV) growth to be consistent in A. albopictus cells but hypervariable in A. aegypti cell lines. As a potential explanation of this variability, we tested the hypothesis that our cells harbored opportunistic viruses. We screened Aedes cell lines for the presence of insect specific viruses (ISVs), Cell-fusing agent virus (CFAV) and Phasi charoen-like virus (PCLV). PCLV was present in the ZIKV-growth-variable A. aegypti cell lines but absent in A. albopictus lines, suggesting that these ISVs may interfere with ZIKV growth. In support of this hypothesis, PCLV infection of CFAV-positive A. albopictus cells inhibited the growth of ZIKV, dengue virus and La Crosse virus. These data suggest ISV infection of cell lines can impact arbovirus growth leading to significant changes in cell permissivity to arbovirus infection. Copyright © 2018 Elsevier Inc. All rights reserved.

  2. Pectinases From Sphenophorus levis Vaurie, 1978 (Coleoptera: Curculionidae): Putative Accessory Digestive Enzymes

    PubMed Central

    Evangelista, Danilo Elton; de Paula, Fernando Fonseca Pereira; Rodrigues, André; Henrique-Silva, Flávio

    2015-01-01

    The cell wall in plants offers protection against invading organisms and is mainly composed of the polysaccharides pectin, cellulose, and hemicellulose, which can be degraded by plant cell wall degrading enzymes (PCWDEs). Such enzymes are often synthesized by free living microorganisms or endosymbionts that live in the gut of some animals, including certain phytophagous insects. Thus, the ability of an insect to degrade the cell wall was once thought to be related to endosymbiont enzyme activity. However, recent studies have revealed that some phytophagous insects are able to synthesize their own PCWDEs by endogenous genes, although questions regarding the origin of these genes remain unclear. This study describes two pectinases from the sugarcane weevil, Sphenophorus levis Vaurie, 1978 (Sl-pectinases), which is considered one of the most serious agricultural pests in Brazil. Two cDNA sequences identified in a cDNA library of the insect larvae coding for a pectin methylesterase (PME) and an endo-polygalacturonase (endo-PG)—denominated Sl-PME and Sl-endoPG, respectively—were isolated and characterized. The quantitative real-time reverse transcriptase polymerase chain reaction expression profile for both Sl-pectinases showed mRNA production mainly in the insect feeding stages and exclusively in midgut tissue of the larvae. This analysis, together Western blotting data, suggests that Sl-pectinases have a digestive role. Phylogenetic analyses indicate that Sl-PME and Sl-endoPG sequences are closely related to bacteria and fungi, respectively. Moreover, the partial genomic sequences of the pectinases were amplified from insect fat body DNA, which was certified to be free of endosymbiotic DNA. The analysis of genomic sequences revealed the existence of two small introns with 53 and 166 bp in Sl-endoPG, which is similar to the common pattern in fungal introns. In contrast, no intron was identified in the Sl-PME genomic sequence, as generally observed in bacteria. These data support the theory of horizontal gene transfer proposed for the origin of insect pectinases, reinforcing the acquisition of PME genes from bacteria and endo-PG genes from fungi. PMID:25673050

  3. The effect of ambient humidity and metabolic rate on the gas-exchange pattern of the semi-aquatic insect Aquarius remigis.

    PubMed

    Contreras, Heidy L; Bradley, Timothy J

    2011-04-01

    We have examined the effects of temperature on metabolic rate and respiratory pattern in the water strider Aquarius remigis. As temperature was increased from 10 to 30°C, the metabolic rate of the insects increased and the respiratory pattern transitioned from discontinuous, to cyclic, to continuous. The discontinuous gas-exchange cycle (DGC) was observed even in insects standing on water when the respirometry chamber was being perfused with humid (>95% relative humidity) air. Comparisons of insects at 20°C in humid and dry air showed no statistically significant differences in metabolic rate or respiratory pattern (P>0.05). The proportion of time that the spiracles were closed was greater at 10°C than at 20°C (P<0.01), and greater at 20°C than at 30°C (P<0.05). These results are compatible with the hypothesis that the respiratory patterns of insects are determined by the relationship between oxygen supply and oxygen demand. There was no evidence in this insect that humidity had any effect on the respiratory pattern. The results are discussed in the context of the ongoing discussion in the literature of the origin, maintenance and adaptive significance of the DGC in insects.

  4. 75 FR 69396 - Availability of an Environmental Assessment for a Biological Control Agent for Arundo donax

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-12

    ... infestations. The proposed biological control agent, Arundo scale, is one of the most damaging insects to A... feeding on cells that carry out photosynthesis and cellular respiration, resulting over time in gradual... donacis (Hemiptera: Diaspididae), an Insect for Biological Control of Arundo donax (Poaceae) in the...

  5. Low temperature stress during pupal development and its effects on adult performance in alfalfa leafcutting bees

    USDA-ARS?s Scientific Manuscript database

    Megachile rotundata develop in brood cells constructed in cavities by adult females. Pre-pupal bees diapause over winter and resume development as temperatures (Ta) increase in spring. While many insects are tolerant of suboptimal Ta in their overwintering stages, insects that initiate active develo...

  6. Hypothesis of snake and insect venoms against Human Immunodeficiency Virus: a review

    PubMed Central

    2009-01-01

    Background Snake and insect venoms have been demonstrated to have beneficial effects in the treatment of certain diseases including drug resistant human immunodeficiency virus (HIV) infection. We evaluated and hypothesized the probable mechanisms of venoms against HIV. Methods Previous literatures published over a period of 30 years (1979-2009) were searched using the key words snake venom, insect venom, mechanisms and HIV. Mechanisms were identified and discussed. Results & Conclusion With reference to mechanisms of action, properties and components of snake venom such as sequence homology and enzymes (protease or L- amino acid oxidase) may have an effect on membrane protein and/or act against HIV at multiple levels or cells carrying HIV virus resulting in enhanced effect of anti-retroviral therapy (ART). This may cause a decrease in viral load and improvement in clinical as well as immunological status. Insect venom and human Phospholipase A2 (PLA2) have potential anti-viral activity through inhibition of virion entry into the cells. However, all these require further evaluation in order to establish its role against HIV as an independent one or as a supplement. PMID:19922674

  7. [Expression of goat IL-18 mature protein in insect/baculovirus and determination of bioactivity of the recombinant protein].

    PubMed

    Wang, Ting-Ting; Wang, Xi-Hui; Fan, Zhong-Ling; Chen, Jin-Long; Cao, Bing-Lei; Kong, Na; Hu, Jing-Dong; Zhao, Hong-Kun

    2011-02-01

    To express goat IL-18 in insect/baculovirus and detect the bioactivity of the recombinant protein. The mature goat interleukin-18(gIL-18) gene was cloned into the baculovirus transfer vector pFastBac Dual, and then the resulting eukaryotic expression plasmid pFastBac Dual-gIL18 was transformed into DH10Bac, followed by the identification of Bacmid-gIL18 recombinat plosmid by three antibiotics and blue-white patch. Finally, the recombinant bacmid was transfected into sf9 insect cells by Cellfectin and the transfected cells were harvested at different times. Then the expressed protein was identified by SDS-PAGE, Western blot and bioactivity assay. The recombinant protein recognized and bound to its specific antibody. Bioactivity assay showed that the recombinant protein stimulated the proliferation of lymphocytes and induced IFN-γproduction in spleen lymphocytes. The mature gIL-18 protein has been expressed successfully in insect/baculovirus expression system, and have good immunogenicity and bioactivity. The study paves a way for application of gIL-18 as an immunomodulator or immune adjuvant.

  8. Biofuel cell backpacked insect and its application to wireless sensing.

    PubMed

    Shoji, Kan; Akiyama, Yoshitake; Suzuki, Masato; Nakamura, Nobuhumi; Ohno, Hiroyuki; Morishima, Keisuke

    2016-04-15

    This study investigated an enzymatic biofuel cell (BFC) which can be backpacked by cockroaches. The BFC generates electric power from trehalose in insect hemolymph by the trehalase and glucose dehydrogenase (GDH) reaction systems which dehydrogenate β-glucose obtained by hydrolyzing trehalose. First, an insect-mountable BFC (imBFC) was designed and fabricated with a 3D printer. The electrochemical reaction of anode-modified poly-L-lysine, vitamin K3, diaphorase, nicotinamide adenine dinucleotide, GDH and poly(sodium 4-styrenesulfonate) in the imBFC was evaluated and an oxidation current of 1.18 mAcm(-2) (at +0.6 V vs. Ag|AgCl) was observed. Then, the performance of the imBFC was evaluated and a maximum power output of 333 μW (285 μW cm(-)(2)) (at 0.5 V) was obtained. Furthermore, driving of both an LED device and a wireless temperature and humidity sensor device were powered by the imBFC. These results indicate that the imBFC has sufficient potential as a battery for novel ubiquitous robots such as insect cyborgs. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. How phloem-feeding insects face the challenge of phloem-located defenses

    PubMed Central

    Will, Torsten; Furch, Alexandra C. U.; Zimmermann, Matthias R.

    2013-01-01

    Due to the high content of nutrient, sieve tubes are a primary target for pests, e.g., most phytophagous hemipteran. To protect the integrity of the sieve tubes as well as their content, plants possess diverse chemical and physical defense mechanisms. The latter mechanisms are important because they can potentially interfere with the food source accession of phloem-feeding insects. Physical defense mechanisms are based on callose as well as on proteins and often plug the sieve tube. Insects that feed from sieve tubes are potentially able to overwhelm these defense mechanisms using their saliva. Gel saliva forms a sheath in the apoplast around the stylet and is suggested to seal the stylet penetration site in the cell plasma membrane. In addition, watery saliva is secreted into penetrated cells including sieve elements; the presence of specific enzymes/effectors in this saliva is thought to interfere with plant defense responses. Here we detail several aspects of plant defense and discuss the interaction of plants and phloem-feeding insects. Recent agro-biotechnological phloem-located aphid control strategies are presented. PMID:24009620

  10. Laboratory and Modeling Studies of Insect Swarms

    DTIC Science & Technology

    2016-03-10

    and measuring the response. These novel methods allowed us for the first time to characterize precisely properties of the swarm at the group level... Time series for a randomly chosen pair as well as its continuous wavelet transform (CWT; bottom panel). Nearly all of the power in the signal for... based time -frequency analysis to identify such transient interactions, as long as they modified the frequency structure of the insect flight

  11. 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.

  12. Metabolic interdependence of obligate intracellular bacteria and their insect hosts.

    PubMed

    Zientz, Evelyn; Dandekar, Thomas; Gross, Roy

    2004-12-01

    Mutualistic associations of obligate intracellular bacteria and insects have attracted much interest in the past few years due to the evolutionary consequences for their genome structure. However, much less attention has been paid to the metabolic ramifications for these endosymbiotic microorganisms, which have to compete with but also to adapt to another metabolism--that of the host cell. This review attempts to provide insights into the complex physiological interactions and the evolution of metabolic pathways of several mutualistic bacteria of aphids, ants, and tsetse flies and their insect hosts.

  13. Adaptation of Listeria monocytogenes in a simulated cheese medium: effects on virulence using the Galleria mellonella infection model.

    PubMed

    Schrama, D; Helliwell, N; Neto, L; Faleiro, M L

    2013-06-01

    The aim of this study was to evaluate the effect of the acid and salt adaptation in a cheese-based medium on the virulence potential of Listeria monocytogenes strains isolated from cheese and dairy processing environment using the Galleria mellonella model. Four L. monocytogenes strains were exposed to a cheese-based medium in conditions of induction of an acid tolerance response and osmotolerance response (pH 5·5 and 3·5% w/v NaCl) and injected in G. mellonella insects. The survival of insects and the L. monocytogenes growth kinetics in insects were evaluated. The gene expression of hly, actA and inlA genes was determined by real-time PCR. The adapted cells of two dairy strains showed reduced insect mortality (P < 0·05) in comparison with nonadapted cells. Listeria monocytogenes Scott A was the least virulent, whereas the cheese isolate C882 caused the highest insect mortality, and no differences (P > 0·05) was found between adapted and nonadapted cells. The gene expression results evidenced an overexpression of virulence genes in cheese-based medium, but not in simulated insect-induced conditions. Our results suggest that adaptation to low pH and salt in a cheese-based medium can affect the virulence of L. monocytogenes, but this effect is strain dependent. In this study, the impact of adaptation to low pH and salt in a cheese-based medium on L. monocytogenes virulence was tested using the Wax Moth G. mellonella model. This model allowed the differentiation of the virulence potential between the L. monocytogenes strains. The effect of adaptation on virulence is strain dependent. The G. mellonella model revealed to be a prompt method to test food-related factors on L. monocytogenes virulence. © 2013 The Society for Applied Microbiology.

  14. Expression and functional characterization of a C-7 cholesterol desaturase from Tetrahymena thermophila in an insect cell line.

    PubMed

    Poklepovich, Tomas J; Urtasun, Nicolás; Miranda, María V; Nusblat, Alejandro D; Nudel, Clara B

    2015-04-01

    Tetrahymena thermophila transforms exogenous cholesterol into pro-vitamin D3 (7-dehydrocholesterol) with remarkable efficiency in a one-step reaction carried out by a C-7 cholesterol desaturase. The enzyme DES7 is encoded by the gene TTHERM_00310640, identified with RNAi and gene knock-out experiments, but has not yet been heterologously expressed actively in any organism. A model derived from its amino acid sequence classified DES7p as a Rieske-type oxygenase with transmembrane localization. The protein has catalytic activity, sequence and topological similarity to DAF-36/Neverland proteins involved in the synthesis of steroid hormones in insects and nematodes. Due to their structural and functional similarity, we analyzed the expression of a codon optimized DES7 gene from Tetrahymena in the insect Sf9 cell line, identified and measured the steroid metabolites formed, and extended the actual knowledge on its localization. We found that the accumulation of 7-dehydrocholesterol could be increased 16-40-fold in Spodopterafrugiperda, depending on physiological conditions, by overexpression of T. thermophila DES7. The protein was detected in the microsomal fraction, in accordance with previous reports. Although the electron transfer chain for Des7p/DAF-36/Neverland Rieske-type oxygenases is presently unknown, we identified possible donors in the ciliate and insect genomes by bioinformatic analysis. In spite of the large evolutionary distance between S. frugiperda and T. thermophila, the results indicate that there is significant functional conservation of the electron donors, since the ciliate's sterol desaturase can function in the context of the insect electron transport system. The results achieved demonstrate that DES7 is the first gene from a ciliate, coding for a microsomal enzyme, expressed in active form in an insect cell line. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. The mitochondria-mediate apoptosis of Lepidopteran cells induced by azadirachtin.

    PubMed

    Huang, Jingfei; Lv, Chaojun; Hu, Meiying; Zhong, Guohua

    2013-01-01

    Mitochondria have been shown to play an important role in apoptosis using mammalian cell lines. However, this seems not to be the case in Drosophila, an insect model organism; thus more in-depth studies of insect cell apoptosis are necessary. In the present study, mitochondrial involvement during azadirachtin- and camptothecin-induced apoptosis in Spodoptera frugiperda Sf9 cells (isolated from Spodoptera frugiperda pupal ovarian tissue) was investigated. The results showed that both azadirachtin and camptothecin could induce apoptosis in Sf9 cells. Reactive oxygen species (ROS) generation, activation of mitochondrial permeability transition pores (MPTPs) and loss of mitochondrial membrane potential (MMP) were observed very early during apoptosis and were followed subsequently by the release of cytochrome-c from the mitochondria. Furthermore, the results also revealed that the opening of MPTPs and the loss of MMP induced by azadirachtin could be significantly inhibited by the permeability transition pore (PTP) inhibitor cyclosporin A (CsA), which was used to identify the key role of mitochondria in the apoptosis of Sf9 cells. However, in camptothecin-treated Sf9 cells, CsA could not suppress the opening of MPTPs and the loss of MMP when apoptosis was induced. The data from caspase-3 and caspase-9 activity assays and detection of apoptosis by morphological observation and flow cytometry also uncovered the different effect of CsA on the two botanical apoptosis inducers. Although different mechanisms of apoptosis induction exist, our study revealed that mitochondria play a crucial role in insect cell line apoptosis.

  16. The Mitochondria-Mediate Apoptosis of Lepidopteran Cells Induced by Azadirachtin

    PubMed Central

    Huang, Jingfei; Lv, Chaojun; Hu, Meiying; Zhong, Guohua

    2013-01-01

    Mitochondria have been shown to play an important role in apoptosis using mammalian cell lines. However, this seems not to be the case in Drosophila, an insect model organism; thus more in-depth studies of insect cell apoptosis are necessary. In the present study, mitochondrial involvement during azadirachtin- and camptothecin-induced apoptosis in Spodoptera frugiperda Sf9 cells (isolated from Spodoptera frugiperda pupal ovarian tissue) was investigated. The results showed that both azadirachtin and camptothecin could induce apoptosis in Sf9 cells. Reactive oxygen species (ROS) generation, activation of mitochondrial permeability transition pores (MPTPs) and loss of mitochondrial membrane potential (MMP) were observed very early during apoptosis and were followed subsequently by the release of cytochrome-c from the mitochondria. Furthermore, the results also revealed that the opening of MPTPs and the loss of MMP induced by azadirachtin could be significantly inhibited by the permeability transition pore (PTP) inhibitor cyclosporin A (CsA), which was used to identify the key role of mitochondria in the apoptosis of Sf9 cells. However, in camptothecin-treated Sf9 cells, CsA could not suppress the opening of MPTPs and the loss of MMP when apoptosis was induced. The data from caspase-3 and caspase-9 activity assays and detection of apoptosis by morphological observation and flow cytometry also uncovered the different effect of CsA on the two botanical apoptosis inducers. Although different mechanisms of apoptosis induction exist, our study revealed that mitochondria play a crucial role in insect cell line apoptosis. PMID:23516491

  17. Display of a maize cDNA library on baculovirus infected insect cells.

    PubMed

    Meller Harel, Helene Y; Fontaine, Veronique; Chen, Hongying; Jones, Ian M; Millner, Paul A

    2008-08-12

    Maize is a good model system for cereal crop genetics and development because of its rich genetic heritage and well-characterized morphology. The sequencing of its genome is well advanced, and new technologies for efficient proteomic analysis are needed. Baculovirus expression systems have been used for the last twenty years to express in insect cells a wide variety of eukaryotic proteins that require complex folding or extensive posttranslational modification. More recently, baculovirus display technologies based on the expression of foreign sequences on the surface of Autographa californica (AcMNPV) have been developed. We investigated the potential of a display methodology for a cDNA library of maize young seedlings. We constructed a full-length cDNA library of young maize etiolated seedlings in the transfer vector pAcTMVSVG. The library contained a total of 2.5 x 10(5) independent clones. Expression of two known maize proteins, calreticulin and auxin binding protein (ABP1), was shown by western blot analysis of protein extracts from insect cells infected with the cDNA library. Display of the two proteins in infected insect cells was shown by selective biopanning using magnetic cell sorting and demonstrated proof of concept that the baculovirus maize cDNA display library could be used to identify and isolate proteins. The maize cDNA library constructed in this study relies on the novel technology of baculovirus display and is unique in currently published cDNA libraries. Produced to demonstrate proof of principle, it opens the way for the development of a eukaryotic in vivo display tool which would be ideally suited for rapid screening of the maize proteome for binding partners, such as proteins involved in hormone regulation or defence.

  18. Magnetoreception in eusocial insects: an update

    PubMed Central

    Wajnberg, Eliane; Acosta-Avalos, Daniel; Alves, Odivaldo Cambraia; de Oliveira, Jandira Ferreira; Srygley, Robert B.; Esquivel, Darci M. S.

    2010-01-01

    Behavioural experiments for magnetoreception in eusocial insects in the last decade are reviewed. Ants and bees use the geomagnetic field to orient and navigate in areas around their nests and along migratory paths. Bees show sensitivity to small changes in magnetic fields in conditioning experiments and when exiting the hive. For the first time, the magnetic properties of the nanoparticles found in eusocial insects, obtained by magnetic techniques and electron microscopy, are reviewed. Different magnetic oxide nanoparticles, ranging from superparamagnetic to multi-domain particles, were observed in all body parts, but greater relative concentrations in the abdomens and antennae of honeybees and ants have focused attention on these segments. Theoretical models for how these specific magnetosensory apparatuses function have been proposed. Neuron-rich ant antennae may be the most amenable to discovering a magnetosensor that will greatly assist research into higher order processing of magnetic information. The ferromagnetic hypothesis is believed to apply to eusocial insects, but interest in a light-sensitive mechanism is growing. The diversity of compass mechanisms in animals suggests that multiple compasses may function in insect orientation and navigation. The search for magnetic compasses will continue even after a magnetosensor is discovered in eusocial insects. PMID:20106876

  19. Overturning dogma: tolerance of insects to mixed-sterol diets is not universal.

    PubMed

    Behmer, Spencer T

    2017-10-01

    Insects cannot synthesize sterols de novo, but like all eukaryotes they use them as cell membrane inserts where they influence membrane fluidity and rigidity. They also use a small amount for metabolic purposes, most notably as essential precursors for steroid hormones. It has been a long-held view that most insects require a small amount of specific sterol (often cholesterol) for metabolic purposes, but for membrane purposes (where the bulk of sterols are used) specificity in sterol structure was less important. Under this model, it was assumed that insects could tolerate mixed-sterol diets as long as a small amount of cholesterol was available. In the current paper this dogma is overturned, using data from plant-feeding insects that were fed mixed-sterol diets with different amounts and ratios of dietary sterols. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Transgenic Plant-Produced Hydrolytic Enzymes and the Potential of Insect Gut-Derived Hydrolases for Biofuels

    PubMed Central

    Willis, Jonathan D.; Mazarei, Mitra; Stewart, C. Neal

    2016-01-01

    Various perennial C4 grass species have tremendous potential for use as lignocellulosic biofuel feedstocks. Currently available grasses require costly pre-treatment and exogenous hydrolytic enzyme application to break down complex cell wall polymers into sugars that can then be fermented into ethanol. It has long been hypothesized that engineered feedstock production of cell wall degrading (CWD) enzymes would be an efficient production platform for of exogenous hydrolytic enzymes. Most research has focused on plant overexpression of CWD enzyme-coding genes from free-living bacteria and fungi that naturally break down plant cell walls. Recently, it has been found that insect digestive tracts harbor novel sources of lignocellulolytic biocatalysts that might be exploited for biofuel production. These CWD enzyme genes can be located in the insect genomes or in symbiotic microbes. When CWD genes are transformed into plants, negative pleiotropic effects are possible such as unintended cell wall digestion. The use of codon optimization along with organelle and tissue specific targeting improves CWD enzyme yields. The literature teaches several important lessons on strategic deployment of CWD genes in transgenic plants, which is the focus of this review. PMID:27303411

  1. The sensitivity of aquatic insects to divalent metals: a comparative analysis of laboratory and field data.

    PubMed

    Brix, Kevin V; DeForest, David K; Adams, William J

    2011-09-15

    Laboratory studies have traditionally indicated that aquatic insects are relatively insensitive to metals while field studies have suggested them to be among the most sensitive aquatic invertebrate taxa. We reviewed and synthesized available studies in the literature to critically assess why this discrepancy exists. Despite the intense effort to study the effects of metals on aquatic biota over the past several decades, we found studies specific to insects to still be relatively limited. In general, the discrepancy between laboratory and field studies continues with few efforts having been made to elucidate the ecological and physiological mechanisms that underlie the relative sensitivity (or insensitivity) of aquatic insects to metals. However, given the limited data available, it appears that aquatic insects are indeed relatively insensitive to acute metal exposures. In contrast, we suggest that some aquatic insect taxa may be quite sensitive to chronic metal exposure and in some cases may not be protected by existing water quality criteria for metals. The discrepancy between laboratory and field studies with respect to chronic sensitivity appears to largely be driven by the relatively short exposure periods in laboratory studies as compared to field studies. It also appears that, in some cases, the sensitivity of aquatic insects in field studies may be the result of direct effects on primary producers, which lead to indirect effects via the food chain on aquatic insects. Finally, available evidence suggests that diet is an important source of metal accumulation in insects, but to date there have been no conclusive studies evaluating whether dietary metal accumulation causes toxicity. There is a clear need for developing a more mechanistic understanding of aquatic insect sensitivity to metals in long-term laboratory and field studies. Copyright © 2011 Elsevier B.V. All rights reserved.

  2. Assembly of insect hormone enthusiasts at Nasu Highland, Japan: Report of the 3rd International Insect Hormone (21st Ecdysone) Workshop.

    PubMed

    Niwa, Ryusuke; Nishimura, Takashi

    2018-01-01

    The 3rd International Insect Hormone (21st Ecdysone) Workshop (IIHW2017) was held in July 2017 at Nasu Highland, Japan. In the 40 years of the workshop's history, this was the first to be held in an Asian country. A total of 109 insect hormone researchers from 18 countries (62 overseas and 47 domestic participants) attended IIHW2017. During the workshop, all participants stayed on-site at the venue's hotel; this was ideal for fostering communication between participants, in particular, interactions between principal investigators and young scientists. The workshop featured one keynote, 64 oral, and 35 poster presentations spanning molecular biology, cell biology, developmental biology, neurobiology, chemical biology, physiology, and ecology of insect hormones, including ecdysteroids, juvenile hormones, and a variety of neuropeptides. The workshop provided an ideal platform for discussing insect hormone biology using not only the typical genetic model insect, the fruit fly Drosophila, but also a diversity of interesting insects, such as the silkworm, the red flour beetle, the cricket, the dragonfly, the social ant, the bloodsucking tick, and so on. The participants succeeded in sharing the latest knowledge in a wide range of insect hormone research fields and in joining active and constructive scientific discussions. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  3. Environmental RNAi in herbivorous insects.

    PubMed

    Ivashuta, Sergey; Zhang, Yuanji; Wiggins, B Elizabeth; Ramaseshadri, Partha; Segers, Gerrit C; Johnson, Steven; Meyer, Steve E; Kerstetter, Randy A; McNulty, Brian C; Bolognesi, Renata; Heck, Gregory R

    2015-05-01

    Environmental RNAi (eRNAi) is a sequence-specific regulation of endogenous gene expression in a receptive organism by exogenous double-stranded RNA (dsRNA). Although demonstrated under artificial dietary conditions and via transgenic plant presentations in several herbivorous insects, the magnitude and consequence of exogenous dsRNA uptake and the role of eRNAi remains unknown under natural insect living conditions. Our analysis of coleopteran insects sensitive to eRNAi fed on wild-type plants revealed uptake of plant endogenous long dsRNAs, but not small RNAs. Subsequently, the dsRNAs were processed into 21 nt siRNAs by insects and accumulated in high quantities in insect cells. No accumulation of host plant-derived siRNAs was observed in lepidopteran larvae that are recalcitrant to eRNAi. Stability of ingested dsRNA in coleopteran larval gut followed by uptake and transport from the gut to distal tissues appeared to be enabling factors for eRNAi. Although a relatively large number of distinct coleopteran insect-processed plant-derived siRNAs had sequence complementarity to insect transcripts, the vast majority of the siRNAs were present in relatively low abundance, and RNA-seq analysis did not detect a significant effect of plant-derived siRNAs on insect transcriptome. In summary, we observed a broad genome-wide uptake of plant endogenous dsRNA and subsequent processing of ingested dsRNA into 21 nt siRNAs in eRNAi-sensitive insects under natural feeding conditions. In addition to dsRNA stability in gut lumen and uptake, dosage of siRNAs targeting a given insect transcript is likely an important factor in order to achieve measurable eRNAi-based regulation in eRNAi-competent insects that lack an apparent silencing amplification mechanism. © 2015 Ivashuta et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  4. Infection Density Dynamics of the Citrus Greening Bacterium "Candidatus Liberibacter asiaticus" in Field Populations of the Psyllid Diaphorina citri and Its Relevance to the Efficiency of Pathogen Transmission to Citrus Plants.

    PubMed

    Ukuda-Hosokawa, Rie; Sadoyama, Yasutsune; Kishaba, Misaki; Kuriwada, Takashi; Anbutsu, Hisashi; Fukatsu, Takema

    2015-06-01

    Huanglongbing, or citrus greening, is a devastating disease of citrus plants recently spreading worldwide, which is caused by an uncultivable bacterial pathogen, "Candidatus Liberibacter asiaticus," and vectored by a phloem-sucking insect, Diaphorina citri. We investigated the infection density dynamics of "Ca. Liberibacter asiaticus" in field populations of D. citri with experiments using field-collected insects to address how "Ca. Liberibacter asiaticus" infection density in the vector insect is relevant to pathogen transmission to citrus plants. Of 500 insects continuously collected from "Ca. Liberibacter asiaticus"-infected citrus trees with pathological symptoms in the spring and autumn of 2009, 497 (99.4%) were "Ca. Liberibacter asiaticus" positive. The infections were systemic across head-thorax and abdomen, ranging from 10(3) to 10(7) bacteria per insect. In spring, the infection densities were low in March, at ∼ 10(3) bacteria per insect, increasing up to 10(6) to 10(7) bacteria per insect in April and May, and decreasing to 10(5) to 10(6) bacteria per insect in late May, whereas the infection densities were constantly ∼ 10(6) to 10(7) bacteria per insect in autumn. Statistical analysis suggested that several factors, such as insect sex, host trees, and collection dates, may be correlated with "Ca. Liberibacter asiaticus" infection densities in field D. citri populations. Inoculation experiments with citrus seedlings using field-collected "Ca. Liberibacter asiaticus"-infected insects suggested that (i) "Ca. Liberibacter asiaticus"-transmitting insects tend to exhibit higher infection densities than do nontransmitting insects, (ii) a threshold level (∼ 10(6) bacteria per insect) of "Ca. Liberibacter asiaticus" density in D. citri is required for successful transmission to citrus plants, and (iii) D. citri attaining the threshold infection level transmits "Ca. Liberibacter asiaticus" to citrus plants in a stochastic manner. These findings provide valuable insights into understanding, predicting, and controlling this notorious citrus pathogen. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  5. Vitellogenin transcytosis in follicular cells of the honeybee Apis mellifera and the wasp Polistes simillimus.

    PubMed

    Dohanik, Virgínia Teles; Gonçalves, Wagner Gonzaga; Oliveira, Leandro Licursi; Zanuncio, José Cola; Serrão, José Eduardo

    2018-05-13

    Vitellogenin receptor (VgR) is a low-density lipoprotein receptor responsible for the mediated endocytosis of vitellogenin (Vg) during egg formation in insects. The maturing oocyte is enveloped by a follicular epithelium, which has large intercellular spaces during Vg accumulation (patency). However, Vg has been reported in the cytoplasm of follicular cells, indicating that there may be a transcellular route for its transport. This study verified the presence of VgR in the follicular cells of the ovaries of the honeybee Apis mellifera and the wasp Polistes simillimus in order to evaluate if Vg is transported via transcytosis in these insects. Antibodies specific for vitellogenin receptor (anti-VgR), vitellogenin (anti-Vg), and clathrin (anti-Clt) were used for immunolocalization. The results showed the presence of VgR on the apical and basal plasma membranes of follicular cells of the vitellogenic follicles in both species, indicating that VgR may have been transported from the basal to the apical cell domain, followed by its release into the perivitelline space, evidenced by the presence of apical plasma membrane projections containing VgR. Co-localization proved that Vg bind to VgR and that the transport of this protein is mediated by clathrin. These data suggest that, in these social insects, Vg is transported via clathrin-mediated VgR transcytosis in follicular cells.

  6. Characterization of canine herpesvirus glycoprotein C expressed by a recombinant baculovirus in insect cells.

    PubMed

    Xuan, X; Maeda, K; Mikami, T; Otsuka, H

    1996-12-01

    The gene encoding the canine herpesvirus (CHV) glycoprotein C (gC) homologue has been identified by sequence homology analyses with other well studied herpesviruses. Previously, we have identified three CHV glycoproteins, gp145/112, gp80 and gp47 using a panel of monoclonal antibodies (MAbs). To determine which CHV glycoprotein corresponds to gC, a recombinant baculovirus which contains the putative CHV gC structural gene under the baculovirus polyhedrin promoter was constructed. The recombinant baculovirus expressed gC-related polypeptides (44-62 kDa), which reacted only with MAbs against CHV gp80, indicating that the previously identified CHV gp80 is the translation product of the gC gene. The baculovirus expressed gC was glycosylated and transported to the surface of infected cells. At least seven neutralizing epitopes were conserved on the gC produced in insect cells. It was found that the recombinant baculovirus infected cells adsorbed murine erythrocytes as is the case for CHV-infected cells. The hemadsorption activity was inhibited by heparin, indicating that the CHV gC binds to heparan sulfate on the surface of murine erythrocytes. Mice immunized with the recombinant gC produced strong neutralizing antibodies. Our results suggest that CHV gC produced in insect cells may be useful as a subunit vaccine to control CHV infections.

  7. Expression, purification and characterization of inactive and active forms of ERK2 from insect expression system.

    PubMed

    Yan, Kelly; Merritt, Hanne; Crawford, Kenneth; Pardee, Gwynn; Cheng, Jan Marie; Widger, Stephania; Hekmat-Nejad, Mohammad; Zaror, Isabel; Sim, Janet

    2015-06-01

    Extracellular signal-regulated kinase 2 (ERK2) is a serine/threonine protein kinase involved in many cellular programs, such as cell proliferation, differentiation, motility and programed cell-death. It is therefore considered an important target in the treatment of cancer. In an effort to support biochemical screening and small molecule drug discovery, we established a robust system to generate both inactive and active forms of ERK2 using insect expression system. We report here, for the first time, that inactive ERK2 can be expressed and purified with 100% homogeneity in the unphosphorylated form using insect system. This resulted in a significant 20-fold yield improvement compared to that previously reported using bacterial expression system. We also report a newly developed system to generate active ERK2 in insect cells through in vivo co-expression with a constitutively active MEK1 (S218D S222D). Isolated active ERK2 was confirmed to be doubly phosphorylated at the correct sites, T185 and Y187, in the activation loop of ERK2. Both ERK2 forms, inactive and active, were well characterized by biochemical activity assay for their kinase function. Inactive and active ERK2 were the two key reagents that enabled successful high through-put biochemical assay screen and structural drug discovery studies. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. 7 CFR 457.148 - Fresh market pepper crop insurance provisions.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... sufficient to directly damage the crop. Freeze. The formation of ice in the cells of the plant or its fruit... (§ 457.8), we will not insure against any loss of production due to: (1) Disease or insect infestation, unless no effective control measure exists for such disease or insect infestation; or (2) Failure to...

  9. 7 CFR 457.148 - Fresh market pepper crop insurance provisions.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... directly damage the crop. Freeze—The formation of ice in the cells of the plant or its fruit, caused by low... against any loss of production due to: (1) Disease or insect infestation, unless no effective control measure exists for such disease or insect infestation; or (2) Failure to market the peppers, unless such...

  10. 7 CFR 457.148 - Fresh market pepper crop insurance provisions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... sufficient to directly damage the crop. Freeze. The formation of ice in the cells of the plant or its fruit... (§ 457.8), we will not insure against any loss of production due to: (1) Disease or insect infestation, unless no effective control measure exists for such disease or insect infestation; or (2) Failure to...

  11. 7 CFR 457.148 - Fresh market pepper crop insurance provisions.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... sufficient to directly damage the crop. Freeze. The formation of ice in the cells of the plant or its fruit... (§ 457.8), we will not insure against any loss of production due to: (1) Disease or insect infestation, unless no effective control measure exists for such disease or insect infestation; or (2) Failure to...

  12. The role of gut bacteria in Schmallenberg virus transmission by Culicoides biting midges

    USDA-ARS?s Scientific Manuscript database

    When an arbo-virus enters a vector it will first enter the gut system of this insect before entering cells of the insect body. Once in the gut-system, arbo-viruses and gut microbiota can interact with each other. We wondered if different gut bacterial communities could influence virus infection of b...

  13. Dramatic transcriptional changes in an intracellular parasite enable host switching between plant and insect.

    PubMed

    Oshima, Kenro; Ishii, Yoshiko; Kakizawa, Shigeyuki; Sugawara, Kyoko; Neriya, Yutaro; Himeno, Misako; Minato, Nami; Miura, Chihiro; Shiraishi, Takuya; Yamaji, Yasuyuki; Namba, Shigetou

    2011-01-01

    Phytoplasmas are bacterial plant pathogens that have devastating effects on the yields of crops and plants worldwide. They are intracellular parasites of both plants and insects, and are spread among plants by insects. How phytoplasmas can adapt to two diverse environments is of considerable interest; however, the mechanisms enabling the "host switching" between plant and insect hosts are poorly understood. Here, we report that phytoplasmas dramatically alter their gene expression in response to "host switching" between plant and insect. We performed a detailed characterization of the dramatic change that occurs in the gene expression profile of Candidatus Phytoplasma asteris OY-M strain (approximately 33% of the genes change) upon host switching between plant and insect. The phytoplasma may use transporters, secreted proteins, and metabolic enzymes in a host-specific manner. As phytoplasmas reside within the host cell, the proteins secreted from phytoplasmas are thought to play crucial roles in the interplay between phytoplasmas and host cells. Our microarray analysis revealed that the expression of the gene encoding the secreted protein PAM486 was highly upregulated in the plant host, which is also observed by immunohistochemical analysis, suggesting that this protein functions mainly when the phytoplasma grows in the plant host. Additionally, phytoplasma growth in planta was partially suppressed by an inhibitor of the MscL osmotic channel that is highly expressed in the plant host, suggesting that the osmotic channel might play an important role in survival in the plant host. These results also suggest that the elucidation of "host switching" mechanism may contribute to the development of novel pest controls.

  14. A spatial model with pulsed releases to compare strategies for the sterile insect technique applied to the mosquito Aedes aegypti.

    PubMed

    Oléron Evans, Thomas P; Bishop, Steven R

    2014-08-01

    We present a simple mathematical model to replicate the key features of the sterile insect technique (SIT) for controlling pest species, with particular reference to the mosquito Aedes aegypti, the main vector of dengue fever. The model differs from the majority of those studied previously in that it is simultaneously spatially explicit and involves pulsed, rather than continuous, sterile insect releases. The spatially uniform equilibria of the model are identified and analysed. Simulations are performed to analyse the impact of varying the number of release sites, the interval between pulsed releases and the overall volume of sterile insect releases on the effectiveness of SIT programmes. Results show that, given a fixed volume of available sterile insects, increasing the number of release sites and the frequency of releases increases the effectiveness of SIT programmes. It is also observed that programmes may become completely ineffective if the interval between pulsed releases is greater that a certain threshold value and that, beyond a certain point, increasing the overall volume of sterile insects released does not improve the effectiveness of SIT. It is also noted that insect dispersal drives a rapid recolonisation of areas in which the species has been eradicated and we argue that understanding the density dependent mortality of released insects is necessary to develop efficient, cost-effective SIT programmes. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  15. Influence of metal exposure history on the bioaccumulation and subcellular distribution of aqueous cadmium in the insect Hydropsyche californica

    USGS Publications Warehouse

    Cain, D.J.; Buchwalter, D.B.; Luoma, S.N.

