Fungal cultivation and identification
Surface sterilized larvae (described above) were dissected straight after collection, their guts were stored in 200 µl of 25% glycerol until further manipulation. Once we obtained enough guts, we crushed and homogenized them by sterile plastic pestle in 1.5 ml Eppendorf tube with 1 ml of sterile 1% Tween 80 in dH2O, and vortexed for approximately 10 s on bench vortex (IKA MS3 vortexer). The vortexed inoculum was spread on 9 cm agar plates with 2% YES medium with antibiotics (5 g/l of yeast extract, 30 g/l of glucose, 15g/l of agar, 60 mg/l of streptomycin and 60 mg/l of chloramphenicol all from Sigma-Aldrich, St. Louis, Missouri, USA). The method of serial dilutions (1, 10, 100 ×) was used to determine the abundance of individual taxa. Agar plates were cultivated at 25 °C for one week in the dark and after this period colonies were morpho-typed and morphologically unique cultures were taken for further identification. In the present study, we describe cultivation and identification only for fungal microbiome as the bacterial part is described elsewhere (Peral-Aranega et al., in press). For the identification of fungal isolates, the DNA was extracted from the fresh pure cultures using a DNeasy PowerSoil Pro Kit (QIAGEN GmbH, Hilden, Germany). Fungi were identified by ITS-LSU rDNA barcode which was amplified using the ITS4 primer (O’Donnell 1992, White et al., 1990). SAP-Exo kit (Jena Bioscience GmbH, Germany) was used for purification of PCR amplicons and sequencing was done at Macrogen Inc. (Seoul, Korea). Obtained sequences were manually aligned in Bioedit v.7.2.5. The obtained sequences were blasted (Altschul et al., 1990) to those of type strains of described species available in public databases to identify the isolates.