Detection and persistence of environmental DNA (eDNA) of a vector mosquito, Culex pipiens pallens

Introduction: Preventing mosquito-borne infectious diseases requires that vector mosquitoes are monitored and controlled. Targeting immature mosquitoes (eggs, larvae, and pupae), which have less mobility than adults, is an effective management approach. However, conducting these surveys is often difficult due to the limitations of morphological classification and survey costs. The application of environmental DNA (eDNA) analysis can solve these issues because it allows easy estimation of species distribution and morphology-independent species identification. Although a few previous studies have reported mosquito eDNA detection, there is a gap in knowledge regarding the dynamics of mosquito eDNA during the developmental stages of immature mosquitoes. Methods: We used Culex pipiens pallens, a vector of West Nile fever, as a model species. First, we developed a species-specific detection assay and confirmed its specificity using in silico and in vitro tests. Next, we conducted laboratory experiments using breeding tanks. Water samples were collected at each developmental stage. In addition, water samples were collected daily until the seventh day after emergence from the pupae. We quantified eDNA using real-time PCR with the developed assay to investigate the dynamics of mosquito eDNA. Results: The specificity of the developed assay was confirmed by in silico and in vitro tests. Mosquito eDNA was detected at all developmental stages and detected up to seven days after emergence of pupae. In particular, high concentrations of eDNA were detected immediately after hatching from eggs and after emergence from pupae. Highly frequent positive eDNA signals were continuously detected between egg hatching and pupa hatching. Conclusions: Mosquito eDNA was detected immediately after the eggs were introduced, and eDNA-positive detections continued until pupae emergence, suggesting that eDNA analysis is useful for monitoring mosquito larvae. The results show that eDNA analysis provides valuable information about the water sources inhabited by immature mosquitoes in mosquito control. In the future, monitoring immature mosquitoes using eDNA analysis will aid in preventing mosquito-borne infectious diseases.