Heat Shock Protein 90 of Bonamia ostreae: Characterization and Possible Correlation with Infection of the Flat Oyster,Ostrea edulis

In this study, we described the cytosolic HSP90 of Bonamia ostreae, an intracellular parasite of Ostrea edulis hemocytes. The complete open reading frame was assembled by Rapid Amplification cDNA Ends reactions on cDNA of B. ostreae‐infected hemocytes. HSP90 amplification was corroborated in infected oysters and B. ostreae purified cells. The functionality of the HSP90, studied by inhibitory assays with radicicol, suggests that this protein may play a role in hemocyte invasion. Our results inform the molecular basis that governs B. ostreae–O. edulis interactions.     

Hemocyte surface changes in the migratory grasshopper,Melanoplus sanguinipes in response to wounding and infection withBeauveria bassiana

Surface changes of hemocytes from the migratory grasshopper,Melanoplus sanguinipes (Fab.), following wounding and/or infection withBeauveria bassiana (Bals.) Vuillemin were assessed using fluorescein labelled wheat germ agglutinin (WGA) and concanavalin A (Con A). Binding was observed on outer wall of hemocytes when either Con A or WGA conjugates were tested. After infection there was an increase in the percentage of hemocytes which bound to WGA and which remained so for 24 h. However, after wounding this increase in binding subsided after only 1 h. Furthermore, it was found that the increase in WGA-binding occurred within the first 15 min of wounding or infection. In addition, ConA binding in males only increased 3-fold in fungus-injected insects within 1 h of treatment and remained high for 24 h. These results indicated the presence of mannose (and/or glucose) residues (Con A specificity) and N-acetyl-D-glucosamine residues (wheat germ agglutinin; WGA specificity) on outer wall surfaces of the hemocytes. The implications of these results in the immune response ofM. sanguinipes to infection withB. bassiana are discussed.     

Release of Mediator Enzyme β-Hexosaminidase and Modulated Gene Expression Accompany Hemocyte Degranulation in Response to Parasitism in the Silkworm Bombyx mori

In insects infections trigger hemocyte-mediated immune reactions including degranulation by exocytosis; however, involvement of mediator enzymes in degranulation process is unknown in insects. We report here that in silkworm Bombyx mori, infection by endoparasitoid Exorista bombycis and microsporidian Nosema bombycis activated granulation in granulocytes and promoted degranulation of accumulated structured granules. During degranulation the mediator lysosomal enzyme β-hexosaminidase showed increased activity and expression of β-hexosaminidase gene was enhanced. The events were confirmed in vitro after incubation of uninfected hemocytes with E. bombycis larval tissue protein. On infection, cytotoxicity marker enzyme lactate dehydrogenase (LDH) was released from the hemocytes illustrating cell toxicity. Strong positive correlation (R²?=?0.71) between LDH activity and β-hexosaminidase released after the infection showed parasitic–protein-induced hemocyte damage and accompanied release of the enzymes. Expression of β-hexosaminidase gene was enhanced in early stages after infection followed by down regulation. The expression showed positive correlation (R²?=?0.705) with hexosaminidase activity pattern. B. mori hexosaminidase showed 98% amino acid similarity with that of B. mandarina showing origin from same ancestral gene; however, 45–60% varied from other lepidopterans showing diversity. The observation signifies the less known association of hexosaminidase in degranulation of hemocytes induced by parasitic infection in B. mori and its divergence in different species.

     

Tissue communication in a systemic immune response of Drosophila

Several signaling pathways, including the JAK/STAT and Toll pathways, are known to activate blood cells (hemocytes) in Drosophila melanogaster larvae. They are believed to regulate the immune response against infections by parasitoid wasps, such as Leptopilina boulardi, but how these pathways control the hemocytes is not well understood. Here, we discuss the recent discovery that both muscles and fat body take an active part in this response. Parasitoid wasp infection induces Upd2 and Upd3 secretion from hemocytes, leading to JAK/STAT activation mainly in hemocytes and in skeletal muscles. JAK/STAT activation in muscles, but not in hemocytes, is required for an efficient encapsulation of wasp eggs. This suggests that Upd2 and Upd3 are important cytokines, coordinating different tissues for the cellular immune response in Drosophila. In the fat body, Toll signaling initiates a systemic response in which hemocytes are mobilized and activated hemocytes (lamellocytes) are generated. However, the contribution of Toll signaling to the defense against wasps is limited, probably because the wasps inject inhibitors that prevent the activation of the Toll pathway. In conclusion, parasite infection induces a systemic response in Drosophila larvae involving major organ systems and probably the physiology of the entire organism.     

