DNA extraction and PCR
Wasp samples in open tubes were submerged in liquid nitrogen and manually homogenized with sterile plastic pestle. Subsequent DNA extractions were performed using DNeasy Blood & Tissue kits (Qiagen) following several modifications of the manufacturer’s protocol. The initial lysis step was done gently at 37°C overnight and finished by 30 minutes incubation with 1 µL of RNase A (Qiagen) per sample. To enhance the yield the final elution step was done with total of 200 µL of deionized water separated in two rounds of column washing. The resulting DNA solution was then dried using vacuum concentrator and resuspended in 38 µL of buffer EB in order to increase the concentration. One microliter was used for Qubit quantification, 2 µL were loaded on 2% agarose gel pre-stained with GelRed® Nucleic Acid Gel Stain (Biotium) and ran at 120V for 70 minutes to assess the quality of extracted DNA. Samples were quantified using a Qubit 3 Fluorometer (ThermoFisher Scientific) and diluted to a total of ~20 ng in 35 µL of EB buffer (Qiagen). We used the primers and protocols of Baldo et al. (Baldo et al. 2006) to amplify the Wolbachia surface protein gene (wsp ) and the five Multi Locus Strain Typing (MLST) genes used for accurate strain typing and better detection of recombination. All PCR products were sequenced using Sanger sequencing, chromatograms were checked for multiple peaks and edited in Finch TV (Geospiza: https://digitalworldbiology.com/FinchTV ) before alignment using BioEdit (Hall 1999).