Mosquito-borne helminth infections are responsible for a significant worldwide disease burden in both humans and animals. Accordingly, development of novel strategies to reduce disease transmission by targeting these pathogens in the vector are of paramount importance. We found that a strain of Aedes aegypti that is refractory to infection by Dirofilaria immitis, the agent of canine heartworm disease, mounts a stronger immune response during infection than does a susceptible strain. Moreover, activation of the Toll immune signaling pathway in the susceptible strain arrests larval development of the parasite, thereby decreasing the number of transmission-stage larvae. Notably, this strategy also blocks transmission-stage Brugia malayi, an agent of human lymphatic filariasis. Our data show that mosquito immunity can play a pivotal role in restricting filarial nematode development and suggest that genetically engineering mosquitoes with enhanced immunity will help reduce pathogen transmission.

Background Mosquitoes transmit filarial nematodes to both human and animal hosts, with 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 produced emerging third-stage larvae. Once D. immitis third-stage larvae emerged at 13 days post infection, the proportion of mosquitoes producing them and the number produced per mosquito remained stable until at least day 21. The prevalence and intensity of emerging third-stage B. malayi were similar on days 12–14 post infection. Increased uptake of D. immitis microfilariae increased the fitness cost to the mosquito but did 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 third-stage larvae 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.

Liverpool strain (LVP, refractory, Ae. aegypti R ), microfilariae (mf), Malpighian tubules (MT), third-stage larvae (L3), emerging third-stage larvae (eL3). AbstractMosquitoes 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 thirdstage 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.