Detection of Culex restuans Environmental RNA (eRNA) Using Droplet Digital PCR: A Proof-of-Concept for Molecular Mosquito Surveillance

ABSTRACT Sensitive surveillance tools are needed to improve detection of emerging mosquito populations relevant to public health. We evaluated an environmental nucleic acid-based approach using droplet digital PCR (ddPCR) to detect Culex restuans in water samples from semi-field conditions. Water samples were collected between July and September 2025, and molecular assays targeting COX1 mRNA were used to detect the presence of Cx. restuans . ddPCR detected Cx. restuans eRNA in water samples with confirmed larvae, with strong signal in semi-field samples and a positive correlation between eRNA copy number and larval abundance under controlled conditions. A time-course experiment indicated rapid loss of eRNA signal after 1 week at room temperature, indicating that eRNA reflects recent mosquito activity. These findings support the operational potential of eRNA as a complement to conventional mosquito surveillance programs, particularly if larval density is high and there is need to confirm findings and/or screen for rare detects.