melanogaster Dm +; D. simulans Ds + and Ds -. B = blank. Note: IS5 primer set does not produce amplicons in all three Glossina samples due to complete absence of this IS element in symbionts of tsetse flies (see discussion). mTOR inhibitor We have recently shown that Wolbachia titers increase in D. paulistorum[11] and Glossina[12] hybrid backgrounds, which should significantly facilitate detection and strain characterization. Such titer increase was sufficient to

detect Wolbachia with the IS5 primer set in A/O hybrids, but the low-titer Wolbachia infection in the AM mother still remained undetected (Figure 2B). Failure of IS5-amplification in the Gs/Gm hybrid plus parents is explained by lacking homology between primer sequences and target, as no matches with the IS5 primer

sequence were found in the wGmm genome [14]. This finding implies that TNF-alpha inhibitor IS5 is not suitable as a general Wolbachia A-supergroup marker. Figure 2A and B show that the ARM-marker system can be applied to address aforementioned problems arising with wsp and IS5 primer: sensitivity during PCR is increased significantly and all tested A-supergroup infections are unambiguously detected. Wolbachia was traced in all low-titer New world Drosophila species (AM1, AM2; CA1, CA2) plus the A/O hybrid. In contrast to IS5, the ARM primer set amplified Wolbachia from all three Glossina samples (Gmm, Gsw and Gs/Gm hybrid). As anticipated, all samples from high-titer Wolbachia infections (OR, Dw + , Dm +, Ds +) showed bright bands with ARM, whereas Wolbachia-uninfected specimens (Dw -, Ds -) did not (Figure 2A,B). This argues for a high specificity of the ARM primer and against mis-amplification of a random Unoprostone host target

rather than the specific symbiont target site. Conclusions We suggest that the new multicopy Wolbachia A-supergroup marker can be used as an ‘ultra-sensitive’ tool to trace low-titer infections by means of classic end-point PCR. First, ARM has the advantage of higher sensitivity compared to classic singlecopy Wolbachia markers like wsp and thus improves detection limit significantly. Particularly, ARM-PCR can be easily applied to screen larger numbers of untyped DNA specimens, even of low quality arising from long-term storage and/or storage in inappropriate media, from laboratory stocks or samples directly from nature. This is of pivotal interest since classical detection tools might yield false negatives when examining species harboring Wolbachia at very low densities, and thereby lead to underestimating natural prevalence of A-supergroup infections. Given that 80% of the Dipteran infections are supergroup A [15], our new method will significantly facilitate and improve the sensitivity of such surveys. In AZD5582 addition our approach is an advantage over the classic IS5-marker, which fails in Wolbachia from the tsetse fly Glossina.