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Molecular phylogeny of Anopheles hyrcanus group members based on ITS2 rDNA.

ABSTRACT: BACKGROUND:The Anopheles hyrcanus group includes 25 species, and is widely distributed in the Oriental and Palaearctic regions. Several species within this group are vectors of malaria, lymphatic filariasis and Japanese encephalitis. It is difficult or impossible to identify cryptic species based on their morphological characteristics, with some closely related species of the Hyrcanus Group have similar adult morphological characteristics. Thus, their molecular identification has been an important complementary method to traditional morphological taxonomy. METHODS:We used 461 ribosomal DNA (rDNA) internal transcribed spacer 2 (ITS2) sequences relating to 19 species to reconstruct the molecular phylogeny of the Hyrcanus Group across its range. In addition, we compared the performance of rDNA ITS2 to that of mitochondrial DNA (mtDNA) cytochrome c oxidase subunit 1 gene (cox1) to assess the genetic divergence of Hyrcanus Group sibling species. RESULTS:Based on Kimura's 2-parameter (K2P) distance model, the average conspecific ITS2 divergence was 0.003, whereas sequence divergence between species averaged 0.480. Average ITS2 sequence divergences were almost 160 times higher among the Hyrcanus Group members than within each species. Two sets of sibling species, An. lesteri Baisas & Hu, 1936 and An. paraliae Sandosham, 1959; and An. sinensis Wiedemann, 1828, An. belenrae Rueda, 2005, and An. kleini Rueda, 2005, were resolved by ITS2. Each of these species was represented as an independent lineage in the phylogenetic tree. Results suggest that An. pseudopictus Grassi, 1899 and An. hyrcanus (Pallas, 1771) are most likely a single species. We uncovered two new ITS2 lineages that require further study before resolving their true taxonomic status, and designed a diagnostic polymerase chain reaction (PCR) assay to distinguish five morphologically similar species. CONCLUSIONS:Nuclear and mitochondrial genes generally provided consistent results for subgroup division. Compared to cox1, ITS2 is a more reliable tool for studying phylogenetic relationships among closely related mosquito taxa. Based on species-specific differences in ITS2 sequences, the multiplex PCR assay developed here can be used to improve the efficiency of vector identification. Thus, this research will promote the progress of malaria vector surveillance in both epidemic and non-epidemic areas of South and East Asia.

SUBMITTER: Fang Y

PROVIDER: S-EPMC5590201 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Molecular phylogeny of Anopheles hyrcanus group members based on ITS2 rDNA.

Fang Yuan Y Shi Wen-Qi WQ Zhang Yi Y

Parasites & vectors 20170907 1

<h4>Background</h4>The Anopheles hyrcanus group includes 25 species, and is widely distributed in the Oriental and Palaearctic regions. Several species within this group are vectors of malaria, lymphatic filariasis and Japanese encephalitis. It is difficult or impossible to identify cryptic species based on their morphological characteristics, with some closely related species of the Hyrcanus Group have similar adult morphological characteristics. Thus, their molecular identification has been an ...[more]

PMID: 28882174

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Project description:BackgroundThe 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.MethodsBased 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.ResultsAverage 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.ConclusionsThe 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.

Project description:BackgroundThe Anopheles hyrcanus group, which includes at least 25 species, is widely distributed in the Oriental and Palearctic regions. Some group members have been incriminated as vectors of malaria and other mosquito-borne diseases. It is difficult to identify Hyrcanus Group members by morphological features. Thus, molecular phylogeny has been proposed as an important complementary method to traditional morphological taxonomy.MethodsBased on the GenBank database and our original study data, we used 466 mitochondrial DNA COI sequences belonging to 18 species to reconstruct the molecular phylogeny of the Hyrcanus Group across its worldwide geographic range.ResultsThe results are as follows. 1) The average conspecific K2P divergence was 0.008 (range 0.002-0.017), whereas sequence divergence between congroup species averaged 0.064 (range 0.026-0.108). 2) The topology of COI tree of the Hyrcanus Group was generally consistent with classical morphological taxonomy in terms of species classification, but disagreed in subgroup division. In the COI tree, the group was divided into at least three main clusters. The first cluster contained An. nimpe; the second was composed of the Nigerrimus Subgroup and An. argyropus; and the third cluster was comprised of the Lesteri Subgroup and other unassociated species. 3) Phylogenetic analysis of COI indicated that ancient hybridizations probably occurred among the three closely related species, An. sinensis, An. belenrae, and An. kleini. 4) The results supported An. paraliae as a probable synonym of An. lesteri, and it was possible that An. pseudopictus and An. hyrcanus were the same species, as evident from their extremely low interspecific genetic divergence (0.020 and 0.007, respectively) and their phylogenetic positions.ConclusionsIn summary, we reconstructed the molecular phylogeny and analysed genetic divergence of the Hyrcanus Group using mitochondrial COI sequences. Our results suggest that in the future of malaria surveillance, we should not only pay much attention to those known vectors of malaria, but also their closely related species.

