Immune challenges trigger cellular and humoral responses in adults of Pterostichus melas italicus (Coleoptera,Carabidae)

The present study focuses on the ability of Pterostichus melas italicus Dejean to mount cellular and humoral immune responses against invading pathogens. Ultrastructural analyses revealed the presence of five morphologically distinct types of hemocytes: prohemocytes, plasmatocytes, granulocytes, oenocytoids and macrophage-like cells. Differential hemocyte counts showed that plasmatocytes and granulocytes were the most abundant circulating cell types and plasmatocytes exhibited phagocytic activity following the latex bead immune challenge. Macrophage-like cells were recruited after the immune challenge to remove exhausted phagocytizing cells, apoptotic cells and melanotic capsules formed to immobilize the latex beads. Total hemocyte counts showed a significant reduction of hemocytes after latex bead treatment. Phenoloxidase (PO) assays revealed an increase of total PO in hemolymph after immune system activation with lipopolysaccharide (LPS). Moreover, the LPS-stimulated hemocytes showed increased protein expression of inducible nitric oxide synthase, indicating that the cytotoxic action of nitric oxide was engaged in this antimicrobial collaborative response. These results provide a knowledge base for further studies on the sensitivity of the P. melas italicus immune system to the environmental perturbation in order to evaluate the effect of chemicals on non-target species in agroecosystems.     

The relative abundance of hemocyte types in a polyphagous moth larva depends on diet

Hemocytes are crucial cells of the insect immune system because of their involvement in multiple immune responses including coagulation, phagocytosis and encapsulation. There are various types of hemocytes, each having a particular role in immunity, such that variation in their relative abundance affects the outcome of the immune response. This study aims to characterize these various types of hemocytes in larvae of the grapevine pest insect Eupoecilia ambiguella, and to assess variation in their concentration as a function of larval diet and immune challenge. Four types of hemocytes were found in the hemolymph of 5th instar larvae: granulocytes, oenocytoids, plasmatocytes and spherulocytes. We found that the total concentration of hemocytes and the concentration of each hemocyte type varied among diets and in response to the immune challenge. Irrespective of the diet, the concentration of granulocytes increased following a bacterial immune challenge, while the concentration of plasmatocytes and spherulocytes differentially varied between larval diets. The concentration of oenocytoids did not vary among diets before the immune challenge but varied between larval diets in response to the challenge. These results suggest that the resistance of insect larvae to different natural enemies critically depends on the effect of larval diet on the larvae’s investment into the different types of hemocytes.     

Hematopoiesis in larval Pseudoplusia includens and Spodoptera frugiperda

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

Losing the battle against fungal infection: suppression of termite immune defenses during mycosis

The dampwood termite, Zootermopsis angusticollis is known to generate humoral immune responses to the entomopathogenic fungus Metarhizium anisopliae. However, little is known about how the termite's cellular immune system reacts to fungal infection. To test the effect of conidia exposure on cellular immunity, we quantified the number and types of hemocytes in the hemolymph of naïve nymphs and compared their circulating counts with those of nestmates exposed to 0, 2 × 10³, 2 × 10⁶ or 2 × 10⁸ conidia/ml doses. These termites were then bled and their hemocytes counted on days 1, 2, 3, 4, 7 post-exposure. Our results show, first, that naïve Z. angusticollis nymphs have three different blood cell types tentatively identified as granular hemocytes, prohemocytes and plasmatocytes. In these individuals, plasmatocytes were on average 13.5 and 3.3 times more numerous than granular hemocytes and prohemocytes, respectively. Second, a full factorial general linear analysis indicated that hemocyte type, time elapsed since conidia exposure and conidia dosage as well as all their interactions explained 43% of the variability in hemocyte density. The numbers of prohemocytes and particularly plasmatocytes, but not granular hemocytes, appear to be affected by the progression of disease. The decline in hemocyte numbers coincided with the appearance of hyphal bodies and the onset of “sluggish” termite behavior that culminated in the insect's death. Hemocyte counts of infected males and females were affected to the same extent. Hence, M. anisopliae overtakes the cellular immune responses of Z. angusticollis mainly by destroying the host's most abundant hemocyte types.     

