1; [30] Number of matches in column four refer to hits of the 315 bp ARM-PCR amplicon in the searched Wolbachia genomes. Hits were produced using the blastn algorithm (megablast) with match/mismatch scores 1,-2. Wolbachia strains are

organized by supergroup (column two). Matches to ARM were only found within the A-supergroup. aMinimum number of ARMs in the corresponding genome. Exact number cannot be given due to the lack of a complete genome. bRefers to no similarity detected between ARM and searched genome (complete/draft). ARM facilitates detection of low-titer Wolbachia from A-supergoup ARM-targeting primer were tested via end-point PCR screen on DNA from high- and low-titer Wolbachia infections in Drosophila and Glossina (tsetse fly) species (Additional file RG7204 concentration 2). As shown in Figure 2, the classic Wolbachia singlecopy ABT-263 ic50 gene marker wsp (Wolbachia outer surface protein gene) is only applicable for samples with high-titer infections, since Wolbachia was only detected in high-titer D. paulistorum Orinocan semispecies (OR, Figure 2A) as well as in D. willistoni (Dw +, Figure 2B), D. melanogaster (Dm +, Figure 2B), D. simulans (Ds +, Figure 2B) and Glossina morsitans morsitans (Gmm, Figure 2B). The wsp primer failed to detect Wolbachia in low-titer strains like D. paulistorum Amazonian (AM) and Centroamerican (CA) semispecies plus Glossina swynnertoni

(Figure 2A,B), indicating that a singlecopy gene like wsp is not suited for tracking low-titer infections. As multicopy gene markers like insertion sequences (IS) can be used to increase the detection limit, we ran PCR using primer for Insertion Sequence 5 (IS5; [8–10] on the same sample set. We observed increased sensitivity compared to wsp-PCR since Wolbachia was detected in low-titer CA2 (Figure 2A) and in the A/O hybrid samples. However, IS5 primer failed at amplifying the target sequence in all three Glossina samples (Gmm, Gsw and Gs/Gm hybrid; Figure 2B) despite the overall high Wolbachia titer in Gmm[12]. Figure 2 Comparison of Wolbachia marker sensitivity by PCR.

(A) The three Wolbachia markers wsp, IS5 and ARM were tested on the following specimens: New world Drosophila species from the Drosophila willistoni group including D. paulistorum Amazonian (AM1, AM2), and Molecular motor Centroamerican (CA1, CA2) semispecies. Orinocan semispecies (OR) served as Wolbachia positive control; Ds – as Wolbachia negative control. B = blank. Quality of DNA was assessed with universal primer set 12SCFR, 12SCRR targeting the mitochondrial 12S rRNA gene [20, 21]. Expected amplicon sizes for Wolbachia positive control (OR) are 631 bp (wsp), 752 bp (IS5), 315 bp (ARM) and 399 bp (12S rRNA). (B) Same markers as above were tested on additional samples including hybrids: A/O hybrid plus parents AM and OR; Glossina Gs/Gm hybrid plus parental strains Gsw and Gmm (Additional file 2). Drosophila New world members include D. willistoni Dw + and Dw -.