Maintenance of redox balance by antioxidants in hemolymph of the greater wax moth Galleria mellonella larvae during encapsulation response

The lipid peroxidation process in hemocytes, activities of phenoloxidase and key enzymatic antioxidants (superoxide dismutase, glutathione‐S‐transferase, catalase) and nonenzymatic antioxidants (thiols, ascorbate) in hemolymph of the greater wax moth Galleria mellonella L. (Lepidoptera: Pyralidae) were studied during the encapsulation process of nylon implants. It has been established that as soon as 15 min after piercing a cuticle with the implant, a capsule is formed on its surface. Active melanization of the capsule has been shown to last for 4 h. During the first hours after incorporating the implant, an increase in phenoloxidase activity and lipid peroxidation in the insect hemocytes has been revealed. Adhesion and degranulation on the surface of foreign object lead to the depletion of total hemocytes count (THC). Our results indicated that thiols and ascorbate molecules take part in the immediate antioxidant response, during later stages of encapsulation process hemolymph glutathione‐S‐transferase detoxifies and protects insect organism thereby restoring the internal redox balance. We suggest that nonenzymatic and enzymatic antioxidants of hemolymph plasma play a key role in the maintenance of redox balance during encapsulation of foreign targets.     

Cellular immune response of brown soft scale Coccus hesperidum L. (Hemiptera: Coccidae) to eggs of Metaphycus luteolus Timberlake (Hymenoptera: Encyrtidae)

The parasitic wasp, Metaphycus luteolus Timberlake, is an endoparasitoid of various soft scale insects including brown soft scale, Coccus hesperidum L. Development of this parasitoid in scale hosts is hindered by encapsulation. In the present study, using transmission and scanning electron microscopy, we show that hemocytes are responsible for encapsulation, which is mediated by the direct deposition of cells and melanin on the surface of M. luteolus eggs. By 12 h post-oviposition, scale hemocytes, presumably granulocytes, aggregate, spread and directly lyse on the surface of parasitoid eggs. This process continues for at least 1 day and results in the gradual formation of a capsule. Two to three days post-oviposition, a melanized capsule is well formed and signs of chemical deposition are evident by examination of the outer surface of the capsule. These results demonstrate that soft scale insects are fully capable of melanotic encapsulation of foreign material mediated by hemocytes.     

Generation of reactive oxygen species and activity of antioxidants in hemolymph of the moth larvae <Emphasis Type="Italic">Galleria mellonella</Emphasis> (L.) (Lepidoptera: Piralidae) at development of the process of encapsulation

Activities of enzymatic antioxidants—superoxide dismutase, glutathione-S-transferase, and catalase—as well as generation of reactive oxygen species (ROS) in lymph of the honeycomb moth Galleria mellonella L. were studied at development of the process of encapsulation of nylon implants. It has been established that as soon as 15 min after piercing of cuticle with the implant the capsule is formed on its surface. Active melanization of the capsule has been shown to last for 4 h. A statistically significant increase of the ROS generation in lymph and a decrease of the enzymatic antioxidant activities in the insect hemocytes have been revealed after the implant incorporation. The authors suggest that the key role in maintenance of the oxidation-reduction balance in hemolymph at development of the encapsulation process is played by non-oxidative antioxidants.     

Prophenoloxidase binds to the surface of hemocytes and is involved in hemocyte melanization in Manduca sexta

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 naïve 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.     

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.

     

The responses of Ligia oceanica (L.) (Crustacea: Isopoda) to infection by Maritrema linguilla Jäg. 1908 and Microphallus similis (Jäg. 1900) (Digenea: Microphallidae) and to abiotic implants

