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

Abstract

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.

Keywords: Alipurduar; An. annularis s.l.; An. culicifacies; An. maculatus s.s.; An. pseudowillmori; An. sawadwongporni; India; first report; malaria.

Figure 1

Map of Alipurduar District, West Bengal, with study areas.

Figure 2

Representative images of malaria vectors of Alipurduar with important identification characters. (A) Adult female Anopheles annularis s.l., (A-1) tarsomeres 3, 4, 5 of the hind leg completely white and (A-2) wing vein 5 dark at the point of branching. (B) Adult female Anopheles (Cellia) maculatus s.l., (B-1) tarsomere 5 (T-5) and part of 4 (T-4) of the hind leg, white. Femora and tibia speckled. (C) Adult female Anopheles (Cellia) culicifacies s.l., (C-1) wing vein 3, black.

Figure 3

Molecular identification of different Anopheles species. (a): Characteristic PCR band of An. annularis s.l. at 387 bp in all the lanes except in lanes 10 and 16. M-Low molecular weight DNA ladder. Faint band in lanes 3 and 4. (b): Agarose gel image of PCR showing amplification of An. maculatus s.s. at 180 bp (lanes 1–6, 8, 11–14, and 17–18, Faint band in lane 6) and An. sawadwongporni at 242 bp (lanes 15 and 16). M-low molecular weight DNA ladder. (c): Agarose gel image of An. aconitus (306) bp in lane 1 and 2, lane 3: An. minimus (184 bp), lane 4: An. varuna (252 bp). M-100 bp DNA ladder, Nc-negative control. (d): Gel image of human blood meal PCR assay showing human blood positive at 519 bp in lanes 1 to 15. M-low molecular weight DNA ladder.

Figure 4

Maximum likelihood bootstrap consensus phylogenetic tree of An. maculatus s.s, An. pseudowillmori, An. vagus, An. annularis s.l., An. culicifacies s.l., and An. fluviatilis s.l. The tree was constructed in MEGA X by applying the general time reversible model (GTR + G + I) with 500 bootstrap values. Bold and coloured are the sequences from the current study.