Characterization of the Hemocytes in Larvae of Protaetia brevitarsis seulensis: Involvement of Granulocyte-Mediated Phagocytosis

Hemocytes are key players in the immune response against pathogens in insects. However, the hemocyte types and their functions in the white-spotted flower chafers, Protaetia brevitarsis seulensis (Kolbe), are not known. In this study, we used various microscopes, molecular probes, and flow cytometric analyses to characterize the hemocytes in P. brevitarsis seulensis. The circulating hemocytes were classified based on their size, morphology, and dye-staining properties into six types, including granulocytes, plasmatocytes, oenocytoids, spherulocytes, prohemocytes, and adipohemocytes. The percentages of circulating hemocyte types were as follows: 13% granulocytes, 20% plasmatocytes, 1% oenocytoids, 5% spherulocytes, 17% prohemocytes, and 44% adipohemocytes. Next, we identified the professional phagocytes, granulocytes, which mediate encapsulation and phagocytosis of pathogens. The granulocytes were immunologically or morphologically activated and phagocytosed potentially hazardous substances in vivo. In addition, we showed that the phagocytosis by granulocytes is associated with autophagy, and that the activation of autophagy could be an efficient way to eliminate pathogens in this system. We also observed a high accumulation of autophagic vacuoles in activated granulocytes, which altered their shape and led to autophagic cell death. Finally, the granulocytes underwent mitotic division thus maintaining their number in vivo.     

Characterization of cell clusters in larval hemolymph of the cabbage armyworm Mamestra brassicae and their role in maintenance of hemocyte populations

Maintenance of hemocyte populations is critical for both development and immune responses. In insects, the maintenance of hemocyte populations is regulated by mitotic division of circulating hemocytes and by discharge from hematopoietic organs. We found cell clusters in the hemolymph of Mamestra brassicae larvae that are composed of small, spherical cells. Microscopic observations revealed that the cells in these clusters are similar to immature or precursor cells present in hematopoietic organs. The results of bromodeoxyuridine (BrdU) incorporation experiments demonstrate that these cells are mitotically active. Furthermore, these cells maintain their immature state and proliferate until late in the last larval instar. The results of in vitro experiments showed that most of the cells changed their morphology to one consistent with plasmatocytes or granulocytes, and that the change was promoted by addition of larval hemolymph to the culture medium, in particular when hemolymph was collected at a prepupal stage. Taken together, our results suggested that cells in clusters may be an additional source of hemocytes during larval development.     

Ultrastructure of the hemocytes of Cetonischema aeruginosa larvae (Coleoptera,Scarabaeidae): involvement of both granulocytes and oenocytoids in in vivo phagocytosis

In the context of comparative studies on immunity defence mechanisms of adults and larvae of the coleopteran Cetonischema aeruginosa (Drury, 1770) the ultrastructure of the circulating hemocytes of the third instar larval stage has been investigated by means of light and transmission electron microscopy (TEM). Six types of hemocytes were found in the hemolymph of C. aeruginosa and they were identified as prohemocytes, granulocytes, plasmatocytes, coagulocytes, oenocytoids and spherule cells. In order to identify the "professional" phagocyte cell, phagocytosis assays were performed in vivo by injection of 0.9 microm carboxylate-modified polystyrene latex beads. It was demonstrated that the granulocytes and the oenocytoids of C. aeruginosa were the only hemocyte types involved in this cellular response.     

Two Hemocyte Lineages Exist in Silkworm Larval Hematopoietic Organ

Background

Insects have multiple hemocyte morphotypes with different functions as do vertebrates, however, their hematopoietic lineages are largely unexplored with the exception of Drosophila melanogaster.

Methodology/Principal Findings

To study the hematopoietic lineage of the silkworm, Bombyx mori, we investigated in vivo and in vitro differentiation of hemocyte precursors in the hematopoietic organ (HPO) into the four mature hemocyte subsets, namely, plasmatocytes, granulocytes, oenocytoids, and spherulocytes. Five days after implantation of enzymatically-dispersed HPO cells from a GFP-expressing transgenic line into the hemocoel of normal larvae, differentiation into plasmatocytes, granulocytes and oenocytoids, but not spherulocytes, was observed. When the HPO cells were cultured in vitro, plasmatocytes appeared rapidly, and oenocytoids possessing prophenol oxidase activity appeared several days later. HPO cells were also able to differentiate into a small number of granulocytes, but not into spherulocytes. When functionally mature plasmatocytes were cultured in vitro, oenocytoids were observed 10 days later. These results suggest that the hemocyte precursors in HPO first differentiate into plasmatocytes, which further change into oenocytoids.

