Repeated thermal stress exposure in Aedes aegypti co-infected with Wolbachia and dengue virus

Climate change is increasing the frequency and intensity of heatwaves, affecting the thermal tolerance of mosquitoes and potentially influencing the efficacy of the biological control agent, Wolbachia. This study investigates the impact of repeated thermal stress on Aedes aegypti mosquitoes co-infected with Wolbachia and dengue virus (DENV). We exposed infected mosquitoes (singly and in co-infection) to varying intensities, frequencies, and durations of thermal stress to assess their thermal sensitivity via a “knockdown assay” compared to uninfected controls. Our results demonstrate that co-infection with Wolbachia and DENV significantly increases thermal sensitivity, with mosquitoes exhibiting a twofold faster median knockdown time than either singly infected or uninfected controls in most cases. A comparison of mosquitoes with no prior heat exposure to those given a single exposure revealed some evidence of heat hardening, or a slight lengthening of time to knockdown. Additional exposures provided no substantial benefit, however. Extended thermal stress (60 mins) also significantlysignificantlysignificantlyreduced DENV loads, while Wolbachia loads remained stable, indicating that prolonged heat may disrupt viral replication without affecting bacterial symbiosis. These findingsfindingssuggest that heatwaves could lower vector competence and disproportionately affectaffectaffectDENV-infected mosquitoes in Wolbachia-release areas, with implications for biocontrol strategies. Field studies should explore how infection affects mosquitoes’ ability to modulate thermal exposure behaviorally, providing insights for optimizing Wolbachia-based control efforts. IMPORTANCE Dengue virus (DENV), spread by the mosquito Aedes aegypti, is a major global health threat affecting millions of people. This study examines how repeated exposures to heat stress affect the thermal tolerance of mosquitoes infected with DENV and/or Wolbachia, a bacterium used for biological control. These repeated exposures mimic the experience of mosquitoes in the wild experiencing heatwaves of increasing frequency under climate change. Our research shows that Ae. aegypti co-infected with Wolbachia and DENV is more susceptible to thermal stress than singly infected or uninfected mosquitoes. We also demonstrate that multiple independent thermal stress exposures do not exacerbate the effect of infection. Understanding these interactions is essential for predicting how climate change may affect dengue transmission and the resilience of Wolbachia-based interventions.