    2006-01-01

    The influence of metal exposure history on rates of aqueous Cd accumulation, elimination, and subcellular distribution was examined in the aquatic insect Hydropsyche californica. Specimens were obtained from a reference site and a metal-contaminated site and returned to the laboratory where they were continuously exposed to aqueous Cd (518 ng/L, nominal) for 6 d, followed by 9 d of depuration. Rates of Cd accumulation and elimination were similar in insects from the two sites. Efflux rate constants, ke, ranged from 0.20 to 0.24/d (t1/2 ??? 3 d). Immediately following exposure, the cytosol accounted for 40% of the body burden in insects from both sites; however, 89 ?? 2% of the cytosolic Cd was associated with metallothionein-like proteins (MTLP) in insects from the contaminated site, compared to 60 ?? 0% in insects from the reference site. The concentration of Cd bound to non-MTLPs (representing potentially Cd-sensitive proteins) was significantly greater in the insects from the reference site (134 ?? 7 ng/g) than in those from the contaminated site (42 ?? 2 ng/g). At the end of the depuration period, 90% of the accumulated Cd body burden had been eliminated, and Cd concentrations in MTLPs and non-MTLPs were similar between the sites. Results suggested that differences in exposure history had no influence on the bioaccumulation of Cd, but did affect the concentrations of Cd bound to MTLP during Cd exposure in these insects. ?? 2006 SETAC.

  16. Minimizing fucosylation in insect cell-derived glycoproteins reduces binding to IgE antibodies from the sera of patients with allergy.

    PubMed

    Palmberger, Dieter; Ashjaei, Kazem; Strell, Stephanie; Hoffmann-Sommergruber, Karin; Grabherr, Reingard

    2014-09-01

    The baculovirus/insect cell system has proven to be a very powerful tool for the expression of several therapeutics. Nevertheless, these products sometimes suffer from reduced biological activity and unwanted side effects. Several studies have demonstrated that glycosylation can greatly influence the structure, function, half-life, antigenicity and immunogenicity of various glycoproteins. Yet, the glycosylation pattern of insect cell-derived products is not favorable for many applications. Especially, the presence of core α1,3-linked fucose bears the risk of causing immediate hypersensitivity reactions in patients with allergy. In this study, we evaluated the impact of fucose residues on the allergenic potential of an insect cell-expressed vaccine candidate. In order to block the GDP-L-fucose de novo synthesis pathway, we integrated the Pseudomonas aeruginosa GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD) gene into a baculovirus backbone. This virus was then used for the expression of soluble influenza A virus hemagglutinin (HA). Expression studies showed that the co-expression of RMD did not influence the overall level of recombinant protein secretion. We confirmed the result of our strategy by analyzing PNGase A-released N-glycans using MALDI-TOF-MS. In order to evaluate the biological impact of defucosylation of influenza HA we tested the binding activity of IgE derived from the sera of patients with allergy to the purified antigen. The non-fucosylated HA showed a 10-fold decrease in IgE binding levels as compared to wildtype variants. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Gene expression analysis predicts insect venom anaphylaxis in indolent systemic mastocytosis.

    PubMed

    Niedoszytko, M; Bruinenberg, M; van Doormaal, J J; de Monchy, J G R; Nedoszytko, B; Koppelman, G H; Nawijn, M C; Wijmenga, C; Jassem, E; Elberink, J N G Oude

    2011-05-01

    Anaphylaxis to insect venom (Hymenoptera) is most severe in patients with mastocytosis and may even lead to death. However, not all patients with mastocytosis suffer from anaphylaxis. The aim of the study was to analyze differences in gene expression between patients with indolent systemic mastocytosis (ISM) and a history of insect venom anaphylaxis (IVA) compared to those patients without a history of anaphylaxis, and to determine the predictive use of gene expression profiling. Whole-genome gene expression analysis was performed in peripheral blood cells. Twenty-two adults with ISM were included: 12 with a history of IVA and 10 without a history of anaphylaxis of any kind. Significant differences in single gene expression corrected for multiple testing were found for 104 transcripts (P < 0.05). Gene ontology analysis revealed that the differentially expressed genes were involved in pathways responsible for the development of cancer and focal and cell adhesion suggesting that the expression of genes related to the differentiation state of cells is higher in patients with a history of anaphylaxis. Based on the gene expression profiles, a naïve Bayes prediction model was built identifying patients with IVA. In ISM, gene expression profiles are different between patients with a history of IVA and those without. These findings might reflect a more pronounced mast cells dysfunction in patients without a history of anaphylaxis. Gene expression profiling might be a useful tool to predict the risk of anaphylaxis on insect venom in patients with ISM. Prospective studies are needed to substantiate any conclusions. © 2010 John Wiley & Sons A/S.

  18. The midgut of the silkmoth Bombyx mori is able to recycle molecules derived from degeneration of the larval midgut epithelium.

    PubMed

    Franzetti, Eleonora; Romanelli, Davide; Caccia, Silvia; Cappellozza, Silvia; Congiu, Terenzio; Rajagopalan, Muthukumaran; Grimaldi, Annalisa; de Eguileor, Magda; Casartelli, Morena; Tettamanti, Gianluca

    2015-08-01

    The midgut represents the middle part of the alimentary canal and is responsible for nutrient digestion and absorption in insect larva. Despite the growing interest in this organ for different purposes, such as studies on morphogenesis and differentiation, stem cell biology, cell death processes and transport mechanisms, basic information on midgut development is still lacking for a large proportion of insect species. Undoubtedly, this lack of data could hinder the full exploitation of practical applications that involve midgut as their primary target. This may represent in particular a significant problem for Lepidoptera, an insect order that includes some of the most important species of high economic importance. With the aim of overcoming this fragmentation of knowledge, we performed a detailed morphofunctional analysis of the midgut of the silkworm, Bombyx mori, a representative model among Lepidoptera, during its development from the larval up to the adult stage, focusing attention on stem cells. Our data demonstrate stem cell proliferation and differentiation, not only in the larval midgut but also in the pupal and adult midgut epithelium. Moreover, we present evidence for a complex trophic relationship between the dying larval epithelium and the new adult one, which is established during metamorphosis. This study, besides representing the first morphological and functional characterization of the changes that occur in the midgut of a lepidopteron during the transition from the larva to the moth, provides a detailed analysis of the midgut of the adult insect, a stage that has been neglected up to now.

  19. The endocrine system controlling sexual reproduction in animals: Part of the evolutionary ancient but well conserved immune system?

    PubMed

    De Loof, Arnold; Schoofs, Liliane; Huybrechts, Roger

    2016-01-15

    Drastic changes in hormone titers, in particular of steroid hormones, are intuitively interpreted as necessary and beneficial for optimal functioning of animals. Peaks in progesterone- and estradiol titers that accompany the estrus cycle in female vertebrates as well as in ecdysteroids at each molt and during metamorphosis of holometabolous insects are prominent examples. A recent analysis of insect metamorphosis yielded the view that, in general, a sharp rise in sex steroid hormone titer signals that somewhere in the body some tissue(s) is undergoing programmed cell death/apoptosis. Increased steroid production is part of this process. Typical examples are ovarian follicle cells in female vertebrates and invertebrates and the prothoracic gland cells, the main production site of ecdysteroids in larval insects. A duality emerges: programmed cell death-apoptosis is deleterious at the cellular level, but it may yield beneficial effects at the organismal level. Reconciling both opposites requires reevaluating the probable evolutionary origin and role of peptidic brain hormones that direct steroid hormone synthesis. Do e.g. Luteinizing Hormone in vertebrates and Prothoracicotropic Hormone (PTTH: acting through the Torso receptor) in insects still retain an ancient role as toxins in the early immune system? Does the functional link of some neuropeptides with Ca(2+)-induced apoptosis make sense in endocrine archeology? The endocrine system as a remnant of the ancient immune system is undoubtedly counterintuitive. Yet, we will argue that such paradigm enables the logical framing of many aspects, the endocrine one inclusive of both male and female reproductive physiology. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Identification and Characteristics of microRNAs from Army Worm, Spodoptera frugiperda Cell Line Sf21

    PubMed Central

    Kakumani, Pavan Kumar; Chinnappan, Mahendran; Singh, Ashok K.; Malhotra, Pawan; Mukherjee, Sunil K.; Bhatnagar, Raj K.

    2015-01-01

    microRNAs play important regulatory role in all intrinsic cellular functions. Amongst lepidopteran insects, miRNAs from only Bombyx mori have been studied extensively with a little focus on Spodoptera sp. In the present study, we identified a total of 226 miRNAs from Spodoptera frugiperda cell line Sf21. Of the total, 116 miRNAs were well conserved within other insects, like B. mori, Drosophila melanogaster and Tribolium castenum while the remaining 110 miRNAs were identified as novel based on comparative analysis with the insect miRNA data set. Landscape distribution analysis based on Sf21 genome assembly revealed clustering of few novel miRNAs. A total of 5 miRNA clusters were identified and the largest one encodes 5 miRNA genes. In addition, 12 miRNAs were validated using northern blot analysis and putative functional role assignment for 6 Sf miRNAs was investigated by examining their relative abundance at different developmental stages of Spodoptera litura and body parts of 6th instar larvae. Further, we identified a total of 809 potential target genes with GO terms for selected miRNAs, involved in different metabolic and signalling pathways of the insect. The newly identified miRNAs greatly enrich the repertoire of insect miRNAs and analysis of expression profiles reveal their involvement at various steps of biochemical pathways of the army worm. PMID:25693181

  1. Identification and characteristics of microRNAs from army worm, Spodoptera frugiperda cell line Sf21.

    PubMed

    Kakumani, Pavan Kumar; Chinnappan, Mahendran; Singh, Ashok K; Malhotra, Pawan; Mukherjee, Sunil K; Bhatnagar, Raj K

    2015-01-01

    microRNAs play important regulatory role in all intrinsic cellular functions. Amongst lepidopteran insects, miRNAs from only Bombyx mori have been studied extensively with a little focus on Spodoptera sp. In the present study, we identified a total of 226 miRNAs from Spodoptera frugiperda cell line Sf21. Of the total, 116 miRNAs were well conserved within other insects, like B. mori, Drosophila melanogaster and Tribolium castenum while the remaining 110 miRNAs were identified as novel based on comparative analysis with the insect miRNA data set. Landscape distribution analysis based on Sf21 genome assembly revealed clustering of few novel miRNAs. A total of 5 miRNA clusters were identified and the largest one encodes 5 miRNA genes. In addition, 12 miRNAs were validated using northern blot analysis and putative functional role assignment for 6 Sf miRNAs was investigated by examining their relative abundance at different developmental stages of Spodoptera litura and body parts of 6th instar larvae. Further, we identified a total of 809 potential target genes with GO terms for selected miRNAs, involved in different metabolic and signalling pathways of the insect. The newly identified miRNAs greatly enrich the repertoire of insect miRNAs and analysis of expression profiles reveal their involvement at various steps of biochemical pathways of the army worm.

  2. Comparative proteomics analysis of apoptotic Spodoptera frugiperda cells during p35 knockout Autographa californica multiple nucleopolyhedrovirus infection.

    PubMed

    Yu, Qian; Xiong, Youhua; Liu, Jianliang; Wang, Qin; Qiu, Yuanxin; Wen, Dongling

    2016-06-01

    Infection with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) mutants lacking a functional p35 gene can induce host cell apoptosis, which provides the possibility to use the potential of these viruses in the biological control of pest insects. Nonetheless, the proteomics or the protein changes of Spodoptera frugiperda (Sf9) cells infected with p35 knockout AcMNPV have not yet been studied. To further improve the use of AcMNPV, we set out to analyze the protein composition and protein changes of Sf9 cells of different infection stages by isobaric tag for relative and absolute quantification (iTRAQ) techniques. A total of 4004 sf9 proteins were identified by iTRAQ. After comparation of the significantly expressed 483 proteins from p35koAcMNPV-infected Sf9 cells and the significantly expressed 413 proteins from wtAcMNPV-infected Sf9 cells, we found that 226 proteins were specific to p35koAcMNPV-infected Sf9 cells. The 226 proteins were categorized according to GO classification for insects and were categorized into: biological processes, molecular functions and cellular components. Of interest, the most up-regulated proteins related to Epstein-Barr virus infection, RNA transport, Calcium signaling pathway, cGMP-PKG signaling pathway, oxidative phosphorylation and N-Glycan biosynthesis. Determination of the protein changes in p35 knockout AcMNPV-infected Sf9 cells would facilitate the better use of this virus-host cell interaction in pest insect control and other related fields. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Advances in allergic skin disease, anaphylaxis, and hypersensitivity reactions to foods, drugs, and insects.

    PubMed

    Sicherer, Scott H; Leung, Donald Y M

    2005-07-01

    This review highlights some of the research advances in allergic skin disease, anaphylaxis, and hypersensitivity reactions to foods, drugs, and insects that were reported primarily in the Journal in 2004. Clinical observations included that gastrointestinal symptoms during anaphylaxis are associated with an increased risk for hypotension; recurrence of peanut allergy can occur for about 8% of children who pass an oral food challenge and is associated with continued avoidance of the food after the challenge; seafood allergy is reported by 2.3% of the US population; and determination of the time to resolution of childhood egg and milk allergy might be predictable by means of serial determination of food-specific IgE levels. The comorbid effects of atopic dermatitis (AD) on asthma and the role of topical calcineurin inhibitors in the therapy of AD were also addressed. Basic and translational research observations indicate that improved diagnosis and therapy might become possible on the basis of reported identification or characterization of allergens such as: lipid transfer proteins and birch pollen-related cross-reactive allergens in plant foods; proteins in scorpion venom that cross-react with proteins from imported fire ant; mosquito saliva proteins responsible for systemic anaphylaxis; and IgE binding to quinolones detectable with an in vitro immunoassay. In addition, advances in understanding immune regulation associated with abrogation of oral tolerance in food allergy and of dendritic cell function, modulation of regulatory T cells, and chemokine expression in AD have elucidated possible targets for future intervention.

  4. Insect immune system maintains long-term resident bacteria through a local response.

    PubMed

    Login, Frédéric H; Heddi, Abdelaziz

    2013-02-01

    Long-term associations between bacteria and animals are widely represented in nature and play an important role in animal adaptation and evolution. In insects thriving on nutritionally unbalanced diets, intracellular symbiotic bacteria (endosymbionts) complement the host nutrients with amino acids and vitamins and interfere with host physiology and reproduction. Endosymbionts permanently infect host cells, called bacteriocytes, which express an adapted local immune response that permits symbiont maintenance and control. Among the immune players in bacteriocytes, the coleoptericin A (ColA) antimicrobial peptide of the cereal weevil, Sitophilus zeamais, was recently found to specifically trigger endosymbionts and to inhibit their cytokinesis, thereby limiting bacterial cell division and dispersion throughout the insect tissues. This review focuses on the biological and evolutionary features of Sitophilus symbiosis, and discusses the possible interactions of ColA with weevil endosymbiont proteins and pathways. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. Infectivity of Sf-rhabdovirus variants in insect and mammalian cell lines.

    PubMed

    Maghodia, Ajay B; Jarvis, Donald L

    2017-12-01

    Sf-rhabdovirus was only recently identified as an adventitious agent of Spodoptera frugiperda (Sf) cell lines used as hosts for baculovirus vectors. As such, we still know little about its genetic variation, infectivity, and the potential impact of variation on the Sf-rhabdovirus-host interaction. Here, we characterized Sf-rhabdoviruses from two widely used Sf cell lines to confirm and extend information on Sf-rhabdovirus variation. We then used our novel Sf-rhabdovirus-negative (Sf-RVN) Sf cell line to assess the infectivity of variants with and without a 320bp X/L deletion and found both established productive persistent infections in Sf-RVN cells. We also assessed their infectivity using heterologous insect and mammalian cell lines and found neither established productive persistent infections in these cells. These results are the first to directly demonstrate Sf-rhabdoviruses are infectious for Sf cells, irrespective of the X/L deletion. They also confirm and extend previous results indicating Sf-rhabdoviruses have a narrow host range. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Room Temperature Operable Autonomously Moving Bio-Microrobot Powered by Insect Dorsal Vessel Tissue

    PubMed Central

    Akiyama, Yoshitake; Hoshino, Takayuki; Iwabuchi, Kikuo; Morishima, Keisuke

    2012-01-01

    Living muscle tissues and cells have been attracting attention as potential actuator candidates. In particular, insect dorsal vessel tissue (DVT) seems to be well suited for a bio-actuator since it is capable of contracting autonomously and the tissue itself and its cells are more environmentally robust under culturing conditions compared with mammalian tissues and cells. Here we demonstrate an autonomously moving polypod microrobot (PMR) powered by DVT excised from an inchworm. We fabricated a prototype of the PMR by assembling a whole DVT onto an inverted two-row micropillar array. The prototype moved autonomously at a velocity of 3.5×10−2 µm/s, and the contracting force of the whole DVT was calculated as 20 µN. Based on the results obtained by the prototype, we then designed and fabricated an actual PMR. We were able to increase the velocity significantly for the actual PMR which could move autonomously at a velocity of 3.5 µm/s. These results indicate that insect DVT has sufficient potential as the driving force for a bio-microrobot that can be utilized in microspaces. PMID:22808004

  7. Development of a solar-powered infrared injection laser microminiature transmitting system

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

    Falter, D.D.; Alley, G.T.; Falter, K.G.

    1989-01-01

    A solar-powered infrared microminiature transmitting system is being developed to provide scientists with a tool to continuously track and study Africanized bees. Present tracking methods have limited ranges and lack the capability of continuously tracking individual insects. Preliminary field tests of a stationary prototypic transmitter have demonstrated a range of 1.1 km. The basic design consists of an array of nine 1-mm{sup 2} solar cells, which collect energy for storage in a 1.0-{mu}F tantalum chip capacitor. When the capacitor has been charged to a sufficient level, the circuitry that monitors the capacitor voltage level wakes up'' and fires a 5-{mu}smore » pulse through an 840-nm GaAlAs injection laser diode. The process is then repeated, making the signal frequency (which ranges from 50 to 300 Hz) dependent on solar luminance. The solar cells, capacitor, and laser diode are mounted in hybrid microcircuit fashion directly on the silicon substrate containing the CMOS control and driver circuitry. The transmitter measures {approximately}4 {times} 6 mm and weighs {approximately}65 mg. The receiving system is based on an 8-in. telescope and a Si PIN diode detector. 8 refs., 10 figs.« less

  8. Advances in allergic skin disease, anaphylaxis, and hypersensitivity reactions to foods, drugs, and insects in 2009.

    PubMed

    Sicherer, Scott H; Leung, Donald Y M

    2010-01-01

    This review highlights some of the research advances in anaphylaxis and hypersensitivity reactions to foods, drugs, and insects, as well as advances in allergic skin disease that were reported in the Journal in 2009. Among key epidemiologic observations, several westernized countries report that more than 1% of children have peanut allergy, and there is some evidence that environmental exposure to peanut is a risk factor. The role of regulatory T cells, complement, platelet-activating factor, and effector cells in the development and expression of food allergy were explored in several murine models and human studies. Delayed anaphylaxis to mammalian meats appears to be related to IgE binding to the carbohydrate moiety galactose-alpha-1,3-galactose, which also has implications for hypersensitivity to murine mAb therapeutics containing this oligosaccharide. Oral immunotherapy studies continue to show promise for the treatment of food allergy, but determining whether the treatment causes tolerance (cure) or temporary desensitization remains to be explored. Increased baseline serum tryptase levels might inform the risk of venom anaphylaxis and might indicate a risk for mast cell disorders in persons who have experienced such episodes. Reduced structural and immune barrier function contribute to local and systemic allergen sensitization in patients with atopic dermatitis, as well as increased propensity of skin infections in these patients. The use of increased doses of nonsedating antihistamines and potential usefulness of omalizumab for chronic urticaria was highlighted. These exciting advances reported in the Journal can improve patient care today and provide insights on how we can improve the diagnosis and treatment of these allergic diseases in the future. Copyright 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  9. Efficacy and non-target impact of spinosad, Bti and temephos larvicides for control of Anopheles spp. in an endemic malaria region of southern Mexico.

    PubMed

    Marina, Carlos F; Bond, J Guillermo; Muñoz, José; Valle, Javier; Novelo-Gutiérrez, Rodolfo; Williams, Trevor

    2014-01-30

    The larvicidal efficacy of the naturally derived insecticide spinosad, for control of immature stages of Anopheles albimanus and associated culicids, was compared to that of synthetic and biological larvicides. Effects on non-target insects were also determined. A field trial was performed in replicated temporary pools during the rainy season, in southern Mexico. Pools were treated with 10 ppm a.i. spinosad (Tracer 480SC), Bti granules applied at 2 kg/ha (VectoBac WDG, ABG-6511), and 100 ml/ha temephos (50 EC), or an untreated control. Numbers of immature mosquitoes, and aquatic insects in pools were monitored for 20 weeks. Samples of immature mosquitoes comprised approximately 10% An. albimanus, 70% Culex spp. (mostly Cx. melanoconion and Cx. coronator) and 20% Uranotaenia lowii. The most effective larvicides were spinosad and temephos that eliminated An. albimanus in 16 out of 20 post-treatment samples, or 9 weeks of continuous control of immature stages, respectively. These larvicides resulted in 15 and 5 weeks of elimination of Culex spp., respectively, or 20 and 4 weeks of continuous elimination of U. lowii, respectively. Bti treatment provided little consistent control. Aquatic insects were recorded comprising 3 orders, 20 families, 40 genera and 44 species. Shannon diversity index values (H') for aquatic insects were highest in the control (0.997) and Bti (0.974) treatments, intermediate in the spinosad treatment (0.638) and lowest in the temephos treatment (0.520). Severely affected non-target insects in the spinosad and temephos treated pools were predatory Coleoptera, Hemiptera and Odonata, which in the case of spinosad was likely due to the high concentration applied. Bti had little effect on aquatic insects. The spinosad treatment retained larvicidal activity for markedly longer than expected. Spinosad is likely to be an effective tool for control of anopheline and other pool-breeding mosquitoes in tropical regions. Non-target effects of spinosad on aquatic insects merit further study, but were likely related to the concentration of the product used.

  10. Spatial Navigation and the Central Complex: Sensory Acquisition, Orientation, and Motor Control

    PubMed Central

    Varga, Adrienn G.; Kathman, Nicholas D.; Martin, Joshua P.; Guo, Peiyuan; Ritzmann, Roy E.

    2017-01-01

    Cockroaches are scavengers that forage through dark, maze-like environments. Like other foraging animals, for instance rats, they must continually asses their situation to keep track of targets and negotiate barriers. While navigating a complex environment, all animals need to integrate sensory information in order to produce appropriate motor commands. The integrated sensory cues can be used to provide the animal with an environmental and contextual reference frame for the behavior. To successfully reach a goal location, navigational cues continuously derived from sensory inputs have to be utilized in the spatial guidance of motor commands. The sensory processes, contextual and spatial mechanisms, and motor outputs contributing to navigation have been heavily studied in rats. In contrast, many insect studies focused on the sensory and/or motor components of navigation, and our knowledge of the abstract representation of environmental context and spatial information in the insect brain is relatively limited. Recent reports from several laboratories have explored the role of the central complex (CX), a sensorimotor region of the insect brain, in navigational processes by recording the activity of CX neurons in freely-moving insects and in more constrained, experimenter-controlled situations. The results of these studies indicate that the CX participates in processing the temporal and spatial components of sensory cues, and utilizes these cues in creating an internal representation of orientation and context, while also directing motor control. Although these studies led to a better understanding of the CX's role in insect navigation, there are still major voids in the literature regarding the underlying mechanisms and brain regions involved in spatial navigation. The main goal of this review is to place the above listed findings in the wider context of animal navigation by providing an overview of the neural mechanisms of navigation in rats and summarizing and comparing our current knowledge on the CX's role in insect navigation to these processes. By doing so, we aimed to highlight some of the missing puzzle pieces in insect navigation and provide a different perspective for future directions. PMID:28174527

  11. Efficacy and non-target impact of spinosad, Bti and temephos larvicides for control of Anopheles spp. in an endemic malaria region of southern Mexico

    PubMed Central

    2014-01-01

    Background The larvicidal efficacy of the naturally derived insecticide spinosad, for control of immature stages of Anopheles albimanus and associated culicids, was compared to that of synthetic and biological larvicides. Effects on non-target insects were also determined. Methods A field trial was performed in replicated temporary pools during the rainy season, in southern Mexico. Pools were treated with 10 ppm a.i. spinosad (Tracer 480SC), Bti granules applied at 2 kg/ha (VectoBac WDG, ABG-6511), and 100 ml/ha temephos (50 EC), or an untreated control. Numbers of immature mosquitoes, and aquatic insects in pools were monitored for 20 weeks. Results Samples of immature mosquitoes comprised approximately 10% An. albimanus, 70% Culex spp. (mostly Cx. melanoconion and Cx. coronator) and 20% Uranotaenia lowii. The most effective larvicides were spinosad and temephos that eliminated An. albimanus in 16 out of 20 post-treatment samples, or 9 weeks of continuous control of immature stages, respectively. These larvicides resulted in 15 and 5 weeks of elimination of Culex spp., respectively, or 20 and 4 weeks of continuous elimination of U. lowii, respectively. Bti treatment provided little consistent control. Aquatic insects were recorded comprising 3 orders, 20 families, 40 genera and 44 species. Shannon diversity index values (H’) for aquatic insects were highest in the control (0.997) and Bti (0.974) treatments, intermediate in the spinosad treatment (0.638) and lowest in the temephos treatment (0.520). Severely affected non-target insects in the spinosad and temephos treated pools were predatory Coleoptera, Hemiptera and Odonata, which in the case of spinosad was likely due to the high concentration applied. Bti had little effect on aquatic insects. Conclusions The spinosad treatment retained larvicidal activity for markedly longer than expected. Spinosad is likely to be an effective tool for control of anopheline and other pool-breeding mosquitoes in tropical regions. Non-target effects of spinosad on aquatic insects merit further study, but were likely related to the concentration of the product used. PMID:24479683

  12. Influence of cell density and phase variants of bacterial symbionts (Xenorhabdus spp.) on dauer juvenile recovery and development of biocontrol nematodes Steinernema carpocapsae and S. feltiae (Nematoda: Rhabditida).

    PubMed

    Hirao, A; Ehlers, R-U

    2009-08-01

    The rhabditid nematodes Steinernema carpocapsae and Steinernema feltiae are used in biological control of insect pests. Mass production is done in liquid culture media pre-incubated with their bacterial symbionts Xenorhabdus nematophila and Xenorhabdus bovienii, respectively, before nematode dauer juveniles (DJs) are inoculated. As a response to food signals produced by the bacterial symbionts, the DJs exit from the developmentally arrested dauer stage (they recover development) and grow to adults, which produce DJ offspring. Variable DJ recovery after inoculation often causes process failure due to non-synchronous population development and low numbers of adult nematodes. This contribution investigated the influence of the bacterial cell density on DJ recovery and development to adults. At higher density of 10(10) bacterial cells ml(-1), a higher percentage of DJ recovery was induced, and adults occurred earlier in both Steinernema spp. than at lower density of 10(9) and 10(8) cells ml(-1). Xenorhabdus symbionts produce phase variants. Recovery in bacteria-free supernatants was lower than in supernatants containing bacterial cells for both primary and secondary phase Xenorhabdus spp. and lower in secondary than in primary phase supernatants or cell suspensions. In general, recovery was lower for Steinernema feltiae and the time at which 50% of the population had recovered after exposure to the food signal was longer (RT(50) = 17.1 h) than for Steinernema carpocapsae (RT(50) = 6.6 h). Whereas >90% S. carpocapsae DJs recovered in hemolymph serum of the lepidopteran insect Galleria mellonella, recovery of S. feltiae only reached 31%. Penetration into a host insect prior to exposure to the insect's food signal did not enhance DJ recovery. Consequences for liquid culture mass production of the nematodes and differences between species of the genera Steinernema and Heterorhabditis are discussed.

  13. Goose parvovirus structural proteins expressed by recombinant baculoviruses self-assemble into virus-like particles with strong immunogenicity in goose

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

    Ju, Huanyu; Wei, Na; Wang, Qian

    Highlights: {yields} All three capsid proteins can be expressed in insect cells in baculovirus expression system. {yields} All three recombinant proteins were spontaneously self-assemble into virus-like particles whose size and appearance were similar to those of native purified GPV virions. {yields} The immunogenicity of GPV-VLPs was better than commercial inactivated vaccine and attenuated vaccine. -- Abstract: Goose parvovirus (GPV), a small non-enveloped ssDNA virus, can cause Derzsy's disease, and three capsid proteins of VP1, VP2, and VP3 are encoded by an overlapping nucleotide sequence. However, little is known on whether recombinant viral proteins (VPs) could spontaneously assemble into virus-like particlesmore » (VLPs) in insect cells and whether these VLPs could retain their immunoreactivity and immunogenicity in susceptible geese. To address these issues, genes for these GPV VPs were amplified by PCR, and the recombinant VPs proteins were expressed in insect cells using a baculovirus expression system for the characterization of their structures, immunoreactivity, and immunogenicity. The rVP1, rVP2, and rVP3 expressed in Sf9 cells were detected by anti-GPV sera, anti-VP3 sera, and anti-His antibodies, respectively. Electron microscopy revealed that these rVPs spontaneously assembled into VLPs in insect cells, similar to that of the purified wild-type GPV virions. In addition, vaccination with individual types of VLPs, particularly with the rVP2-VLPs, induced higher titers of antibodies and neutralized different strains of GPVs in primary goose and duck embryo fibroblast cells in vitro. These data indicated that these VLPs retained immunoreactivity and had strong immunogenicity in susceptible geese. Therefore, our findings may provide a framework for development of new vaccines for the prevention of Derzsy's disease and vehicles for the delivery of drugs.« less

  14. Using pennycress, camelina, and canola cash crops to provision pollinators

    USDA-ARS?s Scientific Manuscript database

    As pollinator decline continues, the need to provide high value forage for insects continues to rise. Finding agricultural crops to diversify the landscape and provide forage is one way to improve pollinator health. Three winter industrial oilseed crops (pennycress, winter camelina, and winter canol...

  15. Responses of tree and insect herbivores to elevated nitrogen inputs: A meta-analysis

    NASA Astrophysics Data System (ADS)

    Li, Furong; Dudley, Tom L.; Chen, Baoming; Chang, Xiaoyu; Liang, Liyin; Peng, Shaolin

    2016-11-01

    Increasing atmospheric nitrogen (N) inputs have the potential to alter terrestrial ecosystem function through impacts on plant-herbivore interactions. The goal of our study is to search for a general pattern in responses of tree characteristics important for herbivores and insect herbivorous performance to elevated N inputs. We conducted a meta-analysis based on 109 papers describing impacts of nitrogen inputs on tree characteristics and 16 papers on insect performance. The differences in plant characteristics and insect performance between broadleaves and conifers were also explored. Tree aboveground biomass, leaf biomass and leaf N concentration significantly increased under elevated N inputs. Elevated N inputs had no significantly overall effect on concentrations of phenolic compounds and lignin but adversely affected tannin, as defensive chemicals for insect herbivores. Additionally, the overall effect of insect herbivore performance (including development time, insect biomass, relative growth rate, and so on) was significantly increased by elevated N inputs. According to the inconsistent responses between broadleaves and conifers, broadleaves would be more likely to increase growth by light interception and photosynthesis rather than producing more defensive chemicals to elevated N inputs by comparison with conifers. Moreover, the overall carbohydrate concentration was significantly reduced by 13.12% in broadleaves while increased slightly in conifers. The overall tannin concentration decreased significantly by 39.21% in broadleaves but a 5.8% decrease in conifers was not significant. The results of the analysis indicated that elevated N inputs would provide more food sources and ameliorate tree palatability for insects, while the resistance of trees against their insect herbivores was weakened, especially for broadleaves. Thus, global forest insect pest problems would be aggravated by elevated N inputs. As N inputs continue to rise in the future, forest ecosystem management should pay more attention to insect pest, especially in the regions dominated by broadleaves.

  16. The study of insect blood-feeding behaviour. 2. Recording techniques and the use of flow charts.

    PubMed

    Smith, J J; Friend, W G

    1987-01-01

    This paper continues a discussion of approaches and methodologies we have used in our studies of feeding in haematophagous insects. Described are techniques for directly monitoring behaviour: electrical recording of feeding behaviour via resistance changes in the food canal, optical methods for monitoring mouthpart activity, and a computer technique for behavioural event recording. Also described is the use of "flow charts" or "decision diagrams" to model interrelated sequences of behaviours.

  17. Molecular interactions and immune responses between maize fine streak virus and the leafhopper vector G. nigrifrons through differential expression and RNA interference

    USDA-ARS?s Scientific Manuscript database

    Maize fine streak virus (MFSV) is an emerging virus of maize that is transmitted by an insect vector, the leafhopper called Graminella nigrifrons. Virus transmission by the leafhopper requires that the virus enter into and multiply in insect cells, tissues and organs before being transmitted to a ne...

  18. Continuous Measurement of Macronutrient Ions in the Transpiration Stream of Intact Plants Using the Meadow Spittlebug Coupled with Ion Chromatography1

    PubMed Central

    Malone, Michael; Herron, Michelle; Morales, M.-Angeles

    2002-01-01

    A method is described for continuous, nondestructive analysis of xylem-borne mineral nutrients in intact transpiring plants. The method uses the xylem-feeding insect the meadow spittlebug (Philaenus spumarius L. [Homoptera: Cercopidae]). This insect will feed from a wide range of plant species and organs. Insect excreta can be collected at all times of the day and night, and its mineral ion content can be analyzed rapidly, and without purification, by ion chromatography. The excreta will have a mineral content virtually identical to that of xylem sap. Cages suitable for containing the insects and collecting excreta from any desired location on plants in both laboratory and greenhouse are described. Even in the greenhouse, evaporation had only a minor effect on the sample ion content. Example results are presented which illustrate dynamics, over several days, in the xylem concentrations of sodium (Na+), potassium (K+), NH4+, magnesium (Mg2+), calcium (Ca2+), chloride (Cl−), NO3−, PO43−, and SO42−. These data were collected from young plants growing in pots of compost in the laboratory and from fully mature pepper (Capsicum annuum L. cv Bellboy) plants growing in hydroponics (rockwool) in the greenhouse. This method should facilitate studies of macronutrient uptake and transport in a range of plants and environments. PMID:12428008

  19. Double abdomen in a short-germ insect: Zygotic control of axis formation revealed in the beetle Tribolium castaneum

    PubMed Central

    Ansari, Salim; Troelenberg, Nicole; Dao, Van Anh; Richter, Tobias; Klingler, Martin

    2018-01-01

    The distinction of anterior versus posterior is a crucial first step in animal embryogenesis. In the fly Drosophila, this axis is established by morphogenetic gradients contributed by the mother that regulate zygotic target genes. This principle has been considered to hold true for insects in general but is fundamentally different from vertebrates, where zygotic genes and Wnt signaling are required. We investigated symmetry breaking in the beetle Tribolium castaneum, which among insects represents the more ancestral short-germ embryogenesis. We found that maternal Tc-germ cell-less is required for anterior localization of maternal Tc-axin, which represses Wnt signaling and promotes expression of anterior zygotic genes. Both RNAi targeting Tc-germ cell-less or double RNAi knocking down the zygotic genes Tc-homeobrain and Tc-zen1 led to the formation of a second growth zone at the anterior, which resulted in double-abdomen phenotypes. Conversely, interfering with two posterior factors, Tc-caudal and Wnt, caused double-anterior phenotypes. These findings reveal that maternal and zygotic mechanisms, including Wnt signaling, are required for establishing embryo polarity and induce the segmentation clock in a short-germ insect. PMID:29432152

  20. New directions in diagnostic evaluation of insect allergy.

    PubMed

    Golden, David B K

    2014-08-01

    Diagnosis of insect sting allergy and prediction of risk of sting anaphylaxis are often difficult because tests for venom-specific IgE antibodies have a limited positive predictive value and do not reliably predict the severity of sting reactions. Component-resolved diagnosis using recombinant venom allergens has shown promise in improving the specificity of diagnostic testing for insect sting allergy. Basophil activation tests have been explored as more sensitive assays for identification of patients with insect allergy and for prediction of clinical outcomes. Measurement of mast cell mediators reflects the underlying risk for more severe reactions and limited clinical response to treatment. Measurement of IgE to recombinant venom allergens can distinguish cross-sensitization from dual sensitization to honeybee and vespid venoms, thus helping to limit venom immunotherapy to a single venom instead of multiple venoms in many patients. Basophil activation tests can detect venom allergy in patients who show no detectable venom-specific IgE in standard diagnostic tests and can predict increased risk of systemic reactions to venom immunotherapy, and to stings during and after stopping venom immunotherapy. The risk of severe or fatal anaphylaxis to stings can also be predicted by measurement of baseline serum tryptase or other mast cell mediators.

  1. Insect-specific flaviviruses, a worldwide widespread group of viruses only detected in insects.

    PubMed

    Calzolari, Mattia; Zé-Zé, Líbia; Vázquez, Ana; Sánchez Seco, Mari Paz; Amaro, Fátima; Dottori, Michele

    2016-06-01

    Several flaviviruses are important pathogens for humans and animals (Dengue viruses, Japanese encephalitis virus, Yellow-fever virus, Tick-borne encephalitis virus, West Nile virus). In recent years, numerous novel and related flaviviruses without known pathogenic capacity have been isolated worldwide in the natural mosquito population. However, phylogenetic studies have shown that genomic sequences of these viruses diverge from other flaviviruses. Moreover, these viruses seem to be exclusive of insects (they do not seem to grow on vertebrate cell lines), and were already defined as mosquito-only flaviviruses or insect-specific flaviviruses. At least eleven of these viruses were isolated worldwide, and sequences ascribable to other eleven putative viruses were detected in several mosquito species. A large part of the cycle of these viruses is not well known, and their persistence in the environment is poorly understood. These viruses are detected in a wide variety of distinct mosquito species and also in sandflies and chironomids worldwide; a single virus, or the genetic material ascribable to a virus, was detected in several mosquito species in different countries, often in different continents. Furthermore, some of these viruses are carried by invasive mosquitoes, and do not seem to have a depressive action on their fitness. The global distribution and the continuous detection of new viruses in this group point out the likely underestimation of their number, and raise interesting issues about their possible interactions with the pathogenic flaviviruses, and their influence on the bionomics of arthropod hosts. Some enigmatic features, as their integration in the mosquito genome, the recognition of their genetic material in DNA forms in field-collected mosquitoes, or the detection of the same virus in both mosquitoes and sandflies, indicate that the cycle of these viruses has unknown characteristics that could be of use to reach a deeper understanding of the cycle of related pathogenic flaviviruses. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. [Recombinant Vp2 protein of infectious bursal disease virus AH1 strain expressed in insect cells: a vaccine candidate].

