A novel assay to isolate and quantify third-stage filarial larvae emerging from individual mosquitoes

Background Mosquitoes transmit filarial nematodes to both human and animal hosts, resulting in worldwide health and economic consequences. Transmission to a vertebrate host requires that ingested microfilariae develop into infective third-stage larvae capable of emerging from the mosquito proboscis onto the skin of the host during blood feeding. Determining the number of microfilariae that successfully develop to infective third-stage larvae in the mosquito host is key to understanding parasite transmission potential and to developing new strategies to block these worms in their vector. Methods We developed a novel method to efficiently assess the number of infective third-stage filarial larvae that emerge from experimentally infected mosquitoes. Following infection, individual mosquitoes were placed in wells of a multi-well culture plate and warmed to 37 °C to stimulate parasite emergence. Aedes aegypti infected with Dirofilaria immitis were used to determine infection conditions and assay timing. The assay was also tested with Brugia malayi infected Ae. aegypti . Results Approximately 30% of Ae. aegypti infected with D. immitis and 50% of those infected with B. malayi produce emerging third-stage larvae. Once D. immitis third-stage larvae emerge at 13 days post infection, the proportion of mosquitoes producing them, and the number produced per mosquito remain stable until at least day 21. The prevalence and intensity of emerging third-stage B. malayi were similar on days 12-14 days post infection. Increased uptake of D. immitis microfilariae increases the fitness cost to the mosquito but does not increase the number of emerging third-stage larvae. Conclusions We provide a new assay with an associated set of infection conditions that will facilitate assessment of the filarial transmission potential of mosquito vectors and promote preparation of uniformly infectious L3 for functional assays. The ability to quantify infection outcome will facilitate analyses of molecular interactions between vectors and filariae, ultimately allowing for the establishment of novel methods to block disease transmission. Graphical Abstract