Conclusions/Significance

From these results, we propose that B. mori hemocytes can be divided into two major lineages, a granulocyte lineage and a plasmatocyte-oenocytoid lineage. The origins of the spherulocytes could not be determined in this study. We construct a model for the hematopoietic lineages at the larval stage of B. mori.     

Hemocytes and hemocytopoiesis in Silkworms.

A brief review is presented of the current state of ultrastructure, cytochemistry, and physiology of the hemocytes and meso- and metathoracic peri-imaginal-wing organs in silkworms. According to the accepted morphological classification, five circulating types of hemocytes are recognized in Bombyx mori as well as in Antheraea pernyi. They are prophemocytes or stem cells, plasmatocytes or pre-differentiated cells and three specialized cells, granulocytes, spherule cells and oenocytoids. During post-embryonic development the last four types are the most common in the circulating hemolymph. Plasmatocytes are considered to be pluripotent cells from which granulocytes, spherule cells and oenocytoids are derived. Contrary to the situation in most insects the plasmatocytes are not phagocytic in Antheraea. The granulocytes are efficient phagocytes. Both plasmatocytes and granulocytes are involved in pinocytosis. Another possible function of the granulocytes is hemolymph coagulation. The function of the spherule cells which contain a paracrystalline material (muco- or glycoproteins) is by no means clear. The phenoloxidase activity found within the cytosol of oenocytoids appears effective against the natural monophenol and diphenol substrates. The involvement of oenocytoids in the complex metabolism of phenols and particularly in the production of plasma phenolases has been reported. The mitotic division of five circulating hemocyte types is well known and was long regarded as the only mechanism of postembryonic hemocyte production. We present for silkworms, experimental evidence of the hemocytopoietic function of the meso- and metathoracic organs surrounding the imaginal wing discs. Ablation experiments demonstrate that the mitotic activity of free hemocytes is unable to maintain the normal hemocytogram in the absence of the two paris of organs. These organs are typically divided into cell islets ensheathed by a connective tissue membrane. Two types of islets may be classified by the disposition of the cells : the compact islets or aggregations of stem cells and the reticulate islets which are mainly composed of hemocytes at different steps of differentiation. The relative number of prohemocytes in the total hemocyte population ranges from 84 to 97 p. cent in organs of Antheraea pernyi. This well-defined cell type appears to be the major hemocyte type in hemocytopoietic organs. In Antheraea, the mitotic index (the relative number of mitotic hemocytes in the total cell population) varies from 0.5 to about 3 p. cent. Finally, our data direct attention to cyclic functional changes such as mitotic divisions and hemocyte differentiation which run parallel to the molting cycle.     

Hemocyte differentiation in the hematopoietic organs of the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>: prohemocytes have the function of phagocytosis

Hemocytes isolated from the larval hematopoietic organs of the silkworm were classified following staining with acridine orange and propidium iodide. Among the hemocytes isolated from the hematopoietic organs of whole fifth larval and wandering stages, most were prohemocytes (60%–70%) and oenocytoids (30%–40%). Granulocytes comprised only about 0.5%–1% at the wandering stage and were even rarer at other stages; no spherulocytes or plasmatocytes were found. Therefore, hemocyte differentiation inside larval hematopoietic organs is not as extensive as previously thought. Following 10–30 min in vitro culture of hemocytes isolated from larval hematopoietic organs, many young granulocytes and plasmatocytes appeared. Furthermore, during phagocytosis assays, prohemocytes were seen to adopt the morphology of plasmatocytes, containing fragments of phagocytosed cells. Our results underline the similarities between Drosophila and Bombyx hematopoiesis.     

胚后发育期臭腹腺蝗中的血细胞种类

应用血球计数器统计了胚后发育期臭腹腺蝗Zonocerus variegatus中存在的血细胞类型和数目。从1龄幼虫至成虫的发育阶段中共观察到6种血细胞类型,即原血细胞 （PRS）、 浆血细胞 （PLS）、粒细胞 （GRS）、珠血细胞 （SPS）、绛色细胞（OES） 和adipohaemocytes （ADS）。不过,在1龄幼虫期未发现OES。在这6种血细胞中,PLS的总平均数最高,OES的总平均数最低。成虫期的血细胞数目显著高于其他发育阶段（P<0.05）,而1龄幼虫和2龄幼虫期的血细胞数目不存在显著差异（P>0.05）。     

