Evidence of Adaptive Evolution in Wolbachia-Regulated Gene DNMT2 and Its Role in the Dipteran Immune Response and Pathogen Blocking.

Eukaryotic nucleic acid methyltransferase (MTase) proteins are essential mediators of epigenetic and epitranscriptomic regulation. DNMT2 belongs to a large, conserved family of DNA MTases found in many organisms, including holometabolous insects such as fruit flies and mosquitoes, where it is the lone MTase. Interestingly, despite its nomenclature, DNMT2 is not a DNA MTase, but instead targets and methylates RNA species. A growing body of literature suggests that DNMT2 mediates the host immune response against a wide range of pathogens, including RNA viruses. Curiously, although DNMT2 is antiviral in Drosophila, its expression promotes virus replication in mosquito species. We, therefore, sought to understand the divergent regulation, function, and evolution of these orthologs. We describe the role of the Drosophila-specific host protein IPOD in regulating the expression and function of fruit fly DNMT2. Heterologous expression of these orthologs suggests that DNMT2's role as an antiviral is host-dependent, indicating a requirement for additional host-specific factors. Finally, we identify and describe potential evidence of positive selection at different times throughout DNMT2 evolution within dipteran insects. We identify specific codons within each ortholog that are under positive selection and find that they are restricted to four distinct protein domains, which likely influence substrate binding, target recognition, and adaptation of unique intermolecular interactions. Collectively, our findings highlight the evolution of DNMT2 in Dipteran insects and point to structural, regulatory, and functional differences between mosquito and fruit fly homologs.

Glyphosate inhibits melanization and increases susceptibility to infection in insects.

Melanin, a black-brown pigment found throughout all kingdoms of life, has diverse biological functions including UV protection, thermoregulation, oxidant scavenging, arthropod immunity, and microbial virulence. Given melanin's broad roles in the biosphere, particularly in insect immune defenses, it is important to understand how exposure to ubiquitous environmental contaminants affects melanization. Glyphosate-the most widely used herbicide globally-inhibits melanin production, which could have wide-ranging implications in the health of many organisms, including insects. Here, we demonstrate that glyphosate has deleterious effects on insect health in 2 evolutionary distant species, Galleria mellonella (Lepidoptera: Pyralidae) and Anopheles gambiae (Diptera: Culicidae), suggesting a broad effect in insects. Glyphosate reduced survival of G. mellonella caterpillars following infection with the fungus Cryptococcus neoformans and decreased the size of melanized nodules formed in hemolymph, which normally help eliminate infection. Glyphosate also increased the burden of the malaria-causing parasite Plasmodium falciparum in A. gambiae mosquitoes, altered uninfected mosquito survival, and perturbed the microbial composition of adult mosquito midguts. Our results show that glyphosate's mechanism of melanin inhibition involves antioxidant synergy and disruption of the reaction oxidation-reduction balance. Overall, these findings suggest that glyphosate's environmental accumulation could render insects more susceptible to microbial pathogens due to melanin inhibition, immune impairment, and perturbations in microbiota composition, potentially contributing to declines in insect populations.

Genetic determinants of antiviral immunity in dipteran insects - Compiling the experimental evidence.

The genetic basis of antiviral immunity in dipteran insects is extensively studied in Drosophila melanogaster and advanced technologies for genetic manipulation allow a better characterization of immune responses also in non-model insect species. Especially, immunity in vector mosquitoes is recently in the spotlight, due to the medical impact that these insects have by transmitting viruses and other pathogens. Here, we review the current state of experimental evidence that supports antiviral functions for immune genes acting in different cellular pathways. We discuss the well-characterized RNA interference mechanism along with the less well-defined JAK-STAT, Toll, and IMD signaling pathways. Furthermore, we highlight the initial evidence for antiviral activity observed for the autophagy pathway, transcriptional pausing, as well as piRNA production from endogenous viral elements. We focus our review on studies from Drosophila and mosquito species from the lineages Aedes, Culex, and Anopheles, which contain major vector species responsible for virus transmission.

A bioinformatic study of antimicrobial peptides identified in the Black Soldier Fly (BSF) Hermetia illucens (Diptera: Stratiomyidae).

