*2.5. Gene Expression*

Wax moth larvae at 96 h after injection (2500 conidia) were dissected on ice cold PBS and midguts without contents and fat bodies were collected. Midguts content were removed by eye forceps. Midguts from ten larvae or fat bodies from five larvae were pooled in each sample. A total of 5–6 samples

(biological replicates) from each treatment were used for analysis. The tissues were frozen in liquid nitrogen and stored at −80 ◦C. Samples were lyophilized at −65 ◦C, 400 mtorr for 15 h and disrupted in liquid nitrogen using micropestles just before RNA isolation. The tissues were homogenized in QIAzol Lysis Reagent (Qiagen, Hilden, Germany) and RNA was isolated according to the manufacturer's instructions. Quantity and quality of the total DNA were estimated by NanoDrop NanoVue Plus (GE Healthcare, Chicago, Illinois, USA). Each sample was normalized to a concentration of 1.5 μg/μ<sup>L</sup> and treated by RQ1 RNase-free DNase (Promega, Madison, WI, USA). RNA was converted to cDNA using 6 μg DNA-free RNA, 3 μL 100 nM random nanomers and 4 μL RevertedAidTM M-MuLV Reverse Transcriptase (Fermentas, Vilnius, Lithuania).

qPCR was carried out using HS-qPCR SYBR Blue (2×) mix (BioLabMix, Novosibirsk, Russia) with a CFX96 Touch (Bio-Rad Laboratories, Inc., Hercules, CA, USA). qPCR was performed in triplicate under the following conditions: 95 ◦C for 3 min, and 40 cycles of 15 s at 94 ◦C and 30 s at 60/62/64 ◦C (depending on the primer Tm), followed by melt curves (70–90 ◦C). Gene expression was estimated by the ΔΔCq protocol with Bio-Rad CFX Manager (Bio-Rad, Laboratories, Inc., Hercules, CA, USA). The following *G. mellonella* genes were used as references: translation elongation factor 1-alpha 1 (eEF1 α1) and the subunit of DNA-directed RNA polymerase II. The expression dynamics of the following ten genes of interest were studied: antimicrobial peptides gallerimycin, galiomycin, gloverin, cecropin-like and lysozyme-like, apoptosis-related IAP, the ROS-related NOX-DUOX domain and heat shock proteins Hsp70 and Hsp90. These genes and primer sequences were from the work of Lange and coauthors [67] and Melo and coauthors [68] or designed by us (Table S1). Primer properties were estimated by IDT OligoAnalyser 3.1 (http://eu.idtdna.com/calc/analyzer). Primers were synthesized by Biosintez, Koltsovo, Russia.

#### *2.6. In Vitro Interaction between Fungi and Bacteria*

For the interaction studies, we used the predominant cultivable bacteria previously isolated from *G. mellonella* midgut, *Enterococcus faecalis* and *Enterobacter* sp. [7] In addition to *C. militaris*, the fungi *M. robertsii* (strain MB-1) and *B. bassiana* (strain Sar-31) from the microorganism collection of the Institute of Systematics and Ecology of Animals SB RAS were used as positive controls. Bacteria were cultivated on nutrient agar (Himedia, Mumbai, India) and fungi were cultivated on SDAY media. One-day-old plugs of bacteria (8 mm) or plugs of nutrient agar (control) were placed on freshly plated cultures of fungi in 90 mm Petri dishes. Zones of mycelial growth inhibition were measured at 4 days of incubation at 25 ◦C. Similarly, four-day-old plugs of fungi were placed on freshly plated bacterial cultures. Zones of growth inhibition were estimated on the first and second day of incubation at 25 ◦C. Radial mycelial growth on cultures of bacteria was recorded over 24 days. As a control, measurements of mycelial growth on bacteria-free nutrient agar were conducted. Three replicates were used in each treatment.
