Optimization of high-throughput methodology for pathogens detection and vectors identification In Culicidae of Northern Italy.

Vector-borne diseases transmitted by mosquitoes represents one of the main globally concerns for human health, due to the large number of people they affect, and the variety of vector species they involve. Investigation of pathogens prevalence in vectors and the distribution of vectors themselves is crucial for preventing and managing disease outbreaks. To achieve that, instruments and methodologies that can analyse large number of samples in a short time are fundamental. Our aim was to develop a high-throughput protocol for both pathogens screening and mosquitoes’ identification, to conduct surveillance in Northern Italy. In this insight we collected adult mosquitoes in 3 different districts of Lombardy Region (Bergamo, Como, Milan) from June to October 2024, with over 8000 individuals collected. We selected a microfluidic tool (Fluidigm), to minimize the starting material required and maximise the number of samples analysed, and we developed a 24.192 chip for screening of 8 species and 15 pathogens. To date, we optimized a commercial kit for quick and simultaneous extraction of DNA and RNA from a single individual. We finalized the concept of the 24.194 chip, completing the panel of target with 3 newly designed sets of primers and TaqMan probes. Specifically, we designed new sets of primers for the identification of Aedes caspius and Culiseta longiareolata, as we morphologically pre-identified a consistent number of them alongside the most common species (e. g. Ae. albopictus, Ae. koreicus, Culex pipiens), and tested them with classical qPCR. Also, we designed primers and probe for pan-Culicidae identification, which we validated against samples of selected Culicidae species established in Italy and other Diptera species (e. g. Chironimidae). Our goal is to enhance our understanding of pathogen spread in Northern Italy while simultaneously assessing the effectiveness of our panel. With the imminent implementation of our chip on morphologically identified mosquitoes, we also aim to establish a rapid and high-throughput methodology for vector and vector-borne disease surveillance in Italy.