Antimicrobial peptides (AMPs) play a key role in the innate immunity, the first line of defense against bacteria, fungi, and viruses. AMPs are small molecules, ranging from 10 to 100 amino acid residues produced by all living organisms. Because of their wide biodiversity, insects are among the richest and most innovative sources for AMPs. In particular, the insect Hermetia illucens (Diptera: Stratiomyidae) shows an extraordinary ability to live in hostile environments, as it feeds on decaying substrates, which are rich in microbial colonies, and is one of the most promising sources for AMPs. The larvae and the combined adult male and female H. illucens transcriptomes were examined, and all the sequences, putatively encoding AMPs, were analysed with different machine learning-algorithms, such as the Support Vector Machine, the Discriminant Analysis, the Artificial Neural Network, and the Random Forest available on the CAMP database, in order to predict their antimicrobial activity. Moreover, the iACP tool, the AVPpred, and the Antifp servers were used to predict the anticancer, the antiviral, and the antifungal activities, respectively. The related physicochemical properties were evaluated with the Antimicrobial Peptide Database Calculator and Predictor. These analyses allowed to identify 57 putatively active peptides suitable for subsequent experimental validation studies.

Tabanidae insect (horsefly and deerfly) allergy in humans: A review of the literature.

Allergy to insects of the family Tabanidae (order Diptera), commonly called horseflies or deerflies, is anecdotally common, although the published literature is limited to case reports and small case series. This review summarizes the available literature, in which there is enormous variability in clinical detail, identification of species or even genus, and means and thoroughness of assessment of sensitization. The clinical utility of in vivo and in vitro assays remains unclear. Investigation and management of patients reporting anaphylaxis to suspected bites must therefore be pragmatic, by considering other insects (eg Hymenoptera), provision of a written action plan and self-injectable adrenaline if appropriate, and advice on avoidance.

Cytokine expression in bovine PBMC cultures stimulated with Hypoderma lineatum antigens.

Hypoderma antigens are involved in host inflammation and immune response, conditioning larvae survival. In this study, peripheral blood mononuclear cell (PBMC) cultures from Hypoderma sensitized and unsensitized cattle were performed to determine the effect of H. lineatum antigens and incubation time (18, 24, 48 h) on IFN-γ, TNF-α, IL-10 and IL-4 mRNA gene expression determined by RT-qPCR. TNF-α and IL-4 gene expression were higher in Hypoderma previously sensitized PBMCs, suggesting that a mixed Th1/Th2 response may play a significant role in host defence reactions against Hypoderma exhibited by previously infested cattle. Incubation time had a significant effect on IL-10 and TNF-α gene expression, which decreased over time. Regarding to H. lineatum antigens, the crude larval extract and the purified fraction hypodermin B (HB) produced a significant reduction of the mRNA expression levels of the proinflammatory cytokine, IFN-γ; moreover, the HB had a stimulating effect on the mRNA gene expression of the anti-inflammatory cytokine IL-10, demonstrating that the parasite would modulate the host defence mechanisms by avoiding harmful immune responses that would limit its survival into the host tissues.

Dynamic Evolution of Antimicrobial Peptides Underscores Trade-Offs Between Immunity and Ecological Fitness.

There is a developing interest in how immune genes may function in other physiological roles, and how traditionally non-immune peptides may, in fact, be active in immune contexts. In the absence of infection, the induction of the immune response is costly, and there are well-characterized trade-offs between immune defense and fitness. The agents behind these fitness costs are less understood. Here we implicate antimicrobial peptides (AMPs) as particularly costly effectors of immunity using an evolutionary framework. We describe the independent loss of AMPs in multiple lineages of Diptera (true flies), tying these observations back to life history. We then focus on the intriguing case of the glycine-rich AMP, Diptericin, and find several instances of loss, pseudogenization, and segregating null alleles. We suggest that Diptericin may be a particularly toxic component of the Dipteran immune response lost in flies either with reduced pathogen pressure or other environmental factors. As Diptericins have recently been described to have neurological roles, these findings parallel a developing interest in AMPs as potentially harmful neuropeptides, and AMPs in other roles beyond immunity.

Susceptibility to entomopathogens and modulation of basal immunity in two insect models at different temperatures.

