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.

Exploring the role of RNASET2 in the immune response of black soldier fly larvae.

T2 RNases are transferase-type enzymes distributed across phyla, crucial for breaking down single-stranded RNA molecules. In addition to their canonical function, several T2 enzymes exhibit pleiotropic roles, contributing to various biological processes, such as the immune response in invertebrates and vertebrates. This study aims at characterizing RNASET2 in the larvae of black soldier fly (BSF), Hermetia illucens, which are used for organic waste reduction and the production of valuable insect biomolecules for feed formulation and other applications. Given the exposure of BSF larvae to pathogens present in the feeding substrate, it is likely that the mechanisms of their immune response have undergone significant evolution and increased complexity. After in silico characterization of HiRNASET2, demonstrating the high conservation of this T2 homolog, we investigated the expression pattern of the enzyme in the fat body and hemocytes, two districts mainly involved in the insect immune response, in larvae challenged with bacterial infection. While no variation in HiRNASET2 expression was observed in the fat body following infection, a significant upregulation of HiRNASET2 synthesis occurred in hemocytes shortly after the injection of bacteria in the larva. The intracellular localization of HiRNASET2 in lysosomes of plasmatocytes, its extracellular association with bacteria, and the presence of a putative antimicrobial domain in the molecule, suggest its potential role in RNA clean-up and as an alarm molecule promoting phagocytosis activation by hemocytes. These insights contribute to the characterization of the immune response of Hermetia illucens larvae and may facilitate the development of animal feedstuff enriched with highly valuable BSF bioactive compounds.

The bug-killer fly Gymnosoma rotundatum (L.) (Diptera: Tachinidae) forms the respiratory funnel independently of the host's immune response.

In internal parasitism, the respiration strategy within the host's body is as essential as evading attack from the host's immune system. Tachinid flies are parasitoids of terrestrial arthropods, mostly insects, during their larval stage. To obtain oxygen while living in the host body, they build a cylindrical structure known as the respiratory funnel at the aperture opened by the tachinid larva on the host integument or trachea. These funnels can be divided morphologically into sheath and cone types. Previous research on sheath-type funnels revealed that they are derived from the encapsulating substance produced by the host's immune system. In contrast, the cone-type funnels cover part of the body of the larval tachinid and may be constructed independently from the host immune system. To determine the mechanisms of cone-type funnel formation, histological observations were carried out on Gymnosoma rotundatum (L.) (Diptera: Tachinidae), which possesses this type of funnel. The respiratory funnel of G. rotundatum was found to be derived from the tube-shaped faeces wrapped with the peritrophic membrane and excreted by the fly larva, not from host tissue or haemocytes. Additionally, secretory glands putatively involved in the funnel formation were discovered around the larval anal plate of G. rotundatum. A comparison of funnel types within Tachinidae revealed that Phasiinae and Dexiinae have cone-type funnels, which may be created by the same mechanism as in G. rotundatum. These new findings suggest that funnel formation that does not use the host immune system is relevant to tachinid phylogeny.

[Impact of black soldier fly (Hermetia illucens) larvae biomass on the immune status of rats].

