Ecotypes, Wolbachia, and urbanization shape Culex pipiens population structure in a West Nile virus hotspot

Culex pipiens is a major vector of West Nile Virus (WNV) in Europe and has a complex evolutionary history that has been linked to the probability of WNV spillover to humans. Here, we present a population genomic analysis of Cx. pipiens from a WNV hotspot in southwestern Spain. Whole-genome sequencing of 217 individuals from 24 localities revealed that population structure is mainly shaped by the coexistence of pipiens and molestus ecotypes. Despite clustering, genome-wide divergence between ecotypes was low, consistent with shared ancestral variation. Furthermore, Wolbachia infection was heterogeneous across the study area, where some Culex pipiens pipiens remain uninfected. We also identified three new polymorphic chromosomal inversions on chromosomes 1 and 3 that segregate independently. Although inversions did not underlie ecotypic differentiation, they were enriched for genes involved in olfaction and insecticide resistance pathways, suggesting potential adaptive and epidemiological relevance. Distance-based redundancy analyses showed that ecotype identity explained the largest fraction of genetic variance, with additional contributions from Wolbachia infection, habitat type (urban versus rural) and geographic distance. These results reveal that partial ecotypic differentiation, symbiont infection, and habitat interact to shape population structure in Cx. pipiens, with implications for vector ecology and disease transmission.