**1. Introduction**

The development of infectious diseases in animals is often accompanied by the proliferation of complex concomitant microorganisms in addition to the development of the main pathogen. In particular, mycoses of insects may develop as mixed infections when opportunistic bacteria are actively involved in the pathogenesis. This occurs due to tissue damage [1] and deregulation of host immune reactions in response to the pathogenic fungi [2,3]. Direct and indirect interactions between fungi and bacteria may lead to both antagonistic and synergistic e ffects on survival [2,4–6]. In addition, interrelations between fungi and bacteria in insect hosts may be mediated by complex environmental factors, such as temperature, chemicals, or parasitoids, that have an influence on the outcome of the disease [7,8]. However, these immune-ecological studies are just beginning to develop.

Temperature is one of the crucial factors that influences the development of mycoses and bacterioses in insects. Temperature acts on both microorganism growth and on insect immune and

behavioral reactions [9–14]. Entomopathogenic ascomycetes usually have optimal growth between 20–30 ◦C [15]. In contrast, many bacteria that are associated with terrestrial insects exhibit more active growth between 28–37 ◦C [16,17]. In many cases, host cellular and humoral antifungal reactions and resistance to fungi are increased with a short or prolonged elevation of temperature [18–24]; however, cold stresses may also activate antifungal systems [18]. Insect antibacterial responses are also dependent on environmental temperatures [25–27] and elevated temperature often promotes bacterial infection [16,17]. There are examples of increased antibacterial responses in insects under short-term or prolonged exposure to low temperatures [16,26,28,29]. Importantly, in a state of cold diapause, cellular immunity continues to work [30] and changes in microbiome composition, immune response and susceptibility to fungal and bacterial infections may also occur [27,31]. It is likely that the outcome of complex infections may be shifted toward mycoses under low temperatures and toward bacterioses under high temperatures. However, the changes in immune response and microbiota composition during complex infections under different temperature conditions are insufficiently understood.

Various antimicrobial peptides (AMPs) of insects have key roles in both antibacterial and antifungal responses [32]. Some AMPs, such as gallerimycin and galiomycin, which are regulated by the Toll immune signaling pathway, exhibit activities against filamentous fungi, but not against bacteria [33,34]. Many AMPs (e.g., cecropins, gloverins, lysozymes) synthesized via the IMD and Toll pathways have a broad spectrum of activities predominantly against gram-positive and gram-negative bacteria as well as against fungi [35,36]. It is likely that AMPs control the proliferation of bacteria during the development of mycoses. In fact, the level of AMP gene expression clearly responds to changes in the microbial community during fungal infections [3]. Moreover, AMP gene expression is dependent on temperature [23,29].

The expression of apoptosis, reactive oxygen species (ROS) and stress-related genes may be crucial in the development of infections caused by entomopathogenic fungi and concomitant bacteria. In particular, a key regulator of programmed cell death, inhibitor of apoptosis (IAP), has paramount physiological importance, including in the antifungal response as was recently shown by Zhang and coworkers [37]. RNAi-mediated knockdown of the IAP homologue in locusts led to a decrease in the total hemocyte count, a degeneration of the gut, a shift in the microbiota, and increased susceptibility to fungal infection. In addition, IAP is involved in immunity to bacterial infections, as shown for *Drosophila* [38]. The generation of ROS has a large impact in reactions against different pathogens [39], as well as in maintaining microbial homeostasis, especially in the insect gu<sup>t</sup> [40–42]. The main source of ROS in insect hemolymph is the prophenoloxidase cascade. As a result of its activation, ROS (primarily semi-quinone radicals and H2O2) are formed [43–45]. In the gut, fat body and in other tissues, the formation of ROS occurs with the participation of members of the NADPH oxidase (NOX) family, such as dual oxidases (DUOX). The enzyme generates superoxide and H2O2, which are powerful oxidants that exhibit microbicidal activity [40]. Both fungal and bacterial infections led to changes in DUOX activity in the gu<sup>t</sup> and hemocoel tissues [2,3,42]. RNAi knockdown of the DUOX system caused a decrease in ROS and uncontrolled proliferation of bacteria [42]. Heat shock proteins (HSPs) have functions in protein folding and unfolding, and participate in immune signaling pathways and other processes [46]. HSPs are important stress markers, which sense different thermal actions, diapause formation [47] and infections [12].

