The entomopathogenic fungus Conidiobolus coronatus produces enzymes that may hydrolyze the cuticle of Galleria mellonella. Of these enzymes, elastase activity was the highest: this figure being 24 times higher than NAGase activity 553 times higher than chitinase activity and 1844 times higher than lipase activity. The present work examines the differences in the hydrolysis of cuticles taken from larvae, pupae and adults (thorax and wings), by C. coronatus enzymes. The cuticles of the larvae and adult thorax were the most susceptible to digestion by proteases and lipases. Moreover, the maximum concentration of free N-glucosamine was in the hydrolysis of G. mellonella thorax. These differences in the digestion of the various types of cuticle may result from differences in their composition. GC-MS analysis of the cuticular fatty acids isolated from pupae of G. mellonella confirmed the presence of C 8:0, C 9:0, C 12:0, C 14:0, C 15:0, C 16:1, C 16:0, C 17:0, C 18:1, C 18:0, with C 16:0 and C 18:0 being present in the highest concentrations. Additional fatty acids were found in extracts from G. mellonella imagines: C 10:0, C 13:0, C 20:0 and C 20:1, with a considerable dominance of C 16:0 and C 18:1. In larvae, C 16:0 and C 18:1 predominated. Statistically significant differences in concentration (p≤0.05) were found between the larvae, pupae and imago for each fatty acid. The qualitative and quantitative differences in the fatty acid composition of G. mellonella cuticle occurring throughout normal development might be responsible for the varied efficiency of fungal enzymes in degrading larval, pupal and adult cuticles.

Entomopathogenic fungi infect insects via penetration through the cuticle, which varies remarkably in chemical composition across species and life stages. Fungal infection involves the production of enzymes that hydrolyse cuticular proteins, chitin and lipids. Host specificity is associated with fungus-cuticle interactions related to substrate utilization and resistance to host-specific inhibitors. The soil fungus Conidiobolus coronatus (Constantin) (Entomophthorales: Ancylistaceae) shows virulence against susceptible species. The larvae and pupae of Calliphora vicina (Robineau-Desvoidy) (Diptera: Calliphoridae), Calliphora vomitoria (Linnaeus), Lucilia sericata (Meigen) (Diptera: Calliphoridae) and Musca domestica (Linnaeus) (Diptera: Muscidae) are resistant, but adults exposed to C. coronatus quickly perish. Fungus was cultivated for 3 weeks in a minimal medium. Cell-free filtrate, for which activity of elastase, N-acetylglucosaminidase, chitobiosidase and lipase was determined, was used for in vitro hydrolysis of the cuticle from larvae, puparia and adults. Amounts of amino acids, N-glucosamine and fatty acids released were measured after 8 h of incubation. The effectiveness of fungal enzymes was correlated with concentrations of compounds detected in the cuticles of tested insects. Positive correlations suggest compounds used by the fungus as nutrients, whereas negative correlations may indicate compounds responsible for insect resistance. Adult deaths result from the ingestion of conidia or fungal excretions.

The evolutionary success of insects is arguably due to their ability to build up a complex, highly-adaptable and very effective defense system against numerous pathogens, including entomopathogenic fungi. This system relies on the humoral immune system and cellular defense reactions. The first line of defense against biological pathogens is a cuticle formed of several layers. The cuticular lipids may contain hydrocarbons, free fatty acids (FFA), alcohols, waxes, glycerides, aldehydes and sterols. Cuticular fatty acids may also play a role in defending against fungal invasion. Our present findings show that the diet of insects can have a significant effect on their sensitivity and defense response to pathogens; for example, while G. mellonella larvae fed on beeswax had a similar appearance to those reared on a semi-artificial diet, they possessed a different cuticular free fatty acid (FFA) profile to those fed on a semi-artificial diet, and were less sensitive to Conidiobolus coronatus infection. It is possible that the presence of heneicosenoic acid (C21:1) and other long-chain free fatty acids (C22:0, C24:0, C26:0), as well as Brevibacillus laterosporus bacteria, on the cuticle of larvae fed on beeswax, plays a protective role against fungal invasion. Insect pests represent a global problem. An understanding of the basic mechanisms underlying the fungal infection of insects might provide a clearer insight into their defenses, thus allowing the design of more effective, and environmentally-friendly, means of controlling them. The greater wax moth is an excellent model for the study of immunology resistance. Knowledge of the influence of diet on pathogen resistance in insects can be also useful for creating a model of human diseases caused by pathogens, such as Candia albicans.