The initial and principal encapsulation response of Ligia oceanica to Araldite implants and to encysted metacercariae of Maritrema linguilla is hemolymph coagulation followed by limited hemocyte agglutination. Granules secreted by isolated granulocytes and semigranulocytes may catalyze coagulation. Isolated hyaline cells explode and make an insignificant contribution to the initial cyst wall. Later, hemocytes agglutinate and some granulocytes retain their granules which become melanized. Eventually, a wide multilayered hos capsule is formed. Unencysted metacercariae of M. linguilla transplanted from the pleopods into the dorsal hemocoel of another specimen of L. oceanica encyst and become encapsulated but are not damaged by encapsulation. Transplanted encysted metacercariae are also encapsulated and unharmed. Cercariae implanted directly into the dorsal hemocoel, however, fail to encyst, become encapsulated, die, and lyse within the capsule. Implanted cercariae and encysted metacercariae of Microphallus similis are also encapsulated and destroyed in the hemocoel of L. oceanica. The absence of host response to the naturally infecting unencysted parasite in the pleopod sinuses may be attributed to rhythmic movement, mucopolysaccharide secretions and to the retention of excreta within the excretory bladder. Once the excreta is released during cyst formation in the dorsal hemocoel, encapsulation occurs but this does not appear to harm the parasite. On the contrary, considerable growth occurs within the cyst which suggests that the parasite may absorb nutrients released from necrotic hemocytes.     

Hemocyte reactions and early cellular changes during melanotic tumor formation in Drosophila melanogaster

In Drosophila melanogaster tu bw larvae melanotic tumors form as a result of a cell-mediated immune response involving the encapsulation and melanization by hemocytes of portions of the caudal adipose tissue. The tissue-specific encapsulation response is not due to the disintegration of the basement membrane surrounding the adipose tissue as is reported to be the case in other melanotic mutants. Prior to encapsulation large numbers of hemocytes appear in the circulation and begin to differentiate into flattened cells termed lamellocytes. This transformation occurs at a time when changes are noted within the adipose cells. The localized accumulation of blood cells near intact basement membrane suggests that abnormally developing adipose cells acquire altered molecular surfaces or release substances to which the hemocytes respond. The initial reaction of the hemocytes with the adipose tissue is cell lysis, and this is rapidly followed by the aggregation of numerous additional blood cells which eventually cohere to one another to form a multilayered capsule. What little evidence there is of disintegration of the basement membrane occurs only after hemocytes have lysed at the surface, and other blood cells begin to invade the adipose tissue. Melanization occurs first in the intercellular spaces along the plasma membranes of the lysed cells, and progresses from the innermost layers toward the periphery of the encapsulating cells. Since the changes observed in the hemocytes and adipose cells are precocious, occurring to a lesser degree later in normal development, the initial effect of the genetic mutation in tu bw larvae may be an endocrine dysfunction which causes an asynchronous and abnormal development of the caudal adipose tissue and/or the hematopoietic organs.     

Effects of parasitism and pesticide exposure on characteristics and functions of hemocyte populations in the freshwater snail Lymnaea palustris (Gastropoda, Pulmonata)

Morphological characteristics and functions of hemocytes were used to compare the immunological effects of biological and chemical stress in the freshwater snailLymnaea palustris. Animals were either infected by a trematode parasite (Metaleptocephalus sp.), or exposed to environmental contaminants, namely atrazine and hexachlorobenzene (HCB). Three populations of circulating hemocytes, morphologically and cytochemically distinct (round cells, hyalinocytes, granulocytes), were identified in both control and parasitized or pesticide-exposed snails. After 6 h of exposure, HCB and atrazine resulted in 8-fold increases in the mean total number of hemocytes, whereas only a 2.2-fold increase was observed 6 h after cercaria emission in parasitized snails. The impact of HCB was limited to the first 24 h of exposure, whereas long-lasting effects of atrazine were observed. Hyalinocytes and, to a lesser extent, round cells contributed most to the increases in hemocyte density in pesticide-exposed snails. Parasitism and atrazine treatment resulted in significant increases of lectin-stained hemocytes, whereas exposure to HCB did not affect the percentages of stained and unstained cells. Hemocyte phagocytic activity increased in HCB-exposed snails but with no concomitant change of the oxidative burst. Opposite results were obtained in atrazine-treated snail hemocytes, with unchanged phagocytosis and decreased phorbol 12-myristate 13-acetate-stimulated production of reactive oxygen intermediates. No increase in phagocytosis, or in the production of reactive oxygen intermediates, was observed in hemocytes from parasitized snails. Infection with the immunologically compatible trematode parasiteMetaleptocephalus sp. and exposure to atrazine generated similar reactions from circulating hemocytes, whereas a different response pattern was observed in HCB-exposed snails. This revised version was published online in July 2006 with corrections to the Cover Date.     

