High-sensitivity detection of cryptic Wolbachia in the African tsetse fly (Glossina spp.).

Background: In African tsetse flies Glossina, spp. detection of bacterial symbionts such as Wolbachia is challenging since their prevalence and distribution are patchy, and natural symbiont titers can range at levels far below detection limit of standard molecular techniques. Reliable estimation of symbiont infection frequency, especially with regard to interrelations between symbionts and their potential impact on host biology, is of pivotal interest in the context of future applications for the control and eradication of Glossina-vectored African trypanosomosis. The presence or absence of symbionts is routinely screened with endpoint polymerase chain reaction (PCR), which has numerous advantages, but reaches its limits, when detecting infections at natural low titer. To not only determine presence of native tsetse symbionts but also to localize them to specific host tissues, fluorescence in situ hybridization (FISH) can be applied. However, classic FISH assays may not detect low-titer infections due to limitations in sensitivity.

Results: We have compared classic endpoint PCR with high-sensitivity blot-PCR. We demonstrate that the latter technique allows for clear detection of low-titer Wolbachia in the morsitans and palpalis groups while classic endpoint PCR does not. In order to localize Wolbachia in situ in high and low-titer Glossina species, we applied high-end Stellaris® rRNA-FISH. We show that with this high sensitivity method, even low amounts of Wolbachia can be traced in specific tissues. Furthermore, we highlight that more tissues and organs than previously recorded are infested with Wolbachia in subspecies of the morsitans and palpalis groups.

Conclusions: Our results demonstrate that overall symbiont infection frequencies as well as the presence in specific host tissues may be underestimated when using low-sensitivity methods. To better understand the complex interrelation of tsetse flies and their native symbionts plus the pathogenic trypanosomes, it is important to consider application of a broader range of high-sensitivity detection tools.