    PubMed

    Ouyang, Wei; Wang, Yongshan; Zhou, Yu; Zhang, Haibin; Tang, Yude

    2010-05-01

    Protective immune response of the available IBD vaccine is insufficient to fully protect against the prevailing strain of the infectious bursal disease virus (IBDV). Such a vaccination escape IBDV field isolate idenfied from Anhui province of China in December 2007, where IBD broke out at 2 weeks post vaccination. The IBDV vp2 gene was cloned into pFastBacHTA donor plasmid, followed by generation of the recombinant bacmid DNA pBac-VP2. The latter was used to transfect insect cell Sf9 with Lipofectamine to produce recombinant baculovirus vBac-VP2. The Sf9 cells infected with vBac-VP2 were stained positive against IBDV antibody using the indirect immunofluorescence assay (IFA), which was also confirmed by the detection of IBDV Vp2 protein in the infected Sf9 cells by IBDV sandwich ELISA. Western blotting revealed that the calculated protein of approximately 53 kDa was in the expressed in the insect cells. Moreover, virus-like particles (VLPs) and "inclusion body-like"structure in the infected Sf9 cells were observed under electron microscopy. We further developed an indirect ELISA for the detection of the IBDV antibodies, which was specific and sensitive. In addition, the lysates of vBac-VP2 infected cells was used to immunize 2-week-old SPF chickens, followed by challenging with the virulent IBDV, the survival rate was 30% at 14 days post primary immunization, however, the survival rate was 100% at 14 d after the booster vaccination. The ELISA antibody titers was up to 3.2 x 10(3) and neutralization antibody titer was 2536, significantly higher than those of one-shot vaccination, 8 x 10(2) and 1106, respectively. The immunized chickens did not show any clinical signs and histopathological changes of infection in 7-days trial time. The bursa/body-weight ratios were higher than those of the unimmunized control (P < 0.05). The virus-like-particle recombinant Vp2 protein expressed in insect cells promises to be a novel subunit vaccine and diagnostic reagent candidate for IBDV.

  3. Participation of 14-3-3ε and 14-3-3ζ proteins in the phagocytosis, component of cellular immune response, in Aedes mosquito cell lines.

    PubMed

    Trujillo-Ocampo, Abel; Cázares-Raga, Febe Elena; Del Angel, Rosa María; Medina-Ramírez, Fernando; Santos-Argumedo, Leopoldo; Rodríguez, Mario H; Hernández-Hernández, Fidel de la Cruz

    2017-08-01

    Better knowledge of the innate immune system of insects will improve our understanding of mosquitoes as potential vectors of diverse pathogens. The ubiquitously expressed 14-3-3 protein family is evolutionarily conserved from yeast to mammals, and at least two isoforms of 14-3-3, the ε and ζ, have been identified in insects. These proteins have been shown to participate in both humoral and cellular immune responses in Drosophila. As mosquitoes of the genus Aedes are the primary vectors for arboviruses, causing several diseases such as dengue fever, yellow fever, Zika and chikungunya fevers, cell lines derived from these mosquitoes, Aag-2 from Aedes aegypti and C6/36 HT from Aedes albopictus, are currently used to study the insect immune system. Here, we investigated the role of 14-3-3 proteins (ε and ζ isoform) in phagocytosis, the main cellular immune responses executed by the insects, using Aedes spp. cell lines. We evaluated the mRNA and protein expression of 14-3-3ε and 14-3-3ζ in C6/36 HT and Aag-2 cells, and demonstrated that both proteins were localised in the cytoplasm. Further, in C6/36 HT cells treated with a 14-3-3 specific inhibitor we observed a notable modification of cell morphology with filopodia-like structure caused through cytoskeleton reorganisation (co-localization of 14-3-3 proteins with F-actin), more importantly the decrease in Salmonella typhimurium, Staphylococcus aureus and E. coli phagocytosis and reduction in phagolysosome formation. Additionally, silencing of 14-3-3ε and 14-3-3ζ expression by mean of specific DsiRNA confirmed the decreased phagocytosis and phagolysosome formation of pHrodo labelled E. coli and S. aureus bacteria by Aag-2 cells. The 14-3-3ε and 14-3-3ζ proteins modulate cytoskeletal remodelling, and are essential for phagocytosis of Gram-positive and Gram-negative bacteria in Aedes spp. cell lines.

  4. Oxidative stress in entomopathogenic fungi grown on insect-like hydrocarbons.

    PubMed

    Huarte-Bonnet, Carla; Juárez, M Patricia; Pedrini, Nicolás

    2015-08-01

    Entomopathogenic fungi mostly attack their insect hosts by penetration through the cuticle. The outermost insect surface is covered by a lipid-rich layer, usually composed of very long chain hydrocarbons. These fungi are apt to grow on straight chain hydrocarbons (alkanes) as the sole carbon source. Insect-like hydrocarbons are first hydroxylated by a microsomal P450 monooxygenase system, and then fully catabolized by peroxisomal β-oxidation reactions in Beauveria bassiana. In this review, we will discuss lipid metabolism adaptations in alkane-grown fungi, and how an oxidative stress scenario is established under these conditions. Fungi have to pay a high cost for hydrocarbon utilization; high levels of reactive oxygen species are produced and a concomitant antioxidant response is triggered in fungal cells to cope with this drawback.

  5. Contribution of different taste cells and signaling pathways to the discrimination of "bitter" taste stimuli by an insect.

    PubMed

    Glendinning, John I; Davis, Adrienne; Ramaswamy, Sudha

    2002-08-15

    Animals can discriminate among many different types of foods. This discrimination process involves multiple sensory systems, but the sense of taste is known to play a central role. We asked how the taste system contributes to the discrimination of different "bitter" taste stimuli in Manduca sexta caterpillars. This insect has approximately eight bilateral pairs of taste cells that respond selectively to bitter taste stimuli. Each bilateral pair of bitter-sensitive taste cells has a different molecular receptive range (MRR); some of these taste cells also contain two signaling pathways with distinctive MRRs and temporal patterns of spiking. To test for discrimination, we habituated the caterpillar's taste-mediated aversive response to one bitter taste stimulus (salicin) and then asked whether this habituation phenomenon generalized to four other bitter taste stimuli (caffeine, aristolochic acid, Grindelia extract, and Canna extract). We inferred that the two compounds were discriminable if the habituation phenomenon failed to generalize (e.g., from salicin to aristolochic acid). We found that M. sexta could discriminate between salicin and those bitter taste stimuli that activate (1) different populations of bitter-sensitive taste cells (Grindelia extract and Canna extract) or (2) different signaling pathways within the same bitter-sensitive taste cell (aristolochic acid). M. sexta could not discriminate between salicin and a bitter taste stimulus that activates the same signaling pathway within the same bitter-sensitive taste cell (caffeine). We propose that the heterogeneous population of bitter-sensitive taste cells and signaling pathways within this insect facilitates the discrimination of bitter taste stimuli.

  6. Cell cycle arrest in the jewel wasp Nasonia vitripennis in larval diapause.

    PubMed

    Shimizu, Yuta; Mukai, Ayumu; Goto, Shin G

    2018-04-01

    Insects enter diapause to synchronise their life cycle with biotic and abiotic environmental conditions favourable for their development, reproduction, and survival. One of the most noticeable characteristics of diapause is the blockage of ontogeny. Although this blockage should occur with the cessation of cellular proliferation, i.e. cell cycle arrest, it was confirmed only in a few insect species and information on the molecular pathways involved in cell cycle arrest is limited. In the present study, we investigated developmental and cell cycle arrest in diapause larvae of the jewel wasp Nasonia vitripennis. Developmental and cell cycle arrest occur in the early fourth instar larval stage of N. vitripennis under short days. By entering diapause, the S fraction of the cell cycle disappears and approximately 80% and 20% of cells arrest their cell cycle in the G0/G1 and G2 phases, respectively. We further investigated expression of cell cycle regulatory genes and some housekeeping genes to dissect molecular mechanisms underlying the cell cycle arrest. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Interactions of predominant insects and diseases with climate change in Douglas-fir forests of Western Oregon and Washington, USA

    EPA Science Inventory

    Forest disturbance regimes are beginning to show evidence of climate-mediated shifts associated with global climate change, and these patterns will likely continue due to continuing changes in environmental conditions. Tree growth is controlled by the physiological constraints o...

  8. Fungal DNA virus infects a mycophagous insect and utilizes it as a transmission vector

    PubMed Central

    Liu, Si; Xie, Jiatao; Cheng, Jiasen; Li, Bo; Chen, Tao; Fu, Yanping; Li, Guoqing; Wang, Manqun; Jin, Huanan; Wan, Hu; Jiang, Daohong

    2016-01-01

    Mycoviruses are usually transmitted horizontally via hyphal anastomosis and vertically via sexual/asexual spores. Previously, we reported that a gemycircularvirus, Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), could infect its fungal host extracellularly. Here, we discovered that SsHADV-1 could infect a mycophagous insect, Lycoriella ingenua, and use it as a transmission vector. Virus acquired by larvae feeding on colonies of a virus-infected strain of S. sclerotiorum was replicated and retained in larvae, pupae, adults, and eggs. Virus could be transmitted to insect offspring when larvae were injected with virus particles and allowed to feed on a nonhost fungus. Virus replication in insect cells was further confirmed by inoculating Spodoptera frugiperda cells with virus particles and analyzing with RT-PCR, Northern blot, immunofluorescence, and flow cytometry assays. Larvae could transmit virus once they acquired virus by feeding on virus-infected fungal colony. Offspring larvae hatched from viruliferous eggs were virus carriers and could also successfully transmit virus. Virus transmission between insect and fungus also occurred on rapeseed plants. Virus-infected isolates produced less repellent volatile substances to attract adults of L. ingenua. Furthermore, L. ingenua was easily observed on Sclerotinia lesions in rapeseed fields, and viruliferous adults were captured from fields either sprayed with a virus-infected fungal strain or nonsprayed. Our findings may facilitate the exploration of mycoviruses for control of fungal diseases and enhance our understanding of the ecology of SsHADV-1 and other newly emerging SsHADV-1–like viruses, which were recently found to be widespread in various niches including human HIV-infected blood, human and animal feces, insects, plants, and even sewage. PMID:27791095

  9. Dramatic Transcriptional Changes in an Intracellular Parasite Enable Host Switching between Plant and Insect

    PubMed Central

    Oshima, Kenro; Ishii, Yoshiko; Kakizawa, Shigeyuki; Sugawara, Kyoko; Neriya, Yutaro; Himeno, Misako; Minato, Nami; Miura, Chihiro; Shiraishi, Takuya; Yamaji, Yasuyuki; Namba, Shigetou

    2011-01-01

    Phytoplasmas are bacterial plant pathogens that have devastating effects on the yields of crops and plants worldwide. They are intracellular parasites of both plants and insects, and are spread among plants by insects. How phytoplasmas can adapt to two diverse environments is of considerable interest; however, the mechanisms enabling the “host switching” between plant and insect hosts are poorly understood. Here, we report that phytoplasmas dramatically alter their gene expression in response to “host switching” between plant and insect. We performed a detailed characterization of the dramatic change that occurs in the gene expression profile of Candidatus Phytoplasma asteris OY-M strain (approximately 33% of the genes change) upon host switching between plant and insect. The phytoplasma may use transporters, secreted proteins, and metabolic enzymes in a host-specific manner. As phytoplasmas reside within the host cell, the proteins secreted from phytoplasmas are thought to play crucial roles in the interplay between phytoplasmas and host cells. Our microarray analysis revealed that the expression of the gene encoding the secreted protein PAM486 was highly upregulated in the plant host, which is also observed by immunohistochemical analysis, suggesting that this protein functions mainly when the phytoplasma grows in the plant host. Additionally, phytoplasma growth in planta was partially suppressed by an inhibitor of the MscL osmotic channel that is highly expressed in the plant host, suggesting that the osmotic channel might play an important role in survival in the plant host. These results also suggest that the elucidation of “host switching” mechanism may contribute to the development of novel pest controls. PMID:21858041

  10. Isolation and determination of ivermectin in post-mortem and in vivo tissues of dung beetles using a continuous solid phase extraction method followed by LC-ESI+-MS/MS

    PubMed Central

    Ortiz, Antonio J.; Cortez, Vieyle; Azzouz, Abdelmonaim

    2017-01-01

    A new analytical method based on solvent extraction, followed by continuous solid-phase extraction (SPE) clean-up using a polymeric sorbent, was demonstrated to be applicable for the detection of ivermectin in complex biological matrices of dung beetles (hemolymph, excreta or dry tissues) using liquid chromatography combined with positive electrospray ionization tandem mass spectrometry (LC/ESI+–MS/MS). Using a signal-to-noise ratio of 3:1, the limit of detection (LOD) in the insect matrices at trace levels was 0.01 ng g–1 and the limit of quantification (LOQ) was 0.1 ng g–1. The proposed method was successfully used to quantitatively determine the levels of ivermectin in the analysis of small samples in in vivo and post mortem samples, demonstrating the usefulness for quantitative analyses that are focused on future pharmacokinetic and bioavailability studies in insects and the establishment of a new protocol to study the impact of ivermectin on non-target arthropods such as dung beetles and other insects that are related with the “dung community”. Because satisfactory precision and accuracy values were obtained in both in vivo matrices, we suggest that the method can be consistently used for quantitative determinations that are focused on future pharmacokinetic and bioavailability studies in insects. Furthermore, this new analytical method was successfully applied to biological samples of dead dung beetles from the field suggesting that the method can be used to establish a new routine analysis of ivermectin residues in insect carcasses that is applied to complement typical mortality tests. PMID:28207908

  11. Mastocytosis

    MedlinePlus

    ... when your body reacts to something like an insect bite or a bee sting (called an allergic reaction). Symptoms of mastocytosis The symptoms are different, depending on where the extra mast cells are. If there are too many mast cells ...

  12. Fluorescence in insects

    NASA Astrophysics Data System (ADS)

    Welch, Victoria L.; Van Hooijdonk, Eloise; Intrater, Nurit; Vigneron, Jean-Pol

    2012-10-01

    Fluorescent molecules are much in demand for biosensors, solar cells, LEDs and VCSEL diodes, therefore, considerable efforts have been expended in designing and tailoring fluorescence to specific technical applications. However, naturally occurring fluorescence of diverse types has been reported from a wide array of living organisms: most famously, the jellyfish Aequorea victoria, but also in over 100 species of coral and in the cuticle of scorpions, where it is the rule, rather than the exception. Despite the plethora of known insect species, comparatively few quantitative studies have been made of insect fluorescence. Because of the potential applications of natural fluorescence, studies in this field have relevance to both physics and biology. Therefore, in this paper, we review the literature on insect fluorescence, before documenting its occurrence in the longhorn beetles Sternotomis virescens, Sternotomis variabilis var. semi rufescens, Anoplophora elegans and Stellognatha maculata, the tiger beetles Cicindela maritima and Cicindela germanica and the weevil Pachyrrhynchus gemmatus purpureus. Optical features of insect fluorescence, including emitted wavelength, molecular ageing and naturally occurring combinations of fluorescence with bioluminescence and colour-producing structures are discussed.

  13. 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.

  14. Expression of full-length HER2 protein in Sf9 insect cells and its presentation on the surface of budded virus-like particles.

    PubMed

    Nika, Lisa; Wallner, Jakob; Palmberger, Dieter; Koczka, Krisztina; Vorauer-Uhl, Karola; Grabherr, Reingard

    2017-08-01

    Biomarkers of cancer are often glycosylated membrane receptor proteins present on the cellular surface. In order to develop new antibodies for cancer diagnostics or treatment, it is a main pre-requisite that these target proteins are available in a native conformation. However, membrane receptor proteins are notoriously difficult to produce due to their hydrophobic nature and complex architecture. Here, we used the baculovirus-insect cell expression system to produce budded virus-like particles (VLPs) as the scaffold for the presentation of complex membrane proteins. Since the human epidermal growth factor receptor 2 (HER2) is known to be overexpressed in a number of cancers it was chosen as model for a tumor antigen. VLPs displaying full-length HER2 on the surface were produced in Spodoptera frugiperda 9 (Sf9) insect cells and purified by sucrose gradient ultracentrifugation. The number of secreted particles was quantified by nanoparticle tracking analysis. To confirm the presence of HER2 protein on the surface, VLPs were labeled with gold-conjugated antibodies and analyzed by transmission electron microscopy. Functionality of displayed HER2 was investigated by ELISA and a newly established biolayer interferometry based technique. Detection was accomplished using the specific monoclonal antibody Herceptin and filamentous phages displaying a single-chain variable fragment of an anti-HER2 antibody. Significant stronger binding of Herceptin and anti-HER2 phages to HER2-displaying VLPs as compared to control VLPs was demonstrated. Thus, we suggest that Sf9 insect cells are highly feasible for the fast and easy production of various budded VLPs that serve as a platform for full-length membrane receptor presentation. Copyright © 2017. Published by Elsevier Inc.

  15. Aquatic Plant Control Research Program. A Survey of the Fauna Associated with Pistia Stratiotes L. (Waterlettuce) in Florida

    DTIC Science & Technology

    1988-04-01

    both north and south Florida were examined for water- lettuce populations during the period June 1985 thrrugh May 1986. A sample of at least 20 plants ...AQUATIC PLANT CONTROL C’ RESEARCH PROGRAM JX"O9 TECHNICAL REPORT A-88-6 A SURVEY OF THE FAUNA ASSOCIATED WITH PISTIA STRATIOTES L. (WATERLETTUCE) IN...and iaoentify by block number) FIELD GROUP SUB-GROIJP quatic plants ,, InsectS, tBiological control/ Waterlettuce d 19. ABSTRACT (Continue on torerse

  16. Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control

    PubMed Central

    Bravo, Alejandra; Gill, Sarjeet S.; Soberón, Mario

    2007-01-01

    Bacillus thuringiensis Cry and Cyt protein families are a diverse group of proteins with activity against insects of different orders - Lepidoptera, Coleoptera, Diptera and also against other invertebrates such as nematodes. Their primary action is to lyse midgut epithelial cells by inserting into the target membrane and forming pores. Among this group of proteins, members of the 3-Domain Cry family are used worldwide for insect control, and their mode of action has been characterized in some detail. Phylogenetic analyses established that the diversity of the 3-Domain Cry family evolved by the independent evolution of the three domains and by swapping of domain III among toxins. Like other pore-forming toxins (PFT) that affect mammals, Cry toxins interact with specific receptors located on the host cell surface and are activated by host proteases following receptor binding resulting in the formation of a pre-pore oligomeric structure that is insertion competent. In contrast, Cyt toxins directly interact with membrane lipids and insert into the membrane. Recent evidence suggests that Cyt synergize or overcome resistance to mosquitocidal-Cry proteins by functioning as a Cry-membrane bound receptor. In this review we summarize recent findings on the mode of action of Cry and Cyt toxins, and compare them to the mode of action of other bacterial PFT. Also, we discuss their use in the control of agricultural insect pests and insect vectors of human diseases. PMID:17198720

  17. Incorporating hemlock woolly adelgid impacts into the Forest Vegetation Simulator model

    Treesearch

    R. Talbot III Trotter; Anthony W. Courter; Richard M. Turcotte; Brad. Onken

    2008-01-01

    The hemlock woolly adelgid (HWA), Adelges tsugae, is a small aphid-like insect that feeds on the xylem ray parenchyma cells near the base of hemlock needles. This insect, a native of Asia and western North America, was first noted in eastern North America in 1951 and has now spread to infest hemlock in at least 17 states. The increased hemlock...

  18. Following insects around: tools and techniques of eighteenth-century natural history.

    PubMed

    Terrall, Mary

    2010-12-01

    This paper examines the movement of the materials, ideas and practices that went into the construction of natural-historical observations in Paris and the French provinces--in particular, observations of insects. The paired notions of circulation and locality expose the complex dynamic at play in the production of knowledge about these mundane creatures. I show how the movement of things and people problematizes the notion of a single 'centre of calculation', even where a dominant figure like Réaumur was managing collections and producing authoritative texts. Réaumur was indeed managing the flow of observations, letters and specimens from his privileged vantage point in Paris, but he was not the only one doing the processing, and the objects and knowledge flowed in all directions. The paper uses correspondence among eighteenth-century naturalists of various sorts to get at the dynamics of circulation, tracing the movements of insects, bits of text or narrative, drawings, letters, questions, apparatus, books and people. My title refers to the activities of naturalists, who had to follow insects around in order to observe them, and to my own activity in following the insects in their movement through letters, conversations, specimen jars, drawings and texts. My research depends on the accumulation of details about experimental and observational practice, culled from the masses of letters that moved continually around Europe, much as the science of insects depended on the accumulation of details about insects--their physiology, habits, metamorphosis and place in the human economy and the economy of nature.

  19. Production of Recombinant Rabies Virus Glycoprotein by Insect Cells in a Single-Use Fixed-Bed Bioreactor.

    PubMed

    Ventini-Monteiro, Daniella C; Astray, Renato M; Pereira, Carlos A

    2018-01-01

    A single-use fixed-bed bioreactor (iCELLis nano) can be used for cultivating non adherent insect cells, which can be then recovered for scaling up or for harvesting a membrane-associated viral glycoprotein with high quality in terms of preserved protein structure and biological function. Here, we describe the procedures for establishing genetically modified Drosophila melanogaster Schneider 2 (S2) cell cultures in the iCELLis nano bioreactor and for quantifying by ELISA the recombinant rabies virus glycoprotein (rRVGP) synthesized. By using the described protocol of production, the following performance can be regularly achieved: 1.7 ± 0.6 × 1E10 total cells; 2.4 ± 0.8 × 1E7 cells/mL and 1.2 ± 0.9 μg of rRVGP/1E7 cells; 1.5 ± 0.8 mg of total rRVGP.

  20. On gonads and gadflies: the estrus angle.

    PubMed

    Hillier, Stephen G

    2017-06-01

    The first sex steroid to be crystallized was the vertebrate ovarian hormone, estrone - a less potent metabolite of 17β-estradiol, which in mammals stimulates the female urge to mate (estrus). The gadfly (Greek oistros ) lent its name to the process of estrus, as an insect that bites and torments in classical Greek mythology. With the purification and crystallization of a moult-inducing steroid (ecdysone) from insects, an interesting parallel emerged between mating and moulting in lower mammals and arthropods. Ecdysterone (potent ecdysone metabolite) has anabolic effects in mammalian muscle cells that can be blocked by selective estrogen receptor antagonists. Insects utilize ecdysteroids in similar ways that vertebrates use estrogens, including stimulation of oocyte growth and maturation. Ecdysteroids also modify precopulatory insect mating behaviour, further reinforcing the gonad-gadfly/mate-moult analogy. © 2017 Society for Endocrinology.

  1. Neurogenesis in the water flea Daphnia magna (Crustacea, Branchiopoda) suggests different mechanisms of neuroblast formation in insects and crustaceans.

    PubMed

    Ungerer, Petra; Eriksson, Bo Joakim; Stollewerk, Angelika

    2011-09-01

    Within euarthropods, the morphological and molecular mechanisms of early nervous system development have been analysed in insects and several representatives of chelicerates and myriapods, while data on crustaceans are fragmentary. Neural stem cells (neuroblasts) generate the nervous system in insects and in higher crustaceans (malacostracans); in the remaining euarthropod groups, the chelicerates (e.g. spiders) and myriapods (e.g. millipedes), neuroblasts are missing. In the latter taxa, groups of neural precursors segregate from the neuroectoderm and directly differentiate into neurons and glial cells. In all euarthropod groups, achaete-scute homologues are required for neuroblast/neural precursor group formation. In the insects Drosophila melanogaster and Tribolium castaneum achaete-scute homologues are initially expressed in clusters of cells (proneural clusters) in the neuroepithelium but expression becomes restricted to the future neuroblast. Subsequently genes such as snail and prospero are expressed in the neuroblasts which are required for asymmetric division and differentiation. In contrast to insects, malacostracan neuroblasts do not segregate into the embryo but remain in the outer neuroepithelium, similar to vertebrate neural stem cells. It has been suggested that neuroblasts are present in another crustacean group, the branchiopods, and that they also remain in the neuroepithelium. This raises the questions how the molecular mechanisms of neuroblast selection have been modified during crustacean and insect evolution and if the segregation or the maintenance of neuroblasts in the neuroepithelium represents the ancestral state. Here we take advantage of the recently published Daphnia pulex (branchiopod) genome and identify genes in Daphnia magna that are known to be required for the selection and asymmetric division of neuroblasts in the fruit fly D. melanogaster. We unambiguously identify neuroblasts in D. magna by molecular marker gene expression and division pattern. We show for the first time that branchiopod neuroblasts divide in the same pattern as insect and malacostracan neuroblasts. Furthermore, in contrast to D. melanogaster, neuroblasts are not selected from proneural clusters in the branchiopod. Snail rather than ASH is the first gene to be expressed in the nascent neuroblasts suggesting that ASH is not required for the selection of neuroblasts as in D. melanogaster. The prolonged expression of ASH in D. magna furthermore suggests that it is involved in the maintenance of the neuroblasts in the neuroepithelium. Based on these and additional data from various representatives of arthropods we conclude that the selection of neural precursors from proneural clusters as well as the segregation of neural precursors represents the ancestral state of neurogenesis in arthropods. We discuss that the derived characters of malacostracans and branchiopods - the absence of neuroblast segregation and proneural clusters - might be used to support or reject the possible groupings of paraphyletic crustaceans. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. Chemical ecology: studies from East Africa.

    PubMed

    Meinwald, J; Prestwich, G D; Nakanishi, K; Kubo, I

    1978-03-17

    The International Centre of Insect Physiology and Ecology (ICIPE), in Nairobi, provides a laboratory at which a multinational group of scientists pursues interdisciplinary research. In collaboration with their colleagues in biology, ICIPE chemists have characterized the sex pheromones of the tick which serves as a vector of East Coast fever and have identified a termite queen-cell-building pheromone. The structure of many anthropod defensive chemicals have been determined; most interesting of these are the trinervitenes, structurally novel diterpenoids from nasute termites. Several highly active insect antifeedants were discovered using a simple bioassay to screen selected East African plants. These antifeedants may provide leads for the development of new insect-control techniques.

  3. DNA extracellular traps are part of the immune repertoire of Periplaneta americana.

    PubMed

    Nascimento, M T C; Silva, K P; Garcia, M C F; Medeiros, M N; Machado, E A; Nascimento, S B; Saraiva, E M

    2018-07-01

    Extracellular traps (ETs), web-like structures composed of DNA and histones, are released by innate immune cells in a wide range of organisms. ETs capture microorganisms, thereby avoiding their spread, and also concentrate antimicrobial molecules, which helps to kill microbes. Although vertebrate innate immune systems share homology with the insect immune system, ETosis have yet to be characterized in insects. Here, we report that the hemocytes of the hemimetabolous insect Periplaneta americana release ETs upon in vitro stimulation. We further discuss the relationship between ETs and nodulation and in controlling bacterial spread in vivo. Copyright © 2018 Elsevier Ltd. All rights reserved.

  4. Hindgut Innate Immunity and Regulation of Fecal Microbiota through Melanization in Insects*

    PubMed Central

    Shao, Qimiao; Yang, Bing; Xu, Qiuyun; Li, Xuquan; Lu, Zhiqiang; Wang, Chengshu; Huang, Yongping; Söderhäll, Kenneth; Ling, Erjun

    2012-01-01

    Many insects eat the green leaves of plants but excrete black feces in an as yet unknown mechanism. Insects cannot avoid ingesting pathogens with food that will be specifically detected by the midgut immune system. However, just as in mammals, many pathogens can still escape the insect midgut immune system and arrive in the hindgut, where they are excreted out with the feces. Here we show that the melanization of hindgut content induced by prophenoloxidase, a key enzyme that induces the production of melanin around invaders and at wound sites, is the last line of immune defense to clear bacteria before feces excretion. We used the silkworm Bombyx mori as a model and found that prophenoloxidase produced by hindgut cells is secreted into the hindgut contents. Several experiments were done to clearly demonstrate that the blackening of the insect feces was due to activated phenoloxidase, which served to regulate the number of bacteria in the hindgut. Our analysis of the silkworm hindgut prophenoloxidase discloses the natural secret of why the phytophagous insect feces is black and provides insight into hindgut innate immunity, which is still rather unclear in mammals. PMID:22375003

  5. Influence of different recombinant systems on the cooperativity exhibited by cytochrome P4503A4.

    PubMed

    Zhang, Z; Li, Y; Shou, M; Zhang, Y; Ngui, J S; Stearns, R A; Evans, D C; Baillie, T A; Tang, W

    2004-05-01

    1. The in vitro cooperativity exhibited by cytochrome P450 (CYP) 3A4 is influenced by the nature of the recombinant system in which the phenomenon is studied. Diclofenac, piroxicam and R-warfarin were used as model substrates, and quinidine was the effector. 2. The 5-, 5'- and 10-hydroxylation of diclofenac, piroxicam and R-warfarin, respectively, were enhanced five- to sevenfold by quinidine in human liver microsomal incubations. Whereas these cooperative drug interactions were apparent in incubations with CYP3A4 expressed in human lymphoblast cells, similar phenomena were not observed with the enzyme expressed in insect cells. 3. Insect cell microsomes were treated with a detergent and CYP3A4 was solubilized into a buffer medium. In incubations with CYP3A4 'freed' from its host membrane, the 5-hydroxylation of diclofenac increased with increasing quinidine concentrations, reaching a maximal eightfold elevation relative to controls. The metabolism of piroxicam and warfarin was similarly enhanced by quinidine. 4. Kinetically, enhancement by quinidine of the 5-hydroxylation of diclofenac in incubations with solubilized CYP3A4 was characterized by increases in the rate of metabolism with little change in the substrate-binding affinity. Conversely, the 3-hydroxylation of quinidine was not affected by diclofenac. 5. The data suggest that certain properties of CYP3A4 are masked by expression of the protein in insect cells and reinforce the concept that the enzyme possesses multiple binding domains. The absence of cooperative drug interactions with quinidine when CYP3A4 was expressed in insect cells might be due to an absence of enzyme conformation changes on quinidine binding, or the inability of quinidine to gain access to a putative effector-binding domain. 6. Caution should be exercised when comparing models for CYP3A4 cooperativity derived from different recombinant preparations of the enzyme.

  6. Humidity-enhanced wet adhesion on insect-inspired fibrillar adhesive pads

    PubMed Central

    Xue, Longjian; Kovalev, Alexander; Eichler-Volf, Anna; Steinhart, Martin; Gorb, Stanislav N.

    2015-01-01

    Many insect species reversibly adhere to surfaces by combining contact splitting (contact formation via fibrillar contact elements) and wet adhesion (supply of liquid secretion via pores in the insects’ feet). Here, we fabricate insect-inspired fibrillar pads for wet adhesion containing continuous pore systems through which liquid is supplied to the contact interfaces. Synergistic interaction of capillarity and humidity-induced pad softening increases the pull-off force and the work of adhesion by two orders of magnitude. This increase and the independence of pull-off force on the applied load are caused by the capillarity-supported formation of solid–solid contact between pad and the surface. Solid–solid contact dominates adhesion at high humidity and capillarity at low humidity. At low humidity, the work of adhesion strongly depends on the amount of liquid deposited on the surface and, therefore, on contact duration. These results may pave the way for the design of insect-inspired adhesive pads. PMID:25791574

  7. Immune modulation enables a specialist insect to benefit from antibacterial withanolides in its host plant

    PubMed Central

    Barthel, Andrea; Vogel, Heiko; Pauchet, Yannick; Pauls, Gerhard; Kunert, Grit; Groot, Astrid T.; Boland, Wilhelm; Heckel, David G.; Heidel-Fischer, Hanna M.

    2016-01-01

    The development of novel plant chemical defenses and counter adaptations by herbivorous insect could continually drive speciation, producing more insect specialists than generalists. One approach to test this hypothesis is to compare closely related generalist and specialist species to reveal the associated costs and benefits of these different adaptive strategies. We use the specialized moth Heliothis subflexa, which feeds exclusively on plants in the genus Physalis, and its close generalist relative H. virescens. Specialization on Physalis plants necessitates the ability to tolerate withanolides, the secondary metabolites of Physalis species that are known to have feeding deterrent and immune inhibiting properties for other insects. Here we find that only H. subflexa benefits from the antibacterial properties of withanolides, and thereby gains a higher tolerance of the pathogen Bacillus thuringiensis. We argue that the specialization in H. subflexa has been guided to a large extent by a unique role of plant chemistry on ecological immunology. PMID:27561781

  8. SIFamide in the brain of the sphinx moth, Manduca sexta.

    PubMed

    Heuer, C M; Binzer, Marlene; Schachtner, J

    2012-01-01

    SIFamides form a group of highly conserved neuropeptides in insects, crustaceans, and chelicerates. Beyond their biochemical commonalities, the neuroanatomical distribution of SIFamide in the insect nervous system also shows a remarkable degree of conservation. Thus, expression of SIFamide has been found to be restricted to four neurons of the pars intercerebralis in different holometabolous species. By means of immunohistological stainings, we here show that in Manduca sexta, those four cells are complemented by additional immunoreactive cells located in the vicinity of the mushroom body calyx. Immunopositive processes form arborizations throughout the brain, innervating major neuropils like the antennal lobes, the central complex, and the optic neuropils.

  9. Adult-specific insulin-producing neurons in Drosophila melanogaster.

    PubMed

    Ohhara, Yuya; Kobayashi, Satoru; Yamakawa-Kobayashi, Kimiko; Yamanaka, Naoki

    2018-06-01

    Holometabolous insects undergo metamorphosis to reorganize their behavioral and morphological features into adult-specific ones. In the central nervous system (CNS), some larval neurons undergo programmed cell death, whereas others go through remodeling of axonal and dendritic arbors to support functions of re-established adult organs. Although there are multiple neuropeptides that have stage-specific roles in holometabolous insects, the reorganization pattern of the entire neuropeptidergic system through metamorphosis still remains largely unclear. In this study, we conducted a mapping and lineage tracing of peptidergic neurons in the larval and adult CNS by using Drosophila genetic tools. We found that Diuretic hormone 44-producing median neurosecretory cells start expressing Insulin-like peptide 2 in the pharate adult stage. This neuronal cluster projects to the corpora cardiaca and dorsal vessel in both larval and adult stages, and also innervates an adult-specific structure in the digestive tract, the crop. We propose that the adult-specific insulin-producing cells may regulate functions of the digestive system in a stage-specific manner. Our study provides a neuroanatomical basis for understanding remodeling of the neuropeptidergic system during insect development and evolution. © 2018 Wiley Periodicals, Inc.

  10. Construction of baculovirus expression vector of miRNAs and its expression in insect cells.

    PubMed

    Huang, Yong; Zou, Quan; Shen, Xing Jia; Yu, Xue Li; Wang, Zhan Bin; Cheng, Xiang Chao

    2012-01-01

    MicroRNAs (miRNAs) are endogenous small non-protein coding RNAs that play important regulatory roles in animals and plants by binding to target transcripts for cleavage or translational repression. The miR-9a is very conservative in animals from flies to humans. Studies indicated that miR-9a is involved in the regulation of neurogenesis in animals. In our study, the baculovirus expression system was used to transcribe a recombinant vector containing miR-9a for further analysis the function ofmiR-9a. The sequence ofpre-miR-9a from silkworm DNA was first cloned into the donor pFastBac. The enhanced green fluorescent protein (EGFP) was used as reporter gene. The recombinant donor plasmid pFastBac-miR-9a was transformed into E.coli DH10Bac/AcNPV forming Bacmid-9a which was transfected into insect cells with cational lipofectin. The transcription of mature miR-9a was detected by Real-time PCR. The results show the recombinant Bacmid-9a was successfully constructed and effectively transcribed miR-9a in infected Sf21 insect cells.

  11. Modulation of plant defense responses to herbivores by simultaneous recognition of different herbivore-associated elicitors in rice

    PubMed Central

    Shinya, Tomonori; Hojo, Yuko; Desaki, Yoshitake; Christeller, John T.; Okada, Kazunori; Shibuya, Naoto; Galis, Ivan

    2016-01-01

    Induced plant defense responses against insect herbivores are triggered by wounding and/or perception of herbivore elicitors from their oral secretions (OS) and/or saliva. In this study, we analyzed OS isolated from two rice chewing herbivores, Mythimna loreyi and Parnara guttata. Both types of crude OS had substantial elicitor activity in rice cell system that allowed rapid detection of early and late defense responses, i.e. accumulation of reactive oxygen species (ROS) and defense secondary metabolites, respectively. While the OS from M. loreyi contained large amounts of previously reported insect elicitors, fatty acid-amino acid conjugates (FACs), the elicitor-active P. guttata’s OS contained no detectable FACs. Subsequently, elicitor activity associated with the high molecular mass fraction in OS of both herbivores was identified, and shown to promote ROS and metabolite accumulations in rice cells. Notably, the application of N-linolenoyl-Gln (FAC) alone had only negligible elicitor activity in rice cells; however, the activity of isolated elicitor fraction was substantially promoted by this FAC. Our results reveal that plants integrate various independent signals associated with their insect attackers to modulate their defense responses and reach maximal fitness in nature. PMID:27581373

  12. Temperature dependent characteristics of a recombinant infectious hematopoietic necrosis virus glycoprotein produced in insect cells.

