Wolbachia 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.

Endosymbiotic Wolbachia bacteria infect divergent arthropod and nematode hosts. Many strains cause cytoplasmic incompatibility (CI) that kills uninfected embryos fertilized by Wolbachia-modified sperm. Infected embryos are protected from CI, promoting Wolbachia spread to high equilibrium frequencies balanced by imperfect maternal transmission. CI strength varies widely in nature and tends to decrease as males age. Understanding the causes of CI-strength variation is crucial to explain Wolbachia prevalence in host populations. Here, we investigate how fast and why CI strength decreases with male age in two model systems: wMel in Drosophila melanogaster and wRi in D. simulans. Average wMel CI strength decreases rapidly (19%/ day), and wRi CI strength decreases slowly (6%/ day) as males age; thus, within three days, wMel-infected males do not cause CI, whereas twelve-day-old wRi-infected males still cause minor, yet significant, CI. We tested if reductions in Wolbachia densities or CI gene expression as males age could explain this pattern. Indeed, wRi densities and CI gene expression decrease in testes as males age, but wMel densities and CI gene expression surprisingly increase with male age as CI strength decreases. Phage WO lytic activity and wMel Octomom copy number-an ampliconic gene region that influences wMel proliferation-do not explain age-dependent Wolbachia densities. However, the expression of Relish, an essential gene in the Drosophila immune deficiency pathway, strongly correlates with wMel densities. Together, these results suggest that testes-wide Wolbachia density and CI gene expression are insufficient to explain age-dependent CI strength across strains and that Wolbachia density is variably impacted by male age across Wolbachia-host associations. We hypothesize that host immunity may underlie variation in age-dependent density dynamics. More broadly, the rapid decline of wMel CI strength during the first week of D. melanogaster life likely contributes to wMel frequency variation observed on several continents.

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 (100–7.4% 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 the efficacy of CI-based biocontrol of arboviruses and agricultural pests globally.

Like all taxa, populations of aquatic insects may respond to climate change by evolving new physiologies or behaviors, shifting their ranges, exhibiting physiological and behavioral plasticity, or by 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 six-week experimental period, and oxygen and temperature interacted to affect growth in complex ways. Survival was lower for the warm treatment, though only four mortalities occurred (91.6 vs 100%). Nymphs acclimated to warmer temperatures did not have higher critical thermal maxima (CTMAX), but those acclimated to hypoxia had CTMAX values (in normoxia) 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 and abdominal 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.