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

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

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.

     

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

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

Role of venom and ovarian proteins in immune suppression of Ostrinia furnacalis （Lepidoptera： Pyralidae） larvae parasitized by Macrocentrus cingulum （Hymenoptera： Braconidae）, a polyembryonic parasitoid

Venom and ovarian proteins in braconid and ichneumonid wasps play an important role in the successful parasitism of their host, especially for immune suppression immediately after oviposition. In this study, we compared the effect of venom and ovarian proteins collected from the female wasps of Macrocentrus cingulum, a polyembryonic parasitoid of the larvae of Ostriniafunacalis, on the encapsulation capacity of Sephadex A- 25 beads at 4 h and 24 h post-injection both in vivo and in vitro. The results showed that the ovarian proteins significantly interfered with the encapsulation capacity of hemocytes in a dose-dependent manner. Spreading and viability of hemocytes in O. furnacalis was not disrupted by venom and ovarian proteins at various concentrations injected. It seems likely that the ovarian proteins from M. cingulum play a role in suppressing the encapsulation capacity of host hemocytes.     

Morphofunctional characterization of hemocytes in black soldier fly larvae

In insects, the cell-mediated immune response involves an active role of hemocytes in phagocytosis, nodulation, and encapsulation. Although these processes have been well documented in multiple species belonging to different insect orders, information concerning the immune response, particularly the hemocyte types and their specific function in the black soldier fly Hermetia illucens, is still limited. This is a serious gap in knowledge given the high economic relevance of H. illucens larvae in waste management strategies and considering that the saprophagous feeding habits of this dipteran species have likely shaped its immune system to efficiently respond to infections. The present study represents the first detailed characterization of black soldier fly hemocytes and provides new insights into the cell-mediated immune response of this insect. In particular, in addition to prohemocytes, we identified five hemocyte types that mount the immune response in the larva, and analyzed their behavior, role, and morphofunctional changes in response to bacterial infection and injection of chromatographic beads. Our results demonstrate that the circulating phagocytes in black soldier fly larvae are plasmatocytes. These cells also take part in nodulation and encapsulation with granulocytes and lamellocyte-like cells, developing a starting core for nodule/capsule formation to remove/encapsulate large bacterial aggregates/pathogens from the hemolymph, respectively. These processes are supported by the release of melanin precursors from crystal cells and likely by mobilizing nutrient reserves in newly circulating adipohemocytes, which could thus trophically support other hemocytes during the immune response. Finally, the regulation of the cell-mediated immune response by eicosanoids was investigated.     

An ultrastructural study on the encapsulation of microfilariae of Brugia pahangi in the haemocoel of Anopheles quadrimaculatus

Chen C. C. and Laurence B. R. 1985. An ultrastructural study on the encapsulation of microfilariae of Brugia pahangi in the haemocoel of Anopheles quadrimaculatus. International Journal for Parasitology15: 421–428. The encapsulation of microfilariae of Brugia pahangi in the haemocoel of Anopheles quadrimaculatus was studied ultrastructurally. The microfilariae was first seen enclosed in an acellular electron dense capsule as early as 10 min after the engorgement of the mosquitoes from a cat parasitized by filariae. Two hours later, the mosquito plasmatocytes spread onto and around the humoral capsule. A completed capsule, which was seen at 24–48 h, was composed of an inner humoral layer and outer cellular layer. After 1 week, some electron dense haemocytes were seen attached to the outer surface of the cellular layer. These results suggested that the encapsulation of microfilariae in the haemocoel of mosquitoes combines both humoral and cellular reaction; humoral encapsulation occurs first and cellular encapsulation takes place later. The significance of combined reactions of humoral and cellular encapsulation in the mosquito-filarial system is discussed with reference to the encapsulation reaction of other insects.     