It is important to note that entomopathogenic fungi produce various secondary metabolites and enzymes (oosporeins, destruxins, different proteinases, AMPs) for inhibiting both host immune responses and competitive microorganisms [36,48,49]. The set of enzymes and secondary metabolites present is significantly different between fungal species and depends on host and habitat specificity. As a rule, generalist species have a broader spectrum of metabolites compared to species with restricted host ranges [50–52].

The ascomycete *Cordyceps militaris* is characterized by a restricted host and habitat range and has a highly reduced number of genes involved in secondary metabolism and the synthesis of proteases compared to generalists such as *Metarhizium robertsii* and *Beauveria bassiana* [50,53]. This fungus mainly infects forest lepidopterans (Lepidoptera, Macroheterocera) in the larval and pupal stages which are located in the soil, forest flour and fallen wood [54–56]. Previously, natural infections of insects with *C. militaris* were studied insufficiently. It is known that larvae and pupae could be infected by topical application with ascospores or conidia in a laboratory [22,57]. However this method is difficult to reproduce, and the outcomes strongly depend on the physiological state of the host [22]. In contrast, injection of lepidopteran larvae and pupae with blastospores or conidia has led to more stable development of the mycosis [55,58,59]. Importantly, spontaneous bacterial infections have been constantly documented after infection of *C. militaris* with insects in laboratory conditions [22,58,59]. Therefore, *C. militaris* is a convenient model to study fungal–bacteria interactions in insects. The optimal temperature for mycelial growth of *C. militaris* palearctic isolates is approximately 20 ◦C [22,60]. In a previous study [22], we showed in a model insect, the wax moth *Galleria mellonella,* that larvae in a state of facultative diapause induced by a low temperature (15 ◦C) are most susceptible to the fungus. Mycosis successfully developed after injection with *C. militaris* blastospores at 15 ◦C. By contrast, at 25 ◦C (active state), larvae were able to overcome the infection and complete metamorphosis, although, the infection may persist in pupae and adults and could still be activated by a low temperature. Activation of the antifungal response (encapsulation and phenoloxidase activity) in response to *C. militaris* infection was observed in wax moth larvae at 25 ◦C, while inhibition of these parameters occurred at 15 ◦C. We suggested that *C. militaris* uses fewer universal tools for evasion and inhibition of host immunity compared to generalists fungi, such as *Metarhizium* and *Beauveria,* that induce prolonged mycosis development, persist in hosts and have a specialization in killing dormant insects with reduced immune activity [22,59]. Moreover, because we registered spontaneous bacterioses in the wax moths post injection of *C. militaris* blastospores and conidia, we hypothesized that *C. militaris* has poorly developed mechanisms for manipulating the host microbiota and requires a low temperature for its normal development.

In the present study we investigated the microbial communities of the wax moth larvae hemolymph and midgut, as well as the expression of AMP, apoptosis, ROS and stress-related genes in the wax moth midgut and fat body after injection with *C. militaris* and incubation under two temperatures, 15 ◦C (state of facultative diapause) and 25 ◦C (active state). We found significant changes in these parameters in response to infection at different temperatures, which support the hypothesis mentioned above.

#### **2. Material and Methods**

#### *2.1. Fungi and Insects*

*C. militaris* isolate CNAp (GenBank No MF073255.1), from the microorganism collection of the Institute of Systematics and Ecology of Animals SB RAS, was used in this work. Conidia had been stored at −80 ◦C since 2015. For infections, conidia were cultivated on Sabouraud dextrose agar with yeas<sup>t</sup> extract (2.5 g/L) (SDAY) for 22 days at 23 ◦C and a photoperiod of 8:16 (light:dark). Conidia were suspended in saline (0.9% NaCl) without any detergents and filtered through a sterile cloth to remove mycelial clumps. Concentrations of conidia were determined using a Neubauer hemocytometer. A Siberian line of wax moth larvae was maintained on artificial media as described previously [61]. Larvae of the sixth instar were used in experiments.

