Morphological and molecular identification reveals a high diversity of Anopheles species in the forest region of the Cambodia–Laos border

Abstract

Background To develop an effective malaria vector intervention method in forested international border regions within the Greater Mekong Subregion (GMS), more in-depth studies should be conducted on local Anopheles species composition and bionomic features. There is a paucity of comprehensive surveys of biodiversity integrating morphological and molecular species identification conducted within the border of Laos and Cambodia. Methods A total of 2394 adult mosquitoes were trapped in the Cambodia–Laos border region. We first performed morphological identification of Anopheles mosquitoes and subsequently performed molecular identification using 412 recombinant DNA–internal transcribed spacer 2 (rDNA-ITS2) and 391 mitochondrial DNA–cytochrome c oxidase subunit 2 (mtDNA-COII) sequences. The molecular and morphological identification results were compared, and phylogenetic analysis of rDNA-ITS2 and mtDNA-COII was conducted for the sequence divergence among species. Results Thirteen distinct species of Anopheles were molecularly identified in a 26,415 km2 border region in Siem Pang (Cambodia) and Pathoomphone (Laos). According to the comparisons of morphological and molecular identity, the interpretation of local species composition for dominant species in the Cambodia–Laos border (An. dirus, An. maculatus, An. philippinensis, An. kochi and An. sinensis) achieved the highest accuracy of morphological identification, from 98.37 to 100%. In contrast, the other species which were molecularly identified were less frequently identified correctly (0–58.3%) by morphological methods. The average rDNA-ITS2 and mtDNA-COII interspecific divergence was respectively 318 times and 15 times higher than their average intraspecific divergence. The barcoding gap ranged from 0.042 to 0.193 for rDNA-ITS2, and from 0.033 to 0.047 for mtDNA-COII. Conclusions The Cambodia–Laos border hosts a high diversity of Anopheles species. The morphological identification of Anopheles species provides higher accuracy for dominant species than for other species. Molecular methods combined with morphological analysis to determine species composition, population dynamics and bionomic characteristics can facilitate a better understanding of the factors driving malaria transmission and the effects of interventions, and can aid in achieving the goal of eliminating malaria. Graphical Abstract

Population genetic structure and evolutionary genetics of Anopheles sinensis based on knockdown resistance (kdr) mutations and mtDNA-COII gene in China–Laos, Thailand–Laos, and Cambodia–Laos borders

“…sinensis sample were paired to determine the species of An. sinensis in accordance with previous studies [ 35 , 36 ] (Additional file 1 : Table S1).…”

A systematic review: is Anopheles vagus a species complex?

“…vagus was the dominant species but only 12 An.vagus were randomly investigated by molecular analysis of ITS 2 and the COII sequence: all are not genetically distinct An. vagus -like species [ 38 ] Hasanah et al . 2022 a An.…”

“…Likewise, there were no genetically distinct An. vagus -like species in twelve specimens from the border between Laos and Cambodia [ 38 ]. In contrast, studies conducted in East Timor [ 44 ] reported two groups of An.…”

Sensitive and rapid detection of Culex pipiens and Aedes albopictus

“…At present, species identification of mosquitoes mostly relies on morphological identification. However, morphological identification methods are limited by many factors, such as damaged specimens, similar morphology of close-related species, and difficult identification of imported species ( 16 ). More importantly, new mosquito species could become vectors of new diseases, and monitoring of mosquito species in a geographical area is crucial ( 17 ).…”