Phenotypical aspects of Culex pipiens biotype pipiens during diapause: Lipid utilization, body size, insemination, and parity.

In temperate regions, female Culex pipiens biotype pipiens mosquitoes undergo diapause in winter. Diapausing biotype pipiens mosquitoes are potentially important winter reservoirs for mosquito-borne viruses, such as West Nile virus (WNV), Usutu virus (USUV), and Sindbis virus (SINV). Mosquitoes in diapause have not taken a bloodmeal prior to winter. Therefore, they do not become infected by taking an infectious bloodmeal and as a consequence, vertical transmission is considered the primary mechanism of mosquito-borne virus overwintering. Prior to winter, biotype pipiens mosquitoes build up fat reserves, which they utilize throughout winter. Furthermore, earlier studies have indicated that larger body size is correlated with increased survival during winter. However, not much is known about lipid utilization and body size of wild biotype pipiens mosquitoes in diapause. Therefore, we performed monthly collections of diapausing biotype pipiens mosquitoes in two consecutive winters (2020/2021 and 2021/2022) in bunkers of the New Hollandic Waterline in the Netherlands. Every month, we checked the proportion of inseminated and parous females via microscopy. In addition, we measured wing length as proxy for body size, and assessed total lipid content. Furthermore, we monitored indoor temperature in the overwintering locations. We show that the overwintering sites in our study provide relatively stable environments, in which temperatures rarely drop below 0 °C. The vast majority of biotype pipiens females were inseminated (84.1 %) and nulliparous (97.5 %). We detected differences in body size between but not within the two years of sampling. Additionally, we detected a difference in lipid content between the sampling years. We confirm that the vast majority of diapausing biotype pipiens females are inseminated and nulliparous. This indicates that they did not blood feed prior to winter, which underscores the likeliness of vertical transmission being the primary mechanism behind virus overwintering. The detected difference in body size between years can most likely be attributed to differences in summer conditions the mosquitoes were exposed to as larvae, although this needs confirmation. The difference in lipid depletion could not be explained by differences in climatic conditions. To shed more light on the links between climatic conditions, body size, lipid depletion and the consequences for mosquito population dynamics and arbovirus transmission, future experimental work, for example by arbovirus exposure followed by artificially induced diapause, is desired.