Culicidae vector ecology in southern Iran: Integrating Faunistics and molecular analysis of blood-feeding preferences to strengthen malaria surveillance in transition zones

Culicidae mosquitoes are pivotal vectors of malaria and arboviral diseases, necessitating continuous surveillance in transitional zones where ecological and anthropogenic factors amplify transmission risks. This study investigated the fauna and blood-feeding preferences of Culicidae in Shiraz, southern Iran-a region adjacent to malaria-endemic provinces-using molecular methods to inform vector control strategies. From April 2023 to January 2024, 1249 adult mosquitoes were collected across urban and rural sites via pyrethrum spray catches, CDC light traps, and manual aspiration. Morphological identification revealed <i>Culex pipiens</i> (39.0 %), <i>Cx. quinquefasciatus</i> (23.6 %), and <i>Aedes caspius</i> (19.6 %) as dominant Culicinae species, while <i>Anopheles dthali</i> (80.6 % of Anophelinae) and <i>An. sacharovi</i> (19.4 %) comprised the primary Anophelinae. PCR-RFLP analysis of mitochondrial "cytochrome <i>b</i>" gene fragments from 50 blood-fed females demonstrated distinct host preferences: <i>Anopheles</i> spp. primarily fed on rodents/dogs (41 %) and humans (28 %), whereas <i>Cx. pipiens</i> exhibited marked anthropophily (54 % human blood meals). Spatial clustering of <i>An. sacharovi</i> in irrigation-rich northern Shiraz (<i>χ</i> ^<i>2</i> = 12.7, *p* < 0.01) and mixed human-livestock blood meals in <i>Cx. pipiens</i> highlighted ecological overlap favoring zoonotic spillover. These findings underscore the dual role of <i>Cx. pipiens</i> as an important vector for arboviruses and the residual malaria risk posed by <i>Anopheles</i> spp. in transitional zones. Molecular techniques proved critical for precise blood meal identification, surpassing traditional serological limitations. The study advocates for integrated vector management-combining targeted insecticide use, environmental modification, and cross-border surveillance-to mitigate malaria resurgence and emerging arboviral threats. Sustained entomological monitoring, leveraging molecular tools, is essential to safeguard Iran's malaria elimination achievements and address evolving public health challenges.