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 wMel Wolbachia in Drosophila melanogaster. These assays count cif transcripts alongside a synthetic spike-in RNA or a D. melanogaster reference gene to normalize for technical or biological variation. These assays have a limit of detection of about one cifA and three cifB copies per reaction. We expect these methods to be useful for mosquito-control programs that use wMel 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 34 Wolbachia strains, suggesting potential utility beyond wMel. 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.IMPORTANCEWolbachia, a maternally transmitted bacterium, is found in over half of all insect species. Its ability to induce cytoplasmic incompatibility (CI), which prevents Wolbachia-free eggs from hatching, significantly contributes to its high prevalence in host populations. Public health experts use CI to spread pathogen-blocking Wolbachia through mosquito populations, thereby controlling pathogen spread. CI is often weak, resulting in few egg deaths and consequently slowing Wolbachia's spread. We recently discovered that weak CI often correlates with low CI factor B (cifB) mRNA levels. However, our understanding of CI-strength variation remains limited because cifB is transcribed at low levels, making it challenging to measure in individual insects. Here, we report four RT-ddPCR assays to overcome this challenge. These assays offer high sensitivity for rare targets and maintain accuracy and precision across a wide dynamic range. We expect these tools will enhance efforts to understand CI-strength variation in both natural and applied populations.