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.