Diapausing adult female mosquitoes (Diptera: Culicidae) in overwintering sites in Estonia in 2023–2024: Species composition and variation

In temperate regions, several mosquito species overwinter as diapausing females in sheltered habitats, enabling them to serve as early-season vectors of mosquito-borne pathogens. While overwintering species compositions have been documented in several parts of Europe, no comprehensive data exist for Estonia or for the Baltic region. This study aimed to investigate the species composition of diapausing female mosquitoes across natural and artificial overwintering sites in Estonia. Mosquitoes were collected during the winter season of 2023/2024 from 18 overwintering locations, including caves, bunkers and dugouts. Specimens were primarily identified morphologically, and species distribution patterns were analysed across sites. However, a subset of the Culex pipiens/Culex torrentium from the Piusa cave system was additionally identified to species level using molecular methods to test the accuracy of morphological identification. The relative temperature and humidity of each collection site were also recorded. A total of 5268 mosquitoes were analysed, of which 5090 (96.6%) were identified as Cx. pipiens/Cx. torrentium. Culiseta annulata (2.0%) and Culex territans (1.3%) were the next most frequent taxa, while species from the Anopheles maculipennis complex and Culiseta alaskaensis were rarely encountered. Species abundance varied among sites, with some, such as Astangu and Tartu Kalmistu paljand, supporting multiple taxa, while some others were dominated exclusively by Cx. pipiens/Cx. torrentium. Further molecular analysis of 300 randomly chosen Cx. pipiens/Cx. torrentium revealed 3/4 of the specimens to be Cx. torrentium, contrasting with the preliminary morphological identification. The highest mosquito abundances were recorded at overwintering sites where temperatures at the time of sampling ranged between 5.3 and 7.5°C and relative humidity ranged between 58% and 70%. Our study provides the first comprehensive overview of overwintering mosquito populations in Estonia. Culex pipiens/Cx. torrentium were the dominant overwintering species across selected hibernation sites, highlighting their ecological adaptability and potential epidemiological importance. The presence of other species was limited and spatially restricted. Based on molecular analysis of a randomly selected subset of specimens from the Piusa cave system, Cx. torrentium may be more abundant than Cx. pipiens at some sites. Furthermore, morphological identification is not suitable for differentiating between the overwintering females of Cx. pipiens and Cx. torrentium. These findings highlight the importance of integrating molecular tools into overwintering mosquito surveys to improve epidemiological assessments. Multi-season continuous microclimatic monitoring is needed to evaluate the relationship between microclimate parameters, mosquito abundance and species variation.