The interactions between entomopathogenic fungi and insects serve a classic example of a co-evolutionary arms race between pathogens and their target host. The cuticle, site of the first contact between insects and entomopathogenic fungus, is an important defensive barrier against pathogens. It is covered by a layer of lipids that appears to play a key role in these processes and cuticular free fatty acid (FFA) profiles are consider as a determinant of susceptibility, or resistance, to fungal infections. These profiles are species-specific. The cockroaches Blattella germanica (Blattodea: Blattidae) and Blatta orientalis (Blattodea: Ectobiidae) are unsusceptible to the soil fungus Conidiobolus coronatus (Entomophthorales: Ancylistaceae) infection, therefore we studied the profiles of FFAs in order to understand the defensive capabilities of the cockroaches. The fungus was cultivated for three weeks in minimal medium. Cell-free filtrate was obtained, assayed for elastase, N-acetylglucosaminidase, chitobiosidase and lipase activity, and then used for in vitro hydrolysis of the cuticle from wings and thoraces of adults and oothecae. The amounts of amino acids, N-glucosamine and FFAs released from the hydrolysed cuticle samples were measured after eight hours of incubation. The FFA profiles of the cuticle of adults, and the wings, thoraces and oothecae of both species were established using GC-MS and the results were correlated with the effectiveness of fungal proteases, chitinases and lipases in the hydrolyzation of cuticle samples. Positive correlations would suggest the existence of compounds used by the fungus as nutrients, whereas negative correlations may indicate that these compounds could be engaged in insect defence.

Invertebrates are becoming more popular models for research on the immune system. The innate immunity possessed by insects shows both structural and functional similarity to the resistance displayed by mammals, and many processes occurring in insect hemocytes are similar to those that occur in mammals. The humoral immune response in insects acts by melanization, clotting and the production of reactive oxygen species and antimicrobial peptides, while the cellular immunity system is based on nodulation, encapsulation and phagocytosis. An increasingly popular insect model in biological research is Galleria mellonella, whose larvae are sensitive to infection by the entomopathogenic fungus Conidiobolus coronatus, which can also be dangerous to humans. One group of factors that modulate the response of the immune system during infection in mammals are heat shock proteins (HSPs). The aim of this study was to investigate whether infection by C. coronatus in G. mellonella hemolymph is accompanied by an increase of HSP90, HSP70, HSP60 and HSP27. Larvae (five-day-old last instar) were exposed for 24 hours to fully-grown and sporulating fungus. Hemolymph was collected either immediately after termination of exposure (F24) or 24 hours later (F48). The concentration of the HSPs in hemolymph was determined using ELISA. Immunolocalization in hemocytes was performed using fluorescence microscopy and flow cytometry. HSP90, HSP70, HSP60 and HSP27 were found to be present in the G. mellonella hemocytes. HSP60 and HSP90 predominated in healthy insects, with HSP70 and HSP27 being found in trace amounts; HSP60 and HSP27 were elevated in F24 and F48, and HSP90 was elevated in F48. The fungal infection had no effect on HSP70 levels. These findings shed light on the mechanisms underlying the interaction between the innate insect immune response and entomopathogen infection. The results of this innovative study may have a considerable impact on research concerning innate immunology and insect physiology.

