<b>Repeated thermal stress exposure in </b><b><i>Aedes aegypti</i></b><b> co-infected with </b><b><i>Wolbachia</i></b><b> and dengue virus</b>

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, <i>Wolbachia</i>. This study investigates the impact of repeated thermal stress on <i>Aedes aegypti</i> mosquitoes co-infected with <i>Wolbachia</i> 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 <i>Wolbachia</i> 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 <i>Wolbachia</i> 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 <i>Wolbachia</i>-release areas, with implications for biocontrol strategies. Field studies should explore how infection affects mosquitoes’ ability to regulate temperature behaviorally, providing insights for optimizing <i>Wolbachia</i>-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<i>olbachia (</i>+ 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/ µl), log 10 DENV (log10 transformation of concentration)."Thermal Knockdown Assay"- Results from thermal knockdown assay by treatment group. Includes columns: Mosquito number, Time to knockdown (seconds), WALB +/- (W<i>olbachia status), DENV+/- (</i>dengue infection status), duration (10 minutes or 60 minutes), Frequency (1, 2, 3 exposures), and replicate number."Thermal Knockdown Assay_0x and 1x"- Results from a secondary knockdown assay by treatment group. Include columns: Mosquito number, Time to knockdown (seconds), WALB +/- (W<i>olbachia status), DENV+/- (</i>dengue infection status), and Frequency (1, 2, 3 exposures)."<i>Wolbachia </i>Load (DPI14)"- Results from <i>Wolbachia</i> quantifications by qPCR measurement. Includes columns: Treatment group, Sample (number and name), walbB quanitification, RSP17 (reference quantification), Target/Reference (walbB/RSP17), Positive/Negative."Survival analysis"- Results from adult survival assay documenting each day mosquitoes died. Include columns: Cup number (replicate cups), Wolb (+ or -), Time_min (10 minutes or 60 minutes), Heat_Shock_Temperature (35°C or 40°C), Frequency (1, 2, 3 exposures), DPI (days post infection), survived (how many individuals survived on a given day), % survived (number of mosquitoes that survived / number of mosquitoes at the start), Number_Dead (number of mosquitoes that died on that day), Status (documents that the given mosquito died, 1=dead, 0=alive)"Survival Analysis R Script"- Code for survival analysis using Kaplan-Meier curves using packages survminer and survival. This code requires "Survival Analysis" data set.All analysis was done in GraphPad Prism, version 10, aside from adult survival analysis which was done in R/Rstudio Version 2023.06.1.<br><br>