Activation of the Toll pathway in Aedes aegypti blocks the development of emerging third-stage larvae of drug-resistant Dirofilaria immitis

•Emergence assay used to study drug-resistant D. immitis development in mosquitoes.•Mosquito Toll signaling blocks eL3 of two drug-resistant D. immitis strains.•Different drug-resistant D. immitis strains are blocked by distinct mechanisms. Dirofilaria immitis is the globally distributed agent of heartworm disease. Infection in canines causes debilitating disease that can be fatal if left untreated. Macrocyclic lactones can prevent heartworm disease in dogs, cats and ferrets by killing larvae before they develop into adult worms in the pulmonary artery. However, administration of prophylactic drugs to wild canids to prevent D. immitis infection is not feasible. Furthermore, a vaccine against heartworm is currently unavailable and drug resistant D. immitis have been identified, highlighting the need for new strategies to prevent parasite transmission. We recently established a method to block development of emerging third-stage larvae (eL3) from the mosquito Aedes aegypti by over-activating the Toll pathway, one of the major innate immune signaling pathways in mosquitoes. Our previous study used a drug-sensitive strain of D. immitis and it remains unknown if the strategy is effective against different D. immitis genotypes and, more importantly, if it would work against drug-resistant genotypes. The purpose of this study was to determine whether Toll pathway activation is capable of blocking eL3 development of D. immitis strains that are resistant to macrocyclic lactones. We infected mosquitoes with two independent strains of D. immitis previously confirmed as being resistant to macrocyclic lactones, and then activated Toll signaling by RNAi-mediated silencing of the pathway inhibitor, IκB/Cactus, and quantitatively measured eL3 development. Similar to the drug-sensitive strain, eL3 were strongly reduced by Toll activation in both drug-resistant strains. Furthermore, similar to the drug-sensitive strain, the reduction of eL3 in both drug-resistant strains suggests a defect in larval invasion of, or development in, the Malpighian tubules – the organ in the mosquito to which microfilariae migrate after ingestion and where the larvae undergo several developmental molts. In summary, Toll pathway activation blocks the development of three distinct D. immitis genotypes, including two different drug-resistant genotypes. If this strategy can be applied to heartworm vectors in the field, it may help reduce the spread of disease and is not predicted to favor the sprea