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Harman and norharman, metabolites of entomopathogenic fungus Conidiobolus coronatus (Entomopthorales), disorganize development of Galleria mellonella (Lepidoptera) and affect serotonin-regulating enzymes.

ABSTRACT: 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.

SUBMITTER: Wronska AK

PROVIDER: S-EPMC6169936 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

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.

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Harman and norharman, metabolites of entomopathogenic fungus Conidiobolus coronatus (Entomopthorales), disorganize development of Galleria mellonella (Lepidoptera) and affect serotonin-regulating enzymes.

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