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.

Ricin-B-lectins (RBLs) have been identified in many groups of organisms, including coleopterans insects, particularly the Colorado potato beetle Leptinotarsa decemlineata (LdRBLs). We hypothesized that one of these LdRBLs (LdRBLk) may be involved in the immune response to fungal infections. We performed a theoretical analysis of the structure of this protein. Additionally, the expression levels of the LdRBlk gene were measured in L. decemlineata in response to infections with the fungi Metarhizium robertsii and Beauveria bassiana. The expression levels of LdRBlk in the L. decemlineata cuticle and fat body were increased in response to both infections. The induction of LdRBlk expression was dependent on the susceptibility of larvae to the fungi. Upregulation of the LdRBlk gene was also observed in response to other stresses, particularly thermal burns. Elevation of LdRBlk expression was frequently observed to be correlated with the expression of the antimicrobial peptide attacin but was not correlated with hsp90 regulation. Commercially available β-lectin of ricin from Ricinuscommunis was observed to inhibit the germination of conidia of the fungi. We suggest that LdRBLk is involved in antifungal immune responses in the Colorado potato beetle, either exerting fungicidal properties directly or acting as a modulator of the immune response.

Entomopathogenic fungi can be inhibited by different soil microorganisms, but the effect of a soil microbiota on fungal growth, survival, and infectivity toward insects is insufficiently understood. We investigated the level of fungistasis toward Metarhizium robertsii and Beauveria bassiana in soils of conventional potato fields and kitchen potato gardens. Agar diffusion methods, 16S rDNA metabarcoding, bacterial DNA quantification, and assays of Leptinotarsa decemlineata survival in soils inoculated with fungal conidia were used. Soils of kitchen gardens showed stronger fungistasis toward M. robertsii and B. bassiana and at the same time the highest density of the fungi compared to soils of conventional fields. The fungistasis level depended on the quantity of bacterial DNA and relative abundance of Bacillus, Streptomyces, and some Proteobacteria, whose abundance levels were the highest in kitchen garden soils. Cultivable isolates of bacilli exhibited antagonism to both fungi in vitro. Assays involving inoculation of nonsterile soils with B. bassiana conidia showed trends toward elevated mortality of L. decemlineata in highly fungistatic soils compared to low-fungistasis ones. Introduction of antagonistic bacilli into sterile soil did not significantly change infectivity of B. bassiana toward the insect. The results support the idea that entomopathogenic fungi can infect insects within a hypogean habitat despite high abundance and diversity of soil antagonistic bacteria.

The Colorado potato beetle is one of the most devastating potato pests widespread in the world. However, its viral pathogens remain highly unexplored. Here using SISPA high-throughput sequencing of Colorado potato beetle (CPB) samples derived from prepupal larvae that died from an unknown infection, we have identified two previously unknown viruses and assembled their full-length genomic sequences. The subsequent genetic and phylogenetic analysis of the obtained sequences demonstrated that the isolated viruses, named Leptinotarsa iflavirus 1 and Leptinotarsa solinvi-like virus 1 , are the novel representatives of Iflaviridae and Solinviviridae viral families, respectively. To the best of our knowledge, these are the first sequencing-confirmed insect viruses derived directly from CPB samples. And we also propose that Leptinotarsa iflavirus 1 may be associated with lethal disease in CPB.