Characterization of overwintering sites (hibernacula) of the West Nile vector Culex pipiens in Central Italy.

Background: In cool-temperate regions, mosquitoes face winter conditions that hinder their development. To cope with cold temperatures, species like Culex pipiens, a major vector of West Nile virus (WNV), diapause as adult females in overwintering shelters known as hibernacula. This study aimed to identify and characterize the overwintering sites of Cx. pipiens in central Italy, analyzing the environmental factors influencing the abundance of the two biological forms, Cx. pipiens pipiens and Cx. pipiens molestus.

Methods: Field surveys were conducted in Lazio and Tuscany over two consecutive winters (2022/2023 and 2023/2024). Overwintering mosquitoes were collected from different hibernacula types, including natural caves, artificial cavities and buildings. Environmental variables such as temperature, humidity, light intensity and vapor pressure deficit (VPD) were recorded in the entrance and inner rooms of each hibernaculum. Mosquito species and Cx. pipiens forms were identified through morphological and molecular analyses. A beta regression model was applied to assess the relationship between environmental factors and Cx. pipiens abundance. Redundancy analysis (RDA) was used to explore the impact of small- and landscape-scale variables on biological forms distribution.

Results: Culex pipiens presence was confirmed in 24 hibernacula and represented the most abundant species, with Anopheles maculipennis s.l., Culiseta annulata and Culex hortensis accounting for 0.4% of the collected individuals. Comparing the entrance and inner rooms, a higher abundance of Cx. pipiens s.s. was observed in the darker environments, characterized by a humidity of 50-75%, a temperature of 10-20 °C and a VPD of 0.3-0.8 kPa. Inside the inner rooms, Cx. pipiens females preferred lower temperatures, light intensity and humidity, combined with higher VPD. The RDA highlighted that Cx. pipiens pipiens was associated with low temperatures and VPD and high humidity levels, preferring semi-natural areas. Culex pipiens molestus was positively associated with artificial areas. Hybrids were observed in several types of hibernacula.

Conclusions: This study provides insights into the overwintering ecology of Cx. pipiens in southern Europe, highlighting the environmental factors driving its abundance. These results may inform future vector control strategies aimed at reducing mosquito populations and limiting WNV diffusion in temperate regions.