In this work, we analysed the efficacy of different commercial bio-insecticides (Steinernema feltiae, Steinernema carpocapsae, Heterorhabditis bacteriophora and Bacillus thuringiensis) by valuating the mortality induced on two insect models, Galleria mellonella (Lepidoptera) and Sarcophaga africa (Diptera) after exposure to different temperatures (10, 20 and 30 °C). Moreover, we investigated the effects of temperature on the basal humoral immunity of the two target insects; particularly, phenoloxidase (PO) and lysozyme activity. Our results show that G. mellonella is susceptible to all bio-insecticides at all the examined temperatures, except when infected at 10 °C with S. carpocapsae and at 30 °C with S. feltiae and B. thuringiensis. S. africa is more susceptible at 30 °C to all bioinsecticides; whereas, when infected at 10 and 20 °C, H. bacteriophora is the most efficient. Temperature modulates PO activity of both G. mellonella and S. africa, otherwise variations in lysozyme activity is observed only in G. mellonella. Except for a possible correlation between the increased lysozyme activity and the delayed Bt efficacy recorded on G. mellonella at 30 °C, a different resistance to bio-insecticides at different temperatures does not seem to be associated to variations of the host basal immunity, probably due to immunoevasive and immunodepressive strategies of these entomopathogens.

Hypodermin C improves the survival of kidney allografts.

Although immunosuppressive therapies have made organ transplantation a common medical procedure worldwide, chronic toxicity is a major issue of long-term treatment. One method to improve such therapies is the application of immunomodulatory agents from parasites, such as Hypoderma lineatum (Diptera: Oestridae). Hypodermin C (HC) is an enzyme secreted by H. lineatum larvae, and our previous study showed that recombinant HC could degrade guinea pig C3 and inhibit the complement pathway in vitro, suggesting potential activity for inhibiting transplant rejection. However, such properties have not been fully demonstrated in vivo. In this study, we investigated the impact of HC on a fully MHC-mismatched, life-sustaining, murine model of kidney allograft rejection using B6 donors and BABL/c (HC transgenic or wild-type) recipients. Kidney grafts were analyzed by histology, immunohistochemistry and western blotting. The results suggested that HC could effectively inhibit kidney allograft rejection. These findings suggest HC is a promising strategy to improve the survival of human implants.

Immunosuppressive mechanism of Hypoderma lineatum secreted serine esterase, a potential modulatory method used to inhibit transplant rejection

Background: Although immunosuppressive therapies have made organ transplantation a common medical procedure worldwide, chronic toxicity has a major issue for long-term treatment. One method to improve therapies and methods is the application of immunomodulatory agents from parasites such as Hypoderma lineatum. Hypodermin A (HA) is a serine esterase secreted by the larvae of Hypoderma lineatum, several studies demonstrated its immunosuppressive mechanism in vitro, and recently we discovered that HA inhibits the expression of interferon (IFN)-γ and interleukin (IL)-2 and activates IL-10 expression. Therefore, we hypothesized that it might be a potential agent used to block allograft rejections. However, most studies of the immunosuppressive mechanisms associated with HA were undertaken at the cellular level. In order to augment these studies, we evaluated the immunosuppressive effects of HA in vivo using an HA transgenic mouse model. Result: Our results revealed similar findings to those reported by in vitro studies, specifically that HA induced prostaglandin E2 expression, downregulated IFN-γ and IL-2 expression, and promoted IL-10 secretion via E-type prostanoid receptor 4. Additionally, we observed that HA overexpression inhibited lipopolysaccharide-induced TLR4 activation. These findings provide insight into a new potential agent capable of blocking graft rejection. Conclusion: Our founding suggested that HA-related treatment could be a promising option to improve the viability of grafts in human.

Nutritional immunology: Diversification and diet-dependent expression of antimicrobial peptides in the black soldier fly Hermetia illucens.

The black soldier fly Hermetia illucens is used for the bioconversion of organic waste into feed for livestock and aquaculture, and is economically among the most important farmed insects in the world. The larvae can be fed on agricultural waste and even liquid manure, resulting in highly unpredictable pathogen levels and dietary conditions. Here we show that H. illucens larvae express a remarkably expanded spectrum of antimicrobial peptides (AMPs), many of which are induced by feeding on a diet containing high bacterial loads. The addition of sulfonated lignin, cellulose, chitin, brewer's grains or sunflower oil revealed the diet-dependent expression profiles of AMPs in the larvae. The highest number of AMPs and the highest levels of AMP expression were induced by feeding larvae on diets supplemented with protein or sunflower oil. Strikingly, the diet-dependent expression of AMPs translated into diet-dependent profiles of inhibitory activities against a spectrum of bacteria, providing an intriguing example for the emerging field of nutritional immunology. We postulate that the fine-tuned expression of the expanded AMP repertoire mediates the adaptation of the gut microbiota to the digestion of unusual diets, and this feature could facilitate the use of H. illucens for the bioconversion of organic waste.

