**4. Discussion**

The development of mycoses in insects is not restricted to fungal monoinfections, and bacterial commensals and pathogens are also involved in this process [1,2,5]. We show that bacterial involvement in fungal pathogenesis and its outcome is dependent on environmental conditions, particularly temperature (Figure 8). The development of *C. militaris* in wax moth larvae was faster and more successful at 15 ◦C compared to 25 ◦C. At 25 ◦C, fungal virulence was decreased however a high frequency of spontaneous bacteriosis was observed, which was caused by the proliferation of enterococci

and enterobacteria in the hemolymph. We also showed that *C. militaris* is a weak competitor of bacteria compared to generalist fungi such as *M. robertsii* and *B. bassiana*. This is consistent with the more specific conditions for cultivation required by *C. militaris* in vivo or in vitro [70]. Occurrence of bacterioses after topical infection or injection of *C. militaris* in insects has been documented previously [22,55,58,71]. We sugges<sup>t</sup> that *C. militaris* has a limited ability to suppress host commensal bacteria, as the fungus is associated with narrow environmental requirements, including a specific temperature range [22]. Low temperatures (~15 ◦C) limit the active influence of bacteria on fungal pathogenesis. This may be explained by the fact that low temperatures are suboptimal for the proliferation of many bacteria. In addition, we showed a stronger antibacterial response in the host during *C. militaris* development under low temperature conditions.

**Figure 8.** Outline of the interactions between *C. militaris* and bacterial associates in wax moth larvae under different temperature conditions.

Consistent with our study of the midgut microbiome, the predominant bacteria in the gu<sup>t</sup> of healthy wax moths are *Enterococcus* species [72,73]. In different pathological states (e.g., toxicosis caused by *Bacillus thuringiensis* or envenomation with parasitoids), a shift in the microbiome structure toward Enterobacteriaceae prevalence occurred in the wax moth gu<sup>t</sup> [7,74]. However, we observed another effect in the present study, the replacement of one *Enterococcus* species with another under the influence of a fungal infection. Change in the dominance between different *Enterococcus* species was also documented previously after injection of wax moth larvae with *C. militaris* blastospores (unpublished [75]). The mechanism of this restructuring is not clear and is likely associated with the selective action of fungal metabolites on different species of enterococci. For example, significant changes in the mouse gu<sup>t</sup> microbiome were observed after feeding mice a major metabolite of *C. militaris* cordycepin [76]. Gamage and coworkers [77] showed that *C. militaris* water extracts exhibited different levels of inhibition of various gram-positive and gram-negative bacteria.

We observed an increase in bacterial CFU counts in the midgut during the development of *C. militaris* infection. This confirmed previous work performed on adult mosquitos following topical infection with *Beauveria* and *Isaria* species [2,3], as well as work on Colorado potato beetle larvae after topical treatment with *Metarhiziun robertsii* [78]. These enhancements may be caused by a disturbance in feeding, gu<sup>t</sup> peristalsis or by a deregulation in immune reactions during mycosis development. It should be noted that significant elevations in enterococci and enterobacteria loads in response to *C. militaris* infection were observed only in warm (25 ◦C) conditions and not in cold (15 ◦C) conditions.

In the hemolymph of uninfected larvae, we observed single colonies of enterococci and enterobacteria. Dramatic (39–54-thousand-fold) elevations in the CFU counts of these bacteria in the hemolymph during fungal infection were observed only in warm conditions (25 ◦C). It should be noted that the enterococci are a prevalent group of bacteria in wax moth integuments and enterobacteria are also present in these tissues [73,79]. However, it is hardly possible that the observed septicemia was the result of an influx through a cuticle puncture since bacterial-induced death began at five days post injection and occurred simultaneously with death due to mycosis. Moreover, the frequency of spontaneous bacterioses at 25 ◦C was positively correlated with the dose of *C. militaris* conidia. The source of bacterial penetration into the hemolymph could be the gu<sup>t</sup> or other organs such as the trachea or excretory organs, the biome of which has not been studied in the wax moth. It is interesting to note that the occurrence of septicemia was less common after injection of wax moth larvae with conidia of the generalist fungi *Metarhizium* or *Beauveria*. For example, injection of the larvae with *B. bassiana* and *M. robertsii* at doses of 2500 conidia per larvae and subsequent incubation at 25 ◦C did not lead to bacterial decomposition and all cadavers were mummified and overgrown with these fungi (Figure S3). Fan and coauthors [48] showed that at the final stages of mycoses, *B. bassiana* suppresses the proliferation of bacteria in the host through the production of secondary metabolites such as oosporeins. However, compared to *Beauveria* and *Metarhizium* species, *C. militaris* has fewer genes involved in secondary metabolism [50,53]. It is likely that the combination of less developed mechanisms for the suppression of bacteria and harsher tools for host tissue destruction caused the septicemia during *C. militaris* infection. In particular, we recently showed that *C. militaris* infection led to necrotic death of hemocytes and a strong elevation in dopamine and ROS in wax moth larvae, which were not observed after *M. robertsii* infection [59].

