The wPip infection was characterized through the analysis of 13 different Wolbachia markers, which were amplified by PCR and Sanger sequenced, according to methodologies previously described. The markers were: (i) the Wolbachia surface protein gene (wsp)44; (ii) the analysis of five house-keeping gene sequences for the Wolbachia MLST methodology according to Baldo et al.38. For that, multiple alignment is performed with the concatenated sequences of the genes (gatB, coxA, hcpA, ftsZ, and fbpA, downloaded from https://pubmlst.org/wolbachia/ and detailed in Table S2). The phylogeny was estimated by Mr Bayes program, using members of all Wolbachia supergroups; (iii) the analysis of two ANK Wolbachia markers (ank2 and pk1) by a specific PCR–RFLP assay described by Dumas et al.41 that allows to distinguish the 5 known wPip groups (wPip-I to wPip-V); and (iv) the identification of wPip haplotypes based on the polymorphism of seven genes: the former two ANK Wolbachia markers plus five additional genes, the DNA mismatch repair protein gene MutL, the ANK gene pk2, the methylase gene GP12, the putative secreted protein gene GP15 and the regulatory protein gene RepA49. In this case, the haplotypes of each gene are identified based on the comparison of the sequence of the gene with all of the sequences of the described haplotypes methodology, according to Atyame et al.50.

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