Parasitism by Cotesia plutellae alters the hemocyte population and immunological function of the diamondback moth, Plutella xylostella

Cotesia plutellae, a solitary endoparasitoid wasp, parasitizes the diamondback moth, Plutella xylostella, and induces host immunosuppression and lethality in the late larval stage. This study focused on changes of cellular immunity in the parasitized P. xylostella in terms of hemocyte composition and cellular functions. In third and fourth instar larvae of nonparasitized P. xylostella, granular cells represented the main hemocyte type (60-70%) and plasmatocytes were also present at around 15% among the total hemocytes. Following parasitization by C. plutellae, the relative proportions of these two major hemocytes changed very little, but the total hemocyte counts exhibited a significant reduction. Functionally, the granular cells played a significant role in phagocytosis based on a fluorescence assay using fluorecein isothiocyanate-labeled bacteria. The phagocytic activity of the granular cells occurred as early as 5 min after incubation with the bacteria, and increased during the first 40 min of incubation. The parasitism by C. plutellae significantly inhibited phagocytosis of the granular cells. Plasmatocytes also exhibited minor phagocytic activity. Moreover, plasmatocyte phagocytosis was not inhibited by parasitism. On the other hand, hemocyte-spreading behavior in response to pathogen infection was significant only for plasmatocytes, which exhibited a characteristic spindle shape upon infection. A significant spreading of the plasmatocytes was found as early as 5 min after pathogen incubation and their ratio increased during the first 40 min.An insect cytokine, plasmatocyte-spreading peptide 1 (PSP1) from Pseudoplusia includens, was highly active in inducing plasmatocyte-spreading behavior of P. xylostella in a dose-dependent manner. P. xylostella parasitized by C. plutella was significantly inhibited in plasmatocyte-spreading in response to an active dose of PSP1. An in vivo encapsulation assay showed that the parasitized P. xylostella could not effectively form the hemocyte capsules around injected agarose beads. This research demonstrates that the parasitism of C. plutellae adversely affects the total hemocyte populations in number and function, which would contribute to host immunosuppression.     

Different responses to temperature in three closely-related sympatric cereal aphids

Cotesia glomerata L. (Hymenoptera: Braconidae) is a parasitoid of early instar larvae of Pieris brassicae L. (Lepidoptera: Pieridae). Late instars of P. brassicae can more often overcome parasitization by hemocytic encapsulation of C. glomerata eggs. Short-term hemocyte responses to parasitization were examined in third and fourth instar larvae of P. brassicae. Total and differential hemocyte counts did not differ between parasitized and unparasitized host larvae. A rapid, but temporary decrease of total hemocyte as well as plasmatocyte numbers was observed immediately after oviposition. Numbers of hemocytes adhering to tissues were shown to be the same in untreated, wounded and parasitized P. brassicae larvae by tracing hemocytes with monoclonal antibodies as markers. The in vitro spreading ability of hemocytes from unparasitized third and fourth instar larvae was lower than that of the last instar's; parasitization, however, had no influence on hemocyte spreading. We therefore suggest that the higher parasitization success of C. glomerata in earlier instars of P. brassicae is mainly due to the low spreading ability of the hemocytes. Abbreviations: ACS – anticoagulant saline; BSA – bovine serum albumin; DABCO – 1,4-diazabicyclo-[2,2,2]-octane; DHC – differential hemocyte count; FITC – fluorescein isothiocyanate; GR – granular cells; LPS – lipopolysaccharide; mAb – monoclonal antibody; OE – oenocytoids; PL – plasmatocytes; PRO – prohemocytes; PS – Pieris saline; PVP – polyvinylpyrrolidone; TBS – tris-buffered saline; THC – total hemocyte count.     

Differences in Cellular Immune Competence Explain Parasitoid Resistance for Two Coleopteran Species

