Counting cytoplasmic incompatibility factor mRNA using digital droplet PCR

Wolbachia bacteria inhabit over half of all insect species and often spread through host populations via efficient maternal transmission and cytoplasmic incompatibility (CI), killing aposymbiotic embryos when fertilized by symbiotic males. Wolbachia 's cifB gene triggers CI in males, while cifA , expressed in females, rescues embryos from CI-induced lethality. In some systems, cifA also contributes to CI induction. CI strength—the percentage of embryos that die from CI—is a key determinant of Wolbachia 's prevalence in host populations, and cifB mRNA levels in testes generally correlate with CI strength. Yet, cifB 's rarity can hamper precise quantification, necessitating tissue pooling for reverse transcription quantitative PCR (RT-qPCR) to achieve reliable measurements, obscuring variation at the level of individual insect tissues. Here, we present four RT digital droplet PCR (RT-ddPCR) assays to count rare cifA and cifB mRNA from w Mel Wolbachia in Drosophila melanogaster . These assays count cif transcripts alongside a synthetic spike-in RNA or a D. melanogaster housekeeping gene to normalize for technical or biological variation. These assays have a limit of detection of about 1 cifA and 3 cifB copies per reaction. We expect these methods to be useful for mosquito-control programs that use w Mel to block the spread of pathogens from Aedes aegypti to humans. Moreover, the oligos were designed with homology to cifA and cifB sequences from at least 33 Wolbachia strains, suggesting utility beyond w Mel. These methods will allow researchers to measure cif mRNA levels from individual insect tissues, enabling efforts to pair molecular and phenotypic data at unprecedented resolutions.