Real-time dynamic monitoring and multiplex PCR identification of vector mosquitoes in Zhejiang, China

The monitoring and identification of mosquito vectors are crucial for controlling the transmission of mosquito-borne diseases. Traditional mosquito morphological identification and surveillance methods, such as human landing catches, human-baited double net traps and BG-Sentinel mosquito traps, require a large amount of manpower but can only provide fragmented data. We utilized the MS-300, an internet-based vector mosquito monitor, to continuously capture and upload real-time data to cloud services across ten monitoring sites located in seven cities in Zhejiang Province, China from May to December 2023. A new multiplex PCR system was developed for amplifying the internal transcribed spacer 2 region, followed by employing both multiplex PCR and DNA barcoding techniques for detecting wild mosquitoes. A comprehensive monitoring of 9749 mosquitoes was conducted. The mosquito density gradually increased from May 2023, peaked around June 22nd, and then declined in a wave-like pattern. The mosquitoes have two peak activity times, the peak times may vary depending on different locations and seasons. The study showed the high specificity of a multiplex PCR system in distinguishing six mosquito species: Aedes albopictus, Aedes aegypti, Culex pipiens pallens, Armigeres subalbatus, Anopheles sinensis and Anopheles anthropophagus. Notably, the sensitivity of detecting An. anthropophagus reached an impressive 1fg/µL. With the exception of Ae. aegypti and An. anthropophagus, all four other mosquito species have been identified in Zhejiang Province, with Cx. p. pallens being the predominant population. The results were highly consistent with DNA barcoding technology. The MS-300 continuously and automatically monitors mosquito population density and activity, providing effective guidance for mosquito control based on the environment and reducing labor costs. Our newly established multiple PCR system enables precise identification of crucial vector mosquitoes, facilitating a comprehensive understanding of population structures across diverse regions for selecting effective control measures.