The immune defence of an organism is evolving continuously, causing counteradaptations in interacting species, which in turn affect other ecological and evolutionary processes. Until recently comparative studies of species interactions and immunity, combining information from both ecological and immunological fields, have been rare. The cellular immune defense in insects, mainly mediated by circulating hemocytes, has been studied primarily in Lepidoptera and Diptera, whereas corresponding information about coleopteran species is still scarce. In the study presented here, we used two closely related chrysomelids, Galerucella pusilla and G. calmariensis (Coleoptera), both attacked by the same parasitoid, Asecodes parviclava (Hymenoptera). In order to investigate the structure of the immune system in Galerucella and to detect possible differences between the two species, we combined ecological studies with controlled parasitism experiments, followed by an investigation of the cell composition in the larval hemolymph. We found a striking difference in parasitism rate between the species, as well as in the level of successful immune response (i.e. encapsulation and melanisation of parasitoid eggs), with G. pusilla showing a much more potent immune defense than G. calmariensis. These differences were linked to differences in the larval cell composition, where hemocyte subsets in both naïve and parasitised individuals differed significantly between the species. In particular, the hemocytes shown to be active in the encapsulation process; phagocytes, lamellocytes and granulocytes, differ between the species, indicating that the cell composition reflects the ability to defend against the parasitoid.     

Ontogeny of tick hemocytes: a comparative analysis of <Emphasis Type="Italic">Ixodes ricinus</Emphasis> and <Emphasis Type="Italic">Ornithodoros moubata</Emphasis>

Hemocytes of two tick species, Ixodes ricinus and Ornithodoros moubata, were investigated with the aim to determine their ultrastructural characteristics and developmental relationships. Only a limited number of ultrastructural features was shown to be unequivocally homological across all hemocyte types. The two species, representing distant groups of ticks, differ in the composition of their circular cell populations. In I. ricinus, three groups of distinct morphological types of hemocytes could be determined according to well-defined ultrastructural features: a typical non-phagocytic granular cell with electron-dense granula and homogeneous cytoplasm (Gr II), and two different types of phagocytic hemocytes, namely plasmatocytes with a low number of granula and phagocytic granolocytes, designated as Gr I. In contrast, an additional cell type resembling insect spherulocytes was determined in O. moubata. This cell type does not seem to be homologous to any I. ricinus hemocyte and may represent a cell type typical of soft ticks only. Possible ontogenetic lineages of the hemocytes of both tick-species were inferred.     

The ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) differentially affects cells mediating the immune response of its flesh fly host,Sarcophaga bullata Parker (Diptera: Sarcophagidae)

In this study, we examined cellular immune responses in the flesh fly, Sarcophaga bullata, when parasitized by the ectoparasitoid Nasonia vitripennis. In unparasitized, young pharate adults and third instar, wandering larvae of S. bullata, four main hemocyte types were identified by light microscopy: plasmatocytes, granular cells, oenocytoids, and pro-hemocytes. Parasitism of young pharate adults had a differential effect on host hemocytes; oenocytoids and pro-hemocytes appeared to be unaltered by parasitism, whereas adhesion and spreading behavior were completely inhibited in plasmatocytes and granular cells by 60 min after oviposition. The suppression of spreading behavior in granular cells lasted the duration of parasitism. Plasmatocytes were found to decline significantly during the first hour after parasitism and this drop was attributed to cell death. Melanization and clotting of host hemolymph did not occur in parasitized flies, or the onset of both events was retarded by several hours in comparison to unparasitized pharate adults. Hemocytes from envenomated flies were altered in nearly identical fashion to that observed for natural parasitism; the total number of circulating hemocytes declined sharply by 60 min post-envenomation, the number of plasmatocytes declined but not granular cells, and the ability of plasmatocytes and granular cells to spread when cultured in vitro was abolished within 1 h. As with parasitized hosts, the decrease in plasmatocytes was due to cell death, and inhibition of spreading lasted until the host died. Isolated crude venom also blocked adhesion and spreading of these hemocyte types in vitro. Thus, it appears that maternally derived venom disrupts host immune responses almost immediately following oviposition and the inhibition is permanent. The possibility that this ectoparasite disables host defenses to afford protection to feeding larvae and adult females is discussed.     

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.     

Microplitis demolitor bracovirus inhibits phagocytosis by hemocytes from Pseudoplusia includens

