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Cuticular fatty acids of Galleria mellonella (Lepidoptera) inhibit fungal enzymatic activities of pathogenic Conidiobolus coronatus.

ABSTRACT: 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.

SUBMITTER: Wronska AK

PROVIDER: S-EPMC5843172 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Cuticular fatty acids of Galleria mellonella (Lepidoptera) inhibit fungal enzymatic activities of pathogenic Conidiobolus coronatus.

Wrońska Anna Katarzyna AK Wrońska Anna Katarzyna AK Boguś Mieczysława Irena MI Włóka Emilia E Kazek Michalina M Kaczmarek Agata A Zalewska Katarzyna K

PloS one 20180308 3

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 ...[more]

PMID: 29518079

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Project description: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.

Project description:Mycoses are a global problem that affects humans and animals. In the present study, the entomopathogenic soil fungus Conidiobolus coronatus (Entomophthorales), infecting in tropics also humans, sheep and horses, was cultivated with the addition of insect cuticular compounds (CCs) previously detected in the cuticle of C. coronatus-resistant fly species (C10-C30 fatty alcohols, butyl oleate, butyl stearate, glycerol oleate, squalene, tocopherol acetate). Our findings indicate that CCs have diversified and complex effects on the growth and sporulation of C. coronatus and its ability to infect the larvae of Galleria mellonella (Lepidoptera). The CCs affected protein content and cuticle-degrading enzymes (CDEs) activity in the conidia. Some CCs inhibited fungal growth (0.1% C10), decreased sporulation (C12, C16, C24, C28, C30, butyl stearate, squalene), virulence (C12, C14, butyl oleate, butyl stearate) and protein content (C18). They also reduced conidial CDE activity: elastase (C24, butyl oleate, butyl stearate, squalene, tocopherol acetate), chitobiosidase (C12, C14, C20) and lipase (C12, C18, C26, squalene, tocopherol acetate). Several CCs enhanced sporulation (C14, C18, C22, C26, C30), virulence (C18, C26, squalene), conidial protein content (C16, C24, C30, squalene) and CDE activity: elastase (C10, C16, C18), NAGase (C16, C20), chitobiosidase (C16) and lipase (C10, C14, C16, C20, butyl oleate). Our findings indicate that C. coronatus colonies grown on media supplemented with CCs employ various compensation strategies: colonies grown with C16 alcohol demonstrated reduced sporulation but greater conidial protein accumulation and increased elastase, NAGase, chitobiosidase and lipase activity, thus preserving high virulence. Also, colonies supplemented with C18 alcohol demonstrated high virulence and enhanced sporulation and elastase activity but slightly decreased conidial protein content. CCs that inhibit the activity of lipases and proteases show promise in the fight against conidiobolomycosis.

Project description:BackgroundAlthough 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.ResultsSignificantly 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.ConclusionOur 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.

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Cuticular fatty acids of Galleria mellonella (Lepidoptera) inhibit fungal enzymatic activities of pathogenic Conidiobolus coronatus.

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Cuticular fatty acids of Galleria mellonella (Lepidoptera) inhibit fungal enzymatic activities of pathogenic Conidiobolus coronatus.

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