Insect Immunity Varies Idiosyncratically During Overwintering.

Overwintering insects face multiple stressors, including pathogen and parasite pressures that shift with seasons. However, we know little of how the insect immune system fluctuates with season, particularly in the overwintering period. To understand how immune activity changes across autumn, winter, and spring, we tracked immune activity of three temperate insects that overwinter as larvae: a weevil (Curculio sp., Coleoptera), gallfly (Eurosta solidaginis, Diptera), and larvae of the lepidopteran Pyrrharctia isabella. We measured baseline circulating hemocyte numbers, phenoloxidase activity, and humoral antimicrobial activity, as well as survival of fungal infection and melanization response at 12°C and 25°C to capture any potential plasticity in thermal performance. In Curculio sp. and E. solidaginis, hemocyte concentrations remained unchanged across seasons and antimicrobial activity against Gram-positive bacteria was lowest in autumn; however, Curculio sp. were less likely to survive fungal infection in autumn, whereas E. solidaginis were less likely to survive infection during the winter. Furthermore, hemocyte concentrations and antimicrobial activity decreased in P. isabella overwintering beneath snow cover. Overall, seasonal changes in activity were largely species dependent, thus it may be difficult to create generalizable predictions about the effects of a changing climate on seasonal immune activity in insects. However, we suggest that the relationship between the response to multiple stressors (e.g., cold and pathogens) drives changes in immune activity, and that understanding the physiology underlying these relationships will inform our predictions of the effects of environmental change on insect overwintering success.

[REGULATION OF ANTIMICROBIAL PEPTIDE SYNTHESIS IN THE LARVAE OF CALLIPHORA VICINA (DIPTERA, CALLIPHORIDAE): DOSE-DEPENDENT EFFECT OF ECDYSTEROIDS].

Ecdysteroids are multifunctional hormones regulating virtually all morphogenetic processes in insects. Their role in stress and immune response regulation is less known. Here we studied 20-hydroxyecdysone effect on synthesis of the antimicrobial peptides in larvae of Calliphora vicina. An inverse correlation was found between 20-hydroxyecdysone titer and the concentration of antimicrobial peptides in the hemolymph of unaffected and bacteria-immunized insects. High and low doses of 20-hydroxyecdysone, injected simultaneously with bacterial cells, had an opposite effect on antimicrobial peptide synthesis in the diapausing larvae. Morphogenetically effective doses of 20-hydroxyecdysone demonstrated immuno-suppressive activity. Low doses of 20-hydroxyecdysone, on the contrary, moderately stimulated synthesis of the antimicrobial peptides. These data suggest that ecdysteroids are directly involved in regulation of the immune system activity and the final effect is dose-dependent.

Use of Monoclonal Antibodies in an ELISA for Detecting an Invasive Pest Insect, Liriomyza trifolii (Diptera: Agromyzidae).

A monoclonal antibody was prepared by the hybridoma technology. It reacted only with the protein of Liriomyza trifolii (Burgess) and not with that of Chromatomyia horticola Goureau or Liriomyza sativae Blanchard in indirect enzyme-linked immunosorbent assay. It was effective even after being diluted more than 8.192×10(6)-fold. The detection sensitivity of the antibody was 31.3 µg/ml under controlled conditions. Positive reaction was achieved with all laboratory-reared L. trifolii samples, including larvae, pupae, and adults. An indirect enzyme-linked immunosorbent assay system was successfully established to detect L. trifolii in the field. This antibody was successfully used to determine the L. trifolii collected in different locations, from different host plants, and in different seasons. More than 50% of leafminers collected on Brassica chinensis var chinensis, Apium graveolens (Miller) Persoon, Vigna unguiculata (L.) Walpers, Phaseolus vulgaris L., Lactuca sativa L., and Chrysanthemum coronarium (L.) Cassini ex Spach were L. trifolii, indicating that those six plant species might be the preference host plants of L. trifolii. Population of L. trifolii peaked in September, October, or November in Hangzhou, Zhejiang. These results suggest a great potential of using this McAb for precisely identifying L. trifolii and monitoring the population dynamics of L. trifolii in the field.

