Genomic DNA Extraction and Genotyping Protocols

Genomic DNA was extracted from dissected reproductive tissues (testes and ovaries) using DNeasy kit (Qiagen), and from carcasses using NucleoSpin 96 Tissue kit (Macherey-Nagel). In 2013, to estimate Wolbachia maternal transmission, DNA was extracted using DNeasy kit (Qiagen) from whole females that were allowed to oviposit, and from their progenies. For M and S genotyping, DNA was extracted from a leg using a fast extraction method. In brief, individual legs were incubated in 40 μl of grinding buffer (10 mM Tris-HCl pH 8.2, 1 mM EDTA, 25 mM NaCl) with 0.2 mg ml⁻¹ proteinase K for 45 min at 37 °C, then 5 min at 95 °C to inactivate the enzyme. DNA extracts (1 μl) were then subjected to PCR amplification targeting the locus S200 × 6.1 using specific primers (FWD: 5′-TCGCCTTAGACCTTGCGTTA-3′; and REV: 5′-CGCTTCAAGAATTCGAGATAC-3′)25 (link). M and S genotyping was also used on larvae and adult carcasses. Larval DNA was used to determine the sex using Y-specific primers (F: 5′-CAAAACGACAGCAGTTCC-3′; and R: 5′-TAAACCAAGTCCGTCGCT-3′).