Gut bacteria influence the development of different pathologies caused by bacteria, fungi and parasitoids in insects. Wax moth larvae became more susceptible to fungal infections after envenomation by the ectoparasitoid Habrobracon hebetor . In addition, spontaneous bacterioses occurred more often in envenomated larvae. We analyzed alterations in the midgut microbiota and immunity of the wax moth in response to H. hebetor envenomation and topical fungal infection ( Beauveria bassiana ) alone or in combination using 16S rRNA sequencing, an analysis of cultivable bacteria and a qPCR analysis of immunity- and stress-related genes. Envenomation led to a predominance shift from enterococci to enterobacteria, an increase in CFUs and the upregulation of AMPs in wax moth midguts. Furthermore, mycosis nonsignificantly increased the abundance of enterobacteria and the expression of AMPs in the midgut. Combined treatment led to a significant increase in the abundance of Serratia and a greater upregulation of gloverin. The oral administration of predominant bacteria ( Enterococcus faecalis, Enterobacter sp. and Serratia marcescens ) to wax moth larvae synergistically increased fungal susceptibility. Thus, the activation of midgut immunity might prevent the bacterial decomposition of envenomated larvae, thus permitting the development of fungal infections. Moreover, changes in the midgut bacterial community may promote fungal killing.

Gut physiology and the bacterial community play crucial roles in insect susceptibility to infections and insecticides. Interactions among Colorado potato beetle Leptinotarsa decemlineata (Say), its bacterial associates, pathogens and xenobiotics have been insufficiently studied. In this paper, we present our study of the survival, midgut histopathology, activity of digestive enzymes and bacterial communities of L. decemlineata larvae under the influence of Bacillus thuringiensis var. tenebrionis (morrissoni) (Bt), a natural complex of avermectins and a combination of both agents. Moreover, we estimated the impact of culturable enterobacteria on the susceptibility of the larvae to Bt and avermectins. An additive effect between Bt and avermectins was established regarding the mortality of the larvae. Both agents led to the destruction of midgut tissues, a decrease in the activity of alpha-amylases and alkaline proteinases, a decrease in the Spiroplasma leptinotarsae relative abundance and a strong elevation of Enterobacteriaceae abundance in the midgut. Moreover, an elevation of the enterobacterial CFU count was observed under the influence of Bt and avermectins, and the greatest enhancement was observed after combined treatment. Insects pretreated with antibiotics were less susceptible to Bt and avermectins, but reintroduction of the predominant enterobacteria Enterobacter ludwigii, Citrobacter freundii and Serratia marcescens increased susceptibility to both agents. We suggest that enterobacteria play an important role in the acceleration of Bt infection and avermectin toxicoses in L. decemlineata and that the additive effect between Bt and avermectin may be mediated by alterations in the bacterial community.

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Fungal infections and toxicoses caused by insecticides may alter microbial communities and immune responses in the insect gut. We investigated the effects of Metarhizium robertsii fungus and avermectins on the midgut physiology of Colorado potato beetle larvae. We analyzed changes in the bacterial community, immunity- and stress-related gene expression, reactive oxygen species (ROS) production, and detoxification enzyme activity in response to topical infection with the M. robertsii fungus, oral administration of avermectins, and a combination of the two treatments. Avermectin treatment led to a reduction in microbiota diversity and an enhancement in the abundance of enterobacteria, and these changes were followed by the downregulation of Stat and Hsp90, upregulation of transcription factors for the Toll and IMD pathways and activation of detoxification enzymes. Fungal infection also led to a decrease in microbiota diversity, although the changes in community structure were not significant, except for the enhancement of Serratia. Fungal infection decreased the production of ROS but did not affect the gene expression of the immune pathways. In the combined treatment, fungal infection inhibited the activation of detoxification enzymes and prevented the downregulation of the JAK-STAT pathway caused by avermectins. The results of this study suggest that fungal infection modulates physiological responses to avermectins and that fungal infection may increase avermectin toxicosis by blocking detoxification enzymes in the gut.

Combination of insect pathogenic fungi and microbial metabolites is a prospective method for mosquito control. The effect of the entomopathogenic fungus Metarhizium robertsii J.F. Bischoff, S.A. Rehner & Humber and avermectins on the survival and physiological parameters of Aedes aegypti (Linnaeus, 1762) larvae (dopamine concentration, glutathione S-transferase (GST), nonspecific esterases (EST), acid proteases, lysozyme-like, phenoloxidase (PO) activities) was studied. It is shown that the combination of these agents leads to a synergistic effect on mosquito mortality. Colonization of Ae. aegypti larvae by hyphal bodies following water inoculation with conidia is shown for the first time. The larvae affected by fungi are characterized by a decrease in PO and dopamine levels. In the initial stages of toxicosis and/or fungal infection (12 h posttreatment), increases in the activity of insect detoxifying enzymes (GST and EST) and acid proteases are observed after monotreatments, and these increases are suppressed after combined treatment with the fungus and avermectins. Lysozyme-like activity is also most strongly suppressed under combined treatment with the fungus and avermectins in the early stages posttreatment (12 h). Forty-eight hours posttreatment, we observe increases in GST, EST, acid proteases, and lysozyme-like activities under the influence of the fungus and/or avermectins. The larvae affected by avermectins accumulate lower levels of conidia than avermectin-free larvae. On the other hand, a burst of bacterial CFUs is observed under treatment with both the fungus and avermectins. We suggest that disturbance of the responses of the immune and detoxifying systems under the combined treatment and the development of opportunistic bacteria may be among the causes of the synergistic effect.

Various insect bacterial associates are involved in pathogeneses caused by entomopathogenic fungi. The outcome of infection (fungal growth or decomposition) may depend on environmental factors such as temperature. The aim of this study was to analyze the bacterial communities and immune response of Galleria mellonella larvae injected with Cordyceps militaris and incubated at 15 °C and 25 °C. We examined changes in the bacterial CFUs, bacterial communities (Illumina MiSeq 16S rRNA gene sequencing) and expression of immune, apoptosis, ROS and stress-related genes (qPCR) in larval tissues in response to fungal infection at the mentioned temperatures. Increased survival of larvae after C. militaris injection was observed at 25 °C, although more frequent episodes of spontaneous bacteriosis were observed at this temperature compared to 15 °C. We revealed an increase in the abundance of enterococci and enterobacteria in the midgut and hemolymph in response to infection at 25 °C, which was not observed at 15 °C. Antifungal peptide genes showed the highest expression at 25 °C, while antibacterial peptides and inhibitor of apoptosis genes were strongly expressed at 15 °C. Cultivable bacteria significantly suppressed the growth of C. militaris. We suggest that fungi such as C. militaris may need low temperatures to avoid competition with host bacterial associates.