Naturally occurring entomopathogenic fungi such as Conidiobolus coronatus are important regulatory factors of insect populations. GC-MS analysis of fungal cell-free filtrates showed that C. coronatus synthesizes two β- carboline alkaloids: harman and norharman. Significantly higher levels of both alkaloids are produced by C. coronatus in minimal postincubation medium than in rich medium. The beta-carboline alkaloids may have an effect on the nervous system of insects and their behavior. Harman and norharman were applied to Galleria mellonella larvae (a parasite of honeybees) either topically or mixed with food. Larvae received alkaloids in three concentrations: 750, 1000 or 1250 ppm. The effect on the survival and further development of larvae was examined. Both harman and norharman delayed pupation and adult eclosion, and inhibit total monoamine oxidase activity. In addition, they increased the serotonin concentration and decreased the monoamine oxidase A level in the heads of the moths. It is likely that the alkaloids were metabolized by the insects, as their effect wore off 24 hours after topical application. This is the first study to show that C. coronatus produces alkaloids. Its aim was to identify the actions of β-carboline alkaloids on insect development and serotonin-regulating enzymes. Knowledge of the potential role of harman and norharman in the process of fungal infection might lead to the development of more effective and environmentally-friendly means of controlling insect pests.

Background: Although the β-carboline alkaloids harman and norharman are considered as plant metabolites, they can also be secreted by fungi such as the entomopathogen Conidiobolus coronatus. Norharman and harman are also known to be reversible competitive monamine oxidase inhibitors, which increase serotonin concentrations in tissues. In addition, these alkaloids are able to bind to serotonin receptors, an important immune regulatory molecule in both vertebrates and invertebrates. In insects, serotonin modulates hemocyte phagocytosis, nodule formation and the populations of hemocyte classes. The present study examines whether harman and norharman may influence the phagocytic activity of insect hemocytes by regulating serotonin levels. Results: Significantly greater serotonin levels and hemocyte phagocytic activity were observed after 24 h of exposure to food contaminated with harman and norharman. Similar responses were noticed 1 h after topical application or addition to in vitro hemocyte cultures. Observations and measurements performed 24 h later revealed decreased responses, suggesting decomposition and/or exertion of alkaloids and/or serotonin. Harman and norharman influenced the activity of Galleria mellonella plasmatocytes and the granulocyte cytoskeleton. Disturbances in hemocyte network formation, abnormal cell shape, naked nuclei, cell aggregates, fragments of disintegrated cells, interrupted cell membrane continuity and actin condensation in cells were observed. Conclusion: Our findings may have a considerable impact on research concerning insect physiology, parasitology, immunology and biocontrol of pests. They confirm for the first time that harman and norharman (metabolites of the entomopathogenic fungus C. coronatus) elevate serotonin levels in G. mellonella hemocytes, thus potentially stimulating their phagocytic activity. Our studies shed light on the mechanisms underlying the interaction between innate insect immune responses and entomopathogen metabolites.

The flies of the Sarcophagidae, widespread throughout the temperate zone, are of great significance in Medicine, Veterinary science, Forensics and Entomotoxicology. Lipids are important elements of cell and organelle membranes and a source of energy for embryogenesis, metamorphosis and flight. Cuticular lipids protect from desiccation and act as recognition cues for species, nest mates and castes, and are a source of various pheromones. The free fatty acid (FFA) profile of cuticular and internal extracts of Sarcophaga (Liopygia) argyrostoma (Robineau-Desvoidy, 1830) larvae, pupae and adults was determined by gas chromatography–mass spectrometry (GC–MS). The larvae, pupae and adults contained FFAs from C5:0 to C28:0. The extracts differed quantitatively and qualitatively from each other: C18:1 > C16:1 > C16:0 > C18:0 predominated in the cuticular and internal extracts from the larvae and adults, while 18:1 > C16:0 > C16:1 > C18:0 predominated in the pupae. The FFA profile of the cuticle varies considerably between each development stage: C23:0 and C25:0 are only present in larvae, C28:0 in the pupal cuticle, and C12:1 and C18:3 in internal extracts from adults. The mechanisms underlying this diversity are discussed herein.