A novel assay to measure the emergence of third-stage filarial nematodes in individual mosquitoes

Abstract 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. An inability to determine what proportion of third-stage larvae are capable of emerging from the mosquito has hindered efforts to assess the efficacy of experimental transmission-blocking strategies and to evaluate the transmission competence of field vectors. To overcome this hurdle, we have developed a novel method to assess the infective third-stage larvae of Dirofilaria immitis that emerge from individual Aedes aegypti and Ae. albopictus mosquitoes. This method does not require specialized equipment, making it amenable for field work. Using this assay, we have determined the proportion of microfilariae that successfully develop into emerging third-stage larvae as well as the fate of those that do not. We have found that 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. However, during this time, the fitness cost of the infection to the mosquito increases, suggesting that larvae continue to either damage the vector, compete for essential nutrients, or produce toxic metabolites. Increasing uptake of D. immitis microfilariae increases the fitness cost to the mosquito but does not increase the number of emerging third-stage larvae. Notably, we find that our assay is also suitable for assessment of emerging third-stage larvae of Brugia malayi, one of the filarial nematodes responsible for human lymphatic filariasis. Thus, it is likely that this assay will be applicable to all mosquito-transmitted filariae and possibly filariae transmitted by other hematophagous arthropods. Together, our results indicate that this new assay will facilitate assessment of the transmission potential of arthropod vectors and promote preparation of uniformly infectious L3 for challenge experiments and other functional assays. It will also facilitate analyses of molecular interactions between vectors and filariae, ultimately allowing for the establishment of novel methods to block disease transmission.