Title - Impact of extrinsic incubation temperature on natural selection during Zika virus infection of Aedes aegypti

Arthropod-borne viruses (arboviruses) require replication across a wide range of temperatures to perpetuate. While vertebrate hosts tend to maintain temperatures of approximately 37{degrees}C - 40{degrees}C, arthropods are subject to ambient temperatures which can have a daily fluctuation of > 10{degrees}C. Temperatures impact vector competence, extrinsic incubation period, and mosquito survival unimodally, with optimum occurring at some intermediate temperature. In addition, the mean and range of daily temperature fluctuations influence arbovirus perpetuation and vector competence. The impact of temperature on arbovirus genetic diversity during systemic mosquito infection, however, is poorly understood. Therefore, we determined how constant extrinsic incubation temperatures of 25{degrees}C, 28{degrees}C, 32{degrees}C, and 35{degrees}C control Zika virus (ZIKV) vector competence and population dynamics within Aedes aegypti and Aedes albopictus mosquitoes. We also examined diurnally fluctuating temperatures which more faithfully mimic field conditions in the tropics. We found that vector competence varied in a unimodal manner for constant temperatures peaking between 28{degrees}C and 32{degrees}C for both Aedes species. Transmission peaked at 10 days post-infection for Aedes aegypti and 14 days for Aedes albopictus. The effect of diurnal temperature was distinct and could not have been predicted from constant temperature-derived data. Using RNA-seq to characterize ZIKV population structure, we identified that temperature alters the selective environment in unexpected ways. During mosquito infection, constant temperatures more often elicited positive selection whereas diurnal temperatures led to strong purifying selection in both Aedes species. These findings demonstrate that temperature has multiple impacts on ZIKV biology within mosquitoes, including major effects on the selective environment within mosquitoes. Author SummaryArthropod-borne viruses (arboviruses) have emerged in recent decades due to complex factors that include increases in international travel and trade, the breakdown of public health infrastructure, land use changes, and many other factors. Climate change also has the potential to shift the geographical ranges of arthropod vectors, consequently increasing the global risk of arbovirus infection. Changing temperatures may also alter the virus-host interaction, ultimately resulting in the emergence of new viruses and virus genotypes in new areas. Therefore, we sought to characterize how temperature (both constant and fluctuating) alters the ability of Aedes aegypti and Aedes albopictus to transmit Zika virus, and how it influences virus populations within mosquitoes. We found that intermediate temperatures maximize virus transmission compared to more extreme and fluctuating temperatures. Constant temperatures increased positive selection on virus genomes, while fluctuating temperatures strengthened purifying selection. Our studies provide evidence that in addition to altering VC, temperature significantly influences the selective environment within mosquitoes.