The ion channel ppk301 controls freshwater egg-laying in the mosquito Aedes aegypti

Aedes aegypti mosquitoes are deadly vectors of arboviral pathogens including Zika, dengue, and yellow fever, and breed in containers of freshwater associated with human habitation1,2. Female Ae. aegypti lay eggs near freshwater because larval and pupal stages are aquatic3. They use volatile cues to locate water at a distance4, while at close-range they contact water to evaluate its suitability for egg-laying4-7. High salinity is lethal to mosquito offspring and therefore correctly laying eggs in freshwater is a crucial parenting decision made by female mosquitoes. Here we show that the DEG/ENaC channel8-10 ppk301 is required for mosquitoes to exploit freshwater egg-laying substrates. When ppk301 mutant females contact water, they do not lay eggs as readily as wild-type animals and are more likely to make aberrant decisions between freshwater and saltwater at concentrations that impair offspring survival. We used a CRISPR-Cas9-based genetic knock-in strategy combined with the Q-binary transactivator system11 to build genetic tools for labelling and imaging neurons in the mosquito. We found that ppk301 is expressed in sensory neurons in legs and proboscis, appendages that directly contact water, and that ppk301-expressing neurons project to central taste centres. Using in vivo calcium imaging with the genetically-encoded calcium sensor GCaMP6s12, we found that ppk301-expressing cells respond to water but, unexpectedly, also to salt. This suggests that ppk301 is instructive for egg-laying at low salt concentrations but that a ppk301-independent pathway is responsible for inhibiting egg-laying at high salt concentrations. Water is a key resource for insect survival and understanding how mosquitoes interact with water to control different behaviours is an opportunity to study the evolution of chemosensory systems. The new genetic tools described here will enable direct study of not only egg-laying, but also other behaviours in mosquitoes that influence disease transmission and enable comparative studies of insect biology more broadly.