Fastprep 24 5g tissue homogenizer

For PCR amplification, DNA was isolated from mycelia and infected larvae of G. mellonella. A piece of pea-broth-cultured mycelium or a cut larvae were transferred into 2 mL tubes with glass beads (425–600 μm) and 800 μL DNA extraction buffer (Sarowar et al., 2014 (link)) containing RNAseA and homogenized in a FastPrep-24™ 5G tissue homogenizer (MP Biomedicals, 4 × 40 s, 6 m/s, 2 min breaks). After disruption samples were incubated at 55 °C for 30 min followed by a centrifugation to remove debris at 10,000 × g for 10 min. The supernatant was used for DNA extraction by a phenol-chloroform method as described elsewhere (Zelaya-Molina et al., 2011 (link)). Briefly, for DNA extraction from insects an additional phenol extraction step was performed. DNA was precipitated in 1 mL isopropanol overnight at −20 °C and pelleted by centrifugation at 4 °C. Pellets were washed twice with 70 % ethanol before air dried at room temperature for 5 min, followed by resuspension in nuclease-free water.

The metabolome profiles in hepatic samples were analyzed by using the AbsoluteIDQ p180 Kit (Biocrates Life Science AG, Innsbruck, Austria) as reported previously [10 (link)], adapted to tissue extracts. The metabolite extraction from the liver samples was carried out following the standard protocol of the AbsoluteIDQ p180 Kit [21 (link)]. Accordingly, liver tissue samples (around 100 mg) were homogenized in ethanol/phosphate buffer (85:15) by mechanical disruption with ceramic beads using a FastPrep-24 5G tissue homogenizer (MP Biomedicals, Inc., Irvine, CA, USA). The supernatants were analyzed by the AbsoluteIDQ p180 Kit, which identifies and quantifies up to 188 metabolites from 5 compound classes: acyl-carnitines (40), proteinogenic and modified amino acids (19), glycerophospho- and sphingolipids (76 phosphatidylcholines, 14 lysophosphatidylcholines, 15 sphingomyelins), biogenic amines (19) and hexoses (1). A detailed list of the compounds is presented in Additional file 1: Table S1. All reagents used in the processing and analysis were of LC–MS grade. The processed concentration data obtained from the LC–MS analysis were first log-transformed, then centered, and Pareto scaled.