Hemocytes from neonates of Biomphalaria glabrata are functionally less mature than those from adult snails

Gastropod molluscs, which serve as obligatory intermediate hosts for digenetic trematodes, possess an internal defense system (IDS) consisting of phagocytic hemocytes and plasma factors. This IDS is responsible for resistance to infection with larval trematodes, which are encapsulated and killed by hemocytes in incompatible snails. Like other physiological systems, the IDS probably undergoes maturation during early stages of life, and the relatively undeveloped state of the IDS in young snails has been hypothesized to be a factor in their increased susceptibility to infection with larval trematodes. In this study, hemocytes were examined in the BS-90 laboratory strain of Biomphalaria glabrata that is resistant to infection with Schistosoma mansoni as adults but susceptible to infection as neonates. Compared with hemocytes from adults, hemocytes from neonates had a smaller perimeter and lower intrinsic directional motility on glass microscope slides. Additionally, in vitro assays showed a lower association with fucoidan-linked polystyrene beads and less ability to produce superoxide anion in hemocytes from neonates compared to hemocytes from adults. These results support the hypothesis that the gastropod IDS undergoes maturation during growth. However, whether the observed differences between hemocytes of neonatal and adult BS-90 snails play a role in the susceptibility of the former and resistance of the latter to infection with S. mansoni is not known.     

In vitro culture of tissue from the tunicateStyela clava

Summary Pharyngeal explants and circulatory hemocytes from the tunicateStyela clava were cultured in a medium containing tunicate plasma, artificial seawater, RPMI 1640, and antibiotics. Pharnngeal tissue remained viable and proliferated for up to 72 d in vitro. Proliferative activity maintained the pool of hemocytes within explants and facilitated the migration of pharyngeal hemocytes from explants into culture supernatants. The diversity of morphologically distinct cell types within the hemocyte pool of pharyngeal cultures indicated that cell division was followed by regulated differentiation. In contrast to pharyngeal cultures, suspensions of circulatory hemocytes did not survive for prolonged periods in vitro. Proliferative activity could not be detected in circulatory hemocyte cultures. These results are discussed in terms of the differentiation state of hemocytes and the efficacy of culture conditions. This study was supported by the National Science Foundation, Washington, DC (grant DCB 85 19848) and by BRSG funds from UCLA Schools of Medicine and Dentistry. Flow cytometric facilities were sponsored in part by a Johnson Cancer Center Core Grant (CA 16042). David A. Raftos is a Fulbright Postdoctoral Fellow and recipient of a Frederik B. Bang Scholarship in Marine Invertebrate Immunology administered by the American Association of Immunologist. Dan L. Stillman was supported by an REU supplement from the National Science Foundation.     

Differential lectin labelling of circulating hemocytes from Biomphalaria glabrata and Biomphalaria tenagophila resistant or susceptible to Schistosoma mansoni infection

Lectins/carbohydrate binding can be involved in the Schistosoma mansoni recognition and activation of the Biomphalaria hemocytes. Therefore, expression of lectin ligands on Biomphalaria hemocytes would be associated with snail resistance against S. mansoni infection. To test this hypothesis, circulating hemocytes were isolated from B. glabrata BH (snail strain highy susceptible to S. mansoni), B. tenagophila Cabo Frio (moderate susceptibility), and B. tenagophila Taim (completely resistant strains), labelled with FITC conjugated lectins (ConA, PNA, SBA, and WGA) and analyzed under fluorescence microscopy. The results demonstrated that although lectin-labelled hemocytes were detected in hemolymph of all snail species tested, circulating hemocytes from both strains of B. tenagophila showed a larger number of lectin-labelled cells than B. glabrata. Moreover, most of circulating hemocytes of B. tenagophila were intensively labelled by lectins PNA-FITC and WGA-FITC, while in B. glabrata small hemocytes were labeled mainly by ConA. Upon S. mansoni infection, lectin-labelled hemocytes almost disappeared from the hemolymph of Taim and accumulated in B. glabrata BH. The role of lectins/carbohydrate binding in resistance of B. tengophila infection to S. mansoni is still not fully understood, but the data suggest that there may be a correlation to its presence with susceptibility or resistance to the parasite.     

