Project SummaryThe mosquito gut is a complex ecosystem containing a variety of bacterial taxa. These microbes play a crucialrole in the biology of the insect. However, it remains unclear how microbes are acquired by mosquitoes andwhat life stages are important in this process. High throughput sequencing studies have shown the microbiomeof the adult is highly variable but comprised of relatively few taxa. The microbiome of aquatic life stages issimilar to the larval habitat, suggesting environmental factors influence acquisition. In addition, some bacteriacan transition between mosquito life stages, suggesting they are highly adapted to the mosquito. Furthermore,it has been postulated that newly emerged adults imbibe larval water soon after emergence, which likely seedthe gut microbiome, particularly with transient microbes. Together, these processes likely account for variationseen in the adult gut and suggest acquisition is a stochastic process. Not surprisingly, lab-reared mosquitoeshave a vastly different microbiome compared to their field caught relatives. Given that the microbiomeinfluences many mosquito phenotypes, including the ability of mosquitoes to transmit pathogens, spuriousresults may be obtained when studying processes with lab-reared individuals. Here, we will address theseissues related to microbiome acquisition and differences between field and lab mosquitoes by exploiting arecently developed gnotobiotic system for rearing mosquitoes that allows for larvae to be reared with a definedenvironmental microbiome. In the first aim, we will examine how the larval habitat affects the adult gutmicrobiome. We will inoculate the larval habitat with a concentration gradient of a single bacterial species andobserve how density in the water affects the density in the adult gut. Using two different fluorescently labeledbacteria (GFP vs mCherry), we will compare gut acquisition of a neutrally competing bacterium at similar ordifferent larval water densities. For the last experiment of this aim, we will seed mosquito gut symbionts atdifferent times in the developmental process to determine if the timing of inoculation influences the gutmicrobiome. In the second specific aim, we will exploit the gnotobiotic system to complete a microbiometransplantation between mosquito cohorts. We will optimize this procedure by transferring a simple microbiomethat is comprised of three gut symbionts (Cedacea, Escherichia-Shigella and Pseudomonas) isolated frommosquitoes. Each of these bacteria will be tagged with a separate fluorescent protein that will allow culture-based validation. We will also develop a cryopreservation technique so the donor microbiome can be storedbefore reinfection. After optimizing the protocol with this tractable system, we will then undertake the transfer ofa more complex microbiome using a donor microbiome sourced from Ae. aegypti collected from the field inGalveston and transferred this microbiome into a lab Ae. aegypti (Galveston strain). These studies will providea more complete understanding of host acquisition of the microbiome and develop important tools formanipulating the microbiome, which will be significant for further studies examining host-microbe interactions.