Brackney & Armstrong Abstract: Aedes aegypti is the primary vector for a number of human pathogens, including dengue virus (DENV; Flaviviridae, Flavivirus), Zika virus (ZIKV; Flaviviridae, Flavivirus), chikungunya virus (CHIKV; Togaviridae, Alphavirus) and yellow fever virus (YFV; Flaviviridae, Flavivirus), all of which present a continued threat to human health worldwide. Understanding the endemic and epidemic risk of these arthropod-borne (arbo-) viruses is critical to the success of public health preparedness and intervention. One key entomological parameter informing risk estimates is vector competence (how able a mosquito is to become infected and transmit an arbovirus; VC). Quantifying the competency of local vector populations can help inform the risk that any one pathogen poses to a given community. This is often quantified in the laboratory by exposing populations of local mosquitoes to an infectious bloodmeal and harvesting tissues at set time-points post infection. While informative, this approach often fails to consider the biology and behavior of the vector mosquito. For example, it is known that wild Ae. aegypti mosquitoes will imbibe several bloodmeals over the course of a traditional laboratory-based vector competence study (e.g. bloodmeal every two to three days). To address these shortcomings, we recently began examining the effects that multiple blood feeding episodes have on the competency of Ae. aegypti mosquitoes for ZIKV. Our preliminary findings reveal that providing a second non-infectious bloodmeal to ZIKV infected Ae. aegypti mosquitoes enhances viral escape from the midgut and significantly shortens the duration between mosquito acquisition of ZIKV to transmission. In this application we will examine the effects that multiple bloodfeeding episodes have on arbovirus infection of and transmission by vector mosquitoes. Specifically, we will be 1) testing this phenomenon in other virus-vector pairings, 2) evaluating the role of the midgut basal lamina in mediating the double-feed phenotype and 3) determining if similar processes are mediating the ability of arboviruses to infect ovarian tissue and be transmitted vertically.