The improvement of the novel foods' safety assessment algorithms is currently one of the food hygiene significant areas. Within the studying of Hermetia illucens insects' effect, the standard in vivo allergological research integrated in the protocol of medical and biological evaluation of genetically modified food has been used. The protocol was supplemented with cytokine profile indicators and pathomorphologic characteristics of immunocompetent organs' lymphoid tissue. The purpose of the research was to study the effect of black soldier fly (Hermetia illucens) larvae biomass on the rats' immune status in the experiment on the induced anaphylactic shock model. Material and methods. The effect of black soldier fly (Hermetia illucens) larvae biomass was studied in a 29-day experiment on growing (43-72 days of life) male Wistar rats fed with Hermetia illucens biomass - main group (n=29) and semi-synthetic casein diet - control group (n=29). The complex assessment of allergenic potential of Hermetia illucens biomass was carried out in the experiment on the induced anaphylactic shock model in Wistar rats. An expanded pool of immune status indicators was studied including active anaphylactic shock severity (lethality, number of severe anaphylaxis reactions, anaphylactic index); cytokine profile (content of proinflammatory and anti-inflammatory cytokines, as well as regulators of cellular and humoral immune response); IgG1 and IgG4 level before and after administration of ovalbumin permissive dose (4 mg/kg b.w.). In addition to this pathomorphologic characteristics of lymphoid tissue of the main immunocompetent organs (thymus, spleen, Payer's patches) have been obtained. Results. The significant systemic anaphylaxis reaction decrease in the main group has been shown. Comparative assessment of the serum cytokines (GM-CSF, IFN-γ, IL-10, IL-12(p70), IL-13, IL-1α, IL-1ß, IL-2, IL-4, IL-5, IL-6, TNF-α) as well as the level of immunoglobulins of the IgG1, IgG4 class before and after administration of ovalbumin permissive dose did not reveal significant differences in rats of the control and main groups. In the main group, there was a decrease in blood serum proallergic cytokines: the level of IL-4 reduced by 1.3 fold, IL-10 - 1.1 and IL-13 - 1.2 fold (p>0.05), and in animals with mild anaphylactic reaction - by 1.8, 1.4 and 1.4 times, respectively (p>0.05). The morphologic studies of the immune system organs showed no intergroup differences. Conclusion. Thus, allergological studies of black soldier fly (Hermetia illucens) larvae in the experiment with the use of systemic anaphylaxis rat model and determination of immune status indicators (anaphylactic shock severity, cytokine profile, IgG1 and IgG4 level, morphologic structure of immunocompetent organs) did not reveal any allergenic effect of the studied product.

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.

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.

[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.

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.

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.

Intracellular immune responses of dipteran insects.

Vector-borne diseases, transmitted by bloodsucking arthropods, pose worldwide socio-medical and economical problems. Some of the major human infectious diseases, such as malaria, Dengue fever, and yellow fever, are transmitted by mosquitoes. While the majority of pathogens enjoy extracellular life styles in insects, viruses and some endosymbionts are strictly intracellular. Here, we summarize our knowledge on defense reactions against intracellular microorganisms in dipteran insects and discuss the potential of insects as models to study human pathogens.

Effects of interaction between temperature conditions and copper exposure on immune defense and other life-history traits of the blow fly Protophormia terraenovae.

Environmental pollution is considered one of the major threats to organisms. Direct effects of heavy metal pollution on various life-history traits are well recognized, while the effects of potential interactions between two distinct environmental conditions on different traits are poorly understood. Here, we have tested the effects of interactions between temperature conditions and heavy metal exposure on innate immunity and other life-history traits. Maggots of the blow fly Protophormia terraenovae were reared on either copper-contaminated or uncontaminated food, under three different temperature environments. Encapsulation response, body mass, and development time were measured for adult flies that were not directly exposed to copper. We found that the effects of copper exposure on immunity and other traits are temperature-dependent, suggesting that the ability to regulate toxic compounds in body tissues might depend on temperature conditions. Furthermore, we found that temperature has an effect on sex differences in immune defense. Males had an encapsulation response at higher temperatures stronger than that of females. Our results indicate that the effects of environmental conditions on different traits are much more intricate than what can be predicted. This is something that should be considered when conducting immunological experiments or comparing results of previous studies.

Antigen characterization from second instars of oestrid bot flies for the detection of anti-Cephenemyia stimulator antibodies by ELISA in roe deer (Capreolus capreolus).

A study to determine the most appropriate antigen for use in the serodiagnosis of Cephenemyia (Diptera: Oestridae) infestation in roe deer (Capreolus capreolus) was carried out using immunoenzymatic tests. Serum samples from 43 roe deer from northern Spain were obtained post-mortem and corresponding numbers of bot fly larvae established. Three antigen complexes were tested, including Cephenemyia stimulator Clark excretory/secretory antigens (CsES), C. stimulator somatic antigens (CsSA) and Oestrus ovis L. (Diptera: Oestridae) excretory/secretory antigens (OoES). In addition, the composition of each antigen was analysed using an electrophoresis system. Cephenemyia stimulator larvae were found in 25% of roe deer; the mean intensity of infection was 24.3 larvae per infested animal. In the antigen analysis, CsSA showed four exclusive bands of molecular weight (17-19, 62, 65 and 67-70 kDa). A positive correlation between immunoglobulin G (IgG) values and total number of larvae was found with CsES and CsSA. The highest sensitivity value, negative predictive value and negative likelihood ratio were obtained using CsES. The highest specificity value, positive likelihood ratio and kappa value were achieved with CsSA. The predictive values of the enzyme-linked immunosorbent assay (ELISA) using CsES and CsSA reached statistical significance and seroprevalence values were 26-44%. The use of ELISA with CsES and CsSA seems promising in the non-invasive diagnosis of Cephenemyia infestation in roe deer.

