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Michael R. Kanost - One of the best experts on this subject based on the ideXlab platform.
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a Hemocyte specific integrin required for hemocytic encapsulation in the tobacco hornworm manduca sexta
Insect Biochemistry and Molecular Biology, 2005Co-Authors: David M Levin, Lisha N Breuer, Shufei Zhuang, Sheri A Anderson, James B Nardi, Michael R. KanostAbstract:Upon encountering an object recognized as foreign, insect Hemocytes aggregate in multiple layers on the surfaces of the object in a process known as encapsulation. For encapsulation to occur, Hemocytes must switch from their usual nonadherent state to an adherent state, presumably by regulating the activity of adhesion proteins. Although detailed knowledge exists regarding the adhesion receptors for cells of the mammalian immune system, comparable information on adhesion molecules of insect Hemocytes and their function in immune responses is extremely limited. We report here the identification of an integrin present exclusively on the surface of Hemocytes in the tobacco hornworm, Manduca sexta. Monoclonal antibodies MS13 and MS34, which bind to plasmatocytes and block encapsulation, were used for immunoaffinity chromatography to isolate their corresponding Hemocyte antigen, which was revealed to be the same integrin β subunit. A cDNA for this M. sexta integrin β1 was cloned and characterized. Integrin-β1 mRNA was detected by Northern analysis in Hemocytes and not in other tissues tested. MS13 and MS34 were demonstrated to bind to a recombinant fragment of integrin β1 consisting of the I-like domain, consistent with their blocking of a ligand-binding site and subsequent disruption of plasmatocyte adhesion. Injection of double stranded integrin-β1 RNA into larvae resulted in decreased integrin β1 expression in plasmatocytes and significantly suppressed encapsulation. These results indicate that activation of ligand-binding by the Hemocyte-specific integrin plays a key role in stimulating plasmatocyte adhesion leading to encapsulation.
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biological mediators of insect immunity
Annual Review of Entomology, 1997Co-Authors: Jeremy P Gillespie, Michael R. Kanost, Tina TrenczekAbstract:▪ Abstract Infection in insects stimulates a complex defensive response. Recognition of pathogens may be accomplished by plasma or Hemocyte proteins that bind specifically to bacterial or fungal polysaccharides. Several morphologically distinct Hemocyte cell types cooperate in the immune response. Hemocytes attach to invading organisms and then isolate them by phagocytosis, by trapping them in Hemocyte aggregates called nodules, or by forming an organized multicellular capsule around large parasites. These responses are often accompanied by proteolytic activation of the phenoloxidase zymogen that is present in the hemolymph. A component of insect immune responses to bacteria is the synthesis by fat body and Hemocytes of a variety of antibacterial proteins and peptides, which are secreted into the hemolymph. These molecules attack bacteria by several mechanisms. Inducible antifungal proteins have also been recently discovered in insect hemolymph. The promoters for several antibacterial protein genes in ins...
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immunochemical identification of insect Hemocyte populations monoclonal antibodies distinguish four major Hemocyte types in manduca sexta
European Journal of Cell Biology, 1994Co-Authors: E Willott, Tina Trenczek, L W Thrower, Michael R. KanostAbstract:: 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.
Rongqing Zhang - One of the best experts on this subject based on the ideXlab platform.
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Hemocytes participate in calcium carbonate crystal formation transportation and shell regeneration in the pearl oyster pinctada fucata
Fish & Shellfish Immunology, 2016Co-Authors: Shiguo Li, Jingliang Huang, Guilan Zheng, Rongqing ZhangAbstract:In this study, light microscope, scanning and transmission electron microscope, hematoxylin-eosin and fluorescent staining, and mass spectrometry methods were employed to observe the calcium carbonate (CaCO3) crystal formation, Hemocyte release and transportation, and Hemocyte distribution at the shell regeneration area and to analyse the proteome of Hemocytes in the pearl oyster, Pinctada fucata. The results indicated that intracellular CaCO3 crystals were observed in circulating Hemocytes in P. fucata, implying that there was a suitable microenvironment for crystal formation in the Hemocytes. This conclusion was further supported by the proteome analysis, in which various biomineralization-related proteins were detected. The crystal-bearing Hemocytes, mainly granulocytes, may be released to extrapallial fluid (EPF) by the secretory cavities distributed on the outer surface of the mantle centre. These granulocytes in the EPF and between the regenerated shells were abundant and free. In the regenerated prismatic layer, the granulocytes were fused into each column and fragmented with the duration of shell maturation, suggesting the direct involvement of Hemocytes in shell regeneration. Overall, this study provided evidence that Hemocytes participated in CaCO3 crystal formation, transportation and shell regeneration in the pearl oyster. These results are helpful to further understand the exact mechanism of Hemocyte-mediated biomineralization in shelled molluscs.
