Genetic variation in olfactory pathways associated with host-seeking behavior in natural populations of Anopheles minimus, a primary malaria vector in western Thailand

Abstract

Background: Malaria transmission hinges on infected Anopheles mosquitoes biting humans, with carbon dioxide (CO<inf>2</inf>), host odor, and body heat acting as key attractants. Along the Thai–Myanmar border, Anopheles minimus (the Funestus Group), a primary malaria vector, exhibits a stronger preference for human hosts than species of the Maculatus Group. Elucidating the genetic basis of this feeding behavior is essential for improving malaria control strategies. Methods: Wild Anopheles mosquitoes were collected in Tha Song Yang district, Tak province, Thailand, from July 2019 to November 2020, using cow-baited traps, human landing catches, and Center for Disease Control (CDC) light traps. Specimens were identified morphologically and confirmed by Sanger sequencing of the cytochrome c oxidase subunit 1 (cox1) gene. We then performed whole-genome sequencing on An. minimus females categorized by host-seeking behavior: cow-baited collection (COW), human landing indoor (HLI), and human landing outdoor (HLO) to investigate the genetic determinants of host preference. Results: Anopheles minimus females accounted for 25% of total samples (504/1,997). Cox1 sequencing revealed 143 unique haplotypes among 287 specimens, forming two major phylogenetic lineages, A (181 sequences) and B (106 sequences), suggestive of potential cryptic diversity. Whole-genome sequencing of An. minimus Lineage A from COW, HLI, and HLO groups yielded 12,659,785 variants. After filtering, 68,975 non-synonymous single-nucleotide polymorphisms (nsSNPs) remained. Comparing allele frequencies across the three pooled groups (FDR-adjusted p-value < 0.001) yielded 2,629, 2,948, and 4,369 significant nsSNPs, respectively. Gene Ontology (GO) analysis of genes harboring these nsSNPs showed strong enrichment for olfaction-related terms. The top six nsSNPs with olfactory annotations from each group comparison were selected for validation; Sanger sequencing confirmed their association with host-seeking preference. The VectorBase gene IDs for these candidate nsSNPs are AMIN001807, AMIN001339, AMIN003886, AMIN000912, AMIN003926, AMIN011060, AMIN002342, and AMIN015480. Conclusions: The observed significant genomic variance in field-collected An. minimus females, categorized by collection methods (reflecting host-seeking behavior), proposes a genetic underpinning for these behavioral variations. Differential nsSNPs within olfactory pathway genes might be functionally linked to host-seeking in this important malaria vector.