[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.

[Cellular defense system of some synanthropic dipterans inhabitant of bacterially aggressive environment].

The hemocytic count and defense reaction within 4 families of higher Diptera: Tabanidae, Syrphidae, Muscidae and Sarcophagidae, whose larvae inhabit bacterially aggressive environment, were investigated. The least hemocytes types (3) were revealed in Tabanidae and Syrphidae larvae--prohemocytes, plasmatocytes and prophenoloxydase-containing unstable hyaline cells (oenocytoids). In Sarcophaga crassipalpis and Musca domestica stable hyaline cells and thrombocytoids or podocytoid-like cells can be added to this set. At the time of pupariation in Sarcophaga, new generation of prohemocytes is segregated into the hemolymph, which form small round or spindle-shaped hyaline cells. So, the number of plasmatocyte types in Sarcophaga increase to six. Typical to Calliphoridae juvenile plasmatocytes in the members of investigated families are absent. Among the one hemocyte type morphology also can vary, especially in unstable prophenoloxydase hyaline cells. In Drosophila there are crystal cells containing in the cytoplasm paracrystalloidal inclusions. In Calliphoridae there are big hyaline cells with homogenous cytoplasm producing circumferential bubbles. Both in Sarcophaga and Tabanidae they contain in their cytoplasm big globules. However in Sarcophaga they rapidly disintegrate, while in Tabanidae are maintained unchanged during hours. In Muscidae and Syrphidae prophenoloxydase extrusion occurs very early and these cells obtain pycnotic nuclei and very liquid cytoplasm with strings of granules. Thrombocytoids in Musca larvae are represented by big flattened anucleated irregular cytoplasm and "naked" nuclei and cytoplasmic fragments often with fan-like projections. Plasmatocytes in all species studied are the cells with pronounced phylopodies. In larvae they contain cytoplasmic catabolic inclusions and in pupa--ragments of apoptotic tissues. Clearance of hemolymph from alien particles in Sarcophagidae and Muscidae occur by thrombocytoides, while in Tabanidae by plasmatocyte nodulation. A differing case is Syrphidae whe-e charcoal injection produce depletion of hemolymph both from particles and all types of hemocytes. So the specimen of different higher Diptera families can use different schemes of cellular defense reaction.

The Allergen Profile of Two Edible Insect Species-Acheta domesticus and Hermetia illucens.

SCOPE: Edible insect proteins are increasingly introduced as an alternative sustainable food source to address the world's need to feed the growing population. Tropomyosin is the main insect allergen; however, additional potential allergens are not well characterized and the impact of extraction procedures on immunological reactivity is unknown. METHODS AND RESULTS: Proteins from different commercial food products derived from cricket (Acheta domesticus) and black soldier fly (BSF) (Hermetia illucens) are extracted using five different extraction buffers. The proteins are analyzed by SDS-PAGE and immunoblotting using allergen-specific antibodies and crustacean allergic patient sera. IgE binding bands are analyzed by mass spectrometry as well as the complete allergen profile of all 30 extracts. Urea-based buffers are most efficient in extracting insect allergens. Shrimp-specific antibody cross-reactivity to tropomyosin from cricket and BSF indicates high sequence and structural similarity between shrimp and insects. Additional unique allergens are identified in both species, including hemocyanin, vitellogenin, HSP20, apolipophorin-III, and chitin-binding protein. CONCLUSIONS: Identifying potential allergenic proteins and their isoforms in cricket and BSF requires specific extraction approaches using urea-based methods. While tropomyosin is the most abundant and immunoreactive allergen, seven unique allergens are identified, highlighting the need for insect species-specific allergen detection in food products.

Molecular Characterization of the Allergenic Arginine Kinase from the Edible Insect Hermetia illucens (Black Soldier Fly).