The hemocytes of Hyalophora cecropia (Lepidoptera)

The hemocytes of selected stages of Hyalophora cecropia from first instar larvae to four-day-old adults were examined and compared with those of Samia cynthia and Antheraea polyphemus. Five classes and two subclasses of hemocytes are described in these moths: (1) prohemocytes, (2) plasmatocytes (of several morphological types), (3) spherule cells, (4) adipohemocytes (two subclasses), and (5) oenocytoids. All types except oenocytoids and subclass II adipohemocytes, are found in all stages examined. Mitotic figures were common among prohemocytes of most stages, but were seen only rarely among plasmatocytes and adipohemocytes, and were not seen among spherule cells or oenocytoids. Prohemocytes and plasmatocytes often contain lipid but rarely PAS positive material. Spherules of spherule cells are PAS positive, as are occasional cytoplasmic inclusions of oenocytoids. Adipohemocytes of both subclasses contain lipid and PAS positive materials in all stages examined. Adipohemocytes and plasmatocytes proved to be most active in phagocytizing ink. Relationships between hemocytes of these and other insects, and some possible functions of hemocytes are discussed.     

Bacterial challenge and eicosanoids act in plasmatocyte spreading

We report on experiments designed to more thoroughly document the roles of eicosanoids as crucial elements in cell spreading and on experiments designed to test the hypothesis that in vivo bacterial infections influence cell spreading on glass surfaces. We used hemocytes prepared from tobacco hornworms, Manduca sexta (L.) (Lepidoptera: Sphingidae) and four species of bacteria (Serratia marcescens, Escherichia coli, Bacillus subtilis, and Micrococcus luteus) in each experiment. Our protocols yielded several important points: (i) hemocytes prepared from hornworms at 15 and 60 min following infection with, separately, each of the four bacterial species were fundamentally altered in size (all less than the 15‐µm counting cut‐off) and none of the hemocytes exhibited cell‐spreading behavior; (ii) the influence of bacterial challenge on cell spreading declined with incubation time post‐challenge; (iii) conditioned medium (CM) prepared by exposing hemocytes to bacterial cells in vitro exerted a strong dose‐dependent influence on cell spreading. Specifically, plasmatocytes increased in length from about 38 µm with 2.5% CM to a maximum of about 54 µm at 100% CM; and (iv) the retarding influence of dexamethasone (an eicosanoid biosynthesis inhibitor) on cell spreading was reversed by arachidonic acid, prostaglandin H₂, and CM. Taken together, these findings indicate that both bacterial infection and eicosanoids influence hemocyte‐spreading processes.     

Effects of different vertebrate growth factors on primary cultures of hemocytes from the gastropod mollusc,Haliotis tuberculata

Summary— A useful experimental system from primary cultures of hemocytes from Haliotis tuberculata has been established. Six days after initiation of the culture, the viability of hemocytes remained constant as measured by the MTT assay. In addition, hemocytes showed physiological responses as judged by protein and DNA syntheses in response to treatment with vertebrate growth factors. Porcine insulin and human epidermal growth factor (EGF) stimulated [³H]-leucine and [³H]-thymidine incorporation in hemocytes in a dose-dependent manner. No additive effect of insulin and EGF is observed either for [³H]-leucine or for [³H]-thymidine incorporation. The response of primary cultures of abalone hemocytes to vertebrate growth factors confirms their growth potential in vitro and provides a suitable model for further studies on regulation of the control of cellular processes such as cell growth, differentiation and migration in invertebrate cells.     

