First Report of Anopheles annularis s.l. , An. maculatus s.s. , and An. culicifacies s.l. as Malaria Vectors and a New Occurrence Record for An. pseudowillmori and An. sawadwongporni in Alipurduar District Villages, West Bengal, India.

A comprehensive entomological survey was undertaken in Alipurduar District, West Bengal, from 2018 to 2020 and in 2022. This study was prompted by reported malaria cases and conducted across nine villages, seven Sub-Centres, and three Primary Health Centres (PHCs). Mosquitoes were hand-collected with aspirators and flashlights from human dwellings and cattle sheds during the daytime. Both morphological and molecular techniques were used for species identification. Additionally, mosquitoes were tested for Plasmodium parasites and human blood presence. Mosquito species such as An. barbirostris s.l. , An. hyrcanus s.l. , An. splendidus , and An. vagus were morphologically identified. For species like An. annularis s.l. , An. minimus s.s. , An. culicifacies s.l. , and An. maculatus s.s ., a combination of morphological and molecular techniques was essential. The mitochondrial cytochrome c oxidase gene subunit 1 (CO1) was sequenced for An. annularis s.l. , An. maculatus s.s. , An. culicifacies s.l. , An. vagus , and some damaged samples, revealing the presence of An. pseudowillmori and An. fluviatilis . The major Anopheles species were An. annularis s.l ., An. culicifacies s.l. , and An. maculatus s.s ., especially in Kumargram and Turturi PHCs. Plasmodium positivity was notably high in An. annularis s.l. and An. maculatus s.s. with significant human blood meal positivity across most species. Morphological, molecular, and phylogenetic analyses are crucial, especially for archived samples, to accurately identify the mosquito fauna of a region. Notably, this study confirms the first occurrence of An. pseudowillmori and An. sawadwongporni in West Bengal and implicates An. maculatus s.s ., An. culicifacies s.l. , and An. annularis s.l. as significant vectors in the Alipurduar region.