SCOPE: Arginine kinase (AK) is an important enzyme for energy metabolism of invertebrate cells by participating in the maintenance of constant levels of ATP. However, AK is also recognized as a major allergen in insects and crustaceans capable of cross-reactivity with sera of patients sensitized to orthologous proteins. In the perspective of introducing insects or their derivatives in the human diet in Western world, it is of primary importance to evaluate possible risks for allergic consumers. METHODS AND RESULTS: This work reports the identification and characterization of AK from Hermetia illucens commonly known as the black soldier fly, a promising insect for human consumption. To evaluate allergenicity of AK from H. illucens, putative linear and conformational epitopes are identified by bioinformatics analyses, and Dot-Blot assays are carried out by using sera of patients allergic to shrimp or mites to validate the cross-reactivity. Gastrointestinal digestion reduces significantly the linear epitopes resulting in lower allergenicity, while the secondary structure is altered at increasing temperatures supporting the possible loss or reduction of conformational epitopes. CONCLUSION: The results indicate that the possible allergenicity of AK should be taken in consideration when dealing with novel foods containing H. illucens or its derivatives.

Serodiagnosis of nasal myasis in camels (Camelus dromedaries) in Egypt using third larval instar affinity-purified glycoprotein.

The larvae of Cephalopina titillator cause nasopharyngeal myiasis in camels, which parasitize the living tissues of the nasal and paranasal sinuses, pharynx, and larynx. C. titillator infestation adversely affects camel health, meat, and milk production, and can even cause death. In our study, to improve the immunodiagnosis of camel nasal myiasis, a sensitive and specific enzyme-linked immunosorbent assay (ELISA) was developed and evaluated using the Concanavalin-A (Con-A) affinity purification for the C. titillator-N-acetylglucosamine (Ct-GlucNAc) glycoprotein fraction from third larval instars as an antigen for detecting C. titillator antibodies. Crude antigens were prepared from larval instars of C. titillator and evaluated by indirect ELISA. The third C. titillator larval antigen (L3Ct) had the highest protein content (P < 0.001) and the best diagnostic value; chi-square = 235 (P < 0.001). Four glycoprotein fractions were purified separately from the L3Ct antigen by Con-A purification and evaluated. The Ct-GlucNAc glycoprotein fraction was the fraction of choice with the highest diagnostic accuracy (P < 0.05). Using Ct-GlucNAc as a coating antigen, indirect ELISA showed a 99.3% sensitivity for positive results in camel myiasis samples and 100% specificity for negative results in healthy camel samples. The diagnostic accuracy was 99.7%, and no cross reactivity was detected for other parasitic diseases. The indirect ELISA results were confirmed by the western immunoblotting which was characterized by comparing sera from naturally infested dromedary camels with C. titillator, sera from healthy camels and sera from camels with other parasitic infections (Echinococcus granulosus, Fasciola gigantica, Hard ticks; Hyalomma dromedarii, Trichostronglid sp., Eimeria spp., and Cryptosporidium sp.). Immunoreactive antigenic bands of 63, 50, 30 and 18 kDa were predominantly detected in sera from camels with nasopharyngeal myiasis and didn't react with healthy and camel's sera from other parasitic infections. However, seven immunoreactive bands appeared at 120, 70, 63, 48, 35, 29, and 19 kDa in the crude L3Ct antigen. In addition, a positive rate of C. titillator immunodiagnosis was detected by indirect ELISA (48.6%, chi-square = 483, P < 0.001), which was significantly greater than that of postmortem diagnosis (31%). In conclusion, the current study introduces a new diagnostic immunoaffinity glycoprotein fraction of C. titillator 3rd larval instar-based ELISA as a highly accurate, simple and fast method to detect specific antibodies of nasal myiasis in camels.

Activation of autophagic programmed cell death and innate immune gene expression reveals immuno-competence of integumental epithelium in Bombyx mori infected by a dipteran parasitoid.

In insects, the integument forms the primary barrier between the environment and internal milieu, but cellular and immune responses of the integumental epithelium to infection by micro- and macro-parasites are mostly unknown. We elucidated cellular and immune responses of the epithelium induced through infection by a dipteran endoparasitoid, Exorista bombycis in the economically important silkworm Bombyx mori. Degradative autophagic vacuoles, lamella-like bodies, a network of cytoplasmic channels with cellular cargo, and an RER network that opened to vacuoles were observed sequentially with increase in age after infection. This temporal sequence culminated in apoptosis, accompanied by the upregulation of the caspase gene and fragmentation of DNA. The infection significantly enhanced the tyrosine level and phenol oxidase activity in the integument. Proteomic analysis revealed enhanced expression of innate immunity components of toll and melanization pathways, cytokines, signaling molecules, chaperones, and proteolytic enzymes demonstrating diverse host responses. qPCR analysis revealed the upregulation of spatzle, BmToll, and NF kappa B transcription factors Dorsal and BmRel. NF kappa B inhibitor cactus showed diminished expression when Dorsal and BmRel were upregulated, revealing a negative correlation (R = (-)0.612). During melanization, prophenol oxidase 2 was expressed, a novel finding in integumental epithelium. The integument showed a low level of melanin metabolism and localized melanism in order to prevent the spreading of cytotoxic quinones. The gene-encoding proteolytic enzyme, beta-N-acetylglucosaminidase, was activated at 24 h post-infection, whereas chitinase, was activated at 96 h post-infection; however, most of the immune genes enhanced their expression in the early stages of infection. Thus the integument contributes to humoral immune responses that enhance resistance against macroparasite invasion.