    PubMed

    Cain, K D; Byrne, K M; Brassfield, A L; LaPatra, S E; Ristow, S S

    1999-04-15

    A recombinant infectious hematopoietic necrosis virus (IHNV) glycoprotein (G protein) was produced in insect cells using a baculovirus vector (Autographa californica nuclear polyhedrosis virus). Characteristics of this protein were evaluated in relation to native viral G protein. A full-length (1.6 kb) cDNA copy of the glycoprotein gene of IHNV was inserted into the baculovirus vector under control of the polyhedrin promoter. High levels of G protein (approximately 0.5 microgram/1 x 10(5) cells) were produced in Spodoptera frugiperda (Sf9) cells following recombinant baculovirus infection. Analysis of cell lysates by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot revealed a recombinant IHNV G of slightly higher mobility on the gel than the viral G protein. Differences in mobility were abrogated by endoglycosidase treatment. When the recombinant G protein was produced in insect cells at 20 degrees C (RecGlow), immunostaining and cell fusion activity demonstrated surface localization of the protein. In contrast, when recombinant protein was produced at 27 degrees C (RecGhigh), G protein was sequestered within the cell, suggesting that at the 2 different temperatures processing differences may exist. Eleven monoclonal antibodies (MAbs) were tested by immunoblotting for reactivity to the recombinant G protein. All 11 MAbs reacted to the reduced proteins. Four MAbs recognized both RecGhigh and RecGlow under non-reducing conditions; however, 1 neutralizing MAb (92A) recognized RecGlow but failed to react to RecGhigh under non-reducing conditions. This suggests that differences exist between RecGlow and RecGhigh which may have implications in the development of a properly folded recombinant G protein with the ability to elicit protective immunity in fish.

  13. Cell-Based Odorant Sensor Array for Odor Discrimination Based on Insect Odorant Receptors.

    PubMed

    Termtanasombat, Maneerat; Mitsuno, Hidefumi; Misawa, Nobuo; Yamahira, Shinya; Sakurai, Takeshi; Yamaguchi, Satoshi; Nagamune, Teruyuki; Kanzaki, Ryohei

    2016-07-01

    The olfactory system of living organisms can accurately discriminate numerous odors by recognizing the pattern of activation of several odorant receptors (ORs). Thus, development of an odorant sensor array based on multiple ORs presents the possibility of mimicking biological odor discrimination mechanisms. Recently, we developed novel odorant sensor elements with high sensitivity and selectivity based on insect OR-expressing Sf21 cells that respond to target odorants by displaying increased fluorescence intensity. Here we introduce the development of an odorant sensor array composed of several Sf21 cell lines expressing different ORs. In this study, an array pattern of four cell lines expressing Or13a, Or56a, BmOR1, and BmOR3 was successfully created using a patterned polydimethylsiloxane film template and cell-immobilizing reagents, termed biocompatible anchor for membrane (BAM). We demonstrated that BAM could create a clear pattern of Sf21 sensor cells without impacting their odorant-sensing performance. Our sensor array showed odorant-specific response patterns toward both odorant mixtures and single odorant stimuli, allowing us to visualize the presence of 1-octen-3-ol, geosmin, bombykol, and bombykal as an increased fluorescence intensity in the region of Or13a, Or56a, BmOR1, and BmOR3 cell lines, respectively. Therefore, we successfully developed a new methodology for creating a cell-based odorant sensor array that enables us to discriminate multiple target odorants. Our method might be expanded into the development of an odorant sensor capable of detecting a large range of environmental odorants that might become a promising tool used in various applications including the study of insect semiochemicals and food contamination.

  14. Establishment of an in vitro transcription system for Peste des petits ruminant virus.

    PubMed

    Yunus, Mohammad; Shaila, Melkote S

    2012-12-05

    Peste-des-petits ruminants virus (PPRV) is a non segmented negative strand RNA virus of the genus Morbillivirus within Paramyxoviridae family. Negative strand RNA viruses are known to carry nucleocapsid (N) protein, phospho (P) protein and RNA polymerase (L protein) packaged within the virion which possess all activities required for transcription, post-transcriptional modification of mRNA and replication. In order to understand the mechanism of transcription and replication of the virus, an in vitro transcription reconstitution system is required. In the present work, an in vitro transcription system has been developed with ribonucleoprotein (RNP) complex purified from virus infected cells as well as partially purified recombinant polymerase (L-P) complex from insect cells along with N-RNA (genomic RNA encapsidated by N protein) template isolated from virus infected cells. RNP complex isolated from virus infected cells and recombinant L-P complex purified from insect cells was used to reconstitute transcription on N-RNA template. The requirement for this transcription reconstitution has been defined. Transcription of viral genes in the in vitro system was confirmed by PCR amplification of cDNAs corresponding to individual transcripts using gene specific primers. In order to measure the relative expression level of viral transcripts, real time PCR analysis was carried out. qPCR analysis of the transcription products made in vitro showed a gradient of polarity of transcription from 3' end to 5' end of the genome similar to that exhibited by the virus in infected cells. This report describes for the first time, the development of an in vitro transcription reconstitution system for PPRV with RNP complex purified from infected cells and recombinant L-P complex expressed in insect cells. Both the complexes were able to synthesize all the mRNA species in vitro, exhibiting a gradient of polarity in transcription.

  15. Evolution of a complex behavior: the origin and initial diversification of foliar galling by Permian insects

    NASA Astrophysics Data System (ADS)

    Schachat, Sandra R.; Labandeira, Conrad C.

    2015-04-01

    A central notion of the early evolution of insect galling is that this unique behavior was uncommon to rare before the diversification of angiosperms 135 to 125 m.yr. ago. However, evidence accumulated during recent years shows that foliar galls were diverse and locally abundant as early as the Permian Period, 299 to 252 m.yr. ago. In particular, a diversity of leaf galling during the Early Permian has recently been documented by the plant-damage record of foliar galls and, now, our interpretation of the body-fossil record of culprit insect gallers. Small size is a prerequisite for gallers. Wing-length measurements of Permian insects indicate that several small-bodied hemipteroid lineages originated early during the Permian, some descendant lineages of which gall the leaves of seed plants to the present day. The earliest foliar gallers likely were Protopsyllidiidae (Hemiptera) and Lophioneuridae (Thripida). Much of the Early Permian was a xeric interval, and modern galls are most common in dry, extra-tropical habitats such as scrubland and deserts. Plant-damage, insect body fossils, and the paleoclimate record collectively support the ecological expansion of foliar galling during the Early Permian and its continued expansion through the Late Permian.

  16. At last, a Pennsylvanian stem-stonefly (Plecoptera) discovered

    PubMed Central

    2011-01-01

    Background Stem-relatives of many winged insect orders have been identified among Pennsylvanian fossils (Carboniferous Period). Owing to their presumed 'basal' position in insect phylogeny, stoneflies were expected to occur at this period. However, no relative has ever been designated convincingly. Results In this paper, we report specimens belonging to a new fossil insect species collected from the Tupo Formation (Pennsylvanian; China). The wing venation of Gulou carpenteri gen. et sp. nov. exhibits character states diagnostic of the order Plecoptera, but lack character states shared by unequivocal representatives of the order. Derived from this identification, the delimitation of the fossil species is ascertained based on comparison of several extant stonefly species. This comparative analysis allowed a trait present in G. carpenteri gen. et sp. nov., but rarely occurring in extant species, to be documented and highlighted as atavistic. Affinities of taxa formerly proposed as putative stem-stoneflies are reconsidered in the light of the new discovery. Conclusions Gulou carpenteri gen. et sp. nov. is considered the only genuine Plecoptera reported from the Pennsylvanian. Continuing efforts on the systematics of Pennsylvanian winged insects indicate a fauna more diverse than previously appreciated. It suggests that insects already had a long, yet undocumented, history by this time. PMID:21880126

  17. A host beetle pheromone regulates development and behavior in the nematode Pristionchus pacificus.

    PubMed

    Cinkornpumin, Jessica K; Wisidagama, Dona R; Rapoport, Veronika; Go, James L; Dieterich, Christoph; Wang, Xiaoyue; Sommer, Ralf J; Hong, Ray L

    2014-10-15

    Nematodes and insects are the two most speciose animal phyla and nematode-insect associations encompass widespread biological interactions. To dissect the chemical signals and the genes mediating this association, we investigated the effect of an oriental beetle sex pheromone on the development and behavior of the nematode Pristionchus pacificus. We found that while the beetle pheromone is attractive to P. pacificus adults, the pheromone arrests embryo development, paralyzes J2 larva, and inhibits exit of dauer larvae. To uncover the mechanism that regulates insect pheromone sensitivity, a newly identified mutant, Ppa-obi-1, is used to reveal the molecular links between altered attraction towards the beetle pheromone, as well as hypersensitivity to its paralyzing effects. Ppa-obi-1 encodes lipid-binding domains and reaches its highest expression in various cell types, including the amphid neuron sheath and excretory cells. Our data suggest that the beetle host pheromone may be a species-specific volatile synomone that co-evolved with necromeny.

  18. Insect-specific flavivirus infection is restricted by innate immunity in the vertebrate host.

    PubMed

    Tree, Maya O; McKellar, Dexter R; Kieft, Kristopher J; Watson, Alan M; Ryman, Kate D; Conway, Michael J

    2016-10-01

    Arboviruses are a large group of viruses that are transmitted by arthropods including ticks and mosquitoes. The global diversity of arboviruses is unknown; however, theoretical studies have estimated that over 2,000 mosquito-borne flaviviruses may exist. An increasing number of flaviviruses can only infect insect cells. We hypothesize that insect-specific flaviviruses (ISFVs) represent model genetic precursors to pathogenic flaviviruses, although the genetic mechanisms required for adaptation to vertebrate hosts are unclear. In this study, we determined that Kamiti River virus (KRV) infection was inhibited by innate immunity pathways in vertebrate cells. KRV infection of IRF3,5,7(-/-) mouse embryonic fibroblasts led to low levels of viral protein production and shedding of infectious progeny. These data suggest that ISFVs cannot evade vertebrate innate immune pathways. Identifying cellular pathways and genetic changes that are required for adaptation of arthropod-specific arboviruses to vertebrate hosts is critical to understanding emerging infectious disease. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. A Regulatory Pathway, Ecdysone-Transcription Factor Relish-Cathepsin L, Is Involved in Insect Fat Body Dissociation

    PubMed Central

    Zhang, Yao; Lu, Yu-Xuan; Liu, Jian; Yang, Cui; Feng, Qi-Li; Xu, Wei-Hua

    2013-01-01

    Insect fat body is the organ for intermediary metabolism, comparable to vertebrate liver and adipose tissue. Larval fat body is disintegrated to individual fat body cells and then adult fat body is remodeled at the pupal stage. However, little is known about the dissociation mechanism. We find that the moth Helicoverpa armigera cathepsin L (Har-CL) is expressed heavily in the fat body and is released from fat body cells into the extracellular matrix. The inhibitor and RNAi experiments demonstrate that Har-CL functions in the fat body dissociation in H. armigera. Further, a nuclear protein is identified to be transcription factor Har-Relish, which was found in insect immune response and specifically binds to the promoter of Har-CL gene to regulate its activity. Har-Relish also responds to the steroid hormone ecdysone. Thus, the dissociation of the larval fat body is involved in the hormone (ecdysone)-transcription factor (Relish)-target gene (cathepsin L) regulatory pathway. PMID:23459255

  20. The response of virally infected insect cells to dissolved oxygen concentration: recombinant protein production and oxidative damage.

    PubMed

    Saarinen, Mark A; Murhammer, David W

    2003-01-05

    The effects of dissolved oxygen (DO) concentration on virally infected insect cells were investigated in 3-L bioreactor culture. Specifically, cultures of Spodoptera frugiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) were infected with Autographa californica multiple nucleopolyhedrovirus expressing secreted alkaline phosphatase (SEAP). Following infection at a DO concentration of 50% air saturation, the DO concentration was adjusted to a final value of either 190%, 50%, or 10% air saturation. Recombinant SEAP production, cell viability, protein carbonyl content, and thiobarbituric acid reactive substances (TBARS) content were monitored. The increases in protein carbonyl and TBARS contents are taken to be indicators of protein oxidation and lipid oxidation, respectively. DO concentration was found to have no noticeable effect on SEAP production or cell viability decline in the Sf-9 cell line. In the Tn-5B1-4 cell line, cells displayed an increased peak SEAP production rate for 190% air saturation and displayed an increased rate of viability decline at increased DO concentration. Protein carbonyl content showed no significant increase in the Sf-9 cell line by 72 h postinfection (pi) at any DO concentration but showed a twofold increase at 10% and 50% DO concentration and a threefold increase at 190% DO concentration by 72 h pi in Tn-5B1-4 cells. TBARS content was found to increase by approximately 50% in Sf-9 cells and by approximately twofold in Tn-5B1-4 cells by 72 h pi with no clear relationship to DO concentration. It is hypothesized that oxygen uptake changes due to the viral infection process may bear a relation to the observed increases in protein and lipid oxidation and that lipid oxidation may play an important role in the death of virally infected insect cells. Copyright 2002 Wiley Periodicals, Inc.

  1. DNA duplication is essential for the repair of gastrointestinal perforation in the insect midgut

    PubMed Central

    Huang, Wuren; Zhang, Jie; Yang, Bing; Beerntsen, Brenda T.; Song, Hongsheng; Ling, Erjun

    2016-01-01

    Invertebrate animals have the capacity of repairing wounds in the skin and gut via different mechanisms. Gastrointestinal perforation, a hole in the human gastrointestinal system, is a serious condition, and surgery is necessary to repair the perforation to prevent an abdominal abscess or sepsis. Here we report the repair of gastrointestinal perforation made by a needle-puncture wound in the silkworm larval midgut. Following insect gut perforation, only a weak immune response was observed because the growth of Escherichia coli alone was partially inhibited by plasma collected at 6 h after needle puncture of the larval midgut. However, circulating hemocytes did aggregate over the needle-puncture wound to form a scab. While, cell division and apoptosis were not observed at the wound site, the needle puncture significantly enhanced DNA duplication in cells surrounding the wound, which was essential to repair the midgut perforation. Due to the repair capacity and limited immune response caused by needle puncture to the midgut, this approach was successfully used for the injection of small compounds (ethanol in this study) into the insect midgut. Consequently, this needle-puncture wounding of the insect gut can be developed for screening compounds for use as gut chemotherapeutics in the future. PMID:26754166

  2. Non-synaptic ion channels in insects--basic properties of currents and their modulation in neurons and skeletal muscles.

    PubMed

    Wicher, D; Walther, C; Wicher, C

    2001-08-01

    Insects are favoured objects for studying information processing in restricted neuronal networks, e.g. motor pattern generation or sensory perception. The analysis of the underlying processes requires knowledge of the electrical properties of the cells involved. These properties are determined by the expression pattern of ionic channels and by the regulation of their function, e.g. by neuromodulators. We here review the presently available knowledge on insect non-synaptic ion channels and ionic currents in neurons and skeletal muscles. The first part of this article covers genetic and structural informations, the localization of channels, their electrophysiological and pharmacological properties, and known effects of second messengers and modulators such as neuropeptides or biogenic amines. In a second part we describe in detail modulation of ionic currents in three particularly well investigated preparations, i.e. Drosophila photoreceptor, cockroach DUM (dorsal unpaired median) neuron and locust jumping muscle. Ion channel structures are almost exclusively known for the fruitfly Drosophila, and most of the information on their function has also been obtained in this animal, mainly based on mutational analysis and investigation of heterologously expressed channels. Now the entire genome of Drosophila has been sequenced, it seems almost completely known which types of channel genes--and how many of them--exist in this animal. There is much knowledge of the various types of channels formed by 6-transmembrane--spanning segments (6TM channels) including those where four 6TM domains are joined within one large protein (e.g. classical Na+ channel). In comparison, two TM channels and 4TM (or tandem) channels so far have hardly been explored. There are, however, various well characterized ionic conductances, e.g. for Ca2+, Cl- or K+, in other insect preparations for which the channels are not yet known. In some of the larger insects, i.e. bee, cockroach, locust and moth, rather detailed information has been established on the role of ionic currents in certain physiological or behavioural contexts. On the whole, however, knowledge of non-synaptic ion channels in such insects is still fragmentary. Modulation of ion currents usually involves activation of more or less elaborate signal transduction cascades. The three detailed examples for modulation presented in the second part indicate, amongst other things, that one type of modulator usually leads to concerted changes of several ion currents and that the effects of different modulators in one type of cell may overlap. Modulators participate in the adaptive changes of the various cells responsible for different physiological or behavioural states. Further study of their effects on the single cell level should help to understand how small sets of cells cooperate in order to produce the appropriate output.

  3. Source-Sink Colonization as a Possible Strategy of Insects Living in Temporary Habitats.

    PubMed

    Frouz, Jan; Kindlmann, Pavel

    2015-01-01

    Continuous colonization and re-colonization is critical for survival of insect species living in temporary habitats. When insect populations in temporary habitats are depleted, some species may escape extinction by surviving in permanent, but less suitable habitats, in which long-term population survival can be maintained only by immigration from other populations. Such situation has been repeatedly described in nature, but conditions when and how this occurs and how important this phenomenon is for insect metapopulation survival are still poorly known, mainly because it is difficult to study experimentally. Therefore, we used a simulation model to investigate, how environmental stochasticity, growth rate and the incidence of dispersal affect the positive effect of permanent but poor ("sink") habitats on the likelihood of metapopulation persistence in a network of high quality but temporary ("source") habitats. This model revealed that permanent habitats substantially increase the probability of metapopulation persistence of insect species with poor dispersal ability if the availability of temporary habitats is spatio-temporally synchronized. Addition of permanent habitats to a system sometimes enabled metapopulation persistence even in cases in which the metapopulation would otherwise go extinct, especially for species with high growth rates. For insect species with low growth rates the probability of a metapopulation persistence strongly depended on the proportions of "source" to "source" and "sink" to "source" dispersal rates.

  4. Goose parvovirus structural proteins expressed by recombinant baculoviruses self-assemble into virus-like particles with strong immunogenicity in goose.

    PubMed

    Ju, Huanyu; Wei, Na; Wang, Qian; Wang, Chunyuan; Jing, Zhiqiang; Guo, Lu; Liu, Dapeng; Gao, Mingchun; Ma, Bo; Wang, Junwei

    2011-05-27

    Goose parvovirus (GPV), a small non-enveloped ssDNA virus, can cause Derzsy's disease, and three capsid proteins of VP1, VP2, and VP3 are encoded by an overlapping nucleotide sequence. However, little is known on whether recombinant viral proteins (VPs) could spontaneously assemble into virus-like particles (VLPs) in insect cells and whether these VLPs could retain their immunoreactivity and immunogenicity in susceptible geese. To address these issues, genes for these GPV VPs were amplified by PCR, and the recombinant VPs proteins were expressed in insect cells using a baculovirus expression system for the characterization of their structures, immunoreactivity, and immunogenicity. The rVP1, rVP2, and rVP3 expressed in Sf9 cells were detected by anti-GPV sera, anti-VP3 sera, and anti-His antibodies, respectively. Electron microscopy revealed that these rVPs spontaneously assembled into VLPs in insect cells, similar to that of the purified wild-type GPV virions. In addition, vaccination with individual types of VLPs, particularly with the rVP2-VLPs, induced higher titers of antibodies and neutralized different strains of GPVs in primary goose and duck embryo fibroblast cells in vitro. These data indicated that these VLPs retained immunoreactivity and had strong immunogenicity in susceptible geese. Therefore, our findings may provide a framework for development of new vaccines for the prevention of Derzsy's disease and vehicles for the delivery of drugs. Copyright © 2011 Elsevier Inc. All rights reserved.

  5. Tomatine-Containing Green Tomato Extracts Inhibit Growth of Human Breast, Colon, Liver, and Stomach Cancer Cells

    USDA-ARS?s Scientific Manuscript database

    Tomato plants (Lycopersicon esculentum) synthesize the glycoalkaloids dehydrotomatine and a–tomatine, possibly as a defense against bacteria, fungi, viruses, and insects. We investigated six green and three red tomato extracts for their ability to induce cell death in human cancer and normal cells ...

  6. Recent advances in phytoplasma research: from genetic diversity and genome evolution to pathogenic redirection of plant stem cell fate

    USDA-ARS?s Scientific Manuscript database

    Parasitizing phloem sieve cells and being transmitted by insects, phytoplasmas are a unique group of cell wall-less bacteria responsible for numerous plant diseases worldwide. Due to difficulties in establishing axenic culture of phytoplasmas, phenotypic characters suitable for conventional microbia...

  7. Virus-like particle of Macrobrachium rosenbergii nodavirus produced in Spodoptera frugiperda (Sf9) cells is distinctive from that produced in Escherichia coli.

    PubMed

    Kueh, Chare Li; Yong, Chean Yeah; Masoomi Dezfooli, Seyedehsara; Bhassu, Subha; Tan, Soon Guan; Tan, Wen Siang

    2017-03-01

    Macrobrachium rosenbergii nodavirus (MrNV) is a virus native to giant freshwater prawn. Recombinant MrNV capsid protein has been produced in Escherichia coli, which self-assembled into virus-like particles (VLPs). However, this recombinant protein is unstable, degrading and forming heterogenous VLPs. In this study, MrNV capsid protein was produced in insect Spodoptera frugiperda (Sf9) cells through a baculovirus system. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) revealed that the recombinant protein produced by the insect cells self-assembled into highly stable, homogenous VLPs each of approximately 40 nm in diameter. Enzyme-linked immunosorbent assay (ELISA) showed that the VLPs produced in Sf9 cells were highly antigenic and comparable to those produced in E. coli. In addition, the Sf9 produced VLPs were highly stable across a wide pH range (2-12). Interestingly, the Sf9 produced VLPs contained DNA of approximately 48 kilo base pairs and RNA molecules. This study is the first report on the production and characterization of MrNV VLPs produced in a eukaryotic system. The MrNV VLPs produced in Sf9 cells were about 10 nm bigger and had a uniform morphology compared with the VLPs produced in E. coli. The insect cell production system provides a good source of MrNV VLPs for structural and immunological studies as well as for host-pathogen interaction studies. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:549-557, 2017. © 2016 American Institute of Chemical Engineers.

  8. Establishment of a Monarch Butterfly (Danaus plexippus, Lepidoptera: Danaidae) Cell Line and its Susceptibility to Insect Viruses

    USDA-ARS?s Scientific Manuscript database

    A cell line from the monarch butterfly Danaus plexippus designated BCIRL-DP-AM/JG was established from adult ovaries. The cell line consisted mainly of round cells and took a prolonged period of time in the growth medium ExCell 401 containing 10% fetal bovine serum and antibiotics before it could be...

  9. In Vitro Iron Availability from Insects and Sirloin Beef.

    PubMed

    Latunde-Dada, Gladys O; Yang, Wenge; Vera Aviles, Mayra

    2016-11-09

    Interest in the consumption of insects (entomophagy) as an alternative environmentally sustainable source of protein in the diet of humans has recently witnessed a surge. Knowledge of the nutrient composition and, in particular, the bioavailability of minerals from insects is currently sparse. This study evaluated the availability of Fe, Ca, Cu, Mg, Mn, and Zn from four commonly eaten insects and compared these to sirloin beef. Soluble iron from the samples was measured by inductively coupled plasma optical emission spectrometry (ICP-OES). Iron bioavailability was determined using an in vitro simulated peptic-pancreatic digestion, followed by measurement of ferritin (a surrogate marker for iron absorption) in Caco-2 cells. Cricket and sirloin beef had comparably higher levels of Fe, Ca, and Mn than grasshopper, meal, and buffalo worms. However, iron solubility was significantly higher from the insect samples than from beef. The complementation of whole-wheat flour with insect or beef protein resulted in overall decreases in mineral content and iron solubility in the composite mixtures. Collectively, the data show that grasshopper, cricket, and mealworms contain significantly higher chemically available Ca, Cu, Mg, Mn, and Zn than sirloin. However, buffalo worms and sirloin exhibited higher iron bioavailability comparable to that of FeSO 4 . Commonly consumed insect species could be excellent sources of bioavailable iron and could provide the platform for an alternative strategy for increased mineral intake in the diets of humans.

  10. Diversity and functions of protein glycosylation in insects.

    PubMed

    Walski, Tomasz; De Schutter, Kristof; Van Damme, Els J M; Smagghe, Guy

    2017-04-01

    The majority of proteins is modified with carbohydrate structures. This modification, called glycosylation, was shown to be crucial for protein folding, stability and subcellular location, as well as protein-protein interactions, recognition and signaling. Protein glycosylation is involved in multiple physiological processes, including embryonic development, growth, circadian rhythms, cell attachment as well as maintenance of organ structure, immunity and fertility. Although the general principles of glycosylation are similar among eukaryotic organisms, insects synthesize a distinct repertoire of glycan structures compared to plants and vertebrates. Consequently, a number of unique insect glycans mediate functions specific to this class of invertebrates. For instance, the core α1,3-fucosylation of N-glycans is absent in vertebrates, while in insects this modification is crucial for the development of wings and the nervous system. At present, most of the data on insect glycobiology comes from research in Drosophila. Yet, progressively more information on the glycan structures and the importance of glycosylation in other insects like beetles, caterpillars, aphids and bees is becoming available. This review gives a summary of the current knowledge and recent progress related to glycan diversity and function(s) of protein glycosylation in insects. We focus on N- and O-glycosylation, their synthesis, physiological role(s), as well as the molecular and biochemical basis of these processes. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Insect Responses to Linearly Polarized Reflections: Orphan Behaviors Without Neural Circuits.

    PubMed

    Heinloth, Tanja; Uhlhorn, Juliane; Wernet, Mathias F

    2018-01-01

    The e-vector orientation of linearly polarized light represents an important visual stimulus for many insects. Especially the detection of polarized skylight by many navigating insect species is known to improve their orientation skills. While great progress has been made towards describing both the anatomy and function of neural circuit elements mediating behaviors related to navigation, relatively little is known about how insects perceive non-celestial polarized light stimuli, like reflections off water, leaves, or shiny body surfaces. Work on different species suggests that these behaviors are not mediated by the "Dorsal Rim Area" (DRA), a specialized region in the dorsal periphery of the adult compound eye, where ommatidia contain highly polarization-sensitive photoreceptor cells whose receptive fields point towards the sky. So far, only few cases of polarization-sensitive photoreceptors have been described in the ventral periphery of the insect retina. Furthermore, both the structure and function of those neural circuits connecting to these photoreceptor inputs remain largely uncharacterized. Here we review the known data on non-celestial polarization vision from different insect species (dragonflies, butterflies, beetles, bugs and flies) and present three well-characterized examples for functionally specialized non-DRA detectors from different insects that seem perfectly suited for mediating such behaviors. Finally, using recent advances from circuit dissection in Drosophila melanogaster , we discuss what types of potential candidate neurons could be involved in forming the underlying neural circuitry mediating non-celestial polarization vision.

  12. Mixed genotype transmission bodies and virions contribute to the maintenance of diversity in an insect virus

    PubMed Central

    Clavijo, Gabriel; Williams, Trevor; Muñoz, Delia; Caballero, Primitivo; López-Ferber, Miguel

    2010-01-01

    An insect nucleopolyhedrovirus naturally survives as a mixture of at least nine genotypes. Infection by multiple genotypes results in the production of virus occlusion bodies (OBs) with greater pathogenicity than those of any genotype alone. We tested the hypothesis that each OB contains a genotypically diverse population of virions. Few insects died following inoculation with an experimental two-genotype mixture at a dose of one OB per insect, but a high proportion of multiple infections were observed (50%), which differed significantly from the frequencies predicted by a non-associated transmission model in which genotypes are segregated into distinct OBs. By contrast, insects that consumed multiple OBs experienced higher mortality and infection frequencies did not differ significantly from those of the non-associated model. Inoculation with genotypically complex wild-type OBs indicated that genotypes tend to be transmitted in association, rather than as independent entities, irrespective of dose. To examine the hypothesis that virions may themselves be genotypically heterogeneous, cell culture plaques derived from individual virions were analysed to reveal that one-third of virions was of mixed genotype, irrespective of the genotypic composition of the OBs. We conclude that co-occlusion of genotypically distinct virions in each OB is an adaptive mechanism that favours the maintenance of virus diversity during insect-to-insect transmission. PMID:19939845

  13. Molecular Biology of Insect Sodium Channels and Pyrethroid Resistance

    PubMed Central

    Dong, Ke; Du, Yuzhe; Rinkevich, Frank; Nomura, Yoshiko; Xu, Peng; Wang, Lingxin; Silver, Kristopher; Zhorov, Boris S.

    2015-01-01

    Voltage-gated sodium channels are essential for the initiation and propagation of the action potential in neurons and other excitable cells. Because of their critical roles in electrical signaling, sodium channels are targets of a variety of naturally occurring and synthetic neurotoxins, including several classes of insecticides. This review is intended to provide an update on the molecular biology of insect sodium channels and the molecular mechanism of pyrethroid resistance. Although mammalian and insect sodium channels share fundamental topological and functional properties, most insect species carry only one sodium channel gene, compared to multiple sodium channel genes found in each mammalian species. Recent studies showed that two posttranscriptional mechanisms, alternative splicing and RNA editing, are involved in generating functional diversity of sodium channels in insects. More than 50 sodium channel mutations have been identified to be responsible for or associated with knockdown resistance (kdr) to pyrethroids in various arthropod pests and disease vectors. Elucidation of molecular mechanism of kdr led to the identification of dual receptor sites of pyrethroids on insect sodium channels. Most of the kdr mutations appear to be located within or close to the two receptor sites. The accumulating knowledge of insect sodium channels and their interactions with insecticides provides a foundation for understanding the neurophysiology of sodium channels in vivo and the development of new and safer insecticides for effective control of arthropod pests and human disease vectors. PMID:24704279

  14. EcR expression in the prothoracicotropic hormone-producing neurosecretory cells of the Bombyx mori brain.

    PubMed

    Hossain, Monwar; Shimizu, Sakiko; Fujiwara, Haruhiko; Sakurai, Sho; Iwami, Masafumi

    2006-08-01

    The steroid hormone 20-hydroxyecdysone (20E) initiates insect molting and metamorphosis through binding with a heterodimer of two nuclear receptors, the ecdysone receptor (EcR) and ultraspiracle (USP). Expression of the specific isoforms EcR-A and EcR-B1 governs steroid-induced responses in the developing cells of the silkworm Bombyx mori. Here, analysis of EcR-A and EcR-B1 expression during larval-pupal development showed that both genes were up-regulated by 20E in the B. mori brain. Whole-mount in situ hybridization and immunohistochemistry revealed that EcR-A and EcR-B1 mRNAs and proteins were exclusively located in two pairs of lateral neurosecretory cells in the larval brain known as the prothoracicotropic hormone (PTTH)- producing cells (PTPCs). In the pupal brain, EcR-A and EcR-B1 expression was detected in tritocerebral cells and optic lobe cells in addition to PTPCs. As PTTH controls ecdysone secretion by the prothoracic gland, these results indicate that 20E-responsive PTPCs are the master cells of insect metamorphosis.

  15. Insect heat shock proteins during stress and diapause.

    PubMed

    King, Allison M; MacRae, Thomas H

    2015-01-07

    Insect heat shock proteins include ATP-independent small heat shock proteins and the larger ATP-dependent proteins, Hsp70, Hsp90, and Hsp60. In concert with cochaperones and accessory proteins, heat shock proteins mediate essential activities such as protein folding, localization, and degradation. Heat shock proteins are synthesized constitutively in insects and induced by stressors such as heat, cold, crowding, and anoxia. Synthesis depends on the physiological state of the insect, but the common function of heat shock proteins, often working in networks, is to maintain cell homeostasis through interaction with substrate proteins. Stress-induced expression of heat shock protein genes occurs in a background of protein synthesis inhibition, but in the course of diapause, a state of dormancy and increased stress tolerance, these genes undergo differential regulation without the general disruption of protein production. During diapause, when ATP concentrations are low, heat shock proteins may sequester rather than fold proteins.

  16. Characterization and functional assay of a fatty acyl-CoA reductase gene in the scale insect, Ericerus pela Chavannes (Hemiptera: Coccoidae).

    PubMed

    Hu, Yan-Hong; Chen, Xiao-Ming; Yang, Pu; Ding, Wei-Feng

    2018-04-01

    Ericerus pela Chavannes (Hemiptera: Coccoidae) is an economically important scale insect because the second instar males secrete a harvestable wax-like substance. In this study, we report the molecular cloning of a fatty acyl-CoA reductase gene (EpFAR) of E. pela. We predicted a 520-aa protein with the FAR family features from the deduced amino acid sequence. The EpFAR mRNA was expressed in five tested tissues, testis, alimentary canal, fat body, Malpighian tubules, and mostly in cuticle. The EpFAR protein was localized by immunofluorescence only in the wax glands and testis. EpFAR expression in High Five insect cells documented the recombinant EpFAR reduced 26-0:(S) CoA and to its corresponding alcohol. The data illuminate the molecular mechanism for fatty alcohol biosynthesis in a beneficial insect, E. pela. © 2017 Wiley Periodicals, Inc.

  17. Recurrent loss of CenH3 is associated with independent transitions to holocentricity in insects.

    PubMed

    Drinnenberg, Ines A; deYoung, Dakota; Henikoff, Steven; Malik, Harmit Singh

    2014-09-23

    Faithful chromosome segregation in all eukaryotes relies on centromeres, the chromosomal sites that recruit kinetochore proteins and mediate spindle attachment during cell division. The centromeric histone H3 variant, CenH3, is the defining chromatin component of centromeres in most eukaryotes, including animals, fungi, plants, and protists. In this study, using detailed genomic and transcriptome analyses, we show that CenH3 was lost independently in at least four lineages of insects. Each of these lineages represents an independent transition from monocentricity (centromeric determinants localized to a single chromosomal region) to holocentricity (centromeric determinants extended over the entire chromosomal length) as ancient as 300 million years ago. Holocentric insects therefore contain a CenH3-independent centromere, different from almost all the other eukaryotes. We propose that ancient transitions to holocentricity in insects obviated the need to maintain CenH3, which is otherwise essential in most eukaryotes, including other holocentrics.

  18. Drosophila TRP channels and animal behavior

    PubMed Central

    Fowler, Melissa A.; Montell, Craig

    2012-01-01

    Multiple classes of cell surface receptors and ion channels participate in the detection of changes in environmental stimuli, and thereby influence animal behavior. Among the many classes of ion channels, Transient Receptor Potential (TRP) cation channels are notable in contributing to virtually every sensory modality, and in controlling a daunting array of behaviors. TRP channels appear to be conserved in all metazoan organisms including worms, insects and humans. Flies encode 13 TRPs, most of which are expressed and function in sensory neurons, and impact behaviors ranging from phototaxis to thermotaxis, gravitaxis, the avoidance of noxious tastants and smells and proprioception. Multiple diseases result from defects in TRPs, and flies provide an excellent animal model for dissecting the mechanisms underlying “TRPopathies.” Drosophila TRPs also function in the sensation of botanically derived insect repellents, and related TRPs in insect pests are potential targets for the development of improved repellents to combat insect-borne diseases. PMID:22877650

  19. An Aphid Effector Targets Trafficking Protein VPS52 in a Host-Specific Manner to Promote Virulence1[OPEN

    PubMed Central

    2017-01-01

    Plant- and animal-feeding insects secrete saliva inside their hosts, containing effectors, which may promote nutrient release and suppress immunity. Although for plant pathogenic microbes it is well established that effectors target host proteins to modulate host cell processes and promote disease, the host cell targets of herbivorous insects remain elusive. Here, we show that the existing plant pathogenic microbe effector paradigm can be extended to herbivorous insects in that effector-target interactions inside host cells modify critical host processes to promote plant susceptibility. We showed that the effector Mp1 from Myzus persicae associates with the host Vacuolar Protein Sorting Associated Protein52 (VPS52). Using natural variants, we provide a strong link between effector virulence activity and association with VPS52, and show that the association is highly specific to M. persicae-host interactions. Also, coexpression of Mp1, but not Mp1-like variants, specifically with host VPS52s resulted in effector relocalization to vesicle-like structures that associate with prevacuolar compartments. We show that high VPS52 levels negatively impact virulence, and that aphids are able to reduce VPS52 levels during infestation, indicating that VPS52 is an important virulence target. Our work is an important step forward in understanding, at the molecular level, how a major agricultural pest promotes susceptibility during infestation of crop plants. We give evidence that an herbivorous insect employs effectors that interact with host proteins as part of an effective virulence strategy, and that these effectors likely function in a species-specific manner. PMID:28100451

  20. Cytotoxic and genotoxic effects of defence secretion of Ulomoides dermestoides on A549 cells.

    PubMed

    Crespo, Rosana; Villaverde, M Luciana; Girotti, Juan R; Güerci, Alba; Juárez, M Patricia; de Bravo, Margarita G

    2011-06-14

    Ulomoides dermestoides (Fairmaire, 1893) is a cosmopolitan tenebrionid beetle reared by Argentine people who consume them alive as an alternative medicine in the treatment of different illnesses such as asthma, Parkinson's, diabetes, arthritis, HIV and specially cancer. To evaluate the cytotoxicity and DNA damage of the major volatile components released by Ulomoides dermestoides on human lung carcinoma epithelial cell line A549. The defence compounds of Ulomoides dermestoides were extracted with dichloromethane and analyzed and quantified by capillary gas chromatography. The toxicity effects of the beetle's extract against A549 cell line were evaluated. Cytotoxicity was evaluated by MTT test and Trypan blue assay and genotoxicity was evaluated by the comet assay. The synthetic compounds, individually or combined, were also tested in A549 cells and normal mononuclear human cells. The defence compounds of Ulomoides dermestoides extracted with dichloromethane (methyl-1,4-benzoquinones, ethyl-1,4-benzoquinones and 1-pentadecene as major components) showed cytotoxic activity on A549 cells demonstrated by MTT test and Trypan blue assay, with IC(50) values of 0.26equivalent/ml and 0.34equivalent/ml, respectively (1equivalent=amount of components extracted per beetle). The inhibition of A549 cell proliferation with the synthetic blend (1,4-benzoquinone and 1-pentadecene) or 1,4-benzoquinone alone was similar to that obtained with the insect extract. 1-Pentadecene showed no inhibitory effect. Low doses of insect extract or synthetic blend (0.15equivalent/ml) inhibited mononuclear cell proliferation by 72.2±2.7% and induced significant DNA damage both in tumor and mononuclear cells. Results of this study demonstrated that defence compounds of Ulomoides dermestoides reduced cell viability and induced DNA damage. We also concluded that the insect benzoquinones are primarily responsible for inducing cytotoxicity and genotoxicity in culture cells. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  1. Meiotic Chromosome Analysis of the Giant Water Bug, Lethocerus indicus

    PubMed Central

    Wisoram, Wijit; Saengthong, Pradit; Ngernsiri, Lertluk

    2013-01-01

    The giant water bug, Lethocerus indicus (Lepeletier and Serville) (Heteroptera: Belostomatidae), a native species of Southeast Asia, is one of the largest insects belonging to suborder Heteroptera. In this study, the meiotic chromosome of L. indicus was studied in insect samples collected from Thailand, Myanmar, Loas, and Cambodia. Testicular cells stained with lacto-acetic orcein, Giemsa, DAPI, and silver nitrate were analyzed. The results revealed that the chromosome complement of L. indicus was 2n = 22A + neo-XY + 2m, which differed from that of previous reports. Each individual male contained testicular cells with three univalent patterns. The frequency of cells containing neo-XY chromosome univalent (∼5%) was a bit higher than that of cells with autosomal univalents (∼3%). Some cells (∼0.5%) had both sex chromosome univalents and a pair of autosomal univalents. None of the m-chromosome univalents were observed during prophase I. In addition, this report presents clear evidence about the existence of m-chromosomes in Belostomatidae. PMID:23895100

  2. The fungal aroma gene ATF1 promotes dispersal of yeast cells through insect vectors.

    PubMed

    Christiaens, Joaquin F; Franco, Luis M; Cools, Tanne L; De Meester, Luc; Michiels, Jan; Wenseleers, Tom; Hassan, Bassem A; Yaksi, Emre; Verstrepen, Kevin J

    2014-10-23

    Yeast cells produce various volatile metabolites that are key contributors to the pleasing fruity and flowery aroma of fermented beverages. Several of these fruity metabolites, including isoamyl acetate and ethyl acetate, are produced by a dedicated enzyme, the alcohol acetyl transferase Atf1. However, despite much research, the physiological role of acetate ester formation in yeast remains unknown. Using a combination of molecular biology, neurobiology, and behavioral tests, we demonstrate that deletion of ATF1 alters the olfactory response in the antennal lobe of fruit flies that feed on yeast cells. The flies are much less attracted to the mutant yeast cells, and this in turn results in reduced dispersal of the mutant yeast cells by the flies. Together, our results uncover the molecular details of an intriguing aroma-based communication and mutualism between microbes and their insect vectors. Similar mechanisms may exist in other microbes, including microbes on flowering plants and pathogens. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  3. A single promoter inversion switches Photorhabdus between pathogenic and mutualistic states.

