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 two-fold faster median knockdown time than either singly infected or uninfected controls. 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 minutes) also significantly reduced DENV loads, while Wolbachia loads remained stable, indicating that prolonged heat may disrupt viral replication without affecting bacterial symbiosis. These findings suggest that heatwaves could lower vector competence and disproportionately eliminate DENV-infected mosquitoes in Wolbachia -release areas, with implications for biocontrol strategies. Field studies should explore how infection affects mosquitoes’ ability to regulate temperature behaviorally, providing insights for optimizing Wolbachia -based control efforts. Data Files and R Script Include: "DENV Load (DPI14)"- Results from dengue virus quantification of dissected mosquito tissue using qRT-PCR. Includes columns sample position and name, W olbachia ( + or -), sample number, Dissected tissue (head/thorax or abdomen), Heat shock temperature (35°C or 40°C), Duration (10 minutes or 60 minutes), DPI (days post infection), Cp (crossing point for PCR), Concentration (copies/ ...