The long‐term immunological effects of alloferon and its analogues in the mealworm Tenebrio molitor

The subject of this article is a search for the long‐term immunological effects of alloferon and 3 structural analogues of alloferon, which were earlier characterized by the highest pro‐apoptotic activity in Tenebrio molitor. The differences in the actions of these peptides on immune response were observed. Alloferon increased nodulation and significantly phenoloxidase activity in the hemolymph of experimentally infected T. molitor. However, [Phe(p‐NH₂)¹]‐ and [Phe(p‐OMe)¹]‐alloferon strongly inhibited cellular and humoral defense of the mealworm against Staphylococcus aureus infection. One day after injection of these peptides, the specific biochemical and morphological hallmarks of apoptosis in bacteria‐challenged hemocytes were visible; in contrast, 3 days after peptides injection in all hemocytes, caspase activation was not observed. However, these new, circulating hemocytes differed from the control and the peptide‐untreated bacteria‐challenged hemocytes. They had an increased adhesion that led to a separation of viable, anucleated fragments of hemocytes that retain the ability to adhere and to form long filopodia. The peptide‐induced separation of hemocyte fragments may resemble the formation of platelets in mammals and perhaps play a role in sealing wounds in insects. The results of in vivo studies may suggest a long half‐life of studied peptides in the hemolymph of mealworm. Moreover, we showed the importance of the N‐terminal histidine residues at position one of the alloferon molecule for its immunological properties in insects. The results obtained here show that alloferon plays pleiotropic functions in insects.     

Cytotoxic effects of parasitism and application of venom from the endoparasitoid Pimpla turionellae on hemocytes of the host Galleria mellonella

In parasitoid species devoid of polydnaviruses and virus‐like particles, venom appears to play a major role in suppression of host immunity. Venom from the pupal endoparasitoid Pimpla turionellae L. (Hymenoptera: Ichneumonidae) has previously been shown to contain a mixture of biologically active components, which display potent paralytic, cytotoxic, and cytolytic effects toward lepidopteran and dipteran hosts. The current study was undertaken to investigate if parasitism and/or envenomation by P. turionellae affects the frequency of apoptotic and necrotic hemocytes, hemocyte viability and mitotic indices in Galleria mellonella L. (Lepidoptera: Pyralidae) pupae and larvae. Our study indicates that parasitism and experimental envenomation of G. mellonella by P. turionellae resulted in markedly different effects on the ratio of apoptotic hemocytes circulating in hemolymph depending on the host developmental stages. The ratio of early and late apoptotic hemocytes increased in G. mellonella pupae and larvae upon parasitization and at high doses of venom when compared to untreated, null and Phosphate Buffered Saline (PBS) injected controls. In contrast, an increase in necrotic hemocytes was only observed in parasitized pupae at 24 h and no difference was observed in larvae. The lowest hemocyte viability values were observed with pupae as 69.87%, 69.80%, and 72.47% at 4, 8, and 24 h post‐parasitism. The ratio of mitotic hemocytes also decreased in pupae and larvae upon parasitization and at high doses of venom. Staining of hemocytes with annexin V‐FITC revealed green fluorescent ‘halos’ along the plasma membranes of venom treated cells within 15 min following exposure to venom. By 1 h post‐venom – treatment, the majority of hemocytes displayed binding of this probe, indicative of early stage apoptosis. These same hemocytes also displayed a loss of plasma membrane integrity at the same time points as evidenced by accumulation of propidium iodide in nuclei.     

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

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

Opsonizing properties of an isolated hemolymph agglutinin and demonstration of lectin-like recognition molecules at the surface of hemocytes fromMytilus edulis

Summary Mytilus hemolymph was found to contain an agglutinin which could be inhibited by mucin. The agglutinin was isolated by affinity chromatography using neuraminidase-treated mucin/Sepharose.In vitro phagocytosis experiments revealed that only about 5% of washed hemocytes phagocytosed yeast cells suspended in a Tris-buffered NaCl-solution, whereas yeast suspended in hemolymph was normally ingested by more than 50% of the hemocytes. This relatively high phagocytic activity was shown to depend on the presence of two serum factors: When purified agglutinin was added to saline-suspended yeast, phagocytosis rates returned to normal, demonstrating opsonizing properties of the purified agglutinin. — On the other hand, addition of Ca⁺⁺-ions to saline caused an increase of the phagocytic activity of hemocytes. This was interpreted to indicate the activation of divalent cation-dependent recognition molecules at the hemocyte surface. The function of these postulated recognition factors was demonstrated by phagocytosis inhibition tests. Their location at the hemocyte membrane became evident by binding of specific antiagglutinin IgG purified by help of an agglutinin/Sepharose column from an antiserum raised againstMytilus serum proteins. Consequently, humoral as well as cell bound agglutinin molecules are involved in the attachment of yeast cells toMytilus hemocytes which subsequently internalize foreign cells.Abbreviations DAB dimethylamino benzaldehyde - PO peroxidase - IgG immunoglobulin G     