    PubMed

    Somvanshi, Vishal S; Sloup, Rudolph E; Crawford, Jason M; Martin, Alexander R; Heidt, Anthony J; Kim, Kwi-suk; Clardy, Jon; Ciche, Todd A

    2012-07-06

    Microbial populations stochastically generate variants with strikingly different properties, such as virulence or avirulence and antibiotic tolerance or sensitivity. Photorhabdus luminescens bacteria have a variable life history in which they alternate between pathogens to a wide variety of insects and mutualists to their specific host nematodes. Here, we show that the P. luminescens pathogenic variant (P form) switches to a smaller-cell variant (M form) to initiate mutualism in host nematode intestines. A stochastic promoter inversion causes the switch between the two distinct forms. M-form cells are much smaller (one-seventh the volume), slower growing, and less bioluminescent than P-form cells; they are also avirulent and produce fewer secondary metabolites. Observations of form switching by individual cells in nematodes revealed that the M form persisted in maternal nematode intestines, were the first cells to colonize infective juvenile (IJ) offspring, and then switched to P form in the IJ intestine, which armed these nematodes for the next cycle of insect infection.

  4. Intracellular survival of Staphylococcus aureus during persistent infection in the insect Tenebrio molitor.

    PubMed

    McGonigle, John E; Purves, Joanne; Rolff, Jens

    2016-06-01

    Survival of bacteria within host cells and tissues presents a challenge to the immune systems of higher organisms. Escape from phagocytic immune cells compounds this issue, as immune cells become potential vehicles for pathogen dissemination. However, the duration of persistence within phagocytes and its contribution to pathogen load has yet to be determined. We investigate the immunological significance of intracellular persistence within the insect model Tenebrio molitor, assessing the extent, duration and location of bacterial recovery during a persistent infection. Relative abundance of Staphylococcus aureus in both intracellular and extracellular fractions was determined over 21 days, and live S. aureus were successfully recovered from both the hemolymph and within phagocytic immune cells across the entire time course. The proportion of bacteria recovered from within phagocytes also increased over time. Our results show that to accurately estimate pathogen load it is vital to account for bacteria persisting within immune cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Cytoplasmic and nuclear localization of cadherin in honey bee (Apis mellifera L.) gonads.

    PubMed

    Florecki, Mônica M; Hartfelder, Klaus

    2011-01-01

    Cadherins are crucial molecules mediating cell-cell interactions between somatic and germline cells in insect and mammalian male and female gonads. We analysed the presence and localization of cadherins in ovaries of honeybee queens and in testes of drones. Transcripts representing two classical cadherins, E-cadherin (shotgun) and N-cadherin, as well as three protocadherins (Starry night, Fat and Fat-like) were detected in gonads of both sexes. Pan-cadherin antibodies, which most probably detect a honeybee N-cadherin, were used in immunolocalization analyses. In the germarium of ovarioles, cadherin-IR (cadherin immunoreactivity) was evidenced as homogeneously distributed in the cytoplasm and as nuclear foci, in both germline and somatic cells. It was also detected in polyfusomes and ring canals. In testiolar tubules, cadherin-IR showed a cytoplasmic and nuclear distributon alike in ovaries. The unexpected nuclear localization and cytoplasmic distribution in ovaries and testes were corroborated by immunogold electron microscopy, which revealed cadherin aggregates associated with electron-dense nuclear structures. With respect to cadherin localization, the honeybee differs from Drosophila, the model for gametogenesis in insects, raising the question as to how differences among solitary and social species may be built into and generated from the general architecture of polytrophic meroistic ovaries. It also indicates the possibility of divergent roles for cadherin in the functional architecture of insect gonads, in general, especially in taxa with high reproductive output.

  6. Genetic compensation of high dose radiation-induced damage in an anhydrobiotic insect

    NASA Astrophysics Data System (ADS)

    Gusev, Oleg; Nakahara, Yuichi; Sakashita, Tetsuya; Kikawada, Takahiro; Okuda, Takashi

    Anhydrobiotic larvae of African chironomid Polypedilum vanderplanki are known to show an extremely high tolerance against a range of stresses. The tolerance against various extreme environments exhibited by that insect might be due to being almost completely desiccated replacing water with trehalose, a state where little or no chemical reactions occur. From 2005 dried larvae of this insect are being used in a number of space experiments, both inside and outside of ISS as a model organism for estimation the limits of higher organisms' resistance to space environment stresses and long-term storage of the alive anhydrobiotic organisms during continues spaceflight. We have shown previously that both hydrated and dried larvae of Polypedilum vanderplanki have very higher tolerance against both highand low-linear energy transfer (LET), surviving after 7000Gy irradiation. It was suggested that the larvae would have effective DNA-reparation system in addition to artificial protection provided by glass-stage without water. In the present study we conducted analysis of stress-related gene expression in the larvae after 70-2000 Gy irradiations. Both DNA damage level and activity of DNA-reparation, anti-apoptotic and protein-damage related genes were analyzed. Direct visualization of DNA damage in the larvae fat body cells using Comet Assay showed that fragmented by radiation DNA is re-arranged within 76-98 hours after exposure. We found that massive overexpression of hsp and anti-oxidant genes occur in larvae entering anhydrobiosis , and provides refolding of proteins after rehydration. In the irradiated larvae overexpression of DNA-reparation enzymes anti-apoptotic genes was confirmed, suggesting that survival after high-dose irradiation is a result of combination of highly effective blocking of entering the apoptosis after severe DNA damage and DNA reparation.

  7. Characterizing Enterovirus 71 and Coxsackievirus A16 virus-like particles production in insect cells.

    PubMed

    Somasundaram, Balaji; Chang, Cindy; Fan, Yuan Y; Lim, Pei-Yin; Cardosa, Jane; Lua, Linda

    2016-02-15

    Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) are two viruses commonly responsible for hand, foot and mouth disease (HFMD) in children. The lack of prophylactic or therapeutic measures against HFMD is a major public health concern. Insect cell-based EV71 and CVA16 virus-like particles (VLPs) are promising vaccine candidates against HFMD and are currently under development. In this paper, the influence of insect cell line, incubation temperature, and serial passaging effect and stability of budded virus (BV) stocks on EV71 and CVA16 VLP production was investigated. Enhanced EV71 and CVA16 VLP production was observed in Sf9 cells compared to High Five™ cells. Lowering the incubation temperature from the standard 27°C to 21°C increased the production of both VLPs in Sf9 cells. Serial passaging of CVA16 BV stocks in cell culture had a detrimental effect on the productivity of the structural proteins and the effect was observed with only 5 passages of BV stocks. A 2.7× higher production yield was achieved with EV71 compared to CVA16. High-resolution asymmetric flow field-flow fractionation couple with multi-angle light scattering (AF4-MALS) was used for the first time to characterize EV71 and CVA16 VLPs, displaying an average root mean square radius of 15±1nm and 15.3±5.8 nm respectively. This study highlights the need for different approaches in the design of production process to develop a bivalent EV71 and CVA16 vaccine. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Re-evaluation of insect melanogenesis research: Views from the dark side.

    PubMed

    Whitten, Miranda M A; Coates, Christopher J

    2017-07-01

    Melanins (eumelanin and pheomelanin) are synthesized in insects for several purposes including cuticle sclerotization and color patterning, clot formation, organogenesis, and innate immunity. Traditional views of insect immunity detail the storage of pro-phenoloxidases inside specialized blood cells (hemocytes) and their release upon recognition of foreign bodies. Activated phenoloxidases convert monophenols into reactive quinones in a two-step enzymatic reaction, and until recently, the mechanism of tyrosine hydroxylation remained a mystery. Herein, we present our interpretations of these enzyme-substrate complexes. The resultant melanins are deposited onto the surface of microbes to immobilize, agglutinate, and suffocate them. Phenoloxidase activity and melanin production are not limited to the blood (hemolymph) or cuticle, as recent evidence points to more diverse, sophisticated interactions in the gut and with the resident symbionts. This review offers insight into the somewhat neglected areas of insect melanogenesis research, particularly in innate immunity, its role in beneficial insects such as pollinators, the functional versatility of phenoloxidases, and the limitations of common experimental approaches that may impede progress inadvertently. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  9. The influence of the atmospheric boundary layer on nocturnal layers of noctuids and other moths migrating over southern Britain.

    PubMed

    Wood, Curtis R; Chapman, Jason W; Reynolds, Donald R; Barlow, Janet F; Smith, Alan D; Woiwod, Ian P

    2006-03-01

    Insects migrating at high altitude over southern Britain have been continuously monitored by automatically operating, vertical-looking radars over a period of several years. During some occasions in the summer months, the migrants were observed to form well-defined layer concentrations, typically at heights of 200-400 m, in the stable night-time atmosphere. Under these conditions, insects are likely to have control over their vertical movements and are selecting flight heights that are favourable for long-range migration. We therefore investigated the factors influencing the formation of these insect layers by comparing radar measurements of the vertical distribution of insect density with meteorological profiles generated by the UK Meteorological Office's (UKMO) Unified Model (UM). Radar-derived measurements of mass and displacement speed, along with data from Rothamsted Insect Survey light traps, provided information on the identity of the migrants. We present here three case studies where noctuid and pyralid moths contributed substantially to the observed layers. The major meteorological factors influencing the layer concentrations appeared to be: (a) the altitude of the warmest air, (b) heights corresponding to temperature preferences or thresholds for sustained migration and (c) on nights when air temperatures are relatively high, wind-speed maxima associated with the nocturnal jet. Back-trajectories indicated that layer duration may have been determined by the distance to the coast. Overall, the unique combination of meteorological data from the UM and insect data from entomological radar described here show considerable promise for systematic studies of high-altitude insect layering.

  10. Practical applications of insects' sexual development for pest control.

    PubMed

    Koukidou, M; Alphey, L

    2014-01-01

    Elucidation of the sex differentiation pathway in insects offers an opportunity to understand key aspects of evolutionary developmental biology. In addition, it provides the understanding necessary to manipulate insects in order to develop new synthetic genetics-based tools for the control of pest insects. Considerable progress has been made in this, especially in improvements to the sterile insect technique (SIT). Large scale sex separation is considered highly desirable or essential for most SIT targets. This separation can be provided by genetic methods based on sex-specific gene expression. Investigation of sex determination by many groups has provided molecular components and methods for this. Though the primary sex determination signal varies considerably, key regulatory genes and mechanisms remain surprisingly similar. In most cases studied so far, a primary signal is transmitted to a basal gene at the bottom of the hierarchy (dsx) through an alternative splicing cascade; dsx is itself differentially spliced in males and females. A sex-specific alternative splicing system therefore offers an attractive route to achieve female-specific expression. Experience has shown that alternative splicing modules can be developed with cross-species function; modularity and standardisation and re-use of parts are key principles of synthetic biology. Both female-killing and sex reversal (XX females to phenotypic males) can in principle also be used as efficient alternatives to sterilisation in SIT-like methods. Sexual maturity is yet another area where understanding of sexual development may be applied to insect control programmes. Further detailed understanding of this crucial aspect of insect biology will undoubtedly continue to underpin innovative practical applications. © 2014 S. Karger AG, Basel.

  11. A 50,000 year insect record from Rancho La Brea, Southern California: Insights into past climate and fossil deposition

    NASA Astrophysics Data System (ADS)

    Holden, Anna R.; Southon, John R.; Will, Kipling; Kirby, Matthew E.; Aalbu, Rolf L.; Markey, Molly J.

    2017-07-01

    Rigorously studied and dated Late Quaternary paleoenvironmental reconstructions from Ranch La Brea (RLB) and the Los Angeles Basin are scarce. Here, we use data from AMS radiocarbon dated insect fragments to infer local climates over the past 50,000 years. Our results indicate: 1) Quaternary insect remains can be located with great accuracy in radiocarbon time, and 2) well-dated and documented climate indicator beetle species are sensitive proxies for environmental change in the Los Angeles Basin. A total of 182 extant RLB ground and darkling beetle species (Coleoptera: Carabidae, Tenebrionidae) were radiocarbon dated. The resulting radiocarbon dates form a semi-continuous range from ∼50 to 28, 16-7.5, and 4 kcal yrs BP to the present. Associated insect climate ranges indicate past conditions consistent with, or very similar to, the current Los Angeles Basin Mediterranean climate. Importantly, these insect data suggest higher temperatures and aridity than inferred previously from other RLB proxies. Furthermore, wider-than-assumed dating spreads for some deposits emphasize the lack of biostratigraphy for RLB, and challenge inferences based on limited sets of radiocarbon dates and assumptions about stratigraphic integrity. Our results demonstrate the necessity to independently radiocarbon date each taxon. The insect paleoclimate interpretations were compared to regional pollen data, primarily from various southern Californian sites including Lake Elsinore and Santa Barbara Basin. These comparisons reveal an important difference in climate interpretations for the last Glacial: the RLB insect data suggest climate similar to the current one, while the regional pollen data have been interpreted as indicating a climate wetter than present.

  12. Establishment and characterization of two new cell lines from the mosquito Armigeres subalbatus (Coquillett) (Diptera: Culicidae).

    PubMed

    Hoshino, Keita; Isawa, Haruhiko; Kuwata, Ryusei; Tajima, Shigeru; Takasaki, Tomohiko; Iwabuchi, Kikuo; Sawabe, Kyoko; Kobayashi, Mutsuo; Sasaki, Toshinori

    2015-08-01

    Armigeres subalbatus (Coquillett) is a medically important mosquito and a model species for immunology research. We successfully established two cell lines from the neonate larvae of A. subalbatus using two different media. To our knowledge, this is the first report of an established Armigeres mosquito cell line. The cell lines, designated as Ar-3 and Ar-13, consist of adherent and diploid cells with compact colonies. Both these cell lines grow slowly after passage at a split ratio of 1:5 and a population doubling time of 2.7 and 3.0 d, respectively. Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) was used to confirm that these lines correspond to the species of origin and are clearly distinct from seven other insect cell lines. Furthermore, reverse-transcription PCR was used to demonstrate that the Ar-3 cell line is susceptible to the Japanese encephalitis virus and two insect flaviviruses associated with Culex and Aedes mosquitoes but relatively insensitive to dengue virus. These data indicate that the newly established cell lines are cellular models of A. subalbatus as well as beneficial tools for the propagation of viruses associated with the Armigeres mosquito.

  13. In vivo production of recombinant proteins using occluded recombinant AcMNPV-derived baculovirus vectors.

    PubMed

    Guijarro-Pardo, Eva; Gómez-Sebastián, Silvia; Escribano, José M

    2017-12-01

    Trichoplusia ni insect larvae infected with vectors derived from the Autographa californica multiple nucleopolyhedrovirus (AcMNPV), are an excellent alternative to insect cells cultured in conventional bioreactors to produce recombinant proteins because productivity and cost-efficiency reasons. However, there is still a lot of work to do to reduce the manual procedures commonly required in this production platform that limit its scalability. To increase the scalability of this platform technology, a current bottleneck to be circumvented in the future is the need of injection for the inoculation of larvae with polyhedrin negative baculovirus vectors (Polh-) because of the lack of oral infectivity of these viruses, which are commonly used for production in insect cell cultures. In this work we have developed a straightforward alternative to obtain orally infective vectors derived from AcMNPV and expressing recombinant proteins that can be administered to the insect larvae (Trichoplusia ni) by feeding, formulated in the insect diet. The approach developed was based on the use of a recombinant polyhedrin protein expressed by a recombinant vector (Polh+), able to co-occlude any recombinant Polh- baculovirus vector expressing a recombinant protein. A second alternative was developed by the generation of a dual vector co-expressing the recombinant polyhedrin protein and the foreign gene of interest to obtain the occluded viruses. Additionally, by the incorporation of a reporter gene into the helper Polh+ vector, it was possible the follow-up visualization of the co-occluded viruses infection in insect larvae and will help to homogenize infection conditions. By using these methodologies, the production of recombinant proteins in per os infected larvae, without manual infection procedures, was very similar in yield to that obtained by manual injection of recombinant Polh- AcMNPV-based vectors expressing the same proteins. However, further analyses will be required for a detailed comparison of production yields reached by injection vs oral infections for different recombinant proteins. In conclusion, these results open the possibility of future industrial scaling-up production of recombinant proteins in insect larvae by reducing manual operations. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. 77 FR 9889 - Black Hills National Forest Advisory Board Public Meeting Dates Announced

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-21

    ... vegetation management (wild and prescribed fire, fuels reduction, controlling insect epidemics, invasive species), travel management (off highway vehicles, the new OHV rule, and related topics), and continuing...

  15. Stinging Insect Matching Game

    MedlinePlus

    ... the AAAAI Foundation Donate Utility navigation Español Journals Pollen Counts Annual Meeting Member Login / My Membership Search ... navigation Donate Annual meeting Browse your conditions Check pollen counts Continuing education center Find an allergist / immunologist ...

  16. OpnS, an outer membrane porin of Xenorhabdus nematophila, confers a competitive advantage for growth in the insect host.

    PubMed

    van der Hoeven, Ransome; Forst, Steven

    2009-09-01

    The gammaproteobacterium Xenorhabdus nematophila engages in a mutualistic association with an entomopathogenic nematode and also functions as a pathogen toward different insect hosts. We studied the role of the growth-phase-regulated outer membrane protein OpnS in host interactions. OpnS was shown to be a 16-stranded beta-barrel porin. opnS was expressed during growth in insect hemolymph and expression was elevated as the cell density increased. When wild-type and opnS deletion strains were coinjected into insects, the wild-type strain was predominantly recovered from the insect cadaver. Similarly, an opnS-complemented strain outcompeted the DeltaopnS strain. Coinjection of the wild-type and DeltaopnS strains together with uncolonized nematodes into insects resulted in nematode progeny that were almost exclusively colonized with the wild-type strain. Likewise, nematode progeny recovered after coinjection of a mixture of nematodes carrying either the wild-type or DeltaopnS strain were colonized by the wild-type strain. In addition, the DeltaopnS strain displayed a competitive growth defect when grown together with the wild-type strain in insect hemolymph but not in defined culture medium. The DeltaopnS strain displayed increased sensitivity to antimicrobial compounds, suggesting that deletion of OpnS affected the integrity of the outer membrane. These findings show that the OpnS porin confers a competitive advantage for the growth and/or the survival of X. nematophila in the insect host and provides a new model for studying the biological relevance of differential regulation of porins in a natural host environment.

  17. Interaction of Phytophagous Insects with Salmonella enterica on Plants and Enhanced Persistence of the Pathogen with Macrosteles quadrilineatus Infestation or Frankliniella occidentalis Feeding

    PubMed Central

    Soto-Arias, José Pablo; Groves, Russell; Barak, Jeri D.

    2013-01-01

    Recently, most foodborne illness outbreaks of salmonellosis have been caused by consumption of contaminated fresh produce. Yet, the mechanisms that allow the human pathogen Salmonella enterica to contaminate and grow in plant environments remain poorly described. We examined the effect of feeding by phytophagous insects on survival of S. enterica on lettuce. Larger S. enterica populations were found on leaves infested with Macrosteles quadrilineatus. In contrast, pathogen populations among plants exposed to Frankliniella occidentalis or Myzus persicae were similar to those without insects. However, on plants infested with F. occidentalis, areas of the infested leaf with feeding damage sustained higher S. enterica populations than areas without damage. The spatial distribution of S. enterica cells on leaves infested with F. occidentalis may be altered resulting in higher populations in feeding lesions or survival may be different across a leaf dependent on local damage. Results suggest the possibility of some specificity with select insects and the persistence of S. enterica. Additionally, we demonstrated the potential for phytophagous insects to become contaminated with S. enterica from contaminated plant material. S. enterica was detected in approximately 50% of all M. quadrilineatus, F. occidentalis, and M. persicae after 24 h exposure to contaminated leaves. Particularly, 17% of F. occidentalis, the smallest of the insects tested, harbored more than 102 CFU/F. occidentalis. Our results show that phytophagous insects may influence the population dynamics of S. enterica in agricultural crops. This study provides evidence of a human bacterial pathogen interacting with phytophagous insect during plant infestation. PMID:24205384

  18. Interaction of phytophagous insects with Salmonella enterica on plants and enhanced persistence of the pathogen with Macrosteles quadrilineatus infestation or Frankliniella occidentalis feeding.

    PubMed

    Soto-Arias, José Pablo; Groves, Russell; Barak, Jeri D

    2013-01-01

    Recently, most foodborne illness outbreaks of salmonellosis have been caused by consumption of contaminated fresh produce. Yet, the mechanisms that allow the human pathogen Salmonella enterica to contaminate and grow in plant environments remain poorly described. We examined the effect of feeding by phytophagous insects on survival of S. enterica on lettuce. Larger S. enterica populations were found on leaves infested with Macrosteles quadrilineatus. In contrast, pathogen populations among plants exposed to Frankliniella occidentalis or Myzus persicae were similar to those without insects. However, on plants infested with F. occidentalis, areas of the infested leaf with feeding damage sustained higher S. enterica populations than areas without damage. The spatial distribution of S. enterica cells on leaves infested with F. occidentalis may be altered resulting in higher populations in feeding lesions or survival may be different across a leaf dependent on local damage. Results suggest the possibility of some specificity with select insects and the persistence of S. enterica. Additionally, we demonstrated the potential for phytophagous insects to become contaminated with S. enterica from contaminated plant material. S. enterica was detected in approximately 50% of all M. quadrilineatus, F. occidentalis, and M. persicae after 24 h exposure to contaminated leaves. Particularly, 17% of F. occidentalis, the smallest of the insects tested, harbored more than 10(2) CFU/F. occidentalis. Our results show that phytophagous insects may influence the population dynamics of S. enterica in agricultural crops. This study provides evidence of a human bacterial pathogen interacting with phytophagous insect during plant infestation.

  19. 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.

  20. Yeast-like microorganisms in the scale insect Kermes quercus (Insecta, Hemiptera, Coccomorpha: Kermesidae). Newly acquired symbionts?

    PubMed

    Podsiadło, Elżbieta; Michalik, Katarzyna; Michalik, Anna; Szklarzewicz, Teresa

    2018-01-01

    Scale insects, like other plant sap-consumers, are host to symbiotic microorganisms which provide them with the substances missing from their diet. In contrast to most scale insects, Kermes quercus (Linnaeus) was regarded as asymbiotic. Our histological and ultrastructural observations show that in the body of the feeding stages of K. quercus collected in two locations (Warsaw and Cracow), numerous yeast-like microorganisms occur. These microorganisms were localized in the cytoplasm of fat body cells. The yeast-like microorganisms were observed neither in other organs of the host insect nor in the eggs. These microorganisms did not cause any damage to the structure of the ovaries and the course of oogenesis of the host insect. The females infected by them produced about 1300 larvae. The lack of these microorganisms in the cytoplasm of eggs indicates that they are not transmitted transovarially from mother to offspring. Molecular analyses indicated that the microorganisms which reside in the body of K. quercus are closely related to the entomopathogenic fungi Cordyceps and Ophiocordyceps, which belong to the Sordariomycetes class within the Ascomycota. The role of yeast-like microorganisms to their host insects remains unknown; however, it has been suggested that they may represent newly acquired symbionts. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Insect Larvae: A New Platform to Produce Commercial Recombinant Proteins.

    PubMed

    Targovnik, Alexandra M; Arregui, Mariana B; Bracco, Lautaro F; Urtasun, Nicolas; Baieli, Maria F; Segura, Maria M; Simonella, Maria A; Fogar, Mariela; Wolman, Federico J; Cascone, Osvaldo; Miranda, Maria V

    2016-01-01

    In Biotechnology, the expression of recombinant proteins is a constantly growing field and different hosts are used for this purpose. Some valuable proteins cannot be produced using traditional systems. Insects from the order Lepidoptera infected with recombinant baculovirus have appeared as a good choice to express high levels of proteins, especially those with post-translational modifications. Lepidopteran insects, which are extensively distributed in the world, can be used as small protein factories, the new biofactories. Species like Bombyx mori (silkworm) have been analyzed in Asian countries to produce a great number of recombinant proteins for use in basic and applied science and industry. Many proteins expressed in this larva have been commercialized. Several recombinant proteins produced in silkworms have already been commercialized. On the other hand, species like Spodoptera frugiperda, Heliothis virescens, Rachiplusia nu, Helicoverpa zea and Trichoplusia ni are widely distributed in both the occidental world and Europe. The expression of recombinant proteins in larvae has the advantage of its low cost in comparison with insect cell cultures. A wide variety of recombinant proteins, including enzymes, hormones and vaccines, have been efficiently expressed with intact biological activity. The expression of pharmaceutically proteins, using insect larvae or cocoons, has become very attractive. This review describes the use of insect larvae as an alternative to produce commercial recombinant proteins.

  2. Ecdysteroid-stimulated synthesis and secretion of an N-acetyl-D-glucosamine-rich glycopeptide in a lepidopteran cell line derived from imaginal discs.

    PubMed

    Porcheron, P; Morinière, M; Coudouel, N; Oberlander, H

    1991-01-01

    Hormone-regulated processing of N-acetyl-D-glucosamine was studied in an insect cell line derived from imaginal wing discs of the Indian meal moth, Plodia interpunctella (Hübner). The cell line, IAL-PID2, responded to treatment with 20-hydroxyecdysone with increased incorporation of GlcNAc into glycoproteins. Cycloheximide and tunicamycin counteracted the action of the hormone. In particular, treatment with 20-hydroxyecdysone resulted in the secretion of a 5,000 dalton N-acetyl-D-glucosamine-rich glycopeptide by the IAL-PID2 cells. Accumulation of this peptide was prevented by the use of teflubenzuron, a potent chitin synthesis inhibitor. A glycopeptide of similar molecular weight was observed in imaginal discs of P. interpunctella treated with 20-hydroxyecdysone in vitro, under conditions that induce chitin synthesis. Although the function of the 5,000 dalton glycopeptide is not known, we believe that the PID2 cell line is a promising model for molecular analysis of ecdysteroid-regulated processing of aminosugars by epidermal cells during insect development.

  3. Avoiding a bad apple: Insect pollination enhances fruit quality and economic value☆

    PubMed Central

    Garratt, M.P.D.; Breeze, T.D.; Jenner, N.; Polce, C.; Biesmeijer, J.C.; Potts, S.G.

    2014-01-01

    Insect pollination is important for food production globally and apples are one of the major fruit crops which are reliant on this ecosystem service. It is fundamentally important that the full range of benefits of insect pollination to crop production are understood, if the costs of interventions aiming to enhance pollination are to be compared against the costs of the interventions themselves. Most previous studies have simply assessed the benefits of pollination to crop yield and ignored quality benefits and how these translate through to economic values. In the present study we examine the influence of insect pollination services on farmgate output of two important UK apple varieties; Gala and Cox. Using field experiments, we quantify the influence of insect pollination on yield and importantly quality and whether either may be limited by sub-optimal insect pollination. Using an expanded bioeconomic model we value insect pollination to UK apple production and establish the potential for improvement through pollination service management. We show that insects are essential in the production of both varieties of apple in the UK and contribute a total of £36.7 million per annum, over £6 million more than the value calculated using more conventional dependence ratio methods. Insect pollination not only affects the quantity of production but can also have marked impacts on the quality of apples, influencing size, shape and effecting their classification for market. These effects are variety specific however. Due to the influence of pollination on both yield and quality in Gala, there is potential for insect pollination services to improve UK output by up to £5.7 million per annum. Our research shows that continued pollinator decline could have serious financial implications for the apple industry but there is considerable scope through management of wild pollinators or using managed pollinator augmentation, to improve the quality of production. Furthermore, we show that it is critically important to consider all production parameters including quality, varietal differences and management costs when valuing the pollination service of any crop so investment in pollinator management can be proportional to its contribution. PMID:24748698

  4. Avoiding a bad apple: Insect pollination enhances fruit quality and economic value.

    PubMed

    Garratt, M P D; Breeze, T D; Jenner, N; Polce, C; Biesmeijer, J C; Potts, S G

    2014-02-01

    Insect pollination is important for food production globally and apples are one of the major fruit crops which are reliant on this ecosystem service. It is fundamentally important that the full range of benefits of insect pollination to crop production are understood, if the costs of interventions aiming to enhance pollination are to be compared against the costs of the interventions themselves. Most previous studies have simply assessed the benefits of pollination to crop yield and ignored quality benefits and how these translate through to economic values. In the present study we examine the influence of insect pollination services on farmgate output of two important UK apple varieties; Gala and Cox. Using field experiments, we quantify the influence of insect pollination on yield and importantly quality and whether either may be limited by sub-optimal insect pollination. Using an expanded bioeconomic model we value insect pollination to UK apple production and establish the potential for improvement through pollination service management. We show that insects are essential in the production of both varieties of apple in the UK and contribute a total of £36.7 million per annum, over £6 million more than the value calculated using more conventional dependence ratio methods. Insect pollination not only affects the quantity of production but can also have marked impacts on the quality of apples, influencing size, shape and effecting their classification for market. These effects are variety specific however. Due to the influence of pollination on both yield and quality in Gala, there is potential for insect pollination services to improve UK output by up to £5.7 million per annum. Our research shows that continued pollinator decline could have serious financial implications for the apple industry but there is considerable scope through management of wild pollinators or using managed pollinator augmentation, to improve the quality of production. Furthermore, we show that it is critically important to consider all production parameters including quality, varietal differences and management costs when valuing the pollination service of any crop so investment in pollinator management can be proportional to its contribution.

  5. Replication of Syngrapha falcifera Multiple-Nuclear Polyhedrosis Virus-D in Different Insect Cells

    NASA Astrophysics Data System (ADS)

    Khalid Nessr Alhag, Sadeq; Xin, Peng Jian

    Six insect cell lines were tested for susceptibility to Syngrapha falcifera multiple nucleocapsid nucleopolyhedrovirus-D (SfaMNPV-D) infection by use of a typical endpoint assay procedure. Cell lines from Trichoplusia ni (Tn5B1-4), (L105-clone), Spodoptera litura (SL-ZSU-1), Spodoptera frugiperda (IPLB-SF-21), Pieris rapaeb (Pr-E-HNU9) and Helicoverpa zea (BCIRL-HZ-AM1) in 96-well tissue culture plates were infected with dilutions of extra cellular virus suspensions of (SfaMNPV-D). Each cell/virus combination was incubated at temperatures 27°C and wells were scored for positive infection at 2 to 4 day intervals. The resulting data were analyzed by Reed and Muench method, providing virus titers for each combination of virus, cell line. The results were categorized by accuracy and by rapidity of maximum titer. Virus titer of Tn5B-4 was higher than other cell lines TCID50 8.7x108, the lowest level detected in infected was in (Pr-E-HNU9) cells TCID50 2.4x108. No Virions or polyhedral inclusion bodies were detected in infected SL-ZSU-1 cells.

  6. Metagenetic analysis of the bacterial communities of edible insects from diverse production cycles at industrial rearing companies.

    PubMed

    Vandeweyer, D; Crauwels, S; Lievens, B; Van Campenhout, L

    2017-11-16

    Despite the continuing development of new insect-derived food products, microbial research on edible insects and insect-based foods is still very limited. The goal of this study was to increase the knowledge on the microbial quality of edible insects by comparing the bacterial community composition of mealworms (Tenebrio molitor) and crickets (Acheta domesticus and Gryllodes sigillatus) from several production cycles and rearing companies. Remarkable differences in the bacterial community composition were found between different mealworm rearing companies and mealworm production cycles from the same company. In comparison with mealworms, the bacterial community composition of the investigated crickets was more similar among different companies, and was highly similar between both cricket species investigated. Mealworm communities were dominated by Spiroplasma and Erwinia species, while crickets were abundantly colonised by (Para)bacteroides species. With respect to food safety, only a few operational taxonomic units could be associated with potential human pathogens such as Cronobacter or spoilage bacteria such as Pseudomonas. In summary, our results implicate that at least for cricket rearing, production cycles of constant and good quality in terms of bacterial composition can be obtained by different rearing companies. For mealworms however, more variation in terms of microbial quality occurs between companies. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Evolution of a complex behavior: the origin and initial diversification of foliar galling by Permian insects.

    PubMed

    Schachat, Sandra R; Labandeira, Conrad C

    2015-04-01

    A central notion of the early evolution of insect galling is that this unique behavior was uncommon to rare before the diversification of angiosperms 135 to 125 m.yr. ago. However, evidence accumulated during recent years shows that foliar galls were diverse and locally abundant as early as the Permian Period, 299 to 252 m.yr. ago. In particular, a diversity of leaf galling during the Early Permian has recently been documented by the plant-damage record of foliar galls and, now, our interpretation of the body-fossil record of culprit insect gallers. Small size is a prerequisite for gallers. Wing-length measurements of Permian insects indicate that several small-bodied hemipteroid lineages originated early during the Permian, some descendant lineages of which gall the leaves of seed plants to the present day. The earliest foliar gallers likely were Protopsyllidiidae (Hemiptera) and Lophioneuridae (Thripida). Much of the Early Permian was a xeric interval, and modern galls are most common in dry, extra-tropical habitats such as scrubland and deserts. Plant-damage, insect body fossils, and the paleoclimate record collectively support the ecological expansion of foliar galling during the Early Permian and its continued expansion through the Late Permian.

  8. Thermal acclimation mitigates cold-induced paracellular leak from the Drosophila gut.

    PubMed

    MacMillan, Heath A; Yerushalmi, Gil Y; Jonusaite, Sima; Kelly, Scott P; Donini, Andrew

    2017-08-18

    Chill susceptible insects suffer tissue damage and die at low temperatures. The mechanisms that cause chilling injury are not well understood but a growing body of evidence suggests that a cold-induced loss of ion and water homeostasis leads to hemolymph hyperkalemia that depolarizes cells, leading to cell death. The apparent root of this cascade is the net leak of osmolytes down their concentration gradients in the cold. Many insects, however, are capable of adjusting their thermal physiology, and cold-acclimated Drosophila can maintain homeostasis and avoid injury better than warm-acclimated flies. Here, we test whether chilling causes a loss of epithelial barrier function in female adult Drosophila, and provide the first evidence of cold-induced epithelial barrier failure in an invertebrate. Flies had increased rates of paracellular leak through the gut epithelia at 0 °C, but cold acclimation reduced paracellular permeability and improved cold tolerance. Improved barrier function was associated with changes in the abundance of select septate junction proteins and the appearance of a tortuous ultrastructure in subapical intercellular regions of contact between adjacent midgut epithelial cells. Thus, cold causes paracellular leak in a chill susceptible insect and cold acclimation can mitigate this effect through changes in the composition and structure of transepithelial barriers.