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morphology and classification of Hemocytes in pinctada fucata and their responses to ocean acidification and warming
Fish & Shellfish Immunology, 2015Co-Authors: Shiguo Li, Jingliang Huang, Guilan Zheng, Rongqing ZhangAbstract:Abstract Hemocytes play important roles in the innate immune response and biomineralization of bivalve mollusks. However, the Hemocytes in pearl oysters are poorly understood. In the present study, we investigated the morphology and classification of Hemocytes in the pearl oyster, Pinctada fucata. Three types of Hemocytes were successfully obtained by light microscopy, electron microscopy and flow cytometry methods: small hyalinocytes, large hyalinocytes and granulocytes. The small hyalinocytes are the major Hemocyte population. Morphological analyses indicated that these Hemocytes have species-specific characterizations. In addition, we assessed the potential effects of ocean acidification (OA) and ocean warming (OW) on the immune parameters and calcium homeostasis of the Hemocytes. OA and OW (31 °C) altered pH value of hemolymph, increased the total Hemocyte count, total protein content, and percentage of large hyalinocytes and granulocytes, while it decreased the neutral red uptake ability, suggesting active stress responses of P. fucata to these stressors. Exposure to OW (25 °C) resulted in no significant differences, indicating an excellent immune defense to heat stress at this level. The outflow of calcium from Hemocytes to hemolymph was also determined, implying the potential impact of OA and OW on Hemocyte-mediated biomineralization. This study, therefore, provides insight into the classification and characterization of Hemocyte in the pearl oyster, P. fucata, and also reveals the immune responses of Hemocytes to OA and OW, which are helpful for a comprehensive understanding of the effects of global climate change on pearl oysters.
Xiaoqiang Yu - One of the best experts on this subject based on the ideXlab platform.
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drosophila c type lectins enhance cellular encapsulation
Molecular Immunology, 2007Co-Authors: Jingqun Ao, Erjun Ling, Xiaoqiang YuAbstract:Abstract C-type lectins are calcium-dependent carbohydrate binding proteins, and animal C-type lectins participate in innate immunity and cell–cell interactions. In the fruit fly Drosophila melanogaster, more than 30 genes encode C-type lectin domains. However, functions of Drosophila C-type lectins in innate immunity are not well understood. This study is to investigate whether two Drosophila C-type lectins, CG33532 and CG33533 (designated as DL2 and DL3, respectively), are involved in innate immune responses. Recombinant DL2 and DL3 were expressed and purified. Both DL2 and DL3 agglutinated Gram-negative Escherichia coli in a calcium-dependent manner. Though DL2 and DL3 are predicted to be secreted proteins, they were detected on the surface of Drosophila Hemocytes, and recombinant DL2 and DL3 also directly bound to Hemocytes. Coating of agarose beads with recombinant DL2 and DL3 enhanced their encapsulation and melanization by Drosophila Hemocytes in vitro. However, Hemocyte encapsulation was blocked when the lectin-coated beads were pre-incubated with rat polyclonal antibody specific for DL2 or DL3. Our results suggest that DL2 and DL3 may act as pattern recognition receptors to mediate Hemocyte encapsulation and melanization by directly recruiting Hemocytes to the lectin-coated surface.
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prophenoloxidase binds to the surface of Hemocytes and is involved in Hemocyte melanization in manduca sexta
Insect Biochemistry and Molecular Biology, 2005Co-Authors: Erjun Ling, Xiaoqiang YuAbstract:Abstract In insects, melanotic encapsulation is an important innate immune response against large pathogens or parasites, and phenoloxidase (PO) is a key enzyme in this process. Activation of prophenoloxidase (proPO) to PO is mediated by a serine proteinase cascade. PO has a tendency to adhere to foreign surfaces including Hemocyte surfaces. In this study, we showed that in the naive larvae of the tobacco hornworm Manduca sexta, hemolymph proPO bound to the surface of granulocytes and spherule cells but not to oenocytoids, and about 10% Hemocytes had proPO on their surfaces. When larvae were injected with water (injury) or microsphere beads (immune-challenge), hemolymph proPO was activated, and the number of Hemocytes with surface proPO/PO increased at 12 h post-injection, but dropped to the normal level at 24 h. Hemocyte surface proPO can be activated in vitro, leading to melanization of these Hemocytes. The number of melanized Hemocytes from the larvae injected with water or microsphere beads significantly increased. We also showed that neither Hemocytes nor cell-free plasma alone triggered melanization of immulectin-2-coated agarose beads in vitro. However, agarose beads were effectively melanized by isolated Hemocytes in the presence of cell-free plasma. Our results suggest that activation of Hemocyte surface proPO may initiate melanization, leading to the systemic melanization of Hemocyte capsules.