Site of hemolymph lectin production and its activation in vitro by 20-hydroxyecdysone

To identify the tissues which produce hemolymph lectin in larvae of Bombyx mori, ovary, testis, fat body, and hemocytes from 5th-instar larvae were cultured in vitro and the culture medium was partially purified and assayed for hemagglutinating activity. Among the tissues tested, hemocytes appeared to be a major source of the hemolymph lectins. Ovary produced lectins to about one-tenth of the amount observed for the hemocytes, whereas testis and fat body were not productive. To study the hormonal control of hemolymph lectin production by hemocytes, hemocytes from 4th-instar larvae were cultured in vitro. Hemagglutinating activity in the hemolymph of 4th-instar larvae was immunostainable with the monoclonal antibody raised against 350,000 dalton lectin found in the 5th-instar hemolymph, but their molecular sizes were larger than the 5th-instar hemolymph lectins. When 20-hydroxyecdysone was added into the medium, production of the lectin by the hemocytes was remarkably enhanced, depending upon the hormone concentration.     

Evasion of host defense by in vivo-produced protoplast-like cells of the insect mycopathogen Beauveria bassiana.

In vivo cells (hyphal bodies) of the hyphomycetous insect pathogen Beauveria bassiana collected from host Spodoptera exigua larval hemolymph were osmotically sensitive and lacked a well-defined cell wall. In light and electron microscope studies, a galactose-specific lectin purified from S. exigua hemolymph, concanavalin A (specific for alpha-mannose), and a polyclonal antibody to B. bassiana cell walls all bound to surfaces of in vitro-produced B. bassiana blastospores; however, none of these probes labelled the thin layer of extracellular material covering the plasma membranes of hyphal bodies. These cells were observed freely circulating in S. exigua hemolymph at 36 h postinfection, although immunocompetent hemocytes were known to be present. Additionally, association of hyphal bodies with hemocytes in monolayers was significantly less than for opsonized in vitro blastospores or submerged conidia. The absence of antigenically important galactomannan components on in vivo cells may therefore allow these cells to escape recognition and phagocytosis. Lack of structural components (e.g., chitin, as evidenced by the absence of binding of wheat germ agglutinin) may also be important with respect to evasion of host cellular defense mechanisms. Production of wall material resumed 48 to 60 h postinfection and therefore may coincide with loss of phagocytic capabilities of the hemocytes due to immunosuppressive effects of fungal metabolites. The protoplast-like cells may be formed by the action of hydrolytic enzymes in the hemocytes or by inhibition of fungal cell wall synthetases.     

Hemocytic defense response to the entomopathogenic fungus Beauveria bassiana in the migratory grasshopper Melanoplus sanguinipes

Hemocytic defense response of the migratory grasshopper, Melanoplus sanguinipes (Fab.), to conidia of the white muscardine fungus, Beauveria bassiana (Bals.) Vuill., was studied using phase-contrast photomicroscopy, hemocyte counting and hemocyte aggregation or nodule formation. Grasshoppers were injected with an aqueous suspension of conidia. Adherence of B. bassiana conidia to granulocytes occurred within 10 min and the conidia were encapsulated by these hemocytes 6 h postinjection. Hemocytopenia was accompanied by nodule formation after injection of B. bassiana conidia into grasshoppers. The conidia germinated within the nodules and grew into hyphal forms within the hemolymph 12 to 24 h postinjection. We conclude that B. bassiana competes well with nodule formation by hemocytes of M. sanguinipes and often escapes complete encapsulation.