  9. The xeroderma pigmentosum group B protein ERCC3 produced in the baculovirus system exhibits DNA helicase activity.

    PubMed Central

    Ma, L; Siemssen, E D; Noteborn, H M; van der Eb, A J

    1994-01-01

    The XPB/ERCC3 gene corrects the nucleotide excision-repair defect in the human hereditary disease xeroderma pigmentosum group B and encodes the largest subunit of the basal transcription factor BTF2/TFIIH. The primary sequence of the XPB/ERCC3 protein features the hallmarks of seven helicase motifs found in many known and putative helicases or helicase-related proteins. Recently, the multiprotein BTF2/TFIIH complex has been found to be associated with DNA helicase activity. To explore the properties and functions of XPB/ERCC3, we have used the baculovirus/insect-cell expression system to produce recombinant protein. We report here the construction and analysis of recombinant baculovirus expressing XPB/ERCC3. The XPB/ERCC3 protein is synthesized at a relatively high level in baculovirus-infected insect cells. While the majority of XPB/ERCC3 end up in the insoluble fraction of insect cell lysates, a minor fraction of recombinant protein is present in soluble form which can be purified under native conditions. We have found that a DNA helicase activity is associated with the purified XPB/ERCC3 protein, suggesting that XPB/ERCC3 may function as a DNA helicase in local unwinding of DNA template both in the context of transcription and nucleotide excision repair. Images PMID:7937133

  10. An Automated Flying-Insect Detection System

    NASA Technical Reports Server (NTRS)

    Vann, Timi; Andrews, Jane C.; Howell, Dane; Ryan, Robert

    2007-01-01

    An automated flying-insect detection system (AFIDS) was developed as a proof-of-concept instrument for real-time detection and identification of flying insects. This type of system has use in public health and homeland-security decision support, agriculture and military pest management, and/or entomological research. Insects are first lured into the AFIDS integrated sphere by insect attractants. Once inside the sphere, the insect s wing beats cause alterations in light intensity that is detected by a photoelectric sensor. Following detection, the insects are encouraged (with the use of a small fan) to move out of the sphere and into a designated insect trap where they are held for taxonomic identification or serological testing. The acquired electronic wing-beat signatures are preprocessed (Fourier transformed) in real time to display a periodic signal. These signals are sent to the end user where they are graphically. All AFIDS data are preprocessed in the field with the use of a laptop computer equipped with LabVIEW. The AFIDS software can be programmed to run continuously or at specific time intervals when insects are prevalent. A special DC-restored transimpedance amplifier reduces the contributions of low-frequency background light signals, and affords approximately two orders of magnitude greater AC gain than conventional amplifiers. This greatly increases the signal-to-noise ratio and enables the detection of small changes in light intensity. The AFIDS light source consists of high-intensity Al-GaInP light-emitting diodes (LEDs). The AFIDS circuitry minimizes brightness fluctuations in the LEDs and when integrated with an integrating sphere, creates a diffuse uniform light field. The insect wing beats isotropically scatter the diffuse light in the sphere and create wing-beat signatures that are detected by the sensor. This configuration minimizes variations in signal associated with insect flight orientation. Preliminary data indicate that AFIDS has sufficient sensitivity and frequency measuring capability to differentiate between male and female mosquitoes (Figure 1, bottom panel) and fruit flies (data not shown). Similar studies show that AFIDS can be utilized to detect discrete differences between two mosquito species, Aedes aegypti and Aedes albopictus. When fully deployable, a wireless network of AFIDS monitors could be used in combination with other remotely sensed data and visually displayed in a geographic information system (GIS) to provide real-time surveillance (see Figure 2). More accurate and sensitive insect population forecasts and effective rapid response and mitigation of insect issues would then be possible.

  11. WHOLE INSECT AND MAMMALIAN EMBRYO IMAGING WITH CONFOCAL MICROSCOPY: MORPHOLOGY AND APOPTOSIS

    EPA Science Inventory

    Background: After fluorochromes are incorporated into cells, tissues, and organisms, confocal microscopy can be used to observe three-dimensional structures. LysoTracker Red (LT) is a paraformaldehyde fixable probe that concentrates into acidic compartments of cells and indicates...

  12. A Baculovirus Immediate-Early Gene, ie1, Promoter Drives Efficient Expression of a Transgene in Both Drosophila melanogaster and Bombyx mori

    PubMed Central

    Masumoto, Mika; Ohde, Takahiro; Shiomi, Kunihiro; Yaginuma, Toshinobu; Niimi, Teruyuki

    2012-01-01

    Many promoters have been used to drive expression of heterologous transgenes in insects. One major obstacle in the study of non-model insects is the dearth of useful promoters for analysis of gene function. Here, we investigated whether the promoter of the immediate-early gene, ie1, from the Bombyx mori nucleopolyhedrovirus (BmNPV) could be used to drive efficient transgene expression in a wide variety of insects. We used a piggyBac-based vector with a 3xP3-DsRed transformation marker to generate a reporter construct; this construct was used to determine the expression patterns driven by the BmNPV ie1 promoter; we performed a detailed investigation of the promoter in transgene expression pattern in Drosophila melanogaster and in B. mori. Drosophila and Bombyx belong to different insect orders (Diptera and Lepidoptera, respectively); however, and to our surprise, ie1 promoter-driven expression was evident in several tissues (e.g., prothoracic gland, midgut, and tracheole) in both insects. Furthermore, in both species, the ie1 promoter drove expression of the reporter gene from a relatively early embryonic stage, and strong ubiquitous ie1 promoter-driven expression continued throughout the larval, pupal, and adult stages by surface observation. Therefore, we suggest that the ie1 promoter can be used as an efficient expression driver in a diverse range of insect species. PMID:23152896

  13. RNA interference: a new strategy in the evolutionary arms race between human control strategies and insect pests.

    PubMed

    Machado, Vilmar; Rodríguez-García, María Juliana; Sánchez-García, Francisco Javier; Galan, Jose

    2014-01-01

    The relationship between humans and the insect pests of cultivated plants may be considered to be an indirect coevolutionary process, i.e., an arms race. Over time, humans have developed several strategies to minimize the negative impacts of insects on agricultural production. However, insects have made adaptive responses via the evolution of resistance to insecticides, and more recently against Bacillus thuriengiensis. Thus, we need to continuously invest resources in the development of new strategies for crop protection. Recent advances in genomics have demonstrated the possibility of a new weapon or strategy in this war, i.e., gene silencing, which involves blocking the expression of specific genes via mRNA inactivation. In the last decade, several studies have demonstrated the effectiveness of this strategy in the control of different species of insects. However, several technical difficulties need to be overcome to transform this potential into reality, such as the selection of target genes, the concentration of dsRNA, the nucleotide sequence of the dsRNA, the length of dsRNA, persistence in the insect body, and the life stage of the target species where gene silencing is most efficient. This study analyzes several aspects related to the use of gene silencing in pest control and it includes an overview of the inactivation process, as well as the problems that need to be resolved to transform gene silencing into an effective pest control method.

  14. Common sense about taste: from mammals to insects.

    PubMed

    Yarmolinsky, David A; Zuker, Charles S; Ryba, Nicholas J P

    2009-10-16

    The sense of taste is a specialized chemosensory system dedicated to the evaluation of food and drink. Despite the fact that vertebrates and insects have independently evolved distinct anatomic and molecular pathways for taste sensation, there are clear parallels in the organization and coding logic between the two systems. There is now persuasive evidence that tastant quality is mediated by labeled lines, whereby distinct and strictly segregated populations of taste receptor cells encode each of the taste qualities.

  15. Ovariole Structure of the Cochineal Scale Insect, Dactylophis coccus

    PubMed Central

    Ramírez-Cruz, A.; Llanderal-Cázares, C.; Racotta, R.

    2008-01-01

    The ovaries of the adult cochineal scale insect, Dactylopius coccus Costa (Hemiptera: Coccoidea: Dactylopiidae) are made up of more than 400 short ovarioles of the telotrophic type. The ovarioles develop asynchronously. The ovarioles consist of a germarium with six or seven nurse cells, a vitellarium with an oocyte, and pedicel. A terminal filament is lacking. A maturing oocyte was attached to the trophic core by the trophic cord during previtellogenesis and most of vitellogenesis. PMID:20337555

  16. MOS1 Osmosensor of Metarhizium anisopliae Is Required for Adaptation to Insect Host Hemolymph▿

    PubMed Central

    Wang, Chengshu; Duan, Zhibing; St. Leger, Raymond J.

    2008-01-01

    Entomopathogenic fungi such as Metarhizium anisopliae infect insects by direct penetration of the cuticle, after which the fungus adapts to the high osmotic pressure of the hemolymph and multiplies. Here we characterize the M. anisopliae Mos1 gene and demonstrate that it encodes the osmosensor required for this process. MOS1 contains transmembrane regions and a C-terminal Src homology 3 domain similar to those of yeast osmotic adaptor proteins, and homologs of MOS1 are widely distributed in the fungal kingdom. Reverse transcription-PCR demonstrated that Mos1 is up-regulated in insect hemolymph as well as artificial media with high osmotic pressure. Transformants containing an antisense vector directed to the Mos1 mRNA depleted transcript levels by 80%. This produced selective alterations in regulation of genes involved in hyphal body formation, cell membrane stiffness, and generation of intracellular turgor pressure, suggesting that these processes are mediated by MOS1. Consistent with a role in stress responses, transcript depletion of Mos1 increased sensitivity to osmotic and oxidative stresses and to compounds that interfere with cell wall biosynthesis. It also disrupted developmental processes, including formation of appressoria and hyphal bodies. Insect bioassays confirmed that Mos1 knockdown significantly reduces virulence. Overall, our data show that M. anisopliae MOS1 mediates cellular responses to high osmotic pressure and subsequent adaptations to colonize host hemolymph. PMID:18055914

  17. Molecular biology of insect sodium channels and pyrethroid resistance.

    PubMed

    Dong, Ke; Du, Yuzhe; Rinkevich, Frank; Nomura, Yoshiko; Xu, Peng; Wang, Lingxin; Silver, Kristopher; Zhorov, Boris S

    2014-07-01

    Voltage-gated sodium channels are essential for the initiation and propagation of the action potential in neurons and other excitable cells. Because of their critical roles in electrical signaling, sodium channels are targets of a variety of naturally occurring and synthetic neurotoxins, including several classes of insecticides. This review is intended to provide an update on the molecular biology of insect sodium channels and the molecular mechanism of pyrethroid resistance. Although mammalian and insect sodium channels share fundamental topological and functional properties, most insect species carry only one sodium channel gene, compared to multiple sodium channel genes found in each mammalian species. Recent studies showed that two posttranscriptional mechanisms, alternative splicing and RNA editing, are involved in generating functional diversity of sodium channels in insects. More than 50 sodium channel mutations have been identified to be responsible for or associated with knockdown resistance (kdr) to pyrethroids in various arthropod pests and disease vectors. Elucidation of molecular mechanism of kdr led to the identification of dual receptor sites of pyrethroids on insect sodium channels. Many of the kdr mutations appear to be located within or close to the two receptor sites. The accumulating knowledge of insect sodium channels and their interactions with insecticides provides a foundation for understanding the neurophysiology of sodium channels in vivo and the development of new and safer insecticides for effective control of arthropod pests and human disease vectors. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Insect Responses to Linearly Polarized Reflections: Orphan Behaviors Without Neural Circuits

    PubMed Central

    Heinloth, Tanja; Uhlhorn, Juliane; Wernet, Mathias F.

    2018-01-01

    The e-vector orientation of linearly polarized light represents an important visual stimulus for many insects. Especially the detection of polarized skylight by many navigating insect species is known to improve their orientation skills. While great progress has been made towards describing both the anatomy and function of neural circuit elements mediating behaviors related to navigation, relatively little is known about how insects perceive non-celestial polarized light stimuli, like reflections off water, leaves, or shiny body surfaces. Work on different species suggests that these behaviors are not mediated by the “Dorsal Rim Area” (DRA), a specialized region in the dorsal periphery of the adult compound eye, where ommatidia contain highly polarization-sensitive photoreceptor cells whose receptive fields point towards the sky. So far, only few cases of polarization-sensitive photoreceptors have been described in the ventral periphery of the insect retina. Furthermore, both the structure and function of those neural circuits connecting to these photoreceptor inputs remain largely uncharacterized. Here we review the known data on non-celestial polarization vision from different insect species (dragonflies, butterflies, beetles, bugs and flies) and present three well-characterized examples for functionally specialized non-DRA detectors from different insects that seem perfectly suited for mediating such behaviors. Finally, using recent advances from circuit dissection in Drosophila melanogaster, we discuss what types of potential candidate neurons could be involved in forming the underlying neural circuitry mediating non-celestial polarization vision. PMID:29615868

  19. Intracellular transport and processing of the Marburg virus surface protein in vertebrate and insect cells.

    PubMed

    Becker, S; Klenk, H D; Mühlberger, E

    1996-11-01

    The surface protein (GP) of Marburg virus (MBG) is synthesized as a 90-kDa precursor protein which is cotranslationally modified by the addition of high-mannose sugars (140 kDa). This step is followed by the conversion of the N-linked sugars to endoglycosidase H (endo H)-resistant species and the addition of O-linked oliosaccharides leading to a mature protein of 170-200 kDa approximately 30 min after pulse labelling. The mature form of GP is efficiently transported to the plasma membrane. GP synthesized using the T7 polymerase-driven vaccinia virus expression system was transported with essentially the same kinetics as the authentic GP. However, the protein that is shown to appear 30 min after pulse labeling at the plasma membrane was slighly smaller (160 kDa) than GP incorporated into the virions (170 kDa). Using a recombinant baculovirus, GP was expressed at high levels in insect cells. Three different species could be identified: a 90-kDa unglycosylated GP localized in the cytoplasm and two 140-kDa glycosylated proteins. Characterization of the glycosylated GPs revealed that processing of the oligosaccharides of GP was less efficient in insect cells than in mammalian cells. The majority of GP remained endo H sensitive containing high-mannose type N-linked glycans, whereas only a small fraction became endo H resistant carrying processed N-glycans and O-glycans. Tunicamycin treatment of the GP-expressing cells demonstrated that N-glycosylation is essential for the transport of the MBG surface protein.

  20. Comparative transcriptome and gene co-expression network analysis reveal genes and signaling pathways adaptively responsive to varied adverse stresses in the insect fungal pathogen, Beauveria bassiana.

    PubMed

    He, Zhangjiang; Zhao, Xin; Lu, Zhuoyue; Wang, Huifang; Liu, Pengfei; Zeng, Fanqin; Zhang, Yongjun

    2018-01-01

    Sensing, responding, and adapting to the surrounding environment are crucial for all living organisms to survive, proliferate, and differentiate in their biological niches. Beauveria bassiana is an economically important insect-pathogenic fungus which is widely used as a biocontrol agent to control a variety of insect pests. The fungal pathogen unavoidably encounters a variety of adverse environmental stresses and defense response from the host insects during application of the fungal agents. However, few are known about the transcription response of the fungus to respond or adapt varied adverse stresses. Here, we comparatively analyzed the transcriptome of B. bassiana in globe genome under the varied stationary-phase stresses including osmotic agent (0.8 M NaCl), high temperature (32 °C), cell wall-perturbing agent (Congo red), and oxidative agents (H 2 O 2 or menadione). Total of 12,412 reads were obtained, and mapped to the 6767 genes of the B. bassiana. All of these stresses caused transcription responses involved in basal metabolism, cell wall construction, stress response or cell rescue/detoxification, signaling transduction and gene transcription regulation, and likely other cellular processes. An array of genes displayed similar transcription patterns in response to at least two of the five stresses, suggesting a shared transcription response to varied adverse stresses. Gene co-expression network analysis revealed that mTOR signaling pathway, but not HOG1 MAP kinase pathway, played a central role in regulation the varied adverse stress responses, which was verified by RNAi-mediated knockdown of TOR1. Our findings provided an insight of transcription response and gene co-expression network of B. bassiana in adaptation to varied environments. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Production of N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) fused with secretory signal Igκ in insect cells.

    PubMed

    Horynová, Milada; Takahashi, Kazuo; Hall, Stacy; Renfrow, Matthew B; Novak, Jan; Raška, Milan

    2012-02-01

    The human UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) is one of the key enzymes that initiate synthesis of hinge-region O-linked glycans of human immunoglobulin A1 (IgA1). We designed secreted soluble form of human GalNAc-T2 as a fusion protein containing mouse immunoglobulin light chain kappa secretory signal and expressed it using baculovirus and mammalian expression vectors. The recombinant protein was secreted by insect cells Sf9 and human HEK 293T cells in the culture medium. The protein was purified from the media using affinity Ni-NTA chromatography followed by stabilization of purified protein in 50mM Tris-HCl buffer at pH 7.4. Although the purity of recombinant GalNAc-T2 was comparable in both expression systems, the yield was higher in Sf9 insect expression system (2.5mg of GalNAc-T2 protein per 1L culture medium). The purified soluble recombinant GalNAc-T2 had an estimated molecular mass of 65.8kDa and its amino-acid sequence was confirmed by mass-spectrometric analysis. The enzymatic activity of Sf9-produced recombinant GalNAc-T2 was determined by the quantification of enzyme-mediated attachment of GalNAc to synthetic IgA1 hinge-region peptide as the acceptor and UDP-GalNAc as the donor. In conclusion, murine immunoglobulin kappa secretory signal was used for production of secreted enzymatically active GalNAc-T2 in insect baculovirus expression system. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. An Aphid Effector Targets Trafficking Protein VPS52 in a Host-Specific Manner to Promote Virulence.

    PubMed

    Rodriguez, Patricia A; Escudero-Martinez, Carmen; Bos, Jorunn I B

    2017-03-01

    Plant- and animal-feeding insects secrete saliva inside their hosts, containing effectors, which may promote nutrient release and suppress immunity. Although for plant pathogenic microbes it is well established that effectors target host proteins to modulate host cell processes and promote disease, the host cell targets of herbivorous insects remain elusive. Here, we show that the existing plant pathogenic microbe effector paradigm can be extended to herbivorous insects in that effector-target interactions inside host cells modify critical host processes to promote plant susceptibility. We showed that the effector Mp1 from Myzus persicae associates with the host Vacuolar Protein Sorting Associated Protein52 (VPS52). Using natural variants, we provide a strong link between effector virulence activity and association with VPS52, and show that the association is highly specific to M persicae -host interactions. Also, coexpression of Mp1, but not Mp1-like variants, specifically with host VPS52s resulted in effector relocalization to vesicle-like structures that associate with prevacuolar compartments. We show that high VPS52 levels negatively impact virulence, and that aphids are able to reduce VPS52 levels during infestation, indicating that VPS52 is an important virulence target. Our work is an important step forward in understanding, at the molecular level, how a major agricultural pest promotes susceptibility during infestation of crop plants. We give evidence that an herbivorous insect employs effectors that interact with host proteins as part of an effective virulence strategy, and that these effectors likely function in a species-specific manner. © 2017 American Society of Plant Biologists. All Rights Reserved.

  3. Differences in dissolved cadmium and zinc uptake among stream insects: Mechanistic explanations

    USGS Publications Warehouse

    Buchwalter, D.B.; Luoma, S.N.

    2005-01-01

    This study examined the extent to which dissolved Cd and Zn uptake rates vary in several aquatic insect taxa commonly used as indicators of ecological health. We further attempted to explain the mechanisms underlying observed differences. By comparing dissolved Cd and Zn uptake rates in several aquatic insect species, we demonstrated that species vary widely in these processes. Dissolved uptake rates were not related to gross morphological features such as body size or gill size-features that influence water permeability and therefore have ionoregulatory importance. However, finer morphological features, specifically, the relative numbers of ionoregulatory cells (chloride cells), appeared to be related to dissolved metal uptake rates. This observation was supported by Michaelis-Menten type kinetics experiments, which showed that dissolved Cd uptake rates were driven by the numbers of Cd transporters and not by the affinities of those transporters to Cd. Calcium concentrations in exposure media similarly affected Cd and Zn uptake rates in the caddisfly Hydropsyche californica. Dissolved Cd and Zn uptake rates strongly co-varied among species, suggesting that these metals are transported by similar mechanisms.

  4. Insecticide resistance and intracellular proteases.

    PubMed

    Wilkins, Richard M

    2017-12-01

    Pesticide resistance is an example of evolution in action with mechanisms of resistance arising from mutations or increased expression of intrinsic genes. Intracellular proteases have a key role in maintaining healthy cells and in responding to stressors such as pesticides. Insecticide-resistant insects have constitutively elevated intracellular protease activity compared to corresponding susceptible strains. This increase was shown for some cases originally through biochemical enzyme studies and subsequently putatively by transcriptomics and proteomics methods. Upregulation and expression of proteases have been characterised in resistant strains of some insect species, including mosquitoes. This increase in proteolysis results in more degradation products (amino acids) of intracellular proteins. These may be utilised in the resistant strain to better protect the cell from stress. There are changes in insect intracellular proteases shortly after insecticide exposure, suggesting a role in stress response. The use of protease and proteasome inhibitors or peptide mimetics as synergists with improved application techniques and through protease gene knockdown using RNA interference (possibly expressed in crop plants) may be potential pest management strategies, in situations where elevated intracellular proteases are relevant. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  5. The mechanisms underlying the production of discontinuous gas exchange cycles in insects.

    PubMed

    Matthews, Philip G D

    2018-03-01

    This review examines the control of gas exchange in insects, specifically examining what mechanisms could explain the emergence of discontinuous gas exchange cycles (DGCs). DGCs are gas exchange patterns consisting of alternating breath-hold periods and bouts of gas exchange. While all insects are capable of displaying a continuous pattern of gas exchange, this episodic pattern is known to occur within only some groups of insects and then only sporadically or during certain phases of their life cycle. Investigations into DGCs have tended to emphasise the role of chemosensory thresholds in triggering spiracle opening as critical for producing these gas exchange patterns. However, a chemosensory basis for episodic breathing also requires an as-of-yet unidentified hysteresis between internal respiratory stimuli, chemoreceptors, and the spiracles. What has been less appreciated is the role that the insect's central nervous system (CNS) might play in generating episodic patterns of ventilation. The active ventilation displayed by many insects during DGCs suggests that this pattern could be the product of directed control by the CNS rather than arising passively as a result of self-sustaining oscillations in internal oxygen and carbon dioxide levels. This paper attempts to summarise what is currently known about insect gas exchange regulation, examining the location and control of ventilatory pattern generators in the CNS, the influence of chemoreceptor feedback in the form of O 2 and CO 2 /pH fluctuations in the haemolymph, and the role of state-dependent changes in CNS activity on ventilatory control. This information is placed in the context of what is currently known regarding the production of discontinuous gas exchange patterns.

  6. Insecticidal spectrum and mode of action of the Bacillus thuringiensis Vip3Ca insecticidal protein.

    PubMed

    Gomis-Cebolla, Joaquín; Ruiz de Escudero, Iñigo; Vera-Velasco, Natalia Mara; Hernández-Martínez, Patricia; Hernández-Rodríguez, Carmen Sara; Ceballos, Tomás; Palma, Leopoldo; Escriche, Baltasar; Caballero, Primitivo; Ferré, Juan

    2017-01-01

    The Vip3Ca protein, discovered in a screening of Spanish collections of Bacillus thuringiensis, was known to be toxic to Chrysodeixis chalcites, Mamestra brassicae and Trichoplusia ni. In the present study, its activity has been tested with additional insect species and we found that Cydia pomonella is moderately susceptible to this protein. Vip3Ca (of approximately 90kDa) was processed to an approximately 70kDa protein when incubated with midgut juice in all tested species. The kinetics of proteolysis correlated with the susceptibility of the insect species to Vip3Ca. The activation was faster to slower in the following order: M. brassicae (susceptible), Spodoptera littoralis (moderately susceptible), Agrotis ipsilon and Ostrinia nubilalis (slightly susceptible). Processing Vip3Ca by O. nubilalis or M. brassicae midgut juice did not significantly changed its toxicity to either insect species, indicating that the low susceptibility of O. nubilalis is not due to a problem in the midgut processing of the toxin. M. brassicae larvae fed with Vip3Ca showed binding of this toxin to the apical membrane of the midgut epithelial cells. Histopathological inspection showed sloughing of the epithelial cells with further disruption, which suggests that the mode of action of Vip3Ca is similar to that described for Vip3Aa. Biotin-labeled Vip3Ca and Vip3Aa bound specifically to M. brassicae brush border membrane vesicles and both toxins competed for binding sites. This result suggests that insects resistant to Vip3A may also be cross-resistant to Vip3C, which has implications for Insect Resistance Management (IRM). Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Insulated piggyBac vectors for insect transgenesis

    PubMed Central

    Sarkar, Abhimanyu; Atapattu, Asela; Belikoff, Esther J; Heinrich, Jörg C; Li, Xuelei; Horn, Carsten; Wimmer, Ernst A; Scott, Maxwell J

    2006-01-01

    Background Germ-line transformation of insects is now a widely used method for analyzing gene function and for the development of genetically modified strains suitable for pest control programs. The most widely used transposable element for the germ-line transformation of insects is piggyBac. The site of integration of the transgene can influence gene expression due to the effects of nearby transcription enhancers or silent heterochromatic regions. Position effects can be minimized by flanking a transgene with insulator elements. The scs/scs' and gypsy insulators from Drosophila melanogaster as well as the chicken β-globin HS4 insulator function in both Drosophila and mammalian cells. Results To minimize position effects we have created a set of piggyBac transformation vectors that contain either the scs/scs', gypsy or chicken β-globin HS4 insulators. The vectors contain either fluorescent protein or eye color marker genes and have been successfully used for germ-line transformation of Drosophila melanogaster. A set of the scs/scs' vectors contains the coral reef fluorescent protein marker genes AmCyan, ZsGreen and DsRed that have not been optimized for translation in human cells. These marker genes are controlled by a combined GMR-3xP3 enhancer/promoter that gives particularly strong expression in the eyes. This is also the first report of the use of the ZsGreen and AmCyan reef fluorescent proteins as transformation markers in insects. Conclusion The insulated piggyBac vectors should protect transgenes against position effects and thus facilitate fine control of gene expression in a wide spectrum of insect species. These vectors may also be used for transgenesis in other invertebrate species. PMID:16776846

  8. Lepidopteran insect susceptibility to silver nanoparticles and measurement of changes in their growth, development and physiology.

    PubMed

    Yasur, Jyothsna; Rani, Pathipati Usha

    2015-04-01

    Increased use of nanomaterials in various fields of science has lead for the need to study the impact of nanomaterial on the environment in general and on insect and plant life in particular. We studied the impact of silver nanoparticles (AgNPs) on growth and feeding responses of two lepidopteran pests of castor plant (Ricinus communis L.) namely Asian armyworm, Spodoptera litura F. and castor semilooper, Achaea janata L. Larvae were fed with PVP coated-AgNPs treated castor leaf at different concentrations and their activity was compared to that of silver nitrate (AgNO3) treated leaf diets. Larval and pupal body weights decreased along with the decrease in the concentrations of AgNPs and AgNO3 in both the test insects. Low amounts of silver were accumulated in the larval guts, but major portion of it was eliminated through the feces. Ultrastructural studies of insect gut cell using Transmission Electron Microscopy (TEM) showed accumulation of silver nanoparticles in cell organelles. Changes in the antioxidative and detoxifying enzymes of the treated larva were estimated. The effect of treatments showed differences in the activities of detoxifying enzymes, carboxylesterases (CarE), glucosidases (Glu) and glutathione S-transferases (GST) in the larval gut. Activities of superoxide dismutase, catalase, and peroxidase were also altered in the larval bodies due to the AgNPs treatments, suggesting that exposure of larvae to nanoparticles induces oxidative stress, which is countered by antioxidant enzymes. Induction of these enzymes may be an effective detoxification mechanism by which the herbivorous insect defends itself against nanoparticle treatment. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Expression of Recombinant Antibodies

    PubMed Central

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with “human-like” post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications. PMID:23908655

  10. The insect repellent N,N-diethyl-m-toluamide (DEET) induces angiogenesis via allosteric modulation of the M3 muscarinic receptor in endothelial cells.

    PubMed

    Legeay, Samuel; Clere, Nicolas; Hilairet, Grégory; Do, Quoc-Tuan; Bernard, Philippe; Quignard, Jean-François; Apaire-Marchais, Véronique; Lapied, Bruno; Faure, Sébastien

    2016-06-27

    The insect repellent N,N-diethyl-m-toluamide (DEET) has been reported to inhibit AChE (acetylcholinesterase) and to possess potential carcinogenic properties with excessive vascularization. In the present paper, we demonstrate that DEET specifically stimulates endothelial cells that promote angiogenesis which increases tumor growth. DEET activates cellular processes that lead to angiogenesis including proliferation, migration and adhesion. This is associated with an enhancement of NO production and VEGF expression in endothelial cells. M3 silencing or the use of a pharmacological M3 inhibitor abrogates all of these effects which reveals that DEET-induced angiogenesis is M3 sensitive. The experiments involving calcium signals in both endothelial and HEK cells overexpressing M3 receptors, as well as binding and docking studies demonstrate that DEET acts as an allosteric modulator of the M3 receptor. In addition, DEET inhibited AChE which increased acetylcholine bioavailability and binding to M3 receptors and also strengthened proangiogenic effects by an allosteric modulation.

  11. A cell line resource derived from honey bee (Apis mellifera) embryonic tissues.

    PubMed

    Goblirsch, Michael J; Spivak, Marla S; Kurtti, Timothy J

    2013-01-01

    A major hindrance to the study of honey bee pathogens or the effects of pesticides and nutritional deficiencies is the lack of controlled in vitro culture systems comprised of honey bee cells. Such systems are important to determine the impact of these stress factors on the developmental and cell biology of honey bees. We have developed a method incorporating established insect cell culture techniques that supports sustained growth of honey bee cells in vitro. We used honey bee eggs mid to late in their embryogenesis to establish primary cultures, as these eggs contain cells that are progressively dividing. Primary cultures were initiated in modified Leibovitz's L15 medium and incubated at 32(°)C. Serial transfer of material from several primary cultures was maintained and has led to the isolation of young cell lines. A cell line (AmE-711) has been established that is composed mainly of fibroblast-type cells that form an adherent monolayer. Most cells in the line are diploid (2n = 32) and have the Apis mellifera karyotype as revealed by Giemsa stain. The partial sequence for the mitochondrial-encoded cytochrome c oxidase subunit I (Cox 1) gene in the cell line is identical to those from honey bee tissues and a consensus sequence for A. mellifera. The population doubling time is approximately 4 days. Importantly, the cell line is continuously subcultured every 10-14 days when split at a 1:3 ratio and is cryopreserved in liquid nitrogen. The cell culture system we have developed has potential application for studies aimed at honey bee development, genetics, pathogenesis, transgenesis, and toxicology.

  12. Characterization of N-Glycan Structures on the Surface of Mature Dengue 2 Virus Derived from Insect Cells.

    PubMed

    Lei, Y; Yu, H; Dong, Y; Yang, J; Ye, W; Wang, Y; Chen, W; Jia, Z; Xu, Z; Li, Z; Zhang, F

    2015-01-01

    DENV envelope glycoprotein (E) is responsible for interacting with host cell receptors and is the main target for the development of a dengue vaccine based on an induction of neutralizing antibodies. It is well known that DENV E glycoprotein has two potential N-linked glycosylation sites at Asn67 and Asn153. The N-glycans of E glycoprotein have been shown to influence the proper folding of the protein, its cellular localization, its interactions with receptors and its immunogenicity. However, the precise structures of the N-glycans that are attached to E glycoprotein remain elusive, although the crystal structure of DENV E has been determined. This study characterized the structures of envelope protein N-linked glycans on mature DENV-2 particles derived from insect cells via an integrated method that used both lectin microarray and MALDI-TOF-MS. By combining these methods, a high heterogeneity of DENV N-glycans was found. Five types of N-glycan were identified on DENV-2, including mannose, GalNAc, GlcNAc, fucose and sialic acid; high mannose-type N-linked oligosaccharides and the galactosylation of N-glycans were the major structures that were found. Furthermore, a complex between a glycan on DENV and the carbohydrate recognition domain (CRD) of DC-SIGN was mimicked with computational docking experiments. For the first time, this study provides a comprehensive understanding of the N-linked glycan profile of whole DENV-2 particles derived from insect cells.

  13. Expression of Biologically Active Human Butyrylcholinesterase in the Cabbage Looper (Trichoplusia ni)

    DTIC Science & Technology

    2000-01-01

    recombinant human BUChE; Sf, Spodoptera frugiperda ; VX, 0-ethyl S-[2-[bis(I -methylethyl)amino]ethyl]methyl phosphonothiolate; wt, wild-type. 1 To whom...ATCC (Rockville, MD, U.S.A.). Insect cells ( Spodoptera frugiperda Sf9 cells and T. ni High 5 cells) and wild-type (wt)-AcNPV were purchased from

  14. Studying the Symbiotic Bacterium Xenorhabdus nematophila in Individual, Living Steinernema carpocapsae Nematodes Using Microfluidic Systems.

    PubMed

    Stilwell, Matthew D; Cao, Mengyi; Goodrich-Blair, Heidi; Weibel, Douglas B

    2018-01-01

    Animal-microbe symbioses are ubiquitous in nature and scientifically important in diverse areas, including ecology, medicine, and agriculture. Steinernema nematodes and Xenorhabdus bacteria compose an established, successful model system for investigating microbial pathogenesis and mutualism. The bacterium Xenorhabdus nematophila is a species-specific mutualist of insect-infecting Steinernema carpocapsae nematodes. The bacterium colonizes a specialized intestinal pocket within the infective stage of the nematode, which transports the bacteria between insects that are killed and consumed by the pair for reproduction. Current understanding of the interaction between the infective-stage nematode and its bacterial colonizers is based largely on population-level, snapshot time point studies on these organisms. This limitation arises because investigating temporal dynamics of the bacterium within the nematode is impeded by the difficulty of isolating and maintaining individual living nematodes and tracking colonizing bacterial cells over time. To overcome this challenge, we developed a microfluidic system that enables us to spatially isolate and microscopically observe individual, living Steinernema nematodes and monitor the growth and development of the associated X. nematophila bacterial communities-starting from a single cell or a few cells-over weeks. Our data demonstrate, to our knowledge, the first direct, temporal, in vivo visual analysis of a symbiosis system and the application of this system to reveal continuous dynamics of the symbiont population in the living host animal. IMPORTANCE This paper describes an experimental system for directly investigating population dynamics of a symbiotic bacterium, Xenorhabdus nematophila , in its host-the infective stage of the entomopathogenic nematode Steinernema carpocapsae . Tracking individual and groups of bacteria in individual host nematodes over days and weeks yielded insight into dynamic growth and topology changes of symbiotic bacterial populations within infective juvenile nematodes. Our approach for studying symbioses between bacteria and nematodes provides a system to investigate long-term host-microbe interactions in individual nematodes and extrapolate the lessons learned to other bacterium-animal interactions.

  15. Phytoplasma protein effector SAP11 enhances insect vector reproduction by manipulating plant development and defense hormone biosynthesis.

    PubMed

    Sugio, Akiko; Kingdom, Heather N; MacLean, Allyson M; Grieve, Victoria M; Hogenhout, Saskia A

    2011-11-29

    Phytoplasmas are insect-transmitted phytopathogenic bacteria that can alter plant morphology and the longevity and reproduction rates and behavior of their insect vectors. There are various examples of animal and plant parasites that alter the host phenotype to attract insect vectors, but it is unclear how these parasites accomplish this. We hypothesized that phytoplasmas produce effectors that modulate specific targets in their hosts leading to the changes in plant development and insect performance. Previously, we sequenced and mined the genome of Aster Yellows phytoplasma strain Witches' Broom (AY-WB) and identified 56 candidate effectors. Here, we report that the secreted AY-WB protein 11 (SAP11) effector modulates plant defense responses to the advantage of the AY-WB insect vector Macrosteles quadrilineatus. SAP11 binds and destabilizes Arabidopsis CINCINNATA (CIN)-related TEOSINTE BRANCHED1, CYCLOIDEA, PROLIFERATING CELL FACTORS 1 and 2 (TCP) transcription factors, which control plant development and promote the expression of lipoxygenase (LOX) genes involved in jasmonate (JA) synthesis. Both the Arabidopsis SAP11 lines and AY-WB-infected plants produce less JA on wounding. Furthermore, the AY-WB insect vector produces more offspring on AY-WB-infected plants, SAP11 transgenic lines, and plants impaired in CIN-TCP and JA synthesis. Thus, SAP11-mediated destabilization of CIN-TCPs leads to the down-regulation of LOX2 expression and JA synthesis and an increase in M. quadrilineatus progeny. Phytoplasmas are obligate inhabitants of their plant host and insect vectors, in which the latter transmits AY-WB to a diverse range of plant species. This finding demonstrates that pathogen effectors can reach beyond the pathogen-host interface to modulate a third organism in the biological interaction.

  16. OpnS, an Outer Membrane Porin of Xenorhabdus nematophila, Confers a Competitive Advantage for Growth in the Insect Host▿ †

    PubMed Central

    van der Hoeven, Ransome; Forst, Steven

    2009-01-01

    The gammaproteobacterium Xenorhabdus nematophila engages in a mutualistic association with an entomopathogenic nematode and also functions as a pathogen toward different insect hosts. We studied the role of the growth-phase-regulated outer membrane protein OpnS in host interactions. OpnS was shown to be a 16-stranded β-barrel porin. opnS was expressed during growth in insect hemolymph and expression was elevated as the cell density increased. When wild-type and opnS deletion strains were coinjected into insects, the wild-type strain was predominantly recovered from the insect cadaver. Similarly, an opnS-complemented strain outcompeted the ΔopnS strain. Coinjection of the wild-type and ΔopnS strains together with uncolonized nematodes into insects resulted in nematode progeny that were almost exclusively colonized with the wild-type strain. Likewise, nematode progeny recovered after coinjection of a mixture of nematodes carrying either the wild-type or ΔopnS strain were colonized by the wild-type strain. In addition, the ΔopnS strain displayed a competitive growth defect when grown together with the wild-type strain in insect hemolymph but not in defined culture medium. The ΔopnS strain displayed increased sensitivity to antimicrobial compounds, suggesting that deletion of OpnS affected the integrity of the outer membrane. These findings show that the OpnS porin confers a competitive advantage for the growth and/or the survival of X. nematophila in the insect host and provides a new model for studying the biological relevance of differential regulation of porins in a natural host environment. PMID:19465651

  17. The Comet assay in insects--Status, prospects and benefits for science.

    PubMed

    Augustyniak, Maria; Gladysz, Marcin; Dziewięcka, Marta

    2016-01-01

    The Comet assay has been recently adapted to investigate DNA damage in insects. The first reports of its use in Drosophila melanogaster appeared in 2002. Since then, the interest in the application of the Comet assay to studies of insects has been rapidly increasing. Many authors see substantial potential in the use of the Comet assay in D. melanogaster for medical toxicology studies. This application could allow the testing of drugs and result in an understanding of the mechanisms of action of toxins, which could significantly influence the limited research that has been performed on vertebrates. The possible perspectives and benefits for science are considered in this review. In the last decade, the use of the Comet assay has been described in insects other than D. melanogaster. Specifically, methods to prepare a cell suspension from insect tissues, which is a difficult task, were analyzed and compared in detail. Furthermore, attention was paid to any differences and modifications in the research protocols, such as the buffer composition and electrophoresis conditions. Various scientific fields in addition to toxicological and ecotoxicological research were considered. We expect the Comet assay to be used in environmental risk assessments and to improve our understanding of many important phenomena of insect life, such as metamorphosis, molting, diapause and quiescence. The use of this method to study species that are of key importance to humans, such as pests and beneficial insects, appears to be highly probable and very promising. The use of the Comet assay for DNA stability testing in insects will most likely rapidly increase in the future. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Codon bias and gene ontology in holometabolous and hemimetabolous insects.