Hypodermin A, a potential agent for prevention of allogeneic acute rejection.

Immunosuppressive agents play an important role in the success of organ transplantation, however the chronic toxicity of these agents is a major issue over the long-term. Hypodermin A (HA) is an enzyme secreted by the larvae of Hypoderma lineatum (Diptera: Oestridae), and has been implicated in immunosuppression in cattle. Malassagne et al. have demonstrated that HA can degrade the C3 protein, and could be used to prevent hyperacute xenogeneic rejection. We found that overexpression of HA in RAW264.7 cells induced significant secretion of prostaglandin E2 (PGE2), which mediates a variety of innate and adaptive immune responses through four E-type prostanoid (EP) receptor subtypes (EP1-4). PGE2 is useful in the management of allogeneic acute rejection. In addition, we found that induction of PGE2 expression downregulates the expression of interferon (IFN)-γ and interleukin (IL)-2, and promotes the secretion of IL-10 in vitro through the EP4 receptor. It was previously shown that activation of IL-2 and IFN-γ is involved in allograft acute rejection. IL-10 is known to prevent inflammation, and can improve allograft survival rates. We concluded that besides preventing hyperacute xenogeneic rejection, HA might also be a potential therapeutic candidate for ameliorating acute rejection during allotransplantation.

Selection and Evaluation of Tissue Specific Reference Genes in Lucilia sericata during an Immune Challenge.

The larvae of the common green bottle fly Lucilia sericata (Diptera: Calliphoridae) have been used for centuries to promote wound healing, but the molecular basis of their antimicrobial, debridement and healing functions remains largely unknown. The analysis of differential gene expression in specific larval tissues before and after immune challenge could be used to identify key molecular factors, but the most sensitive and reproducible method qRT-PCR requires validated reference genes. We therefore selected 10 candidate reference genes encoding products from different functional classes (18S rRNA, 28S rRNA, actin, ß-tubulin, RPS3, RPLP0, EF1α, PKA, GAPDH and GST1). Two widely applied algorithms (GeNorm and Normfinder) were used to analyze reference gene candidates in different larval tissues associated with secretion, digestion, and antimicrobial activity (midgut, hindgut, salivary glands, crop and fat body). The Gram-negative bacterium Pseudomonas aeruginosa was then used to boost the larval immune system and the stability of reference gene expression was tested in comparison to three immune genes (lucimycin, defensin-1 and attacin-2), which target different pathogen classes. We observed no differential expression of the antifungal peptide lucimycin, whereas the representative targeting Gram-positive bacteria (defensin-1) was upregulated in salivary glands, crop, nerve ganglion and reached its maximum in fat body (up to 300-fold). The strongest upregulation in all immune challenged tissues (over 50,000-fold induction in the fat body) was monitored for attacin-2, the representative targeting Gram-negative bacteria. Here we identified and validated a set of reference genes that allows the accurate normalization of gene expression in specific tissues of L. sericata after immune challenge.

Histological and immunohistochemical characterization of Hypoderma lineatum (Diptera: oestridae) warbles.

Hypoderma larvae are tissue invading parasites which spend several months migrating within the host tissues before completing their development in the sub-dermal tissues of the back. Subcutaneous stages of the parasite produce an inflammatory reaction in the skin called "warbles", as well as holes through which larvae breathe. In order to elucidate the microscopical structure of the warbles, three hides from warbled cows were collected in a slaughterhouse in Lugo (NW, Spain) between March and May 2012. A total of 60 skin samples, including warbles at different phases of development, were chosen for histopathological and immunohistochemical examination. Microscopic lesions were classified into three groups, according to the predominance and distribution of different cell populations. In warbles containing living or recently dead larvae with apparently well preserved cuticle (type 1), plasma cells were observed in high number. However, macrophages and lymphocytes were the predominant cells in granulomas (type 2) formed in relation to remnants of the dead parasite, containing or not remains of the altered cuticle. Scars (type 3) were characterized by granulation tissue. Immunohistochemistry showed that B lymphocytes and IgG(+) cells were predominant in the lesions, as long as the cuticle of the larvae is intact. On the other side, CD3(+) lymphocytes increased once cuticle is destroyed and a granuloma is formed. Macrophages, revealed by CD68(+), MAC387(+) and lysozyme immunolabelling, were detected in all types of lesions, but they were more abundant in type 2 and scarce in scars. These cells appeared isolated around the intact larvae or forming aggregates around its remains in the granuloma. Moreover, a strong immunolabelling against MAC387 antibody was registered in the squamous epithelium covering the breathing pore. This finding may be associated with the expression of calprotectin, a molecule involved on the healing process of the skin after larvae outcome. Our results suggest the predominance of a humoral response inside the warble as long as larvae are intact. Once they are destroyed, cellular response occurred, isolating and destroying the remains of the larvae until healing process completes and scars with low numbers of inflammatory cells appear.

