Quantification of Mosquito Microbiome

Both culture-dependent and culture-independent assays were conducted in parallel to quantify live and total bacterial loads in mosquitoes and their rearing water. Culture-dependent quantification of microbes was performed by culturing 40 μl of rearing water or 40 μl of 10-fold serial dilutions from individual mosquitoes (3 to 5 per treatment) homogenized in 500 μl PBS on LB plates at 37°C for 5 days. Plated dilutions that yielded distinct, countable colonies were enumerated for each mosquito sample. Each sample was plated in technical triplicates, and the mean colony count is reported. Culture-independent quantification of bacteria was performed by SYBR green real-time PCR (Thermo Fisher Scientific, Emeryville, CA) to amplify the 16S rRNA gene in samples from mosquitoes and water (5 to 10 per treatment). Bacterial culturing and quantitative PCR (qPCR) of mosquitoes were repeated twice for each rearing experiment. The mosquito data were normalized to an A. aegypti reference ribosomal protein S17 (RPS17) gene (82 (link)).