#### *2.2. Procedures for Infection and Bioassays*

Larvae were injected with 4 μL of a suspension containing 1250, 2500 or 5000 conidia. Control larvae were injected with saline. Punctures were made between the sixth and seventh abdominal segments using a microinjector with an insulin syringe. The needle was sterilized with 96% ethanol before each injection. Infected and control larvae were placed at two constant temperatures (25 ◦C and 15 ◦C) immediately after injection. Larvae were maintained in 90 mm glass Petri dishes (12 larvae per dish) with artificial media (3 g per one Petri dish) in the dark. Temperature in the Petri dishes at such an insect density corresponded with environmental temperatures. Ventilation of the Petri dishes and

registration of mortality was conducted every day over 10 days. To determine the causes of death, cadavers were placed on moist filter paper in the Petri dishes and maintained at the temperatures indicated above. Three replicates (one replicate = 12 larvae) were used to assay mortality after injection with each dose, and the whole experiment was repeated twice.

For detection of hyphal bodies and bacteria in the hemolymph of infected larvae, we used gradient centrifugation of the hemolymph in Percol followed by electron transmission microscopy as described previously [59]. To determine the yield of conidia on the larvae, the cadavers were incubated for 30 days in moist chambers at 25 ◦C and 15 ◦C. Then, each cadaver was placed in a tube with 20 mL of a 0.1% water-Tween 20 solution and vortexed for 3 min until the mycelia and conidia were completely washed <sup>o</sup>ff. Conidia were counted using a hemocytometer and the concentrations were calculated for each cadaver.

#### *2.3. Bacterial Colony Forming Unit (CFU) Counts*

At 96 h after injection with a dose of 2500 conidia per larvae, control and infected insects were surface sterilized by 3% H2O2 and 70% ethanol. Forty five μL of hemolymph from three larvae were placed in 100 μL of 150 mM cool NaCl and immediately homogenized using an ultrasonic homogenizer (Sonopuls, Bandelin electronic GmbH & Co. KG, Berlin, Germany). Midguts were pooled in the same NaCl (one sample = three larvae) and homogenized by the same technique. Samples were diluted with the same NaCl by 10, 100, and 1000-fold and a 100 μL aliquot was plated on media (Bile esculin azide agar for enterococci and Endo agar for enterobacteria) in 90 mm Petri dishes. The cultures were incubated for 2 days at 35 ◦C and then CFUs were counted. CFU counts were calculated for each midgut or 10 μL of hemolymph. A total of 5–6 samples from each treatment were used for analysis.

#### *2.4. Analysis of Bacterial Communities*

At 96 h post-treatment, infected (2500 conidia) and control larvae were surface-sterilized by 3% H2O2 and 70% ethanol and dissected. Midguts with content were isolated and frozen in liquid nitrogen (one sample = 5 midguts). In addition, decomposed cadavers (6–7 d post infection) were analyzed. Whole cadavers were frozen in liquid nitrogen (one sample = 3 whole bodies). Three biological replicates from each treatment were used.

DNA was isolated using a DNeasy PowerSoil DNA Isolation Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The 16S rRNA region was amplified with the primer pair V3–V4 combined with Illumina adapter sequences [62]. PCR amplification was performed as described previously [63]. A total of 200 ng of PCR product from each sample was pooled together and purified using a MinElute Gel Extraction Kit (Qiagen, Hilden, Germany). The obtained libraries were sequenced with 2 × 300 bp paired-end reagents on a MiSeq (Illumina Inc., San-Diego, California, USA) in the SB RAS Genomics Core Facility (ICBFM SB RAS, Novosibirsk, Russia). The sequencing data reported in this study were submitted to GenBank under the study accession PRJNA650299.

Raw sequences were analyzed with the UPARSE pipeline [64] using Usearch v11.0. The UPARSE pipeline included a merging of paired reads, read quality filtering, length trimming, merging of identical reads (dereplication), discarding singleton reads, removing chimeras, and operational taxonomic unit (OTU) clustering using the UPARSE-OTU algorithm. The OTU sequences were assigned a taxonomy using the SINTAX [65] and 16S RDP training set v.16 [66]. The final dataset included 384,875 reads (mean ± SE = 20,532 ± 630 per midgut sample and 46,162 ± 1453 per cadaver sample, see Dataset). All rarefaction curves showed a trend of approaching the saturation plateau (Figure S1), which indicated a reasonable volume of the sequenced reads.
