Rapid-Response Vector Surveillance and Emergency Control During the Largest West Nile Virus Outbreak in Southern Spain.

West Nile Virus (WNV) is an emerging arboviral threat in Europe, with rising incidence in Spain since 2004. In 2024, Spain experienced its largest outbreak, primarily in small urban areas of south-western regions. We report a subset of an emergency integrated vector management program, focusing on six municipalities accounting for one-third of all human WNV cases nationwide. Over four months, 725 potential larval sites were inspected during 4026 visits. Adult mosquitoes (n = 2553) were collected with suction traps, and immature stages (n = 4457) with dipper techniques, yielding 11 species. Culex pipiens s.l. was predominant, while Cx. perexiguus, though less abundant, was epidemiologically significant. Cytochrome Oxidase I (COI) gene phylogenetic analysis confirmed Cx. perexiguus, forming a distinct clade from Cx. univittatus. Immature mosquitoes were found in 18.6% of sites, especially irrigation canals, ditches, and backwaters near urban areas. Habitat differences in larval abundance were analyzed using generalized linear mixed models. Targeted larviciding with Bacillus thuringiensis var. israelensis (Bti) and focal adulticiding with cypermethrin totaled 259 interventions (70.4% larviciding, 29.6% adulticiding). A significant 63.9% reduction in larval abundance was observed after five consecutive Bti treatments, with some variation among treatment cycles (52.2-75.5%). Adult activity persisted into late autumn. This study provides the first comprehensive characterization of larval mosquitoes in Spain's main WNV hotspot, highlighting the need for rapid, coordinated expert interventions and extended seasonal control to prevent future outbreaks.