Christophe Lambert - One of the best experts on this subject based on the ideXlab platform.
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Hemocyte characteristics in families of oysters crassostrea gigas selected for differential survival during summer and reared in three sites
Aquaculture, 2007Co-Authors: Maryse Delaporte, Christophe Lambert, Jeanne Moal, Philippe Soudant, Lionel Degremont, Pierre Boudry, Frederic JeanAbstract:High variability among individuals is often encountered when Hemocyte characteristics are measured in bivalves. Such variability is suspected to result partly from genetic factors. In this study, Hemocyte characteristics of six families of Crassostrea gigas were compared by flow cytometry at one sampling date in October 2001. These families were obtained from a nested, halfsibling cross design, and reared from July to October 2001 at three sites distributed along the French Atlantic coast from north to south: Baie des Veys (Normandy), Riviere d'Auray (Brittany) and Ronce (Marennes-oleron Basin, Poitou Charentes). Among the 15 measured Hemocyte characteristics, production of reactive oxygen species (ROS) of untreated Hemocytes (maintained in filtered sterile seawater) and treated Hemocytes (zymosan at 20 particles per Hemocyte, and with Vibrio sp. S322 at 50 bacteria per Hemocyte) was the most notable differences between families. This supports the existence of a genetic basis, at least partly, for the Hemocyte characteristics of oysters, and especially for ROS production. Among the six families analyzed, three have shown high survival during summer (named as "resistant", mean mortality 5.2%) and three experienced high mortality during summer (named as "susceptible", 30.6% mean mortality). Families showing high or low survival to summer mortality had similar Hemocyte characteristics, regardless of the environmental conditions or reproductive state. Resistant families were observed to have higher total Hemocyte counts and lower production of ROS than susceptible families. Moreover, ROS production of Hemocytes from susceptible families was diminished significantly more by pathogenic Vibrio than that of resistant families. However, this study demonstrates also that rearing site strongly affected the Hemocyte characteristics of all families of oysters, most notably Hemocyte concentration and morphology (size and granularity), production of reactive oxygen species (ROS), and susceptibility to the cytotoxic activity of the pathogenic Vibrio sp. S322 (50 bacteria/ Hemocyte). Food availability and reproductive state are the most probable explanations for the site differences observed. Finally, it appeared difficult to link oyster survival during summer mortality to Hemocyte profiles evaluated at one sampling date; other relevant indicators would probably help explaining oyster survival during summer mortality events.
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toxic dinoflagellates alexandrium fundyense and a catenella have minimal apparent effects on oyster Hemocytes
Marine Biology, 2007Co-Authors: Helene Hegaret, Christophe Lambert, Gary H. Wikfors, Philippe Soudant, Sandra E Shumway, Jeanbaptiste Berard, Patrick LassusAbstract:The possible effect of Alexandrium spp. containing paralytic shellfish poisoning (PSP) toxins on the Hemocytes of oysters was tested experimentally. In one trial, eastern oysters, Crassostrea virginica Gmelin, were exposed to bloom concentrations of the sympatric dinoflagellate, Alexandrium fundyense Balech, alone and in a mixture with a non-toxic diatom, Thalassiosira weissflogii (Grun) Fryxell et Hasle. Subsequently, another experiment exposed Pacific oysters, Crassostrea gigas Thunberg, to a mixed suspension of the sympatric, toxic species Alexandrium catenella (Whedon et Kofoid) Balech, with T. weissflogii. Measurements of numbers of oyster Hemocytes, percentages of different cell types, and functions (phagocytosis, reactive oxygen species (ROS) production, and mortality) were made using flow-cytometry. During and after exposure, almost no significant effects of Alexandrium spp. upon Hemocyte numbers, morphology, or functions were detected, despite observations of adductor-muscle paralysis in C. virginica and measured toxin accumulation in C. gigas. The only significant correlation found was between toxin accumulation at one temperature and higher numbers of circulating live and dead Hemocytes in C. gigas. The PSP toxins are known to interfere specifically with sodium-channel function; therefore, the finding that the toxins had no effect on measured Hemocyte functions suggests that sodium-channel physiology is not important in these Hemocyte functions. Finally, because oysters were exposed to the living algae, not purified toxins, there was no evidence of bioactive compounds other than PSP toxins affecting Hemocytes in the two species of Alexandrium studied.
