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Abstract Background Vector control is still a pivotal method for preventing malaria, and its potency is weakened by the increasing resistance of vectors to chemical insecticides. As the most abundant and vital malaria vector in Southeast Asia, the chemical insecticide resistance status in Anopheles sinensis remains elusive in Laos, which makes it imperative to evaluate the true nature of chemical insecticide resistance-associated genetic mutations in An. sinensis in Laos. Methods Adult An. sinensis were collected from three border regions in Laos. DNA was extracted from individual mosquitoes. PCR amplification and DNA sequencing of a fragment containing codon 1014 of the voltage-gated sodium channel (vgsc) gene were completed to study the kdr allele frequency distribution, kdr intron polymorphism, population genetic diversity, and the evolutionary status of the kdr codon. The mitochondrial cytochrome c oxidase subunit II gene (COII) was amplified and sequenced to examine population variations, genetic differentiation, spatial population structure, population expansion, and gene flow patterns. Results Nine wild kdr haplotypes of the vgsc gene were detected in this study, and eight of them, namely 1014L1, 1014L2, 1014L4, 1014L7, 1014L9, 1014L10, 1014L11, and 1014L21, were discovered in the China–Laos border (northern Laos), while 1014L3 was only detected in the Thailand–Laos border (northwestern Laos) and Cambodia–Laos border (southern Laos). The newly identified haplotype, 1014L21, was uniquely distributed in the China–Laos border and was not identified in other countries. Based on sequence analysis of the mitochondrial COII genes, significant genetic differentiation and limited gene flow were detected between the China–Laos and Cambodia–Laos An. sinensis populations, which suggested that those two regions were genetically isolated. The distinct distribution of the kdr haplotype frequencies is probably the result of geographical isolation in mosquito populations. Conclusions Lack of kdr mutations in the vgsc gene was probably due to genetic isolation and the absence of intense selection pressure in the three border regions of Laos. This study reveals that pyrethroid-based chemical insecticides are still appropriate for battling An. sinensis in parts of Laos, and routine monitoring of chemical insecticide resistance should be continuously implemented and focused on more restricted areas as part of chemical insecticide resistance management. Graphical Abstract

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

Abstract Background The Anopheles hyrcanus group, which includes 25 species, is widely distributed in the Oriental and Palaearctic regions. Given the difficulty in identifying cryptic or sibling species based on their morphological characteristics, molecular identification is regarded as an important complementary approach to traditional morphological taxonomy. The aim of this study was to reconstruct the phylogeny of the Hyrcanus group using DNA barcoding markers in order to determine the phylogenetic correlations of closely related taxa and to compare these markers in terms of identification efficiency and genetic divergence among species. Methods Based on data extracted from the GenBank database and data from the present study, we used 399 rDNA–ITS2 sequences of 19 species and 392 mtDNA–COII sequences of 14 species to reconstruct the molecular phylogeny of the Hyrcanus group across its worldwide range. We also compared the performance of rDNA–ITS2 against that of mtDNA–COII to assess the genetic divergence of closely related species within the Hyrcanus group. Results Average interspecific divergence for the rDNA–ITS2 sequence (0.376) was 125-fold higher than the average intraspecies divergence (0.003), and average interspecific divergence for the mtDNA–COII sequence (0.055) was eightfold higher than the average intraspecies divergence (0.007). The barcoding gap ranged from 0.015 to 0.073 for rDNA–ITS2, and from 0.017 to 0.025 for mtDNA–COII. Two sets of closely related species, namely, Anophels lesteri and An. paraliae, and An. sinensis, An. belenrae and An. kleini, were resolved by rDNA–ITS2. In contrast, the relationship of An. sinensis/An. belenrae/An. kleini was poorly defined in the COII tree. The neutrality test and mismatch distribution revealed that An. peditaeniatus, An. hyrcanus, An. sinensis and An. lesteri were likely to undergo hitchhiking or population expansion in accordance with both markers. In addition, the population of an important vivax malaria vector, An. sinensis, has experienced an expansion after a bottleneck in northern and southern Laos. Conclusions The topology of the Hyrcanus group rDNA–ITS2 and mtDNA–COII trees conformed to the morphology-based taxonomy for species classification rather than for that for subgroup division. rDNA–ITS2 is considered to be a more reliable diagnostic tool than mtDNA–COII in terms of investigating the phylogenetic correlation between closely related mosquito species in the Hyrcanus group. Moreover, the population expansion of an important vivax malaria vector, An. sinensis, has underlined a potential risk of malaria transmission in northern and southern Laos. This study contributes to the molecular identification of the Anopheles hyrcanus group in vector surveillance. Graphical abstract