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°C–26°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. ### Competing Interest Statement The authors have declared no competing interest. U.S. National Science Foundation, 2145195, DBI-2010210 National Institutes of Health, https://ror.org/01cwqze88, R35GM124701, P20GM103474 M J Murdock Charitable Trust, https://ror.org/02hxgd925, 202324717 Lehigh University, https://ror.org/012afjb06 University of Montana, https://ror.org/0078xmk34