The braconid wasp Microplitis demolitor carries Microplitis demolitor bracovirus (MdBV) and parasitizes the larval stage of several noctuid moths. A key function of MdBV in parasitism is suppression of the host's cellular immune response. Prior studies in the host Pseudoplusia includens indicated that MdBV blocks encapsulation by preventing two types of hemocytes, plasmatocytes and granulocytes, from adhering to foreign targets. The other main immune response mediated by insect hemocytes is phagocytosis. The goal of this study was to determine which hemocyte types were phagocytic in P. includens and to assess whether MdBV infection affects this defense response. Using the bacterium Escherichia coli and inert polystyrene beads as targets, our results indicated that the professional phagocyte in P. includens is granulocytes. The phagocytic responses of granulocytes were very similar to those of High Five cells that prior studies have suggested are a granulocyte-like cell line. MdBV infection dose-dependently disrupted phagocytosis in both cell types by inhibiting adhesion of targets to the cell surface. The MdBV glc1.8 gene encodes a cell surface glycoprotein that had previously been implicated in disruption of adhesion and encapsulation responses by immune cells. Knockdown of glc1.8 expression by RNA interference (RNAi) during the current study rescued the ability of MdBV-infected High Five cells to phagocytize targets. Collectively, these results indicate that glc1.8 is a key virulence determinant in disruption of both adhesion and phagocytosis by insect immune cells.     

Integument and hemocyte peptides

There are four routing classes of integument peptide in the caterpillar of Calpodes ethlius. The epidermis secretes peptides apically into the cuticle (C), basally into the hemolymph (H) and in both directions (BD). Peptides in a 4th class (T), are presumed to be transported across the epidermis, because the epidermis does not synthesize them although they occur in both cuticle and hemolymph. In a search for the origin of the presumed transepidermal peptides we found that hemocytes contain some peptides from all four routing classes. Peptides prepared from washed hemocytes reacted in immunoblots to antibodies against integument peptides prepared from hemolymph and cuticle. These peptides are probably synthesized by hemocytes because they matched those from medium containing [³⁵S]methionine in which hemocytes had been incubated. Calpodes hemolymph contains four hemocyte types. Immunogold labelling localized integument peptides in the secretory pathway of granulocytes and spherulocytes and in the cytosol of oenocytoids but not in plasmatocytes. Each peptide was localized in a particular kind or kinds of hemocyte. Granulocyte secretory vesicles reacted with antibodies to C180, C55 and BD82 kDa peptides. Spherulocytes secretory vesicles reacted with antibodies to C180, C55, BD89, BD82 and a 78 kDa peptide presumed to be the precursor of T66. Oenocyotoids reacted with antibodies to H45, 38, 32, 23 and BD89 kDa peptides. Spherulocytes were the only tissue to react with antibodies to the T66 kDa peptide that is found abundantly in cuticle and hemolymph. Spherulocytes are therefore presumed to secrete the 66 kDa peptide into the hemolymph from where it is transported to the cuticle. The C180 and C55 kDa peptides do not occur in hemolymph. Their presence in granulocytes and spherulocytes may be associated with hemocyte functions such as basal lamina formation, since immunogold localized them in that part of the basal lamina next to the hemolymph, as would be expected if hemocytes deposited components onto the exposed hemolymph surface. The presence of hemolymph peptides in oenocytoids is more difficult to interpret, since the antigenic reactions are localized in the cytosol rather than in the secretory pathway expected for exported proteins. We conclude that integument peptides are not secreted only by the epidermis, nor is the cuticle their only destination.     

Hemocytes of Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) and their response to Saccharomyces cerevisiae and Bacillus thuringiensis

Originally from tropical Asia, the Red Palm Weevil (RPW Rhynchophorus ferrugineus (Olivier)) is the most dangerous and deadly pest of many palm trees, and there have been reports of its recent detection in France, Greece and Italy. At present, emphasis is on the development of integrated pest management based on biological control rather than on chemical insecticides, however the success of both systems is often insufficient. In this regard, RPW appears to be one pest that is very difficult to control. Thus investigations into the natural defences of this curculionid are advisable. RPW hemocytes, the main immunocompetent cells in the insect, are described for the first time. We identified five hemocyte cell types from the hemolymph of R. ferrugineus: plasmatocytes (∼50%), granulocytes (∼35%), prohemocytes (∼8%), oenocytes (∼4%) and spherulocytes (∼3%). SEM observations were also carried out. Some aspects of RPW interaction with non-self organisms, such as Saccharomyces cerevisiae and the entomopathogen bacterium, Bacillus thuringiensis (Bt), are discussed. Plasmatocytes and granulocytes were involved in nodules and capsule formation as well as in the phagocytosis of yeast. The hemocyte response of RPW larvae to sub-lethal doses of commercial products containing Bt was examined. In vivo assays were carried out and Bt in vegetative form was found in the hemolymph. After a diet containing Bt, the number of total hemocytes, mainly plasmatocytes, in the RPW larva hemolymph declined sharply (∼12%) and then remained at a low level, while the number of other circulating cells was almost unchanged.