Surface characteristics of foreign targets that elicit an encapsulation response by the moth Pseudoplusia includens

Hemocytes from the moth Pseudoplusia includens encapsulate a variety of biotic and abiotic targets. Prior studies indicated that granular cells are usually the first hemocyte type to attach to foreign targets. Thereafter, large numbers of plasmatocytes attach to the target and form a capsule. To identify surface features that induce an encapsulation response, chromatography beads that differed in matrix composition, charge, and functional groups were tested using in vitro and in vivo bioassays. We first conducted in vitro assays using hemocytes with no plasma components present. These experiments indicated that bead types having sulfonic, diethylaminoethyl, and quaternary amine functional groups were encapsulated significantly more often than beads with other functional groups. Charge also significantly affected encapsulation with positively charged beads being encapsulated more often than negatively charged or neutral beads. In vitro assays using purified populations of hemocytes confirmed that these targets were recognized as foreign by granular cells, and that plasmatocytes only formed capsules after granular cells attached to the target. Bead types that were encapsulated under these in vitro conditions were always rapidly encapsulated when injected into P. includens larvae. However, some bead types, like CM-Sephadex, not encapsulated in vitro were encapsulated in vivo if left in the insect hemocoel for a longer period of time (ca. 24 h). Purified plasmatocytes encapsulated these beads in vitro if they were preincubated in plasma. Basic characterization studies suggest these humoral recognition molecules are proteins or small peptides. Comparative studies with other species of noctuid moths also indicated that encapsulation of some bead types differed significantly among species. Collectively, these results reveal that P. includens recognizes some targets as foreign by pattern recognition receptors on granular cells, whereas others are recognized by pattern recognition molecules in plasma. The binding affinities of these recognition molecules also appear to differ among closely related species of Lepidoptera.     

Expression and hemocyte-targeting of a Campoletis sonorensis polydnavirus cysteine-rich gene in Heliothis virescens larvae

The polydnavirus associated with the parasitic wasp Campoletis sonorensis is injected into the lepidopteran insect, Heliothis virescens, during parasitization, after which viral gene products suppress the cellular immune system of the hosts. Four related cysteine-rich polydnavirus genes have been identified in parasitized H. virescens larvae and grouped into a family. In this study, we investigated the expression and hemocyte targeting of the cysteine-rich Vhv1.4 protein. Full- length and truncated Vhv1.4 proteins were produced in a bacterial expression system, and the purified proteins were used to raise polyclonal antisera. In immunoblots the Vhv1.4 protein was detected in parasitized insects as early as 6 h and throughout the entire course of parasitism. The Vhv1.4 protein appeared predominantly in the plasma fraction of hemolymph from parasitized larvae, suggesting that this protein is secreted. The Vhv1.4 protein expressed from a recombinant baculovirus was secreted in two lepidopteran cell lines and in larvae injected with the recombinant virus. Digestion with endoglycosidases suggests that the Vhv1.4 protein is glycosylated at multiple N-glycosylation sites. Immunofluorescence assays showed that the Vhv1.4 protein binds to the hemocytes, most notably the granulocytes, in H. virescens larvae. After binding, the Vhv1.4 protein was internalized, probably by endocytosis. Specific binding of the Vhv1.4 to granulocytes implies an important function in the suppression of host cellular encapsulation response. Arch. Insect Biochem. Physiol. 36:251–271, 1997. © 1997 Wiley-Liss, Inc.     

