DNA extraction
DNA was extracted from the surface sterilized beetle samples (parental
adults, larvae, pupae, and teneral adults) and infested and intact
phloem. Although we were focused on intestinal microbiota, we isolated
DNA from the whole individuals not directly from guts as the softness of
pupal bodies impede such manipulation. DNA was extracted following the
phenol-chloroform protocol (Sagova-Mareckova et al., 2008) using the
phenol-chloroform–isoamyl alcohol (25:24:1) premixed solution
(Sigma-Aldrich, St. Louis, Missouri, USA). The initial homogenization
was done on FastPrep-24™ 5G Instrument (Irvine, California, USA) using
wolfram beads in combination with glass beads (BioSpec Products, Inc.,
Bartlesville USA). DNA yield was quantified on Qubit 2.0 Fluorometer
(Thermo Fisher Scientific, Waltham, Massachusetts, USA) using Qubit
dsDNA BR Assay Kit and DNA quality was checked on Nanodrop (NanoDrop™
2000c, ThermoFisher scientific).