**5. Conclusions**

Bacterial associates of insects may influence the development and outcome of fungal infections. Using a model system of *C. militaris* and *G. mellonella*, in the present study we found that these interactions are significantly dependent on temperature. At high temperatures, these relationships develop in favor of spontaneous bacterioses, while under low temperatures they develop in favor of mycoses. The explanation for these outcomes may lie in the properties of the fungus, as well as in the immune reactions of the host during mycosis development. *C. militaris* is a weak competitor of bacteria and therefore it requires low temperatures to avoid antagonism with bacterial associates of the host to complete its development successfully. In addition, we observed weakened antifungal responses along with increased antibacterial responses in wax moths at a low temperature, which should be beneficial for the development of the fungus. We confirmed the previous works that have shown that AMP expression in *G. mellonella* is temperature-dependent [18,23,28]. However, a comparison of AMP

expression in response to fungal infection at constant low (15 ◦C) and moderate (25 ◦C) temperatures was performed for the first time. Our results are consistent with previous studies in which short cooling of *G. mellonella* [28] and prolonged cooling of other lepidopterans [27] have led to an increase in antibacterial response. Moreover, we found increased expression of stress-related genes in the midgut under the constant low temperature, which may prevent the disruption of gu<sup>t</sup> tissues and penetration of bacteria from the gu<sup>t</sup> into the hemocoel. Further studies should focus on the interaction between bacterial growth and fungal infections using histopathological and histomolecular approaches, as well as on development of *C. militaris* in natural hosts using natural methods of infection. Our research may promote physiological and ecological studies into the interactions between pathogenic fungi, insect hosts and bacterial associates.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2309-608X/6/3/170/s1, Figure S1. Rarefaction curves of the OTU numbers for each sample. Table S1. List and description of genes and primer sequences used in qPCR. Figure S2. Diversity indexes of bacterial communities in the midgut of wax moth larvae at 96 h post injection of *C. militaris* (2500 conidia per larva) with incubation at 15 ◦C and 25 ◦C. Indexes were calculated for OTU levels. Di fferent letters indicate significant di fferences between treatments (Dunn's test, *p* < 0.05). Figure S3. Wax moth larvae overgrown with *B. bassiana* (A) and *M. robertsii* (B) at four days after injection with 2500 conidia per larva and incubation at 25 ◦C.

**Author Contributions:** V.Y.K. and O.T. designed experiments, performed bioassays, analyzed data, prepared figures and tables and wrote the main manuscript text; E.K. and U.R. performed gene expression analysis and wrote the manuscript; O.P., M.T. and O.Y. performed bioassays and sample preparation, and approved the final draft; T.A. and M.K. performed 16S rRNA sequence analyses and wrote the manuscript; O.Y. and V.G. managed the project and approved the final draft. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was initiated within the framework of the Russian Science Foundation project (No. 18-74-00085) and continued within the project of the same fund (No. 20-74-10043). The metagenomic study was supported by the Russian Foundation for Basic Research (No. 18-34-20060).

**Acknowledgments:** We express our special thanks to Natalia Kryukova, Tatyana Marchenko and Evgenia Buntova for their help with lab work. We also thank Mikhail Beketov for English corrections.

**Conflicts of Interest:** All authors declare that they have no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