Early-life temperature modifies adult encapsulation response in an invasive ectoparasite.

Immunity of parasites has been studied amazingly little, in spite of the fact that parasitic organisms, especially the arthropod parasites, need immunity to survive their own infections to successfully complete life cycles. Long-term effects of challenging environmental temperatures on immunity have remained unstudied in insects and parasites. Our study species, the deer ked (Lipoptena cervi; Linnaeus 1758), is an invasive, blood-feeding parasitic fly of cervids. Here, it was studied whether thermal stress during the pupal diapause stage could modify adult immunity (encapsulation capacity) in L. cervi. The effect of either a low temperature or high temperature peak, experienced during winter dormancy, on encapsulation response of active adult was tested. It was found that low temperature exposure during diapause, as long as the temperature is not too harsh, had a favourable effect on adult immunity. An abnormal, high temperature peak during pupal winter diapause significantly deteriorated the encapsulation capacity of emerged adults. The frequency and intensity of extreme weather events such as high temperature fluctuations are likely to increase with climate change. Thus, the climate change might have previously unknown influence on host-ectoparasite interactions, by affecting ectoparasite's immune defence and survival.

Purification and characterization of a novel antibacterial peptide from black soldier fly (Hermetia illucens) larvae.

In this study, we induced and purified a novel antimicrobial peptide exhibiting activity against Gram-positive bacteria from the immunized hemolymph of Hermetia illucens larvae. The immunized hemolymph was extracted, and the novel defensin-like peptide 4 (DLP4) was purified using solid-phase extraction and reverse-phase chromatography. The purified DLP4 demonstrated a molecular weight of 4267 Da, as determined using the matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) method. From analysis of DLP4 by N-terminal amino acid sequencing using Edman degradation, combined with MALDI-TOF and rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR), the amino acid sequence of the mature peptide was determined to be ATCDLLSPFKVGHAACAAHCIARGKRGGWCDKRAVCNCRK. In NCBI BLAST, the amino acid sequence of DPL4 was found to be 75% identical to the Phlebotomus duboscqi defensin. Analysis of the minimal inhibitory concentration (MIC) revealed that DLP4 have antibacterial effects against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA). The expression of DLP4 transcripts in several tissues after bacterial challenge was measured by quantitative real-time PCR. Expression of the DLP4 gene hardly occurred throughout the body before immunization, but was mostly evident in the fat body after immunization.

[Cellular immune system of surgical maggots Lucilia sericata (Diptera, Calliphoridae)].

In the hemolymph of surgical maggots Lucilia sericata seven types of hemocytes were revealed. These are prohemocytes, stable and unstable hyaline cells, thrombocytoids, spindle cells, larval plasmatocytes and plasmatocytes I-IV, which represent sequential stages of one cell line differentiation. In contrast to Calliphora hyaline cells, this type of hemocytes in cropemptying larvae of Lucilia is elongated or vermiform in shape. Hyaline cells may be transformed to both prothrombocytoids and unstable prophenoloxydase-producing cells. Appearance and differentiation of each hemocyte type is rigidly linked with a definite stage of development. In cellular defense the main role play juvenile plasmatocytes, plasmatocytes II and III and trombocytoides. Juvenile plasmatocytes are the most active ones. After charcoal particles injection they were instantly surrounded by the thick envelope of adhered alien particles and form uniform morules aggregations or conglomerates together with thrombocytoidal agglutinates. Plasmatocytes II and III during the early stages of differentiation may be involved in adhesion and phagocytosis of alien particles and during the last stages in the engulfing of apoptose desintegrated tissues. Thus the cellular defense reaction is assisted by 4 hemocyte types--prophenoloxydase-unstable hyaline cells, thrombocytoids, juvenile plasmatocytes and plasmatocytes I-IV.