A Neurotoxic Insecticide Promotes Fungal Infection in Aedes aegypti Larvae by Altering the Bacterial Community.

Symbiotic bacteria have a significant impact on the formation of defensive mechanisms against fungal pathogens and insecticides. The microbiome of the mosquito Aedes aegypti has been well studied; however, there are no data on the influence of insecticides and pathogenic fungi on its structure. The fungus Metarhizium robertsii and a neurotoxic insecticide (avermectin complex) interact synergistically, and the colonization of larvae with hyphal bodies is observed after fungal and combined (conidia + avermectins) treatments. The changes in the bacterial communities (16S rRNA) of Ae. aegypti larvae under the influence of fungal infection, avermectin toxicosis, and their combination were studied. In addition, we studied the interactions between the fungus and the predominant cultivable bacteria in vitro and in vivo after the coinfection of the larvae. Avermectins increased the total bacterial load and diversity. The fungus decreased the diversity and insignificantly increased the bacterial load. Importantly, avermectins reduced the relative abundance of Microbacterium (Actinobacteria), which exhibited a strong antagonistic effect towards the fungus in in vitro and in vivo assays. The avermectin treatment led to an increased abundance of Chryseobacterium (Flavobacteria), which exerted a neutral effect on mycosis development. In addition, avermectin treatment led to an elevation of some subdominant bacteria (Pseudomonas) that interacted synergistically with the fungus. We suggest that avermectins change the bacterial community to favor the development of fungal infection.

[Phenotypic and genetic changes of entomopathogenic ascomycete Beauveria Bassiana under passaging through various hosts].

Phenotypic and genetic estimations of entomopathogenic ascomycete B.bassiana (strain Sar-31) after 6-passaging through four hosts were shown. Increasing of virulence, changes in morpho-cultural characteristics and variations in Inter Simple Sequence Repeats (ISSR) assay between initial and reisolated cultures were registered. Six passages of entomopathogenic ascomycete Beauveria bassiana (strain Sar-31) through four hosts (Galleria mellonella, Tenebrio molitor, Leptinotarsa decemlineata, Locusta migratoria) and following estimation of phenotypic and genetic differences of the initial strain and reisolated cultures were conducted. The passaging of strain through certain host led to increasing of virulence for both this host and other test-insects. Unidirectional changes of morpho-cultural characteristics: colonies pigmentation and relief strengthening, increasing of conidia production and lipolytic activity were registered in all passaged cultures. Genetic analysis with 6 ISSR markers revealed variations between initial and reisolated cultures in 3 markers. Taken together, the results of this study help us understand potential ways of fungi strains changes during epizootic process and possibilities of ISSR assay applying for investigation of pathogen transmission.

[COMPARATIVE ANALYSIS OF THE IMMUNE REACTIONS IN COLORADO POTATO BEETLE LARVAE UNDER DEVELOPMENT OF MYCOSES CAUSED BY METARHIZIUM ROBERTSII, M. BRUNNEUM AND M. PEMPHIGI].

A comparative investigation of humoral and cellular immune response in larvae of Colorado potato beetle Leptinotarsa decemlineata was conducted under development of mycoses caused by entomopatho- genic fungi Metarhizium robertsii, M. brunneum and M. pemphigi. The larvae were found highly suscep- tible to M. robertsii, M. brunneum and less susceptible to M. pemphigi. The susceptibility to the fungi was not correlated with the rate of conidia germination in epicuticular extracts of larvae. A non-specific for Colorado beetle pathogen M. pemphigi did not cause significant changes in the immune response and did not result in colonization of larvae hemocoel. Infection with M. robertsi and M. brunneum led to an increase in total hemocyte count at the initial stages of mycoses (day 2) followed by a sharp decrease on day 3. The strongest decrease was observed for the immunocompetent cells - plasmatocytes and granu- locytes. Enhanced phenoloxidase activity in hemolymph and cuticle was found on days 2 and 3 after in- fection. These changes in immune reactions correlated with the level of virulence of the strains. Thus, the immune response in Colorado potato beetle larvae is an important factor determining differences in the development of mycoses caused by different Metarhizium species.

More than a colour change: insect melanism, disease resistance and fecundity.

A 'dark morph' melanic strain of the greater wax moth, Galleria mellonella, was studied for its atypical, heightened resistance to infection with the entomopathogenic fungus, Beauveria bassiana. We show that these insects exhibit multiple intraspecific immunity and physiological traits that distinguish them from a non-melanic, fungus-susceptible morph. The melanic and non-melanic morphs were geographical variants that had evolved different, independent defence strategies. Melanic morphs exhibit a thickened cuticle, higher basal expression of immunity- and stress-management-related genes, higher numbers of circulating haemocytes, upregulated cuticle phenoloxidase (PO) activity concomitant with conidial invasion, and an enhanced capacity to encapsulate fungal particles. These insects prioritize specific augmentations to those frontline defences that are most likely to encounter invading pathogens or to sustain damage. Other immune responses that target late-stage infection, such as haemolymph lysozyme and PO activities, do not contribute to fungal tolerance. The net effect is increased larval survival times, retarded cuticular fungal penetration and a lower propensity to develop haemolymph infections when challenged naturally (topically) and by injection. In the absence of fungal infection, however, the heavy defence investments made by melanic insects result in a lower biomass, decreased longevity and lower fecundity in comparison with their non-melanic counterparts. Although melanism is clearly correlated with increased fungal resistance, the costly mechanisms enabling this protective trait constitute more than just a colour change.

[An increase in the immune system activity of the wax moth Galleria mellonella and of the Colorado potato beetle Leptinotarsa decemlineata under effect of organophosphorus insecticide].

There has been performed evaluation of the effect of the organophosphorus insecticide (pirimifos-methyl) on some components of the insect immune response. The cellular (a change of the number of hemocytes and of intensity of incapsulation) and the humoral (a change of phenoloxidase activity) components of the immune response were studied in larvae of representatives of two orders--the Colorado potato beetle (Leptinotarsa decemlineata, Chrysomelidae, Coleoptera) and the wax moth (Galeriia mellonella, Pyralidae, Lepidoptera). The action of the insecticide has been found to lead to stimulation of immune reactions (an increase of phenoloxidase activities and of intensity of incapsulation, a rise of the number of hemocytes) at the contact treatment of both sublethal and the half-lethal doses of pirimifos-methyl.