Allergen profile of Protophormia terraenovae, other species of calliphoridae, and Lumbricus terrestris in anglers allergic to maggots in Cáceres, Spain.

BACKGROUND: Our group previously found that up to 7% of amateur anglers in Caceres, Spain may be allergic to the larvae of Protophormia terraenovae (order Diptera, family Calliphoridae) used as live bait for fishing. OBJECTIVE: To identify the pattern of major allergens in P terraenovae and other species of Calliphoridae. MATERIALS AND METHODS: Extracts of P terraenovae, Calliphora vomitoria, Lucilia sericata and Lumbricus terrestris were characterized using sodium dodecyl sulfate polyacrylamide gel electrophoresis and IgE-immunoblotting techniques in individual sera from 24 patients with a positive skin test result and/or specific IgE determination (enzyme-linked immunosorbent assay [ELISA]) to P terraenovae. ELISA and IgE-immunoblotting inhibition studies were also performed to identify potential cross-reactive allergens between these species. RESULTS: IgE-immunoblotting with P terraenovae showed a band of 15.3 kDa recognized by 15 patients, in addition to 2 further allergens of 22.8 kDa and 69 kDa. For C vomitoria, 5 bands of 73, 46, 40, 28, and 14 kDa were observed. For L sericata, 2 major allergens of 73 kDa and 14 kDa were observed. In the case of L terrestris, IgE from 13 patients recognized 1 allergen of around 15.5 kDa. IgE-immunoblotting and ELISA inhibition revealed the presence of cross-reactivity, mainly between L terrestris and P terraenovae. CONCLUSIONS: P terraenovae appears to have species-specific allergens and allergens shared with C vomitoria and L sericata. Striking immunological cross-reactivity was observed between P terraenovae and L terrestris. An allergen of 15-16 kDa could be involved in this phenomenon.

[Functional morphology of blowfly Calliphora vicina hemocytes].

In the hemolymph of Calliphora seven types of hemocytes were revealed. These are prohemocytes, which are the stem cells, stable and unstable hyaline cells, thrombocytoids, spindle cells, juvenile plasmatocytes and plasmatocytes I-IV, which represent sequential stages of one cell line differentiation were registered. The margin between them is completion of the crop emptying and beginning of wandering stage. In the feeding and crop emptying larvae take place rising of hyaline cells, thrombocytoids and hyaline cells amount with parallel growth of their defense function. The second wave of hemogenesis occur in the end of crop emptying period. It is accompanied by burst of plasmatocyte I production with their subsequent differentiation to plasmatocytes II-IV. Production of stable hyaline cells and respectively prothrombocytoids may be regulated not only by hormonal background but also by inorganic or organic particles invaded into the hemocel. Three types of hemocytes are involved in loosing of hemolymph from alien particles, notably thrombocytoids, juvenile plasmatocytes and plasmatocytes I and II. Thrombocytoids are responsible for parasitic eggs encapsulation. In addition they can phagocytize tiny organic and inorganic particles. Juvenile plasmatocytes respond to alien invasion almost as quickly as thrombocytoids at the onset of invasion. Plasmatocytes I and II start phagocytosis more slowly, hours post invasion, frequently accumulating the particles previously catched by thrombocytoids. Plasmatocytes I can absorb foreign particles and group in morules and can also surround filled thrombocytoids forming distinctive capsules. Both morules and capsules are temporary structures and disintegrate some hours lately. It is supposed the existence of three levels of immune defence: the fast response reaction of thrombocytoids and juvenile plasmatocytes and slow cellular reactions of plasmatocytes I. They are prerequisites for more extensive humoral response.