The development and outcome of the fungal infections can also be mediated by di fferences in host immune responses at 15 ◦C and 25 ◦C. Antifungal peptides (gallerimycin and galiomycin) more actively responded to *C. militaris* infection at a higher temperature. This is consistent with previous investigations in which we showed a stronger elevation in phenoloxidase and encapsulation levels in wax moths in response to *C. militaris* infection at 25 ◦C compared to 15 ◦C [22], and this correlated with a greater survival of the infected insects at 25 ◦C. It is interesting that the antifungal peptide genes were actively expressed at 25 ◦C, not only in the fat body but also in the midgut. This may be due to a systemic immune response or an attack of lateral midgut tissues by the fungus. Unlike the antifungal response, the expression of antibacterial peptide genes (gloverin, cecropin, lysozyme) was more active in the fat body at 15 ◦C, which correlated with the absence of elevated CFUs and bacterial decomposition at this temperature. It was previously shown that a short exposure of *G. mellonella* to low temperatures led to an increase in AMP expression in response to *B. thuringiensis* infection [28]. Similar exposure led to enhanced AMP expression in *Ostrinia furnacalis* in the absence of infection [29]. Elevated antibacterial responses were also observed under prolonged cooling. For example, Ferguson and Sinclair [27] showed that overwintering *Eurosta solidagnis* larvae were characterized by an increased clearance of the gram-positive bacteria *Bacillus subtilis* in the hemolymph compared to autumn and spring larvae. According the present study, under cold conditions, insects may exhibit increased antibacterial responses during fungal infections.

We observed an increase in the expression of the gloverin and lysozyme genes in the midgut in response to fungal infection. This elevation was obviously caused by changes in the microbiota structure and the elevation in the bacterial load in the midgut during the development of mycosis. Similar changes were observed by Ramirez and coworkers [3] in the midgut of adult *Aedes aegypti* mosquitos in the acute stages of mycoses caused by *Beauveria* and *Isaria* species. However, we did not observe general temperature-dependent trends in the expression of antibacterial genes in the midgut.

The IAP gene was upregulated in the fat body at a low temperature and its upregulation in response to the infection was also observed only at a low temperature. Previous studies showed that IAP is linked to the IMD immune signaling pathway in insects [38]. In particular, knockdown of this gene in *D. melanogaster* led to confined expression of AMPs in response to bacterial infections and increased susceptibility to gram-negative bacteria [38]. In locusts, IAP knockdown led to blocked defensin expression, which was induced by *Metarhizium acridum* infection [37]. In our experiments, a lack of IAP expression at 25 ◦C was associated with a lower upregulation of antibacterial peptides and an active proliferation of bacteria in the hemolymph, which is consistent with the abovementioned studies.

The NOX-DUOX domain gene displayed an interesting pattern of expression. This system functions in the regulation of bacterial homeostasis, as has been shown in *Drosophila* and mosquitoes [40,42,80]. In our study, the gene was upregulated slightly in the fat body in response to fungal infection at both temperatures. In the midgut, we observed a significant downregulation of this gene at 15 ◦C (Table 2). This was correlated with a trend toward increased enterococci CFU counts in the midgut at 15 ◦C compared to 25 ◦C (Figure 3). This gene was downregulated in response to *C. militaris* infection only at 15 ◦C. This may be caused by the high acuity of mycosis at this temperature and it may be a consequence of the prioritization in immune reactions between the hemocoel and gut, as was suggested by Wei and coworkers [2]. However, this decrease in gene expression and the elevation in the enterococci load at 15 ◦C did not lead to the colonization of the hemocoel by bacteria, i.e., the proliferation of bacteria occurred only in the gu<sup>t</sup> lumen under this temperature. Further immunological and histopathological studies are needed to establish the mechanisms of septicemia development during fungal infections.

We observed an upregulation of HSP70 in both tissues and an upregulation of HSP90 in the midgut at a low temperature. This result was expected because an increase in the expression of these genes during cold diapause has been observed in various insect taxa [46]. We also observed a downregulation in HSP70 expression in the fat body in response to *C. militaris* infection. Previous studies reported either an increase in HSP expression in different tissues of *G. mellonella* after infection with *B. bassiana* and *Conidiobolus coronatus*, or no change compared to uninfected insects [7,61,81]. These inconstancies may be caused by differences in pathogenesis that occur after infection with different fungal species and strains. Regarding the antibacterial response, it was shown that HSP70 transcripts were highly induced in arthropods (*Penaeus monodon*) after injection with bacteria *Vibrio* [82]. In wax moths, an increase in HSP90 expression was observed in response to *Bacillus thuringiensis* infection [83] and mixed (bacteria and yeast) infections [84]. Linder and coworkers [16] sugges<sup>t</sup> that HSPs may improve immune functions against bacterioses at cool temperatures in *Drosophila melanogaster*. The authors have shown elevated expression of HSP83, PGRP-LS and AMPs, and increased resistance to bacteria (*Pseudomonas aeruginosa* and *Lactococcus lactis*) in cold conditions (17 ◦C) compared to warm conditions (29 ◦C). Similarly, in our work, septicemia was observed most often with the lowest levels of HSP expression (fungal infection at 25 ◦C), although we did not observe any correlations between HSP and AMP expression. It is possible that increased expression of HSPs at low temperature may help maintain tissue integrity in gu<sup>t</sup> and other organs and prevent penetration of bacteria into hemolymph.