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Impact of 20∶4n−6 supplementation on the fatty acid composition and Hemocyte parameters of the pacific oyster crassostrea gigas
Lipids, 2006Co-Authors: Maryse Delaporte, Emilie Giudicelli, Catherine Séguineau, Christophe Lambert, Jeanne Moal, Philippe Soudant, Jean-francois SamainAbstract:Arachidonic acid (20∶4n−6, ArA) and its eicosanoid metabolites have been demonstrated to be implicated in immune functions of vertebrates, fish, and insects. Thus, the aim of this study was to assess the impact of ArA supplementation on the FA composition and Hemocyte parameters of oysters Crassostrea gigas. Oyster dietary conditioning consisted of direct addition of ArA solutions at a dose of 0, 0.25, or 0.41 μg ArA per mL of seawater into tanks in the presence or absence of T-Iso algae. Results showed significant incorporation of ArA into gill polar lipids when administered with algae (up to 19.7%) or without algae (up to 12.1%). ArA supplementation led to an increase in Hemocyte numbers, phagocytosis, and production of reactive oxygen species by Hemocytes from ArA-supplemented oysters. Moreover, the inhibitory effect of Vibrio aestuarianus extracellular products on the adhesive proprieties of Hemocytes was lessened in oysters fed ArA-supplemented T-Iso. All changes in oyster Hemocyte parameters reported in the present study suggest that ArA and/or eicosanoid metabolites affect oyster Hemocyte functions.
Shiguo Li - One of the best experts on this subject based on the ideXlab platform.
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Hemocytes participate in calcium carbonate crystal formation transportation and shell regeneration in the pearl oyster pinctada fucata
Fish & Shellfish Immunology, 2016Co-Authors: Shiguo Li, Jingliang Huang, Guilan Zheng, Rongqing ZhangAbstract:In this study, light microscope, scanning and transmission electron microscope, hematoxylin-eosin and fluorescent staining, and mass spectrometry methods were employed to observe the calcium carbonate (CaCO3) crystal formation, Hemocyte release and transportation, and Hemocyte distribution at the shell regeneration area and to analyse the proteome of Hemocytes in the pearl oyster, Pinctada fucata. The results indicated that intracellular CaCO3 crystals were observed in circulating Hemocytes in P. fucata, implying that there was a suitable microenvironment for crystal formation in the Hemocytes. This conclusion was further supported by the proteome analysis, in which various biomineralization-related proteins were detected. The crystal-bearing Hemocytes, mainly granulocytes, may be released to extrapallial fluid (EPF) by the secretory cavities distributed on the outer surface of the mantle centre. These granulocytes in the EPF and between the regenerated shells were abundant and free. In the regenerated prismatic layer, the granulocytes were fused into each column and fragmented with the duration of shell maturation, suggesting the direct involvement of Hemocytes in shell regeneration. Overall, this study provided evidence that Hemocytes participated in CaCO3 crystal formation, transportation and shell regeneration in the pearl oyster. These results are helpful to further understand the exact mechanism of Hemocyte-mediated biomineralization in shelled molluscs.
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morphology and classification of Hemocytes in pinctada fucata and their responses to ocean acidification and warming
Fish & Shellfish Immunology, 2015Co-Authors: Shiguo Li, Jingliang Huang, Guilan Zheng, Rongqing ZhangAbstract:Abstract Hemocytes play important roles in the innate immune response and biomineralization of bivalve mollusks. However, the Hemocytes in pearl oysters are poorly understood. In the present study, we investigated the morphology and classification of Hemocytes in the pearl oyster, Pinctada fucata. Three types of Hemocytes were successfully obtained by light microscopy, electron microscopy and flow cytometry methods: small hyalinocytes, large hyalinocytes and granulocytes. The small hyalinocytes are the major Hemocyte population. Morphological analyses indicated that these Hemocytes have species-specific characterizations. In addition, we assessed the potential effects of ocean acidification (OA) and ocean warming (OW) on the immune parameters and calcium homeostasis of the Hemocytes. OA and OW (31 °C) altered pH value of hemolymph, increased the total Hemocyte count, total protein content, and percentage of large hyalinocytes and granulocytes, while it decreased the neutral red uptake ability, suggesting active stress responses of P. fucata to these stressors. Exposure to OW (25 °C) resulted in no significant differences, indicating an excellent immune defense to heat stress at this level. The outflow of calcium from Hemocytes to hemolymph was also determined, implying the potential impact of OA and OW on Hemocyte-mediated biomineralization. This study, therefore, provides insight into the classification and characterization of Hemocyte in the pearl oyster, P. fucata, and also reveals the immune responses of Hemocytes to OA and OW, which are helpful for a comprehensive understanding of the effects of global climate change on pearl oysters.