Sydney rock oyster (Saccostrea glomerata) hemocytes: morphology and function

In this study, three major hemocyte types were identified in the Sydney rock oyster. They were characterized primarily by light and electron microscopy based on the presence or absence of granules and nucleus to cytoplasm ratios. Hemoblast-like cells were the smallest cell type 4.0+/-0.4microm and comprised 15+/-3% of the hemocyte population. They had large nuclei and scanty basic cytoplasm. This cell type also had some endoplasmic reticuli and mitochondria. The second major type were hyalinocytes. Hyalinocytes represented 46+/-6% of all hemocytes. They were large cells (7.1+/-1.0microm) that had low nucleus:cytoplasm ratios and agranular basic or acidic cytoplasm. Hyalinocytes had the ability to phagocytose yeast cells and formed the core of hemocyte aggregates associated with agglutination. Four discrete sub-populations of hyalinocytes were identified. The third major cell type were the granulocytes, comprising 38+/-1% of the hemocyte population. These cells were large (9.3+/-0.3microm) and were characterized by cytoplasm containing many acidic or basic granules. Granulocytes were more phagocytic than hyalinocytes and they formed the inner layer of hemocytes during the encapsulation of fungal hyphae. Five discrete sub-populations of granulocytes were identified based on the types of granules in their cytoplasm. Flow cytometry showed that the hemocytes of rock oysters could be divided into between two and four major cell types based on their light scattering properties. The most common of the cell types identified by flow cytometry corresponded to hyalinocytes and granulocytes. Cytochemical assays showed that most enzymes associated with immunological activity were localized in granulocytes. Their granules contained acid phosphatase, peroxidase, phenoloxidase, superoxide and melanin. Hyalinocytes were positive only for acid phosphatase. All of these observations suggest that Sydney rock oysters have a broad variety of functionally specialized hemocytes, many of which are involved in host defense.     

Cellular and humoral immune defenses of Oxya japonica (Orthoptera: Acrididae) to entomopathogenic fungi Metarhizium anisopliae

One Indonesian isolate of the fungus Metarhizium anisopliae, named Majalengka strain, was evaluated not only for its virulence but also for the immune response of rice grasshopper Oxya japonica (Orthoptera: Acrididae) as a target organism. Five aqueous suspensions with different conidia concentrations in logarithmic series were prepared. The fungus showed high virulence as it caused 100% mortality at low conidia concentration (1.5 × 10² conidia/mL). Remarkable changes in the cellular and humoral responses were also observed when adult grasshoppers were infected with the fungus. The number of hemocytes decreased significantly within 12 h after infection. In addition, the total number of granulocytes increased rapidly in the first 12 h then gradually decreased 24 and 48 h after infections, while the number of coagulocytes fluctuated over time. The infection influenced the humoral response by increasing the phenoloxidase activity.     

A hemocyte-specific integrin required for hemocytic encapsulation in the tobacco hornworm, Manduca sexta

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 beta subunit. A cDNA for this M. sexta integrin beta1 was cloned and characterized. Integrin-beta1 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 beta1 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-beta1 RNA into larvae resulted in decreased integrin beta1 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.     

Primary culture of hemocytes from the Caribbean spiny lobster, Panulirus argus, and their susceptibility to Panulirus argus Virus 1 (PaV1)

Primary cultures of hemocytes from the Caribbean spiny lobster Panulirus argus were developed for studies on the in vitro propagation of Panulirus argus Virus 1 (PaV1). A modified Leibovitz L-15 medium supported the best survival of hemocytes in in vitro primary cultures. However, degradation of the cultures occurred rapidly in the presence of granulocytes. A Percoll step gradient was used to separate hemocytes into three subpopulations enriched in hyalinocytes, semigranulocytes, and granulocytes, respectively. When cultured separately, hyalinocytes and semigranulocytes maintained higher viability ( approximately 80%) after 18 days incubation compared with granulocytes, which degraded over 2-3 days. Susceptibility of the cell types was investigated in challenge studies with PaV1. Hyalinocytes and semigranulocytes were susceptible to PaV1. Cytopathic effects (CPE) were observed as early as 12h post-inoculation, and as the infection progressed, CPE became more apparent, with cell debris and cellular exudates present in inoculated cultures. Cell lysis was noticeable within 24h of infection. The presence of virus within cells was further confirmed by in situ hybridization using a specific DNA probe. The probe gave a unique staining pattern to cells infected with PaV1 24-h post-inoculation. Cells in the control treatment were intact and negative to hybridization. This assay was further applied to the quantification of infectious virus in hemolymph using a 50% tissue culture infectious dose assay (TCID(50)) based on CPE. These tools will now allow the quantification of PaV1 using established culture-based methods.