Metaviromic profiling of mosquito excreta using superhydrophobic collection devices expands the known RNA virome of North America

ABSTRACT Nearly 30% of emerging infectious disease events worldwide are transmitted by arthropod vectors, and this proportion continues to rise. Rapid and accurate detection is critical for directing vector control interventions, thereby reducing the likelihood of widespread transmission. Surveillance of infected mosquitoes can provide an early warning of impending human infection; however, conventional virus testing relies on processing large pools of mosquitoes and requires labor-intensive pre-processing. During rapidly developing epidemic or panzootic events, these delays may limit the effectiveness of public health responses. Mosquito excreta has recently emerged as a promising alternative substrate for pathogen detection. Sugar-fed mosquitoes regularly excrete gut contents, offering a rich source of nucleic acids. In this study, we developed and applied custom superhydrophobic excreta-collection funnels that efficiently aggregate excreta produced by field-collected Culex mosquitoes into attached microcentrifuge tubes. Shotgun metagenomic sequencing of this material revealed a diverse RNA virome, including both globally distributed viruses and those reported here for the first time from the Americas. Beyond virus detection, additional analyses enabled confirmation of host mosquito species and identification of trypanosomatid parasites, demonstrating the broader utility of mosquito excreta for integrated surveillance. We anticipate that methods and devices of this type will become valuable components of vector surveillance programs, particularly in remote or resource-limited settings where repeated collections are challenging. Overall, our findings highlight the potential of excreta-based monitoring to improve early detection of emerging or unknown pathogens of One Health importance, refine our understanding of mosquito virome biogeography, and facilitate the discovery of previously undescribed viruses. IMPORTANCE Many infectious diseases that affect people and animals are spread by mosquitoes and other biting insects, and the number of these outbreaks is increasing. Detecting pathogens in mosquito populations early can provide a critical warning before human cases begin, allowing health officials to act quickly. However, traditional surveillance requires collecting and processing large numbers of mosquitoes, which can be slow and labor-intensive during fast-moving outbreaks. Here we demonstrate a simpler approach: testing mosquito waste. When mosquitoes feed on sugar, they excrete material that contains genetic traces of viruses and other organisms. Using specially designed collection devices and modern genetic sequencing, we show that mosquito excreta can reveal a wide range of viruses and parasites while also identifying the mosquito species present. This method could make disease surveillance faster and more practical in remote or resource-limited settings, improving our ability to detect emerging pathogens that threaten human, animal, and environmental health.