The persistence of low-titer Wolbachia pipientis infection in antibiotic-treated Drosophila

We have found evidence of recurrent low-abundance infections of Wolbachia in our laboratory Drosophila fly stocks. These fly lines were previously “cleared” of infection by the conventional method of tetracycline treatment and should not display evidence of the bacteria. Regardless, sporadic positive results have been consistently produced when screening fly stocks for infection via endpoint PCR. Follow up analyses with qPCR confirmed these results.

Extra care was taken to ensure that positive-testing samples were not a result of amplicon contamination. “Clean” PCR techniques were performed, yet positive results persisted. Water samples tested negative.

We were confident there was an actual infection that was not being eliminated by tetracycline, so we wanted to characterize it.

We tried to increase the titer of Wolbachia in these lowly-detectible fly lines so that it could be more easily characterized. Based on the literature, we tried raising flies on high sucrose food, which has been previously been shown to increase Wolbachia titer, but it failed in our flies. We also tried raising flies on food containing cycloheximide, which has been hypothesized to affect Wolbachia titer through translational repression, with no success. Finally, we attempted to rear gnotobiotic flies based on a previous observation in our lab that this may cause Wolbachia titer to increase or become more easily detectable, however this attempt also failed.

We have begun analysis of our low-titer Wolbachia strain using multilocus sequence typing. In this poster we describe our efforts to characterize this Wolbachia infection as well as our recommendations for screening Drosophila lines for low Wolbachia titer. We advise against the use of endpoint PCR for low titer strains and suggest qPCR and cytological imaging as alternatives.