    PubMed

    Carlini, David B; Makowski, Matthew

    2015-12-01

    The relationship between preferred codon use (PCU), developmental mode, and gene ontology (GO) was investigated in a sample of nine insect species with sequenced genomes. These species were selected to represent two distinct modes of insect development, holometabolism and hemimetabolism, with an aim toward determining whether the differences in developmental timing concomitant with developmental mode would be mirrored by differences in PCU in their developmental genes. We hypothesized that the developmental genes of holometabolous insects should be under greater selective pressure for efficient translation, manifest as increased PCU, than those of hemimetabolous insects because holometabolism requires abundant protein expression over shorter time intervals than hemimetabolism, where proteins are required more uniformly in time. Preferred codon sets were defined for each species, from which the frequency of PCU for each gene was obtained. Although there were substantial differences in the genomic base composition of holometabolous and hemimetabolous insects, both groups exhibited a general preference for GC-ending codons, with the former group having higher PCU averaged across all genes. For each species, the biological process GO term for each gene was assigned that of its Drosophila homolog(s), and PCU was calculated for each GO term category. The top two GO term categories for PCU enrichment in the holometabolous insects were anatomical structure development and cell differentiation. The increased PCU in the developmental genes of holometabolous insects may reflect a general strategy to maximize the protein production of genes expressed in bursts over short time periods, e.g., heat shock proteins. J. Exp. Zool. (Mol. Dev. Evol.) 324B: 686-698, 2015. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  19. Same host-plant, different sterols: variation in sterol metabolism in an insect herbivore community.

    PubMed

    Janson, Eric M; Grebenok, Robert J; Behmer, Spencer T; Abbot, Patrick

    2009-11-01

    Insects lack the ability to synthesize sterols de novo, which are required as cell membrane inserts and as precursors for steroid hormones. Herbivorous insects typically utilize cholesterol as their primary sterol. However, plants rarely contain cholesterol, and herbivorous insects must, therefore, produce cholesterol by metabolizing plant sterols. Previous studies have shown that insects generally display diversity in phytosterol metabolism. Despite the biological importance of sterols, there has been no investigation of their metabolism in a naturally occurring herbivorous insect community. Therefore, we determined the neutral sterol profile of Solidago altissima L., six taxonomically and ecologically diverse herbivorous insect associates, and the fungal symbiont of one herbivore. Our results demonstrated that S. altissima contained Delta(7)-sterols (spinasterol, 22-dihydrospinasterol, avenasterol, and 24-epifungisterol), and that 85% of the sterol pool existed in a conjugated form. Despite feeding on a shared host plant, we observed significant variation among herbivores in terms of their qualitative tissue sterol profiles and significant variation in the cholesterol content. Cholesterol was absent in two dipteran gall-formers and present at extremely low levels in a beetle. Cholesterol content was highly variable in three hemipteran phloem feeders; even species of the same genus showed substantial differences in their cholesterol contents. The fungal ectosymbiont of a dipteran gall former contained primarily ergosterol and two ergosterol precursors. The larvae and pupae of the symbiotic gall-former lacked phytosterols, phytosterol metabolites, or cholesterol, instead containing an ergosterol metabolite in addition to unmetabolized ergosterol and erogsterol precursors, thus demonstrating the crucial role that a fungal symbiont plays in their nutritional ecology. These data are discussed in the context of sterol physiology and metabolism in insects, and the potential ecological and evolutionary implications.

  20. Feasibility Study of Odor Biosensor Using Dissociate Neuronal Culture with Gene Expression of Ionotropic Odorant Receptors

    NASA Astrophysics Data System (ADS)

    Tanada, Norio; Sakurai, Takeshi; Mitsuno, Hidefumi; Bakkum, Douglas; Kanzaki, Ryohei; Takahashi, Hirokazu

    We propose a highly sensitive and real-time odor biosensor by expressing ionotropic odorant receptors of insects into dissociated cultures of neurons of rats. The odorant-gated ion channel structure of insect odorant receptor is expected to allow easy functional expression into cells. The neuronal dissociated cultures of rats have two significant advantages: a long lifetime comparable to rats, i.e., a few years; and amplification ability from weak ionic currents of odorant receptors into easily detectable action potentials of neurons. In the present work, in order to show the feasibility of the proposed sensor, we attempt to express the pheromone receptors of silkmoth, Bombyx mori, into cultured neurons of rats. We demonstrate that 10% of neuronal cells transfected using Lipofectamine successfully expressed pheromone receptors, and that these cells showed significant increase of calcium signals by 50% at the presentation of pheromone.

  1. Ionotropic crustacean olfactory receptors.

    PubMed

    Corey, Elizabeth A; Bobkov, Yuriy; Ukhanov, Kirill; Ache, Barry W

    2013-01-01

    The nature of the olfactory receptor in crustaceans, a major group of arthropods, has remained elusive. We report that spiny lobsters, Panulirus argus, express ionotropic receptors (IRs), the insect chemosensory variants of ionotropic glutamate receptors. Unlike insects IRs, which are expressed in a specific subset of olfactory cells, two lobster IR subunits are expressed in most, if not all, lobster olfactory receptor neurons (ORNs), as confirmed by antibody labeling and in situ hybridization. Ligand-specific ORN responses visualized by calcium imaging are consistent with a restricted expression pattern found for other potential subunits, suggesting that cell-specific expression of uncommon IR subunits determines the ligand sensitivity of individual cells. IRs are the only type of olfactory receptor that we have detected in spiny lobster olfactory tissue, suggesting that they likely mediate olfactory signaling. Given long-standing evidence for G protein-mediated signaling in activation of lobster ORNs, this finding raises the interesting specter that IRs act in concert with second messenger-mediated signaling.

  2. Immunological recognition of different forms of the neurotensin receptor in transfected cells and rat brain.

    PubMed Central

    Boudin, H; Grauz-Guyon, A; Faure, M P; Forgez, P; Lhiaubet, A M; Dennis, M; Beaudet, A; Rostene, W; Pelaprat, D

    1995-01-01

    In this work, the molecular forms of the rat neurotensin receptor (NTR) expressed in transfected Chinese hamster ovary (CHO) cells, in infected Sf9 insect cells and in rat cerebral cortex were immunologically detected by means of an anti-peptide antibody raised against a fragment of the third intracellular loop of the receptor. Immunoblot experiments against a fusion protein indicated that the anti-peptide antibody recognized, under denaturing conditions, the corresponding amino acid sequence within the NTR. In immunoblot analysis of membranes from NTR-transfected CHO cells, high levels of immunoreactivity were observed between 60 and 72 kDa, while only a faint labelling was observed at 47 kDa, the molecular mass deduced for the rat NTR cDNA. The bands of high molecular mass were no longer observed after deglycosylation of membrane proteins by peptide N-glycosidase F, indicating that they represented glycosylated forms of the receptor. Extracts of membranes derived from baculovirus-infected Sf9 insect-cells expressing the NTR provided a quite different immunoblot pattern, since the major band detected in that case was at 47 kDa, the molecular size of the non-glycosylated receptor. Taken together, these data show that, while most of the NTR protein was glycosylated in CHO cells, it was unglycosylated in Sf9 insect-cells. In addition, molecular sizes of the receptor proteins observed in these two cell lines differed from those obtained for the NTR endogenously expressed in the rat cerebral cortex of 7 day-old rats, where bands at 56 and 54 kDa were detected. Binding experiments carried out on membrane preparations obtained from baculovirus-infected Sf9 cells demonstrated that the immunogenic sequence was still accessible to the antibody when the receptor was embedded in the cell membrane. Immunohistochemical studies carried out on both transfected CHO cells and infected Sf9 cells confirmed this interpretation and further indicated that the antibody could be applied in the visualization of the receptor. Images Figure 1 Figure 2 Figure 3 Figure 5 PMID:7826341

  3. Distribution and Spectroscopy of Green Fluorescent Protein and Acyl-CoA: Cholesterol Acytransferase in Sf21 Insect Cells

    NASA Technical Reports Server (NTRS)

    Richmond, R. C.; Mahtani, H.; Lu, X.; Chang, T. Y.; Malak, H.; Rose, M. Franklin (Technical Monitor)

    2001-01-01

    Acyl-CoA: cholesterol acyltransferase (ACAT) is thought to significantly participate in the pathway of cholesterol esterification that underlies the pathology of artherosclerosis. This enzyme is a membrane protein known to be preferentially bound within the endoplasmic reticulum of mammalian cells, from which location it esterifies cholesterol derived from low density lipoprotein. Cultures of insect cells were separately infected with baculovirus containing the gene for green fluroescent protein (GFP) and with baculovirus containing tandem genes for GFP and ACAT. These infected cultures expressed GFP and the fusion protein GCAT, respectively, with maximum expression occurring on the fourth day after infection. Extraction of GFP- and of GCAT-expressing cells with urea and detergent resulted in recovery of fluorescent protein in aqueous solution. Fluorescence spectra at neutral pH were identical for both GFP and GCAT extracts in aqueous solution, indicating unperturbed tertiary structure for the GFP moiety within GCAT. In a cholesterol esterification assay, GCAT demonstrated ACAT activity, but with less efficiency compared to native ACAT. It was hypothesized that the membrane protein ACAT would lead to differences in localization of GCAT compared to GFP within the respective expressing insect cells. The GFP marker directly and also within the fusion protein GCAT was accordingly used as the intracellular probe that was fluorescently analyzed by the new biophotonics technique of hyperspectral imaging. In that technique, fluorescence imaging was obtained from two dimensional arrays of cells, and regions of interest from within those images were then retrospectively analyzed for the emission spectra that comprises the image. Results of hyperspectral imaging of insect cells on day 4 postinfection showed that GCAT was preferentially localized to the cytoplasm of these cells compared to GFP. Furthermore, the emission spectra obtained for the localized GCAT displayed a peak blue shift from 518nm obtained in neutral aqueous solution to 505nm obtained in localized regions within the cells. This blue shift indicates change in the fluorescence coupling of the GFP moiety of GCAT. It is hypothesized that change in tertiary environment of GCAT, coincident with intracellular deposition of GCAT, follows from intracellular trafficking of GCAT leading to membrane interactions with the ACAT moiety, and/or self-assembly of GCAT, that alters the chromophore environment of the GFP moiety of GCAT. These findings introduce a new technique of biophotonic imaging to studies of intracellular protein trafficking and interactions. This technique of hyperspectral imaging could contribute to advancing the emergent field of proteomics. Because of the noninvasive nature of this technique, kinetic processes associated with intracellular protein trafficking, and interactions of proteins within cellular domains, can be considered for investigation within a single cell as well as a cell population.

  4. The NS3 protein of Rice hoja blanca tenuivirus suppresses RNA silencing in plant and insect hosts by efficiently binding both siRNAs and miRNAs

    PubMed Central

    Hemmes, Hans; Lakatos, Lóránt; Goldbach, Rob; Burgyán, József; Prins, Marcel

    2007-01-01

    RNA silencing plays a key role in antiviral defense as well as in developmental processes in plants and insects. Negative strand RNA viruses such as the plant virus Rice hoja blanca tenuivirus (RHBV) replicate in plants and in their insect transmission vector. Like most plant-infecting viruses, RHBV encodes an RNA silencing suppressor, the NS3 protein, and here it is demonstrated that this protein is capable of suppressing RNA silencing in both plants and insect cells. Biochemical analyses showed that NS3 efficiently binds siRNA as well as miRNA molecules. Binding of NS3 is greatly influenced by the size of small RNA molecules, as 21 nucleotide (nt) siRNA molecules are bound > 100 times more efficiently than 26 nt species. Competition assays suggest that the activity of NS3 is based on binding to siRNAs prior to strand separation during the assembly of the RNA-induced silencing complex. In addition, NS3 has a high affinity for miRNA/miRNA* duplexes, indicating that its activity might also interfere with miRNA-regulated gene expression in both insects and plants. PMID:17513697

  5. Metabolic Interplay between the Asian Citrus Psyllid and Its Profftella Symbiont: An Achilles’ Heel of the Citrus Greening Insect Vector

    PubMed Central

    Ramsey, John S.; Johnson, Richard S.; Hoki, Jason S.; Kruse, Angela; Mahoney, Jaclyn; Hilf, Mark E.; Hunter, Wayne B.; Hall, David G.; Schroeder, Frank C.; MacCoss, Michael J.; Cilia, Michelle

    2015-01-01

    ‘Candidatus Liberibacter asiaticus’ (CLas), the bacterial pathogen associated with citrus greening disease, is transmitted by Diaphorina citri, the Asian citrus psyllid. Interactions among D. citri and its microbial endosymbionts, including ‘Candidatus Profftella armatura’, are likely to impact transmission of CLas. We used quantitative mass spectrometry to compare the proteomes of CLas(+) and CLas(-) populations of D. citri, and found that proteins involved in polyketide biosynthesis by the endosymbiont Profftella were up-regulated in CLas(+) insects. Mass spectrometry analysis of the Profftella polyketide diaphorin in D. citri metabolite extracts revealed the presence of a novel diaphorin-related polyketide and the ratio of these two polyketides was changed in CLas(+) insects. Insect proteins differentially expressed between CLas(+) and CLas(-) D. citri included defense and immunity proteins, proteins involved in energy storage and utilization, and proteins involved in endocytosis, cellular adhesion, and cytoskeletal remodeling which are associated with microbial invasion of host cells. Insight into the metabolic interdependence between the insect vector, its endosymbionts, and the citrus greening pathogen reveals novel opportunities for control of this disease, which is currently having a devastating impact on citrus production worldwide. PMID:26580079

  6. Prophenoloxidase-Mediated Ex Vivo Immunity to Delay Fungal Infection after Insect Ecdysis.

    PubMed

    Zhang, Jie; Huang, Wuren; Yuan, Chuanfei; Lu, Yuzhen; Yang, Bing; Wang, Cheng-Yuan; Zhang, Peng; Dobens, Leonard; Zou, Zhen; Wang, Chengshu; Ling, Erjun

    2017-01-01

    Skin immunity protects animals from airborne pathogen infection. Unlike mammals, arthropods, including insects, undergo periodic ecdysis to grow and develop. Newly molted insects emerge with unsclerotized thin cuticles but successfully escape pathogenic infections during the post-molt period. Here we show that prophenoloxidases (PPOs) in molting fluids remain bioactive on the integument and impede fungal infection after ecdysis. We found that the purified plasma PPOs or recombinant PPOs could effectively bind to fungal spores (conidia) by targeting the cell wall components chitin and β-1,3-glucan. Pretreatment of the spores of the fungal pathogen Beauveria bassiana with PPOs increased spore hydrophilicity and reduced spore adhesion activity, resulting in a significant decrease in virulence as compared with mock infection. We also identified a spore-secreted protease BPS8, a member of peptidase S8 family of protease that degrade PPOs at high levels to benefit fungal infection, but which at lower doses activate PPOs to inhibit spore germination after melanization. These data indicate that insects have evolved a distinct strategy of ex vivo immunity to survive pathogen infections after ecdysis using PPOs in molting fluids retained on the underdeveloped and tender integument of newly molted insects for protection against airborne fungal infection.

  7. 7 CFR 29.3032 - Injury.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Injury. Hurt or impairment from any cause except the fungous or bacterial diseases which attack tobacco..., insects, or weather conditions; insecticides, fungicides, or cell growth inhibitors; nutritional...

  8. 7 CFR 29.3032 - Injury.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Injury. Hurt or impairment from any cause except the fungous or bacterial diseases which attack tobacco..., insects, or weather conditions; insecticides, fungicides, or cell growth inhibitors; nutritional...

  9. 7 CFR 29.3032 - Injury.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... Injury. Hurt or impairment from any cause except the fungous or bacterial diseases which attack tobacco..., insects, or weather conditions; insecticides, fungicides, or cell growth inhibitors; nutritional...

  10. 7 CFR 29.3032 - Injury.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Injury. Hurt or impairment from any cause except the fungous or bacterial diseases which attack tobacco..., insects, or weather conditions; insecticides, fungicides, or cell growth inhibitors; nutritional...

  11. 7 CFR 29.3032 - Injury.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Injury. Hurt or impairment from any cause except the fungous or bacterial diseases which attack tobacco..., insects, or weather conditions; insecticides, fungicides, or cell growth inhibitors; nutritional...

  12. Deuteration and selective labeling of alanine methyl groups of β2-adrenergic receptor expressed in a baculovirus-insect cell expression system.

    PubMed

    Kofuku, Yutaka; Yokomizo, Tomoki; Imai, Shunsuke; Shiraishi, Yutaro; Natsume, Mei; Itoh, Hiroaki; Inoue, Masayuki; Nakata, Kunio; Igarashi, Shunsuke; Yamaguchi, Hideyuki; Mizukoshi, Toshimi; Suzuki, Ei-Ichiro; Ueda, Takumi; Shimada, Ichio

    2018-03-08

    G protein-coupled receptors (GPCRs) exist in equilibrium between multiple conformations, and their populations and exchange rates determine their functions. However, analyses of the conformational dynamics of GPCRs in lipid bilayers are still challenging, because methods for observations of NMR signals of large proteins expressed in a baculovirus-insect cell expression system (BVES) are limited. Here, we report a method to incorporate methyl- 13 C 1 H 3 -labeled alanine with > 45% efficiency in highly deuterated proteins expressed in BVES. Application of the method to the NMR observations of β 2 -adrenergic receptor in micelles and in nanodiscs revealed the ligand-induced conformational differences throughout the transmembrane region of the GPCR.

  13. Stanislaw Smreczynskis legacy and the Department of Zoology of the Jagiellonian University of Krakow (Poland).

    PubMed

    Jaglarz, Mariusz K

    2008-01-01

    This article covers the origin and development of scientific interest in insect and amphibian developmental biology at the Department of Systematic Zoology and Zoogeography of the Jagiellonian University. The greater part of this historical account is devoted to Professor Stanislaw Smreczynski (1899-1975), the founding father of the Department, and comments on his biography and research achievements in the field of animal experimental embryology. A particular emphasis is on Smreczynski's contributions to contemporary understanding of early embryonic development of amphibians and insects as well as his expertise in Pleistocene and extant weevils (Curculionidae). A concise survey of developmental phenomena studied by some of Smreczynski's co-workers and followers is also presented, including the early embryogenesis of entognathans as well as germ cell determination and gonad formation in Drosophila virilis conducted by Jura; analysis of oogenesis in Collembola carried out by Krzysztofowicz; investigations of insects and tradigrades by Weglarska, and finally research into various aspects of ovary structure in diverse insect taxa by the Bilinski group.

  14. Common Sense about Taste: From Mammals to Insects

    PubMed Central

    Yarmolinsky, David A.; Zuker, Charles S.; Ryba, Nicholas J.P.

    2013-01-01

    The sense of taste is a specialized chemosensory system dedicated to the evaluation of food and drink. Despite the fact that vertebrates and insects have independently evolved distinct anatomic and molecular pathways for taste sensation, there are clear parallels in the organization and coding logic between the two systems. There is now persuasive evidence that tastant quality is mediated by labeled lines, whereby distinct and strictly segregated populations of taste receptor cells encode each of the taste qualities. PMID:19837029

  15. Expanding the host range of small insect RNA viruses: Providence virus (Carmotetraviridae) infects and replicates in a human tissue culture cell line.

    PubMed

    Jiwaji, Meesbah; Short, James Roswell; Dorrington, Rosemary Ann

    2016-10-01

    Tetraviruses are small, positive (+ve)-sense ssRNA viruses that infect the midgut cells of lepidopteran larvae. Providence virus (PrV) is the only member of the family Carmotetraviridae (previously Tetraviridae). PrV particles exhibit the characteristic tetraviral T=4 icosahedral symmetry, but PrV is distinct from other tetraviruses with respect to genome organization and viral non-structural proteins. Currently, PrV is the only tetravirus known to infect and replicate in lepidopteran cell culture lines. In this report we demonstrate, using immunofluorescence microscopy, that PrV infects and replicates in a human tissue culture cell line (HeLa), producing infectious virus particles. We also provide evidence for PrV replication in vitro in insect, mammalian and plant cell-free systems. This study challenges the long-held view that tetraviruses have a narrow host range confined to one or a few lepidopteran species and highlights the need to consider the potential for apparently non-infectious viruses to be transferred to new hosts in the laboratory.

  16. Specification of anteroposterior cell fates in Caenorhabditis elegans by Drosophila Hox proteins.

    PubMed

    Hunter, C P; Kenyon, C

    1995-09-21

    Antennapedia class homeobox (Hox) genes specify cell fates in successive anteroposterior body domains in vertebrates, insects and nematodes. The DNA-binding homeodomain sequences are very similar between vertebrate and Drosophila Hox proteins, and this similarity allows vertebrate Hox proteins to function in Drosophila. In contrast, the Caenorhabditis elegans homeodomains are substantially divergent. Further, C. elegans differs from both insects and vertebrates in having a non-segmented body as well as a distinctive mode of development that involves asymmetric early cleavages and invariant cell lineages. Here we report that, despite these differences, Drosophila Hox proteins expressed in C. elegans can substitute for C. elegans Hox proteins in the control of three different cell-fate decisions: the regulation of cell migration, the specification of serotonergic neurons, and the specification of a sensory structure. We also show that the specificity of one C. elegans Hox protein is partly determined by two amino acids that have been implicated in sequence-specific DNA binding. Together these findings suggest that factors important for target recognition by specific Hox proteins have been conserved throughout much of the animal kingdom.

  17. Physiological requirements for 20-hydroxyecdysone-induced rectal sac distention in the pupa of the silkworm, Bombyx mori.

    PubMed

    Suzuki, Takumi; Sakurai, Sho; Iwami, Masafumi

    2010-06-01

    Successful insect development is achieved via appropriate fluctuation of ecdysteroid levels. When an insect's ecdysteroid level is disrupted, physiological and developmental defects occur. In the pupa of the silkworm, Bombyx mori, the rectal sac is an essential organ that operates as a repository for degraded ecdysteroids, and it can be distended by administration of 20-hydroxyecdysone (20E). Our previous study showed that rectal sac distention appears 4 days after 20E administration. Hemolymph ecdysteroid levels, however, decrease to lower level during this period. Thus, the timing of the rectal sac distention does not match with that of ecdysteroid elevation. Here, we examine how 20E induces rectal sac distention. A ligature experiment and ecdysteroid quantification showed that continuous 20E stimulation induces rectal sac distention. Thorax tissue contributed to the continuous 20E stimulation needed to induce distention. Ecdysteroid released from the thorax tissue may be converted to 20E by ecdysone 20-hydroxylase to produce continuous 20E stimulation. Thus, the ecdysone metabolic pathway plays a critical role in rectal sac distention. Copyright 2010 Elsevier Ltd. All rights reserved.

  18. Induction of Autophagy and Apoptosis via PI3K/AKT/TOR Pathways by Azadirachtin A in Spodoptera litura Cells.

    PubMed

    Shao, Xuehua; Lai, Duo; Zhang, Ling; Xu, Hanhong

    2016-10-18

    Azadirachtin is one of the most effective botanical insecticides and has been widely used in pest control. Toxicological reports show that azadirachtin can induce apoptosis in various insect cell lines. However, studies of azadirachtin-induced autophagy in cultured insect cells are lacking. This study reports that azadirachtin A significantly inhibits cell proliferation by inducing autophagic and apoptotic cell death in Spodoptera litura cultured cell line (SL-1 cell). Characteristic autophagolysosome and Atg8-PE (phosphatidylethanolamine) accumulation were observed by electron microscopy and western blotting, indicating that azadirachtin triggered autophagy in SL-1 cell. Furthermore, azadirachtin inhibited survival signaling by blocking the activation of PI3K, AKT and the down-stream target of rapamycin. Similar to the positive control of starvation, azadirachtin induced the activation of insulin receptor (InR) via a cellular feedback mechanism. In addition, the autophagy-related 5 (Atg5), a molecular switch of autophagy and apoptosis, was truncated (tAtg5) to trigger cytochrome c release into the cytoplasm under azadirachtin stress, which indicated that azadirachtin induced apoptosis through autophagy. Our findings suggest that azadirachtin primarily induced autophagy in SL-1 cell by dysregulating InR- and PI3K/AKT/TOR pathways, then stimulated apoptosis by activating tAtg5.

  19. Induction of Autophagy and Apoptosis via PI3K/AKT/TOR Pathways by Azadirachtin A in Spodoptera litura Cells

    PubMed Central

    Shao, Xuehua; Lai, Duo; Zhang, Ling; Xu, Hanhong

    2016-01-01

    Azadirachtin is one of the most effective botanical insecticides and has been widely used in pest control. Toxicological reports show that azadirachtin can induce apoptosis in various insect cell lines. However, studies of azadirachtin-induced autophagy in cultured insect cells are lacking. This study reports that azadirachtin A significantly inhibits cell proliferation by inducing autophagic and apoptotic cell death in Spodoptera litura cultured cell line (SL-1 cell). Characteristic autophagolysosome and Atg8-PE (phosphatidylethanolamine) accumulation were observed by electron microscopy and western blotting, indicating that azadirachtin triggered autophagy in SL-1 cell. Furthermore, azadirachtin inhibited survival signaling by blocking the activation of PI3K, AKT and the down-stream target of rapamycin. Similar to the positive control of starvation, azadirachtin induced the activation of insulin receptor (InR) via a cellular feedback mechanism. In addition, the autophagy-related 5 (Atg5), a molecular switch of autophagy and apoptosis, was truncated (tAtg5) to trigger cytochrome c release into the cytoplasm under azadirachtin stress, which indicated that azadirachtin induced apoptosis through autophagy. Our findings suggest that azadirachtin primarily induced autophagy in SL-1 cell by dysregulating InR- and PI3K/AKT/TOR pathways, then stimulated apoptosis by activating tAtg5. PMID:27752103

  20. 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...

  1. PhaR, a Negative Regulator of PhaP, Modulates the Colonization of a Burkholderia Gut Symbiont in the Midgut of the Host Insect, Riptortus pedestris.

    PubMed

    Jang, Seong Han; Jang, Ho Am; Lee, Junbeom; Kim, Jong Uk; Lee, Seung Ah; Park, Kyoung-Eun; Kim, Byung Hyun; Jo, Yong Hun; Lee, Bok Luel

    2017-06-01

    Five genes encoding PhaP family proteins and one phaR gene have been identified in the genome of Burkholderia symbiont strain RPE75. PhaP proteins function as the surface proteins of polyhydroxyalkanoate (PHA) granules, and the PhaR protein acts as a negative regulator of PhaP biosynthesis. Recently, we characterized one phaP gene to understand the molecular cross talk between Riptortus insects and Burkholderia gut symbionts. In this study, we constructed four other phaP gene-depleted mutants (Δ phaP1 , Δ phaP2 , Δ phaP3 , and Δ phaP4 mutants), one phaR gene-depleted mutant, and a phaR -complemented mutant (Δ phaR/phaR mutant). To address the biological roles of four phaP family genes and the phaR gene during insect-gut symbiont interaction, these Burkholderia mutants were fed to the second-instar nymphs, and colonization ability and fitness parameters were examined. In vitro , the Δ phaP3 and Δ phaR mutants cannot make a PHA granule normally in a stressful environment. Furthermore, the Δ phaR mutation decreased the colonization ability in the host midgut and negatively affected the host insect's fitness compared with wild-type Burkholderia -infected insects. However, other phaP family gene-depleted mutants colonized well in the midgut of the fifth-instar nymph insects. However, in the case of females, the colonization rate of the Δ phaP3 mutant was decreased and the host's fitness parameters were decreased compared with the wild-type-infected host, suggesting that the environment of the female midgut may be more hostile than that of the male midgut. These results demonstrate that PhaR plays an important role in the biosynthesis of PHA granules and that it is significantly related to the colonization of the Burkholderia gut symbiont in the host insects' midgut. IMPORTANCE Bacterial polyhydroxyalkanoate (PHA) biosynthesis is a complex process requiring several enzymes. The biological roles of PHA granule synthesis enzymes and the surface proteins of PHA granules during host-gut symbiont interactions are not fully understood. Here, we report the effects on colonization ability in the host midguts and the fitness of host insects after feeding Burkholderia mutant cells (four phaP -depleted mutants and one phaR -depleted mutant) to the host insects. Analyses of both synthesized PHA granule amounts and CFU numbers suggest that the phaR gene is closely related to synthesis of the PHA granule and the colonization of the Burkholderia gut symbiont in the host insect's midgut. Like our previous report, this study also supports the idea that the environment of the host midgut may not be favorable to symbiotic Burkholderia cells and that PHA granules may be required to adapt in the host midgut. Copyright © 2017 American Society for Microbiology.

  2. PhaR, a Negative Regulator of PhaP, Modulates the Colonization of a Burkholderia Gut Symbiont in the Midgut of the Host Insect, Riptortus pedestris

    PubMed Central

    Jang, Seong Han; Jang, Ho Am; Lee, Junbeom; Kim, Jong Uk; Lee, Seung Ah; Park, Kyoung-Eun; Kim, Byung Hyun; Jo, Yong Hun

    2017-01-01

    ABSTRACT Five genes encoding PhaP family proteins and one phaR gene have been identified in the genome of Burkholderia symbiont strain RPE75. PhaP proteins function as the surface proteins of polyhydroxyalkanoate (PHA) granules, and the PhaR protein acts as a negative regulator of PhaP biosynthesis. Recently, we characterized one phaP gene to understand the molecular cross talk between Riptortus insects and Burkholderia gut symbionts. In this study, we constructed four other phaP gene-depleted mutants (ΔphaP1, ΔphaP2, ΔphaP3, and ΔphaP4 mutants), one phaR gene-depleted mutant, and a phaR-complemented mutant (ΔphaR/phaR mutant). To address the biological roles of four phaP family genes and the phaR gene during insect-gut symbiont interaction, these Burkholderia mutants were fed to the second-instar nymphs, and colonization ability and fitness parameters were examined. In vitro, the ΔphaP3 and ΔphaR mutants cannot make a PHA granule normally in a stressful environment. Furthermore, the ΔphaR mutation decreased the colonization ability in the host midgut and negatively affected the host insect's fitness compared with wild-type Burkholderia-infected insects. However, other phaP family gene-depleted mutants colonized well in the midgut of the fifth-instar nymph insects. However, in the case of females, the colonization rate of the ΔphaP3 mutant was decreased and the host's fitness parameters were decreased compared with the wild-type-infected host, suggesting that the environment of the female midgut may be more hostile than that of the male midgut. These results demonstrate that PhaR plays an important role in the biosynthesis of PHA granules and that it is significantly related to the colonization of the Burkholderia gut symbiont in the host insects' midgut. IMPORTANCE Bacterial polyhydroxyalkanoate (PHA) biosynthesis is a complex process requiring several enzymes. The biological roles of PHA granule synthesis enzymes and the surface proteins of PHA granules during host-gut symbiont interactions are not fully understood. Here, we report the effects on colonization ability in the host midguts and the fitness of host insects after feeding Burkholderia mutant cells (four phaP-depleted mutants and one phaR-depleted mutant) to the host insects. Analyses of both synthesized PHA granule amounts and CFU numbers suggest that the phaR gene is closely related to synthesis of the PHA granule and the colonization of the Burkholderia gut symbiont in the host insect's midgut. Like our previous report, this study also supports the idea that the environment of the host midgut may not be favorable to symbiotic Burkholderia cells and that PHA granules may be required to adapt in the host midgut. PMID:28341680

  3. Injection of insect membrane in Xenopus oocyte: An original method for the pharmacological characterization of neonicotinoid insecticides.

    PubMed

    Crespin, Lucille; Legros, Christian; List, Olivier; Tricoire-Leignel, Hélène; Mattei, César

    2016-01-01

    Insect nicotinic acetylcholine receptors (nAChRs) represent a major target of insecticides, belonging to the neonicotinoid family. However, the pharmacological profile of native nAChRs is poorly documented, mainly because of a lack of knowledge of their subunit stoichiometry, their tissue distribution and the weak access to nAChR-expressing cells. In addition, the expression of insect nAChRs in heterologous systems remains hard to achieve. Therefore, the structure-activity characterization of nAChR-targeting insecticides is made difficult. The objective of the present study was to characterize insect nAChRs by an electrophysiological approach in a heterologous system naturally devoid of these receptors to allow a molecular/cellular investigation of the mode of action of neonicotinoids. Methods To overcome impediments linked to the expression of insect nAChR mRNA or cDNA, we chose to inject insect membranes from the pea aphid (Acyrthosiphon pisum) into Xenopus oocytes. This microtransplantation technique was designed to gain access to native nAChRs embedded in their membrane, through direct stimulation with nicotinic agonists. Results We provide evidence that an enriched-nAChR membrane allows us to characterize native receptors. The presence of such receptors was confirmed with fluorescent α-BgTX labeling. Electrophysiological recordings of nicotine-induced inward currents allowed us to challenge the presence of functional nAChR. We compared the effect of nicotine (NIC) with clothianidin (CLO) and we assessed the effect of thiamethoxam (TMX). Discussion This technique has been recently highlighted with mammalian and human material as a powerful functional approach, but has, to our knowledge, never been used with insect membrane. In addition, the use of the insect membrane microtransplantation opens a new and original way for pharmacological screening of neurotoxic insecticides, including neonicotinoids. Moreover, it might also be a powerful tool to investigate the pharmacological properties of insect nAChR. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. A simple cryo-holder facilitates specimen observation under a conventional scanning electron microscope.

    PubMed

    Tang, Chih-Yuan; Huang, Rong-Nan; Kuo-Huang, Ling-Long; Kuo, Tai-Chih; Yang, Ya-Yun; Lin, Ching-Yeh; Jane, Wann-Neng; Chen, Shiang-Jiuun

    2012-02-01

    A pre-cryogenic holder (cryo-holder) facilitating cryo-specimen observation under a conventional scanning electron microscope (SEM) is described. This cryo-holder includes a specimen-holding unit (the stub) and a cryogenic energy-storing unit (a composite of three cylinders assembled with a screw). After cooling, the cryo-holder can continue supplying cryogenic energy to extend the observation time for the specimen in a conventional SEM. Moreover, the cryogenic energy-storing unit could retain appropriate liquid nitrogen that can evaporate to prevent frost deposition on the surface of the specimen. This device is proved feasible for various tissues and cells, and can be applied to the fields of both biology and material science. We have employed this novel cryo-holder for observation of yeast cells, trichome, and epidermal cells in the leaf of Arabidopsis thaliana, compound eyes of insects, red blood cells, filiform papillae on the surface of rat tongue, agar medium, water molecules, penicillium, etc. All results suggested that the newly designed cryo-holder is applicable for cryo-specimen observation under a conventional SEM without cooling system. Most importantly, the design of this cryo-holder is simple and easy to operate and could adapt a conventional SEM to a plain type cryo-SEM affordable for most laboratories. Copyright © 2011 Wiley Periodicals, Inc.

  5. Bacillus thuringiensis insecticidal three-domain Cry toxins: mode of action, insect resistance and consequences for crop protection.

    PubMed

    Pardo-López, Liliana; Soberón, Mario; Bravo, Alejandra

    2013-01-01

    Bacillus thuringiensis bacteria are insect pathogens that produce different Cry and Cyt toxins to kill their hosts. Here we review the group of three-domain Cry (3d-Cry) toxins. Expression of these 3d-Cry toxins in transgenic crops has contributed to efficient control of insect pests and a reduction in the use of chemical insecticides. The mode of action of 3d-Cry toxins involves sequential interactions with several insect midgut proteins that facilitate the formation of an oligomeric structure and induce its insertion into the membrane, forming a pore that kills midgut cells. We review recent progress in our understanding of the mechanism of action of these Cry toxins and focus our attention on the different mechanisms of resistance that insects have evolved to counter their action, such as mutations in cadherin, APN and ABC transporter genes. Activity of Cry1AMod toxins, which are able to form toxin oligomers in the absence of receptors, against different resistant populations, including those affected in the ABC transporter and the role of dominant negative mutants as antitoxins, supports the hypothesis that toxin oligomerization is a limiting step in the Cry insecticidal activity. Knowledge of the action of 3d-Cry toxin and the resistance mechanisms to these toxins will set the basis for a rational design of novel toxins to overcome insect resistance, extending the useful lifespan of Cry toxins in insect control programs. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  6. The vacuolar protein sorting genes in insects: A comparative genome view.

    PubMed

    Li, Zhaofei; Blissard, Gary

    2015-07-01

    In eukaryotic cells, regulated vesicular trafficking is critical for directing protein transport and for recycling and degradation of membrane lipids and proteins. Through carefully regulated transport vesicles, the endomembrane system performs a large and important array of dynamic cellular functions while maintaining the integrity of the cellular membrane system. Genetic studies in yeast Saccharomyces cerevisiae have identified approximately 50 vacuolar protein sorting (VPS) genes involved in vesicle trafficking, and most of these genes are also characterized in mammals. The VPS proteins form distinct functional complexes, which include complexes known as ESCRT, retromer, CORVET, HOPS, GARP, and PI3K-III. Little is known about the orthologs of VPS proteins in insects. Here, with the newly annotated Manduca sexta genome, we carried out genomic comparative analysis of VPS proteins in yeast, humans, and 13 sequenced insect genomes representing the Orders Hymenoptera, Diptera, Hemiptera, Phthiraptera, Lepidoptera, and Coleoptera. Amino acid sequence alignments and domain/motif structure analyses reveal that most of the components of ESCRT, retromer, CORVET, HOPS, GARP, and PI3K-III are evolutionarily conserved across yeast, insects, and humans. However, in contrast to the VPS gene expansions observed in the human genome, only four VPS genes (VPS13, VPS16, VPS33, and VPS37) were expanded in the six insect Orders. Additionally, VPS2 was expanded only in species from Phthiraptera, Lepidoptera, and Coleoptera. These studies provide a baseline for understanding the evolution of vesicular trafficking across yeast, insect, and human genomes, and also provide a basis for further addressing specific functional roles of VPS proteins in insects. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Evolution of the Insect Desaturase Gene Family with an Emphasis on Social Hymenoptera

    PubMed Central

    Helmkampf, Martin; Cash, Elizabeth; Gadau, Jürgen

    2015-01-01

    Desaturase genes are essential for biological processes, including lipid metabolism, cell signaling, and membrane fluidity regulation. Insect desaturases are particularly interesting for their role in chemical communication, and potential contribution to speciation, symbioses, and sociality. Here, we describe the acyl-CoA desaturase gene families of 15 insects, with a focus on social Hymenoptera. Phylogenetic reconstruction revealed that the insect desaturases represent an ancient gene family characterized by eight subfamilies that differ strongly in their degree of conservation and frequency of gene gain and loss. Analyses of genomic organization showed that five of these subfamilies are represented in a highly microsyntenic region conserved across holometabolous insect taxa, indicating an ancestral expansion during early insect evolution. In three subfamilies, ants exhibit particularly large expansions of genes. Despite these expansions, however, selection analyses showed that desaturase genes in all insect lineages are predominantly undergoing strong purifying selection. Finally, for three expanded subfamilies, we show that ants exhibit variation in gene expression between species, and more importantly, between sexes and castes within species. This suggests functional differentiation of these genes and a role in the regulation of reproductive division of labor in ants. The dynamic pattern of gene gain and loss of acyl-CoA desaturases in ants may reflect changes in response to ecological diversification and an increased demand for chemical signal variability. This may provide an example of how gene family expansions can contribute to lineage-specific adaptations through structural and regulatory changes acting in concert to produce new adaptive phenotypes. PMID:25425561

  8. 7 CFR 29.6021 - Injury.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... may be caused by field diseases, insects, or weather conditions; insecticides, fungicides, or cell... Rule 16), sunburned, sun-scalded, bulk-burnt, pole-burnt, shed-burnt, pole-sweated, stem-rotted...

