Wolbachia

Detection of Wolbachia View in CoL infection

PCR methods were employed to detect Wolbachia infection in each population using genomic DNA extracted from Cx. tritaeniorhynchus . Te detection of Wolbachia infection was performed in two steps. Initially, PCR was used to amplify the Wolbachia surface protein gene (wsp). For specimens that did not yield amplification of the wsp gene, additional PCR targeting the 16S rDNA region of Wolbachia was conducted. Samples that failed to amplify for 16S rDNA were considered to be negative for Wolbachia infection.

Te PCR protocol followed for amplification of the wsp gene was that reported by Zhou et al. [ 23], using the primer pairs wsp_81F (5 ′ -TGG TCC AAT AAG TGA TGA TGA AGA AAC- 3 ′) and wsp_691R (5 ′ -AAA AAT TAA ACG CTA CTC CA- 3 ′). Te PCR reaction mixture (total volume 25.0 μl) consisted of 1× PCR buffer, 0.2 mM dNTPs, 0.4 μM of each primer, 0.5 units of Taq HotStart DNA polymerase (TaKaRa) and 2 μl of extracted genomic DNA. Te PCR cycling conditions consisted of an initial denaturation at 94 °C for 5 min, followed by 40 cycles at 94 °C for 30 s, 53 °C for 30 s and 72 °C for 1 min, with a final extension at 72 °C for 5 min.

PCR amplification of the 16S rDNA of Wolbachia was performed using primers reported by Werren and Windsor [ 37] (WF: 5 ′ -CAT ACC TAT TCG AAG GGA TAG- 3 ′; WR: 5 ′ -AGC TTC GAG TGA AAC CAA TTC- 3 ′). Te reaction mixture for PCR amplification (total volume 25.0 μl) consisted of 1× PCR buffer, 0.2 mM dNTPs, 0.4 μM of each primer, 0.5 units of Taq HotStart DNA polymerase (TaKaRa) and 2 μl of extracted genomic DNA. Te PCR cycling conditions consisted of an initial denaturation at 94 °C for 5 min, followed by 40 cycles at 94 °C for 30 s, 60 °C for 30 s and 72 °C for 1 min, with a final extension at 72 °C for 5 min.

Amplification of the wsp gene and 16S rDNA was confirmed by electrophoresis in 1.5% agarose gels. Samples that were successfully amplified were subjected to Sanger dideoxy sequencing (Macrogen). Te sequences of all wsp genes detected in Cx. tritaeniorhynchus obtained in this study have been deposited in NCBI GenBank (GenBank accession numbers: wsp: PQ014158–PQ014189; 16S: PQ625809–PQ625837; PQ579157–PQ579160).