Molecular phylogeny of the Anopheles hyrcanus group (Diptera: Culicidae) based on rDNA–ITS2 and mtDNA–COII

Zhang, Canglin; Yang, Rui; Wu, Lan; Luo, Chunhai; Guo, Xin; Deng, Yan; Zhou, Hongning; Zhang, Yilong

doi:10.1186/s13071-021-04971-4

Cited by 6 publications

(7 citation statements)

References 54 publications

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“…The comparison of intra- and interspecific ITS2/ COII variation in the present study revealed that ITS2 may be a more effective marker for differentiating species than COII , which is consistent with previous findings that an effective DNA marker should have a small intraspecific distance and a large interspecific distance [ 109 ]. The major downside of using COII for phylogenetic analysis is that COII may be unable to distinguish between closely related species [ 110 ]. Thus, additional research in the Cambodia–Laos border using nuclear and mtDNA sequencing is necessary to accurately identify species.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2022

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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

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Section: Discussionmentioning

confidence: 99%

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

et al. 2022

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“…Using overnight trapping with battery-operated Centers for Disease Control and Prevention (CDC) light traps (model 1012, John W. Hock Inc., USA) in cattle/pig pens or human rooms from 8:00 pm to 8:00 am, adult mosquitoes were collected in Pathoomphone County (Champasak Province) in 2017, as well as in Pak lay County (Xayabuli Province) and Yot Ou County (Phongsaly Province) in 2019 in accordance with our previous study [ 33 ]. The live adult mosquitoes were killed by freezing in a refrigerator.…”

Section: Methodsmentioning

confidence: 63%

“…The extraction of genomic DNA in individual mosquitoes was carried out following the manufacturer’s instructions (QIAamp ® DNA Mini Kit, Germany). The amplification for approximately 650 bp of the COII gene was carried out using primers LEU-F (5′-TCTAATATGGCAGATTAGTGCA-3′) and LYS-R (5′-ACTTGCTTTCAGTCATCTAATG-3′) [ 33 ]; a fragment containing codon 1014 of the An. sinensis vgsc gene (325-bp polymerase chain reaction [PCR] product of the kdr gene) was amplified using primers KDR-F (5′-TGCCACTCCGTGTGTTTAGA-3′) and KDR-R (5′-GAGCGATGATGATCCGAAAT-3′) [ 24 ].…”

Section: Methodsmentioning

confidence: 99%

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

Zhang

et al. 2022

Parasites Vectors

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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

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“…Fang et al studied the phylogenetic relationships of 18 species of the Hyrcanus group based on COI sequences [47]. Zhang et al compared ITS2 and COII fragments in constructing phylogenetic trees and suggested that ITS2 sequences could be a better molecular marker to distinguish closely related mosquito species [48]. However, the lengths of ITS2 sequence fragments are different, and thus it is difficult to compare them in the reconstruction of phylogenetic relationships.…”

Section: Discussionmentioning

confidence: 99%

Phylogenetic Analysis of Some Species of the Anopheles hyrcanus Group (Diptera: Culicidae) in China Based on Complete Mitochondrial Genomes

Dong¹,

Yuan

Yang³

et al. 2023

Genes

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Some species of the Hyrcanus group are vectors of malaria in China. However, the member species are difficult to identify accurately by morphology. The development of sequencing technologies offers the possibility of further studies based on the complete mitochondrial genome. In this study, samples of mosquitoes of the Hyrcanus group were collected in China between 1997 and 2015. The mitochondrial genomes of ten species of the Hyrcanus group were analyzed, including the structure and base composition, codon usage, secondary structure of tRNA, and base difference sites in protein coding regions. Phylogenetic analyses using maximum-likelihood and Bayesian inference were performed based on mitochondrial genes and complete mitochondrial genomes The mitochondrial genome of 10 Hyrcanus group members ranged from 15,403 bp to 15,475 bp, with an average 78.23% (A + T) content, comprising of 13 PCGs (protein coding genes), 22 tRNAs, and 2 rRNAs. Site differences between some closely related species in the PCGs were small. There were only 36 variable sites between Anopheles sinensis and Anopheles belenrae for a variation ratio of 0.32% in all PCGs. The pairwise interspecies distance based on 13 PCGs was low, with an average of 0.04. A phylogenetic tree constructed with the 13 PCGs was consistent with the known evolutionary relationships. Some phylogenetic trees constructed by single coding regions (such as COI or ND4) or combined coding regions (COI + ND2 + ND4 + ND5 or ND2 + ND4) were consistent with the phylogenetic tree constructed using the 13 PCGs. The phylogenetic trees constructed using some coding genes (COII, ND5, tRNAs, 12S rRNA, and 16S rRNA) differed from the phylogenetic tree constructed using PCGs. The difference in mitochondrial genome sequences between An. sinensis and An. belenrae was very small, corresponding to intraspecies difference. The combination of all 13 PCG sequences was demonstrated to be optimal for phylogenetic analysis in closely related species.

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Molecular phylogeny of the Anopheles hyrcanus group (Diptera: Culicidae) based on rDNA–ITS2 and mtDNA–COII

Cited by 6 publications

References 54 publications

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

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

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

Phylogenetic Analysis of Some Species of the Anopheles hyrcanus Group (Diptera: Culicidae) in China Based on Complete Mitochondrial Genomes

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