  9. 7 CFR 29.6021 - Injury.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... may be caused by field diseases, insects, or weather conditions; insecticides, fungicides, or cell... Rule 16), sunburned, sun-scalded, bulk-burnt, pole-burnt, shed-burnt, pole-sweated, stem-rotted...

  10. 7 CFR 29.6021 - Injury.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... may be caused by field diseases, insects, or weather conditions; insecticides, fungicides, or cell... Rule 16), sunburned, sun-scalded, bulk-burnt, pole-burnt, shed-burnt, pole-sweated, stem-rotted...

  11. 7 CFR 29.6021 - Injury.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... may be caused by field diseases, insects, or weather conditions; insecticides, fungicides, or cell... Rule 16), sunburned, sun-scalded, bulk-burnt, pole-burnt, shed-burnt, pole-sweated, stem-rotted...

  12. 7 CFR 29.6021 - Injury.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... may be caused by field diseases, insects, or weather conditions; insecticides, fungicides, or cell... Rule 16), sunburned, sun-scalded, bulk-burnt, pole-burnt, shed-burnt, pole-sweated, stem-rotted...

  13. Valuing Insect Pollination Services with Cost of Replacement

    PubMed Central

    Allsopp, Mike H.; de Lange, Willem J.; Veldtman, Ruan

    2008-01-01

    Value estimates of ecosystem goods and services are useful to justify the allocation of resources towards conservation, but inconclusive estimates risk unsustainable resource allocations. Here we present replacement costs as a more accurate value estimate of insect pollination as an ecosystem service, although this method could also be applied to other services. The importance of insect pollination to agriculture is unequivocal. However, whether this service is largely provided by wild pollinators (genuine ecosystem service) or managed pollinators (commercial service), and which of these requires immediate action amidst reports of pollinator decline, remains contested. If crop pollination is used to argue for biodiversity conservation, clear distinction should be made between values of managed- and wild pollination services. Current methods either under-estimate or over-estimate the pollination service value, and make use of criticised general insect and managed pollinator dependence factors. We apply the theoretical concept of ascribing a value to a service by calculating the cost to replace it, as a novel way of valuing wild and managed pollination services. Adjusted insect and managed pollinator dependence factors were used to estimate the cost of replacing insect- and managed pollination services for the Western Cape deciduous fruit industry of South Africa. Using pollen dusting and hand pollination as suitable replacements, we value pollination services significantly higher than current market prices for commercial pollination, although lower than traditional proportional estimates. The complexity associated with inclusive value estimation of pollination services required several defendable assumptions, but made estimates more inclusive than previous attempts. Consequently this study provides the basis for continued improvement in context specific pollination service value estimates. PMID:18781196

  14. Plant stress signalling: understanding and exploiting plant-plant interactions.

    PubMed

    Pickett, J A; Rasmussen, H B; Woodcock, C M; Matthes, M; Napier, J A

    2003-02-01

    When plants are attacked by insects, volatile chemical signals can be released, not only from the damaged parts, but also systemically from other parts of the plant and this continues after cessation of feeding by the insect. These signals are perceived by olfactory sensory mechanisms in both the herbivorous insects and their parasites. Molecular structures involved can be characterized by means of electrophysiological assays, using the insect sensory system linked to chemical analysis. Evidence is mounting that such signals can also affect neighbouring intact plants, which initiate defence by the induction of further signalling systems, such as those that increase parasitoid foraging. Furthermore, insect electrophysiology can be used in the identification of plant compounds having effects on the plants themselves. It has been found recently that certain plants can release stress signals even when undamaged, and that these can cause defence responses in intact plants. These discoveries provide the basis for new crop protection strategies, that are either delivered by genetic modification of plants or by conventionally produced plants to which the signal is externally applied. Delivery can also be made by means of mixed seed strategies in which the provoking and recipient plants are grown together. Related signalling discoveries within the rhizosphere seem set to extend these approaches into new ways of controlling weeds, by exploiting the elusive potential of allelopathy, but through signalling rather than by direct physiological effects.

  15. Death eaters respond to the dark mark of decomposition day and night: observations of initial insect activity on piglet carcasses.

    PubMed

    Weidner, Lauren M; Monzon, Michael A; Hamilton, George C

    2016-11-01

    Some insect taxa can be of critical importance for criminal investigations because they can be used to assist with a time since death determination. Blow flies (Diptera: Calliphoridae) often are the initial colonizers of a carcass, usually arriving within minutes to hours after carcass exposure during the day. Other insects, such as coleopterans and hymenopterans, can arrive to a carcass during early colonization and affect blow fly development. However, the extent of these interactions remains unclear. This study analyzed the initial 6 h after a piglet carcass was placed out in two locations (rural and urban) in diurnal and nocturnal conditions with continuous video recording and hourly observations. Four piglets were placed out every 2 weeks over the summer of 2014. Initial blow fly arrivals to the carcasses were only recorded during diurnal conditions, and a checklist of orders associated with each environment (time and location) was created. During diurnal conditions, initial blow fly arrival times in rural environments were significantly faster than those in urban, arriving as quickly as 23 s after exposure. These observations also included a novel interaction with Vespidae, which to the best of our knowledge has not been seen in the literature before. This experiment provides baseline data on early insect colonization in two environments in New Jersey, and lends insight into insect interactions that could affect initial colonization.

  16. Effects of periplocoside X on midgut cells and digestive enzymes activity of the soldiers of red imported fire ant.

    PubMed

    Li, Yan; Zeng, Xin-Nian

    2013-07-01

    The pathological effects of ingested periplocoside X, an insecticidal component isolated from the root of Periploca sepium Bunge, on the midgut epithelial cells of the soldiers of red imported fire ant were studied and the symptom was described. The results showed that periplocoside X could induce a severe, time-dependent cytotoxicity in the midgut epithelial cells. An optical microscopy showed that epithelial cells swelled firstly and then lysed. Transmission electron microscopy (TEM) showed that numerous swollen lysosomes were appeared, microvilli were disrupted and sloughed off, and the numbers of the rough endoplasmic reticulum and the mitochondria decreased sharply in earlier stage. Numerous vacuoles were observed in the later stage. Finally, periplocoside X resulted in cell death by cytolysis. Assay of main three digestive enzymes activity indicated that amylase activity was significantly inhibited, but no significant changes were seen for lipase activity and total protease activity. So it is suggested that periplocoside X induced mainly to organic damage of midgut epithelium cells of insect. In all, insect midgut is one of targets for periplocoside X. Copyright © 2013 Elsevier Inc. All rights reserved.

  17. Midgut epithelium in molting silkworm: A fine balance among cell growth, differentiation, and survival.

    PubMed

    Franzetti, Eleonora; Casartelli, Morena; D'Antona, Paola; Montali, Aurora; Romanelli, Davide; Cappellozza, Silvia; Caccia, Silvia; Grimaldi, Annalisa; de Eguileor, Magda; Tettamanti, Gianluca

    2016-07-01

    The midgut of insects has attracted great attention as a system for studying intestinal stem cells (ISCs) as well as cell death-related processes, such as apoptosis and autophagy. Among insects, Lepidoptera represent a good model to analyze these cells and processes. In particular, larva-larva molting is an interesting developmental phase since the larva must deal with nutrient starvation and its organs are subjected to rearrangements due to proliferation and differentiation events. Several studies have analyzed ISCs in vitro and characterized key factors involved in their division and differentiation during molt. However, in vivo studies performed during larva-larva transition on these cells, and on the whole midgut epithelium, are fragmentary. In the present study, we analyzed the larval midgut epithelium of the silkworm, Bombyx mori, during larva-larva molting, focusing our attention on ISCs. Moreover, we investigated the metabolic changes that occur in the epithelium and evaluated the intervention of autophagy. Our data on ISCs proliferation and differentiation, autophagy activation, and metabolic and functional activities of the midgut cells shed light on the complexity of this organ during the molting phase. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. An invitation to die: initiators of sociality in a social amoeba become selfish spores

    PubMed Central

    Kuzdzal-Fick, Jennie J.; Queller, David C.; Strassmann, Joan E.

    2010-01-01

    Greater size and strength are common attributes of contest winners. Even in social insects with high cooperation, the right to reproduce falls to the well-fed queens rather than to poorly fed workers. In Dictyostelium discoideum, formerly solitary amoebae aggregate when faced with starvation, and some cells die to form a stalk which others ride up to reach a better location to sporulate. The first cells to starve have lower energy reserves than those that starve later, and previous studies have shown that the better-fed cells in a mix tend to form disproportionately more reproductive spores. Therefore, one might expect that the first cells to starve and initiate the social stage should act altruistically and form disproportionately more of the sterile stalk, thereby enticing other better-fed cells into joining the aggregate. This would resemble caste determination in social insects, where altruistic workers are typically fed less than reproductive queens. However, we show that the opposite result holds: the first cells to starve become reproductive spores, presumably by gearing up for competition and outcompeting late starvers to become prespore first. These findings pose the interesting question of why others would join selfish organizers. PMID:20504816

  19. Laboratory evaluation on a potential birth control diet for fruit fly sterile insect technique (SIT).

    PubMed

    Chang, Chiou Ling

    2017-08-01

    Sterile insect technique (SIT) is one of the most effective fruit fly control technologies. Irradiation has been used to sterilize male fruit flies before release to the field to compete with the wild males for females. Imagine an environmental and cost effective method using a rearing diet that can make insects sterile indefinitely, by feeding for 7days before release. This could replace costly irradiation process. A potential birth control diet was evaluated on fertility, mating, survival, and protein analysis for fruit fly species in Hawaii. Insects were continuously fed an agar diet with lufenuron (LFN) for 7d after emergence and then switched to a control diet to simulate the actual field condition. The influence on egg hatch was dose dependent. With dose of 2-4mg/g in the diet, egg hatch from LFN-fed was almost 100% suppressed for 24 experimental days if adults of Ceratitia capitate (Widemann), Bactrocera dorsalis (Hendel), and B. latifrons (Hendel) continued to feed on LFN diet. B. cucurbitae (Coquillett) was not affected by LFN. However, egg hatch from LFN fed B. latifrons and B. dorsalis were suppressed for at least 2weeks after switching to the control diet at 7d. Egg hatch did not recover >4% up to 24d. Proteome analysis revealed that ABD-4 protein was under expressed by 70-83% on LFN fed females and males of B. latifrons and B. dorsalis while Pbprp2 protein was significantly over expressed by 6-12 fold on LFN fed males only. These two proteins were not expressed in C. capitata and B. cucurbitae. Therefore, this report focused more on B. latifrons and B. dorsalis. This finding suggested a great potential for one alternative to sterilize fruit flies for SIT without irradiation. Published by Elsevier Inc.

  20. Enhancing the Breadth of Efficacy of Therapeutic Vaccines for Breast Cancer

    DTIC Science & Technology

    2014-10-01

    4b. Generate specific tumor antigen lysates from recombinant baculovirus- infected insect cells. *Currently expressing antigens in C1R cells since... Immunology Conference, Breckenridge CO (invited by Jim Hagman). Sept 20, 2014, Analysis of the T cell repertoire in breast cancer using emulsion...by Jonathan Bramson).   13 Nov 11, 2014 (anticipated), Elimination of the bottlenecks in T cell receptor antigen discovery, Immunology Forum

  1. Studying the organization of genes encoding plant cell wall degrading enzymes in Chrysomela tremula provides insights into a leaf beetle genome.

    PubMed

    Pauchet, Y; Saski, C A; Feltus, F A; Luyten, I; Quesneville, H; Heckel, D G

    2014-06-01

    The ability of herbivorous beetles from the superfamilies Chrysomeloidea and Curculionoidea to degrade plant cell wall polysaccharides has only recently begun to be appreciated. The presence of plant cell wall degrading enzymes (PCWDEs) in the beetle's digestive tract makes this degradation possible. Sequences encoding these beetle-derived PCWDEs were originally identified from transcriptomes and strikingly resemble those of saprophytic and phytopathogenic microorganisms, raising questions about their origin; e.g. are they insect- or microorganism-derived? To demonstrate unambiguously that the genes encoding PCWDEs found in beetle transcriptomes are indeed of insect origin, we generated a bacterial artificial chromosome library from the genome of the leaf beetle Chrysomela tremula, containing 18 432 clones with an average size of 143 kb. After hybridizing this library with probes derived from 12 C. tremula PCWDE-encoding genes and sequencing the positive clones, we demonstrated that the latter genes are encoded by the insect's genome and are surrounded by genes possessing orthologues in the genome of Tribolium castaneum as well as in three other beetle genomes. Our analyses showed that although the level of overall synteny between C. tremula and T. castaneum seems high, the degree of microsynteny between both species is relatively low, in contrast to the more closely related Colorado potato beetle. © 2014 The Royal Entomological Society.

  2. Validation of membrane vesicle-based breast cancer resistance protein and multidrug resistance protein 2 assays to assess drug transport and the potential for drug-drug interaction to support regulatory submissions.

    PubMed

    Elsby, Robert; Smith, Veronica; Fox, Lisa; Stresser, David; Butters, Caroline; Sharma, Pradeep; Surry, Dominic D

    2011-09-01

    Breast cancer resistance protein (BCRP) and multidrug resistance protein 2 (MRP2) can play a role in the absorption, distribution, metabolism, and excretion of drugs, impacting on the potential for drug-drug interactions. This study has characterized insect cell- and mammalian cell-derived ABC-transporter-expressing membrane vesicle test systems and validated methodologies for evaluation of candidate drugs as substrates or inhibitors of BCRP or MRP2. Concentration-dependent uptake of BCRP ([³H]oestrone 3-sulfate, [³H]methotrexate, [³H]rosuvastatin) and MRP2 ([³H]oestradiol 17β-glucuronide, [³H]pravastatin, carboxydichlorofluorescein) substrates, and inhibitory potencies (IC₅₀) of BCRP (sulfasalazine, novobiocin, fumitremorgin C) and MRP2 (benzbromarone, MK-571, terfenadine) inhibitors were determined. The apparent K(m) for probes [³H]oestrone 3-sulfate and [³H]oestradiol 17β-glucuronide was determined in insect cell vesicles to be 7.4 ± 1.7 and 105 ± 8.3 µM, respectively. All other substrates exhibited significant uptake ratios. Positive control inhibitors sulfasalazine and benzbromarone gave IC₅₀ values of 0.74 ± 0.18 and 36 ± 6.1 µM, respectively. All other inhibitors exhibited concentration-dependent inhibition. There was no significant difference in parameters generated between test systems. On the basis of the validation results, acceptance criteria to identify substrates/inhibitors of BCRP and MRP2 were determined for insect cell vesicles. The approach builds on earlier validations to support drug registration and extends from those cell-based systems to encompass assay formats using membrane vesicles.

  3. The Repellent DEET Potentiates Carbamate Effects via Insect Muscarinic Receptor Interactions: An Alternative Strategy to Control Insect Vector-Borne Diseases.

    PubMed

    Abd-Ella, Aly; Stankiewicz, Maria; Mikulska, Karolina; Nowak, Wieslaw; Pennetier, Cédric; Goulu, Mathilde; Fruchart-Gaillard, Carole; Licznar, Patricia; Apaire-Marchais, Véronique; List, Olivier; Corbel, Vincent; Servent, Denis; Lapied, Bruno

    2015-01-01

    Insect vector-borne diseases remain one of the principal causes of human mortality. In addition to conventional measures of insect control, repellents continue to be the mainstay for personal protection. Because of the increasing pyrethroid-resistant mosquito populations, alternative strategies to reconstitute pyrethroid repellency and knock-down effects have been proposed by mixing the repellent DEET (N,N-Diethyl-3-methylbenzamide) with non-pyrethroid insecticide to better control resistant insect vector-borne diseases. By using electrophysiological, biochemichal, in vivo toxicological techniques together with calcium imaging, binding studies and in silico docking, we have shown that DEET, at low concentrations, interacts with high affinity with insect M1/M3 mAChR allosteric site potentiating agonist effects on mAChRs coupled to phospholipase C second messenger pathway. This increases the anticholinesterase activity of the carbamate propoxur through calcium-dependent regulation of acetylcholinesterase. At high concentrations, DEET interacts with low affinity on distinct M1/M3 mAChR site, counteracting the potentiation. Similar dose-dependent dual effects of DEET have also been observed at synaptic mAChR level. Additionally, binding and in silico docking studies performed on human M1 and M3 mAChR subtypes indicate that DEET only displays a low affinity antagonist profile on these M1/M3 mAChRs. These results reveal a selective high affinity positive allosteric site for DEET in insect mAChRs. Finally, bioassays conducted on Aedes aegypti confirm the synergistic interaction between DEET and propoxur observed in vitro, resulting in a higher mortality of mosquitoes. Our findings reveal an unusual allosterically potentiating action of the repellent DEET, which involves a selective site in insect. These results open exciting research areas in public health particularly in the control of the pyrethroid-resistant insect-vector borne diseases. Mixing low doses of DEET and a non-pyrethroid insecticide will lead to improvement in the efficiency treatments thus reducing both the concentration of active ingredients and side effects for non-target organisms. The discovery of this insect specific site may pave the way for the development of new strategies essential in the management of chemical use against resistant mosquitoes.

  4. The Repellent DEET Potentiates Carbamate Effects via Insect Muscarinic Receptor Interactions: An Alternative Strategy to Control Insect Vector-Borne Diseases

    PubMed Central

    Abd-Ella, Aly; Stankiewicz, Maria; Mikulska, Karolina; Nowak, Wieslaw; Pennetier, Cédric; Goulu, Mathilde; Fruchart-Gaillard, Carole; Licznar, Patricia; Apaire-Marchais, Véronique; List, Olivier; Corbel, Vincent; Servent, Denis; Lapied, Bruno

    2015-01-01

    Insect vector-borne diseases remain one of the principal causes of human mortality. In addition to conventional measures of insect control, repellents continue to be the mainstay for personal protection. Because of the increasing pyrethroid-resistant mosquito populations, alternative strategies to reconstitute pyrethroid repellency and knock-down effects have been proposed by mixing the repellent DEET (N,N-Diethyl-3-methylbenzamide) with non-pyrethroid insecticide to better control resistant insect vector-borne diseases. By using electrophysiological, biochemichal, in vivo toxicological techniques together with calcium imaging, binding studies and in silico docking, we have shown that DEET, at low concentrations, interacts with high affinity with insect M1/M3 mAChR allosteric site potentiating agonist effects on mAChRs coupled to phospholipase C second messenger pathway. This increases the anticholinesterase activity of the carbamate propoxur through calcium-dependent regulation of acetylcholinesterase. At high concentrations, DEET interacts with low affinity on distinct M1/M3 mAChR site, counteracting the potentiation. Similar dose-dependent dual effects of DEET have also been observed at synaptic mAChR level. Additionally, binding and in silico docking studies performed on human M1 and M3 mAChR subtypes indicate that DEET only displays a low affinity antagonist profile on these M1/M3 mAChRs. These results reveal a selective high affinity positive allosteric site for DEET in insect mAChRs. Finally, bioassays conducted on Aedes aegypti confirm the synergistic interaction between DEET and propoxur observed in vitro, resulting in a higher mortality of mosquitoes. Our findings reveal an unusual allosterically potentiating action of the repellent DEET, which involves a selective site in insect. These results open exciting research areas in public health particularly in the control of the pyrethroid-resistant insect-vector borne diseases. Mixing low doses of DEET and a non-pyrethroid insecticide will lead to improvement in the efficiency treatments thus reducing both the concentration of active ingredients and side effects for non-target organisms. The discovery of this insect specific site may pave the way for the development of new strategies essential in the management of chemical use against resistant mosquitoes. PMID:25961834

  5. Wolbachia as a bacteriocyte-associated nutritional mutualist

    PubMed Central

    Hosokawa, Takahiro; Koga, Ryuichi; Kikuchi, Yoshitomo; Meng, Xian-Ying; Fukatsu, Takema

    2009-01-01

    Many insects are dependent on bacterial symbionts that provide essential nutrients (ex. aphid–Buchnera and tsetse–Wiglesworthia associations), wherein the symbionts are harbored in specific cells called bacteriocytes that constitute a symbiotic organ bacteriome. Facultative and parasitic bacterial symbionts like Wolbachia have been regarded as evolutionarily distinct from such obligate nutritional mutualists. However, we discovered that, in the bedbug Cimex lectularius, Wolbachia resides in a bacteriome and appears to be an obligate nutritional mutualist. Two bacterial symbionts, a Wolbachia strain and an unnamed γ-proteobacterium, were identified from different strains of the bedbug. The Wolbachia symbiont was detected from all of the insects examined whereas the γ-proteobacterium was found in a part of them. The Wolbachia symbiont was specifically localized in the bacteriomes and vertically transmitted via the somatic stem cell niche of germalia to oocytes, infecting the incipient symbiotic organ at an early stage of the embryogenesis. Elimination of the Wolbachia symbiont resulted in retarded growth and sterility of the host insect. These deficiencies were rescued by oral supplementation of B vitamins, confirming the essential nutritional role of the symbiont for the host. The estimated genome size of the Wolbachia symbiont was around 1.3 Mb, which was almost equivalent to the genome sizes of parasitic Wolbachia strains of other insects. These results indicate that bacteriocyte-associated nutritional mutualism can evolve from facultative and prevalent microbial associates like Wolbachia, highlighting a previously unknown aspect of the parasitism-mutualism evolutionary continuum. PMID:20080750

  6. Insulin-like peptide genes in honey bee fat body respond differently to manipulation of social behavioral physiology.

    PubMed

    Nilsen, Kari-Anne; Ihle, Kate E; Frederick, Katy; Fondrk, M Kim; Smedal, Bente; Hartfelder, Klaus; Amdam, Gro V

    2011-05-01

    Nutrient sensitive insulin-like peptides (ILPs) have profound effects on invertebrate metabolism, nutrient storage, fertility and aging. Many insects transcribe ILPs in specialized neurosecretory cells at changing levels correlated with life history. However, the major site of insect metabolism and nutrient storage is not the brain, but rather the fat body, where functions of ILP expression are rarely studied and poorly understood. Fat body is analogous to mammalian liver and adipose tissue, with nutrient stores that often correlate with behavior. We used the honey bee (Apis mellifera), an insect with complex behavior, to test whether ILP genes in fat body respond to experimentally induced changes of behavioral physiology. Honey bee fat body influences endocrine state and behavior by secreting the yolk protein precursor vitellogenin (Vg), which suppresses lipophilic juvenile hormone and social foraging behavior. In a two-factorial experiment, we used RNA interference (RNAi)-mediated vg gene knockdown and amino acid nutrient enrichment of hemolymph (blood) to perturb this regulatory module. We document factor-specific changes in fat body ilp1 and ilp2 mRNA, the bee's ILP-encoding genes, and confirm that our protocol affects social behavior. We show that ilp1 and ilp2 are regulated independently and differently and diverge in their specific expression-localization between fat body oenocyte and trophocyte cells. Insect ilp functions may be better understood by broadening research to account for expression in fat body and not only brain.

  7. Insulin-like peptide genes in honey bee fat body respond differently to manipulation of social behavioral physiology

    PubMed Central

    Nilsen, Kari-Anne; Ihle, Kate E.; Frederick, Katy; Fondrk, M. Kim; Smedal, Bente; Hartfelder, Klaus; Amdam, Gro V.

    2011-01-01

    SUMMARY Nutrient sensitive insulin-like peptides (ILPs) have profound effects on invertebrate metabolism, nutrient storage, fertility and aging. Many insects transcribe ILPs in specialized neurosecretory cells at changing levels correlated with life history. However, the major site of insect metabolism and nutrient storage is not the brain, but rather the fat body, where functions of ILP expression are rarely studied and poorly understood. Fat body is analogous to mammalian liver and adipose tissue, with nutrient stores that often correlate with behavior. We used the honey bee (Apis mellifera), an insect with complex behavior, to test whether ILP genes in fat body respond to experimentally induced changes of behavioral physiology. Honey bee fat body influences endocrine state and behavior by secreting the yolk protein precursor vitellogenin (Vg), which suppresses lipophilic juvenile hormone and social foraging behavior. In a two-factorial experiment, we used RNA interference (RNAi)-mediated vg gene knockdown and amino acid nutrient enrichment of hemolymph (blood) to perturb this regulatory module. We document factor-specific changes in fat body ilp1 and ilp2 mRNA, the bee's ILP-encoding genes, and confirm that our protocol affects social behavior. We show that ilp1 and ilp2 are regulated independently and differently and diverge in their specific expression-localization between fat body oenocyte and trophocyte cells. Insect ilp functions may be better understood by broadening research to account for expression in fat body and not only brain. PMID:21490257

  8. Interplay between calcineurin and the Slt2 MAP-kinase in mediating cell wall integrity, conidiation and virulence in the insect fungal pathogen Beauveria bassiana.

    PubMed

    Huang, Shuaishuai; He, Zhangjiang; Zhang, Shiwei; Keyhani, Nemat O; Song, Yulin; Yang, Zhi; Jiang, Yahui; Zhang, Wenli; Pei, Yan; Zhang, Yongjun

    2015-10-01

    The entomopathogenic fungus, Beauveria bassiana, is of environmental and economic importance as an insect pathogen, currently used for the biological control of a number of pests. Cell wall integrity and conidiation are critical parameters for the ability of the fungus to infect insects and for production of the infectious propagules. The contribution of calcineurin and the Slt2 MAP kinase to cell wall integrity and development in B. bassiana was investigated. Gene knockouts of either the calcineurin CNA1 subunit or the Slt2 MAP kinase resulted in decreased tolerance to calcofluor white and high temperature. In contrast, the Δcna1 strain was more tolerant to Congo red but more sensitive to osmotic stress (NaCl, sorbitol) than the wild type, whereas the Δslt2 strain had the opposite phenotype. Changes in cell wall structure and composition were seen in the Δslt2 and Δcna1 strains during growth under cell wall stress as compared to the wild type. Both Δslt2 and Δcna1 strains showed significant alterations in growth, conidiation, and viability. Elevation of intracellular ROS levels, and decreased conidial hydrophobicity and adhesion to hydrophobic surfaces, were also seen for both mutants, as well as decreased virulence. Under cell wall stress conditions, inactivation of Slt2 significantly repressed CN-mediated phosphatase activity suggesting some level of cross talk between the two pathways. Comparative transcriptome profiling of the Δslt2 and Δcna1 strains revealed alterations in the expression of distinct gene sets, with overlap in transcripts involved in cell wall integrity, stress response, conidiation and virulence. These data illustrate convergent and divergent phenotypes and targets of the calcineurin and Slt2 pathways in B. bassiana. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Modulation of allergy incidence in icelandic horses is associated with a change in IL-4-producing T cells.

    PubMed

    Hamza, E; Doherr, M G; Bertoni, G; Jungi, T W; Marti, E

    2007-01-01

    Equine insect bite hypersensitivity (IBH) is an immediate-type hypersensitivity reaction provoked by insect-derived allergens. Icelandic horses living in Iceland do not have IBH due to absence of relevant insects, but acquire it at high frequency after being imported to mainland Europe. In contrast, their offspring born in mainland Europe has reduced IBH incidence. T helper 1 (Th1) and Th2 cells and cytokines were determined in Icelandic horses born in Iceland and on the continent and which either have IBH or are healthy. Peripheral blood mononuclear cells (PBMC) from these horses were stimulated for 18 h during summer and winter with polyclonal T cell stimuli, IBH allergen(s) or irrelevant allergen(s). Cells were analysed by flow cytometry for interferon-gamma (IFN-gamma) and interleukin-4 (IL-4); RNA was analysed for IFN-gamma, IL-4, IL-5 and IL-13 mRNA. During summer, but not during winter, IBH PBMC stimulated polyclonally showed reduced IFN-gamma mRNA and IFN-gamma-producing cells when compared with those of healthy horses, regardless of origin. PBMC stimulated polyclonally or with IBH allergen showed increased IL-4 mRNA levels and higher numbers of IL-4-producing cells when born in Iceland or showing IBH symptoms. IL-5 and IL-13 mRNA were modulated neither by disease nor by origin. Abrogation of IL-4 production in healthy horses born in mainland Europe may be due, at least in part, to IL-10. There was an increased level of IL-10 in supernatants from PBMC of healthy horses born in mainland Europe and stimulated polyclonally or with IBH allergen. Modulation of IBH incidence is governed by altered Th1/Th2 ratio, which might be influenced by IL-10. Copyright 2007 S. Karger AG, Basel.

  10. The disulfide-rich Metridia luciferase refolded from E. coli inclusion bodies reveals the properties of a native folded enzyme produced in insect cells.

    PubMed

    Markova, Svetlana V; Larionova, Marina D; Gorbunova, Darya A; Vysotski, Eugene S

    2017-10-01

    The bioluminescence of a marine copepod Metridia longa is determined by a small secreted coelenterazine-dependent luciferase that uses coelenterazine as a substrate of enzymatic reaction to generate light (λ max =480nm). To date, four different isoforms of the luciferase differing in size, sequences, and properties have been cloned by functional screening. All of them contain ten conserved Cys residues that suggests up to five SS intramolecular bonds per luciferase molecule. Whereas the use of copepod luciferases as bioluminescent reporters in biomedical research in vivo is growing from year to year, their application for in vitro assays is still limited by the difficulty in obtaining significant amounts of luciferase. The most cost-effective host for producing recombinant proteins is Escherichia coli. However, prokaryotic and eukaryotic cells maintain the reductive environment in cytoplasm that hinders the disulfide bond formation and consequently the proper folding of luciferase. Here we report the expression of the MLuc7 isoform of M. longa luciferase in E. coli cells and the efficient procedure for refolding from inclusion bodies yielding a high-active monomeric protein. Furthermore, in a set of identical experiments we demonstrate that bioluminescent and structural features of MLuc7 produced in bacterial cells are identical to those of MLuc7 isoform produced from culture medium of insect cells. Although the yield of high-purity protein is only 6mg/L, the application of E. coli cells to produce the luciferase is simpler and more cost-effective than the use of insect cells. We expect that the suggested technology of Metridia luciferase production allows obtaining of sufficient amounts of protein both for the development of novel in vitro analytical assays with the use of MLuc7 as a label and for structural studies. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Invertebrate hematopoiesis: an anterior proliferation center as a link between the hematopoietic tissue and the brain.

    PubMed

    Noonin, Chadanat; Lin, Xionghui; Jiravanichpaisal, Pikul; Söderhäll, Kenneth; Söderhäll, Irene

    2012-11-20

    During evolution, the innate and adaptive immune systems were developed to protect organisms from non-self substances. The innate immune system is phylogenetically more ancient and is present in most multicellular organisms, whereas adaptive responses are restricted to vertebrates. Arthropods lack the blood cells of the lymphoid lineage and oxygen-carrying erythrocytes, making them suitable model animals for studying the regulation of the blood cells of the innate immune system. Many crustaceans have a long life span and need to continuously synthesize blood cells, in contrast to many insects. The hematopoietic tissue (HPT) of Pacifastacus leniusculus provides a simple model for studying hematopoiesis, because the tissue can be isolated, and the proliferation of stem cells and their differentiation can be studied both in vivo and in vitro. Here, we demonstrate new findings of a physical link between the HPT and the brain. Actively proliferating cells were localized to an anterior proliferation center (APC) in the anterior part of the tissue near the area linking the HPT to the brain, whereas more differentiated cells were detected in the posterior part. The central areas of HPT expand in response to lipopolysaccharide-induced blood loss. Cells isolated from the APC divide rapidly and form cell clusters in vitro; conversely, the cells from the remaining HPT form monolayers, and they can be induced to differentiate in vitro. Our findings offer an opportunity to learn more about invertebrate hematopoiesis and its connection to the central nervous system, thereby obtaining new information about the evolution of different blood and nerve cell lineages.

  12. Lettuce and spinach breeding

    USDA-ARS?s Scientific Manuscript database

    Lettuce and spinach production is beset by numerous biotic an abiotic challenges, thus the leafy-vegetable industry of California requires continued development of improved, adapted cultivars to meet new disease and insect problems, changes in the market, and changes in growing procedures. The lettu...

  13. Climate change and temperate zone insects: the tyranny of thermodynamics meets the world of limited resources.

    PubMed

    Adamo, Shelley A; Baker, Jillian L; Lovett, Maggie M E; Wilson, Graham

    2012-12-01

    Climate change will result in warmer temperatures and an increase in the frequency and severity of extreme weather events. Given that higher temperatures increase the reproductive rate of temperate zone insects, insect population growth rates are predicted to increase in the temperate zone in response to climate. This consensus, however, rests on the assumption that food is freely available. However, under conditions of limited food, the reproductive output of the Texan cricket Gryllus texensis (Cade and Otte) was highest at its current normal average temperature and declined with increasing temperature. Moreover, low food availability decreased survival during a simulated heat wave. Therefore, the effects of climate change on this species, and possibly on many others, are likely to hinge on food availability. Extrapolation from our data suggests that G. texensis will show larger yearly fluctuations in population size as climate change continues, and this will also have ecological repercussions. Only those temperate zone insects with a ready supply of food (e.g., agricultural pests) are likely to experience the predicted increase in population growth in response to climate change; food-limited species are likely to experience a population decline.

  14. 7 CFR 29.3524 - Injury.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Type 95) § 29.3524 Injury. Hurt or impairment from any cause except the fungus or bacterial diseases... caused by field diseases, insects, or weather conditions; insecticides, fungicides, or cell growth...

  15. 7 CFR 29.3524 - Injury.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Type 95) § 29.3524 Injury. Hurt or impairment from any cause except the fungus or bacterial diseases... caused by field diseases, insects, or weather conditions; insecticides, fungicides, or cell growth...

  16. 7 CFR 29.3524 - Injury.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Type 95) § 29.3524 Injury. Hurt or impairment from any cause except the fungus or bacterial diseases... caused by field diseases, insects, or weather conditions; insecticides, fungicides, or cell growth...

  17. 7 CFR 29.3524 - Injury.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Type 95) § 29.3524 Injury. Hurt or impairment from any cause except the fungus or bacterial diseases... caused by field diseases, insects, or weather conditions; insecticides, fungicides, or cell growth...

  18. 7 CFR 29.3524 - Injury.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... Type 95) § 29.3524 Injury. Hurt or impairment from any cause except the fungus or bacterial diseases... caused by field diseases, insects, or weather conditions; insecticides, fungicides, or cell growth...

  19. Use of an insect cell culture growth medium to isolate bacteria from horses with effusive, fibrinous pericarditis: a preliminary study.

    PubMed

    Jones, Samuel L; Valenzisi, Amy; Sontakke, Sushama; Sprayberry, Kimberly A; Maggi, Ricardo; Hegarty, Barbara; Breitschwerdt, Edward

    2007-03-31

    Effusive, fibrinous pericarditis is an uncommon disease entity in horses. In 2001, pericarditis occurred in conjunction with an epizootic in central Kentucky that was associated with exposure to eastern tent caterpillars (ETCs). Bacterial isolation from equine pericardial fluid samples was attempted using an insect cell culture growth medium (ICCGM). Using previously cultured, stored frozen samples from four horses with fibrinous pericarditis, inoculation of 10% blood agar plates yielded no growth, whereas simultaneous inoculation of ICCGM resulted in the isolation of Proprionibacterium acnes, Staphylococcus equorum, a Streptococcus sp. and Pseudomonas rhodesiae from pericardial fluid samples. A similar or novel caterpillar-associated bacteria was not identified; however, use of an ICCGM might enhance isolation of bacteria from equine pericardial fluid.

  20. Study on bird's & insect's wing aerodynamics and comparison of its analytical value with standard airfoil

    NASA Astrophysics Data System (ADS)

    Ali, Md. Nesar; Alam, Mahbubul; Hossain, Md. Abed; Ahmed, Md. Imteaz

    2017-06-01

    Flight is the main mode of locomotion used by most of the world's bird & insect species. This article discusses the mechanics of bird flight, with emphasis on the varied forms of bird's & insect's wings. The fundamentals of bird flight are similar to those of aircraft. Flying animals flap their wings to generate lift and thrust as well as to perform remarkable maneuvers with rapid accelerations and decelerations. Insects and birds provide illuminating examples of unsteady aerodynamics. Lift force is produced by the action of air flow on the wing, which is an airfoil. The airfoil is shaped such that the air provides a net upward force on the wing, while the movement of air is directed downward. Additional net lift may come from airflow around the bird's & insect's body in some species, especially during intermittent flight while the wings are folded or semi-folded. Bird's & insect's flight in nature are sub-divided into two stages. They are Unpowered Flight: Gliding and Soaring & Powered Flight: Flapping. When gliding, birds and insects obtain both a vertical and a forward force from their wings. When a bird & insect flaps, as opposed to gliding, its wings continue to develop lift as before, but the lift is rotated forward to provide thrust, which counteracts drag and increases its speed, which has the effect of also increasing lift to counteract its weight, allowing it to maintain height or to climb. Flapping flight is more complicated than flight with fixed wings because of the structural movement and the resulting unsteady fluid dynamics. Flapping involves two stages: the down-stroke, which provides the majority of the thrust, and the up-stroke, which can also (depending on the bird's & insect's wings) provide some thrust. Most kinds of bird & insect wing can be grouped into four types, with some falling between two of these types. These types of wings are elliptical wings, high speed wings, high aspect ratio wings and soaring wings with slots. Hovering is used by several species of birds. Hovering, which is generating only lift through flapping alone rather than as a product of thrust, demands a lot of energy. On the other hand, for practical knowledge we also fabricate the various bird's, insect's & fighter jet wing by using random value of parameter & test those airfoil in wind tunnel. Finally for comparison & achieving analytical knowledge we also test those airfoil model in various simulation software.

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