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

## TL;DR

A new system using internet-connected mosquito monitors and PCR testing was used to track and identify mosquitoes in Zhejiang, China, improving disease control efforts.

## Contribution

A novel multiplex PCR system and internet-based mosquito monitor were developed for real-time, precise mosquito identification and monitoring.

## Key findings

- The MS-300 monitor captured 9749 mosquitoes, showing population density peaks and activity patterns.
- The multiplex PCR system accurately identified six mosquito species with high sensitivity.
- The system reduced labor costs and provided continuous data for effective mosquito control.

## Abstract

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.

Surveillance of mosquito vectors has always been an indispensable component in the prevention and control of mosquito-borne viral and parasitic diseases. However, traditional mosquito surveillance methods like human landing catches, human-baited double net traps, BG-Sentinel mosquito traps are labor-intensive and provide limited data. In particular, the method of human landing catches is easily influenced by human and environmental factors, posing infection risks. We deployed an internet-based vector mosquito monitor called MS-300 to continuously track vector populations at ten locations in Zhejiang province over eight months. The data revealed variations in mosquito vector activity across different regions and seasons, likely influenced by population structure. The initial stages of mosquito vector identification primarily relied on morphological methods; however, inaccurate identification remains likely due to phenotypic plasticity and genetic variability. To enhance species identification, we have developed a multiplex PCR system targeting key vectors including Aedes albopictus and Aedes aegypti, which transmit Dengue virus or Zika virus, as well as Anopheles sinensis known as a malaria vector. This approach not only reduces the manpower requirements for mosquito vector control but also provides continuous and accurate data that offers crucial support for the prevention and management of mosquito-borne diseases.