cifB-transcript levels largely explain cytoplasmic incompatibility variation across divergent Wolbachia.

Divergent hosts often associate with intracellular microbes that influence their fitness. Maternally transmitted Wolbachia bacteria are the most common of these endosymbionts, due largely to cytoplasmic incompatibility (CI) that kills uninfected embryos fertilized by Wolbachia-infected males. Closely related infections in females rescue CI, providing a relative fitness advantage that drives Wolbachia to high frequencies. One prophage-associated gene (cifA) governs rescue, and two contribute to CI (cifA and cifB), but CI strength ranges from very strong to very weak for unknown reasons. Here, we investigate CI-strength variation and its mechanistic underpinnings in a phylogenetic context across 20 million years (MY) of Wolbachia evolution in Drosophila hosts diverged up to 50 MY. These Wolbachia encode diverse Cif proteins (100% to 7.4% pairwise similarity), and AlphaFold structural analyses suggest that CifB sequence similarities do not predict structural similarities. We demonstrate that cifB-transcript levels in testes explain CI strength across all but two focal systems. Despite phylogenetic discordance among cifs and the bulk of the Wolbachia genome, closely related Wolbachia tend to cause similar CI strengths and transcribe cifB at similar levels. This indicates that other non-cif regions of the Wolbachia genome modulate cif-transcript levels. CI strength also increases with the length of the host's larval life stage, presumably due to prolonged cif action. Our findings reveal that cifB-transcript levels largely explain CI strength, while highlighting other covariates. Elucidating CI's mechanism contributes to our understanding of Wolbachia spread in natural systems and to improving the efficacy of CI-based biocontrol of arboviruses and agricultural pests globally.

Plasticity of salmonfly (Pteronarcys californica) respiratory phenotypes in response to changes in temperature and oxygen.

Like all taxa, populations of aquatic insects may respond to climate change by evolving new physiologies or behaviors, shifting their range, exhibiting physiological and behavioral plasticity, or going extinct. We evaluated the importance of plasticity by measuring changes in growth, survival and respiratory phenotypes of salmonfly nymphs (the stonefly Pteronarcys californica) in response to experimental combinations of dissolved oxygen and temperature. Overall, smaller individuals grew more rapidly during the 6-week experimental period, and oxygen and temperature interacted to affect growth in complex ways. Survival was lower for the warm treatment, although only four mortalities occurred (91.6% versus 100%). Nymphs acclimated to warmer temperatures did not have higher critical thermal maxima (CTmax), but those acclimated to hypoxia had CTmax values (in normoxia) that were higher by approximately 1°C. These results suggest possible adaptive plasticity of systems for taking up or delivering oxygen. We examined these possibilities by measuring the oxygen sensitivity of metabolic rates and the morphologies of tracheal gill tufts located ventrally on thoracic segments. Mass-specific metabolic rates of individuals acclimated to warmer temperatures were higher in acute hypoxia but lower in normoxia, regardless of their recent history of oxygen exposure during acclimation. The morphology of gill filaments, however, changed in ways that appeared to depress rates of oxygen delivery in functional hypoxia. Our combined results from multiple performance metrics indicate that rising temperatures and hypoxia may interact to magnify the risks to aquatic insects, but that physiological plasticity in respiratory phenotypes may offset some of these risks.

Male Age and Wolbachia Dynamics: Investigating How Fast and Why Bacterial Densities and Cytoplasmic Incompatibility Strengths Vary.

Endosymbionts can influence host reproduction and fitness to favor their maternal transmission. For example, endosymbiotic Wolbachia bacteria often cause cytoplasmic incompatibility (CI) that kills uninfected embryos fertilized by Wolbachia-modified sperm. Infected females can rescue CI, providing them a relative fitness advantage. Wolbachia-induced CI strength varies widely and tends to decrease as host males age. Since strong CI drives Wolbachia to high equilibrium frequencies, understanding how fast and why CI strength declines with male age is crucial to explaining age-dependent CI's influence on Wolbachia prevalence. Here, we investigate if Wolbachia densities and/or CI gene (cif) expression covary with CI-strength variation and explore covariates of age-dependent Wolbachia-density variation in two classic CI systems. wRi CI strength decreases slowly with Drosophila simulans male age (6%/day), but wMel CI strength decreases very rapidly (19%/day), yielding statistically insignificant CI after only 3 days of Drosophila melanogaster adult emergence. Wolbachia densities and cif expression in testes decrease as wRi-infected males age, but both surprisingly increase as wMel-infected males age, and CI strength declines. We then tested if phage lysis, Octomom copy number (which impacts wMel density), or host immune expression covary with age-dependent wMel densities. Only host immune expression correlated with density. Together, our results identify how fast CI strength declines with male age in two model systems and reveal unique relationships between male age, Wolbachia densities, cif expression, and host immunity. We discuss new hypotheses about the basis of age-dependent CI strength and its contributions to Wolbachia prevalence. IMPORTANCEWolbachia bacteria are the most common animal-associated endosymbionts due in large part to their manipulation of host reproduction. Many Wolbachia cause cytoplasmic incompatibility (CI) that kills uninfected host eggs. Infected eggs are protected from CI, favoring Wolbachia spread in natural systems and in transinfected mosquito populations where vector-control groups use strong CI to maintain pathogen-blocking Wolbachia at high frequencies for biocontrol of arboviruses. CI strength varies considerably in nature and declines as males age for unknown reasons. Here, we determine that CI strength weakens at different rates with age in two model symbioses. Wolbachia density and CI gene expression covary with wRi-induced CI strength in Drosophila simulans, but neither explain rapidly declining wMel-induced CI in aging D. melanogaster males. Patterns of host immune gene expression suggest a candidate mechanism behind age-dependent wMel densities. These findings inform how age-dependent CI may contribute to Wolbachia prevalence in natural systems and potentially in transinfected systems.