Counting cytoplasmic incompatibility factor mRNA using digital droplet PCR

<i>Wolbachia</i> 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. <i>Wolbachia</i>'s <i>cifB</i> gene triggers CI in males, while <i>cifA</i>, expressed in females, rescues embryos from CI-induced lethality. In some systems, <i>cifA</i> also contributes to CI induction. CI strength-the percentage of embryos that die from CI-is a key determinant of <i>Wolbachia</i>'s prevalence in host populations, and <i>cifB</i>-mRNA levels in testes generally correlate with CI strength. Yet, <i>cifB</i>'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 <i>cifA</i> and <i>cifB</i> mRNA from <i>w</i>Mel <i>Wolbachia</i> in <i>Drosophila melanogaster</i>. These assays count <i>cif</i> transcripts alongside a synthetic spike-in RNA or a <i>D. melanogaster</i> reference gene to normalize for technical or biological variation. These assays have a limit of detection of about one <i>cifA</i> and three <i>cifB</i> copies per reaction. We expect these methods to be useful for mosquito-control programs that use <i>w</i>Mel to block the spread of pathogens from <i>Aedes aegypti</i> to humans. Moreover, the oligos were designed with homology to <i>cifA</i> and <i>cifB</i> sequences from at least 34 <i>Wolbachia</i> strains, suggesting potential utility beyond <i>w</i>Mel. These methods will allow researchers to measure <i>cif</i>-mRNA levels from individual insect tissues, enabling efforts to pair molecular and phenotypic data at unprecedented resolutions.IMPORTANCE<i>Wolbachia</i>, a maternally transmitted bacterium, is found in over half of all insect species. Its ability to induce cytoplasmic incompatibility (CI), which prevents <i>Wolbachia</i>-free eggs from hatching, significantly contributes to its high prevalence in host populations. Public health experts use CI to spread pathogen-blocking <i>Wolbachia</i> through mosquito populations, thereby controlling pathogen spread. CI is often weak, resulting in few egg deaths and consequently slowing <i>Wolbachia</i>'s spread. We recently discovered that weak CI often correlates with low CI factor B (<i>cifB</i>) mRNA levels. However, our understanding of CI-strength variation remains limited because <i>cifB</i> 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.