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Résumé Le mécanisme de défense du hémocyte de la sauterelle migratrice Melanoplus sanguinipes envers le champignon pathogène Beauveria bassiana a été etudié a l'aide du photomicroscope a contrase de phase, par dénombrement des hemocytes, ainsi que des nodules formées par l'agrégation des hémocytes. L'adhérence des conidies de B. bassiana aux hemocytes a été observée 10 min après l'injection et leur encapsulement après 1 h. Une baisse des taux d'hémocytes a fait suite a la formation de nodules apres l'injection de conidies dans les sauterelles. Après le déclin initial du taux des hémocytes, une hausse s'est produite dans les sauterelles auquelles on a injecté 10⁶ conidies. Les conidies ont germé dans les nodules et la croissance du mycélium s'est produite dans l'hémolymphe 24 h après avoir injecté. Cette étude a revelé que M. sanguinipes peut exercer temporairement un mécanisme de défense cellulaire contre des conidies fongiques a une concentration de 10⁶ conidies.

     

Hemocytes of the palaemonids Macrobrachium rosenbergiiand M. acanthurus,and of the Penaeid Penaeus paulensis

The hemocytes of two palaemonids and one penaeid were characterized using light and transmission electron microscopy (TEM). The blood cells in all three species were classified as hyaline hemocytes (HH), small granule hemocytes (SGH), and large granule hemocytes (LGH). The HH are unstable hemocytes with a characteristic high nucleo-cytoplasmic ratio. Their cytoplasm appears particularly dense and has from few to numerous granules that often exhibit a typical striated substructure. In both palaemonids, the great majority of the HH contain numerous granules, whereas in Penaeus paulensis, a small number of these cells have few or no granules. The cytoplasm of some HH of the penaeid exhibits typical electron-dense deposits. The granulocytes, LGH and SGH, contain abundant electron-dense granules that are usually smaller in the SGH. In both hemocyte types, the cytosol, but not the granules, is rich in carbohydrates (PAS positive) and numerous vesicles contain acid phosphatase (Gomori reactive). In all studied shrimps, the SGH and LGH were actively phagocytic when examined on blood cell monolayers incubated with the yeast Saccharomyces cerevisiae. A few mitotic figures (less than 1%) were observed in the granulocytes of P. paulensis, but not in the palaemonids. SGH is the main circulating blood cell type in both palaemonids, whereas HH is predominant in the penaeid. Based on morphological and functional features, it appears that the hyaline and the granular hemocytes of the three shrimp species represent different cell lineages. J. Morphol. 236:209–221, 1998. © 1998 Wiley-Liss, Inc.     

Surface antigens of Xenorhabdus nematophila (F. Enterobacteriaceae) and Bacillus subtilis (F. Bacillaceae) react with antibacterial factors of Malacosoma disstria (C. Insecta: O. Lepidoptera) hemolymph

Previous research established different interactions of the insect pathogen, Xenorhabdus nematophila and nonpathogen, Bacillus subtilis, with antimicrobial hemocytes and humoral factors of larval Malacosoma disstria [Giannoulis, P., Brooks, C.L., Dunphy, G.B., Mandato, C.A., Niven, D.F., Zakarian, R.J., 2007. Interaction of the bacteria Xenorhabdus nematophila (Enterobacteriaceae) and Bacillus subtilis (Bacillaceae) with the hemocytes of larval Malacosoma disstria (Insecta: Lepidoptera: Lasicocampidae). J. Invertebr. Pathol. 94, 20-30]. The antimicrobial systems were inhibited by X. nematophila and stimulated by B. subtilis. The bacterial surface antigens participating in these reactions were unknown. Thus, herein the effects of lipopolysaccharide (endotoxin) from X. nematophila and lipoteichoic acid from B. subtilis on the larval M. disstria immune factors, the hemocytes and phenoloxidase, were determined. Endotoxin elevated the level of damaged hemocytes limiting the removal of X. nematophila from the hemolymph and enhancing the rapid release of bacteria trapped by nodulation. Similar effects were observed with the lipid A moiety of the endotoxin. The effects of lipopolysaccharide and lipid A on the hemocyte activities were abrogated by polymyxin B (an antibiotic that binds to lipid A) confirming lipopolysaccharide as the hemocytotoxin by virtue of the lipid A moiety. Lipoteichoic acid elicited nodulation and enhanced phenoloxidase activation and/or activity. Although lipoidal endotoxin and lipid A inhibited phenoloxidase activation they enhanced the activity of the enzyme. Apolipophorin-III precluded the effects of lipopolysaccharide, lipid A, and lipoteichoic acid on the hemocytes and prophenoloxidase until the antigens exceeded a critical threshold.