Optimizing environmental DNA (eDNA) methods for Culex mosquito surveillance in artificial container habitats

Vector-borne diseases account for 30% of global emerging infectious diseases, with arboviruses contributing the highest public health burden. Effective mosquito-borne disease prevention in human populations relies on surveillance of vector species. Current surveillance methods are resource intensive and rely on taxonomic expertise for species identification. Additionally, these methods are ineffective for surveillance of invasive species, which may only be detected at extremely fine spatial scales. A potential alternative to surveillance methods for disease vectors and invasive mosquitoes is the application of environmental DNA (eDNA) methods, an established tool used in aquatic systems. Organisms shed DNA into their environment, and environmental samples (e.g., water) can be tested for known DNA sequences. The purpose of this study is to operationalize eDNA methods for Culex species surveillance in the Northeast US, targeting the larval stage and their preferred artificial-container habitats. Three experiments were deployed between June-August 2022. First, optimization of water sample collection was tested across residential properties within the known target species distribution. Second, quantification of mosquitoes from eDNA was tested by sampling buckets with known concentrations of mosquito larvae. Third, a natural experiment was deployed across residential sites, encompassing an urban-rural gradient to validate the optimization and quantification experiments. DNA extraction and qPCR amplification were performed to test the 802 samples for target DNA. Target species assays were developed and have successfully detected target DNA within samples. Experimental conclusions have the potential for application in widespread surveillance efforts, without the need for taxonomic expertise, contributing to the protection of public health.