Van Vlaenderen, L. | bioRxiv Science

bioRxiv2026

Temperature-sensitive cytoplasmic incompatibility across divergent Wolbachia partly reflects cifB transcription, not endosymbiont density

Maternally transmitted Wolbachia bacteria are common in insects, with many strains altering host reproduction through cytoplasmic incompatibility (CI). CI kills embryos fertilized by Wolbachia-bearing males unless those embryos also carry Wolbachia, which favors females with Wolbachia and drives the endosymbiont to higher frequencies in host populations. Strong CI now underpins successful applications that rely on maintaining pathogen-blocking Wolbachia transinfections in vector populations to reduce arboviral disease transmission. Temperature modulates CI strength (the proportion of embryos killed), with consequences for Wolbachia prevalence in natural and transinfected populations. Yet the mechanisms regulating temperature-sensitive CI-strength variation are poorly understood. We quantified CI strength across eight divergent Drosophila-associated Wolbachia strains at four temperatures (18{degrees}C-26{degrees}C), while characterizing development time, Wolbachia and Wovirus densities, and transcription of the CI-inducing gene cifB. Four of eight Wolbachia strains exhibited temperature-sensitive CI, three of which induced CI at multiple temperatures. Of these three, two expressed significantly more cifB at the temperature yielding stronger CI, whereas testes Wolbachia density did not predict CI strength. Notably, cifB-transcript levels were consistently decoupled from Wolbachia and Wovirus densities, suggesting that cifB transcription is not regulated solely by symbiont abundance. We also report temperature-sensitive rescue of CI, Wolbachia-associated developmental acceleration, and strain-specific Wovirus-Wolbachia covariance. Our findings reveal temperature as a pervasive modulator of Wolbachia-host interactions at multiple levels and extend evidence that cifB transcription partly predicts variable CI strength across strain identities, male ages, and now temperatures. CI variation unaccounted for by cifB transcription points toward additional regulatory or post-transcriptional mechanisms that we discuss.

molecular biology↗

bioRxiv2025

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. Wolbachias 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 Wolbachias prevalence in host populations, and cifB mRNA levels in testes generally correlate with CI strength. Yet, cifBs 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 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 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 33 Wolbachia strains, suggesting 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 Wolbachias 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.

microbiology↗