Project description:BACKGROUND:Species of the Anopheles hyrcanus group are widely distributed in Palearctic and Oriental regions and some of them are important malaria vectors. The cryptic species of An. hyrcanus group was almost impossible to identify based only on their morphology. The phylogenetic relationship of An. hyrcanus group was also not clear. METHODS:Five members of An. hyrcanus group were identified by rDNA ITS2 sequencing as An. yatsushiroensis, An. belenrae, An. kleini, An. lesteri and An. sineroides. The mitochondrial genome fragments were sequenced and annotated using the mitochondrial genome of An. sinensis as reference. Based on the four segments and Joint Data sequences of these species, and other four anopheline species downloaded from GenBank, intraspecific as well as interspecific genetic distances were calculated and the phylogenetic trees were reconstructed by the methods of neighbor joining, maximum parsimony, minimum evolution and maximum likelihood. FINDINGS:Four parts of mitochondrial genomes, which were partial fragments COI + tRNA + COII (F5), ATP6 + COIII(F7 + F8), ND1(F19) and lrRNA (F21), were obtained. All fragments were connected as one sequence (referred as Joint Data), which had a total length of 3393 bp. All fragment sequences were highly conservative within species, with the maximum p distance (0.026) calculated by F19 of An. belenrae. The pairwise interspecific p distance calculated by each fragment showed minor or even no difference among An. sinensis, An. kleini and An. belenrae. However, interspecific p distances calculated by the Joint Data sequence ranged from 0.004 (An. belenrae vs An. kleini) to 0.089 (An. sineroides vs An. minimus), and the p distances of the six members of An. hyrcanus group were all less than 0.029. The phylogenetic tree showed two major clades: all subgenus Anopheles species (including six members of An. hyrcanus group, An. atroparvus and An. quadrimaculatus A) and subgenus Cellia (including An. dirus and An. minimus). The An. hyrcanus group was divided into two clusters as ((An. lesteri, An. sineroides) An. yatsushiroensis) and ((An. belenrae, An. sinensis) An. kleini)). CONCLUSIONS:The An. hyrcanus group in this study could be divided into two clusters, in one of which An. belenrae, An. sinensis and An. kleini were most closely related. More molecular markers would make greater contribution to phylogenetic analysis.

Project description:BackgroundMembers of the Anopheles hyrcanus group have been incriminated as important malaria vectors. This study aims to identify the species and explore the insecticide susceptibility profile within the Anopheles hyrcanus group in Ubon Ratchathani Province, northeastern Thailand where increasing numbers of malaria cases were reported in 2014.MethodsBetween 2013 and 2015, five rounds of mosquito collections were conducted using human landing and cattle bait techniques during both the rainy and dry seasons. Anopheles mosquitoes were morphologically identified and their insecticide susceptibility status was investigated. Synergist bioassays were carried out with An. hyrcanus (s.l.) due to their resistance to all insecticides. An ITS2-PCR assay was conducted to identify to species the Hyrcanus group specimens.ResultsOut of 10,361 Anopheles females collected, representing 18 taxa in 2 subgenera, 71.8% were morphologically identified as belonging to the Hyrcanus Group (subgenus Anopheles), followed by An. barbirostris group (7.9%), An. nivipes (6.5%), An. philippinensis (5.9%) and the other 14 Anopheles species. Specimens of the Hyrcanus Group were more prevalent during the rainy season and were found to be highly zoophilic. Anopheles hyrcanus (s.l.) was active throughout the night, with an early peak of activity between 18:00 h and 21:00 h. ITS2-PCR assay conducted on 603 DNA samples from specimens within the Hyrcanus Group showed the presence of five sisters species. Anopheles peditaeniatus was the most abundant species (90.5%, n = 546), followed by An. nitidus (4.5%, n = 27), An. nigerrimus (4.3%, n = 26), An. argyropus (0.5%, n = 3), and An. sinensis (0.2%, n = 1). All An. hyrcanus (s.l.) specimens that were found resistant to insecticides (deltamethrin 0.05%, permethrin 0.75% and DDT 4% and synergist tests) belonged to An. peditaeniatus. The degree of resistance in An. peditaeniatus to each of these three insecticides was approximately 50%. Addition of PBO (Piperonyl butoxide), but not DEF (S.S.S-tributyl phosphotritioate), seemed to restore susceptibility, indicating a potential role of oxidases as a detoxifying enzyme resistance mechanism.ConclusionsA better understanding of mosquito diversity related to host preference, biting activity and insecticide resistance status will facilitate the implementation of locally adapted vector control strategies.

Project description:BackgroundPlasmodium vivax is transmitted by members of the Anopheles Hyrcanus Group that includes six species in the Republic of Korea: Anopheles sinensis sensu stricto (s.s.), Anopheles pullus, Anopheles kleini, Anopheles belenrae, Anopheles lesteri, and Anopheles sineroides. Individual Anopheles species within the Hyrcanus Group demonstrate differences in their geographical distributions, vector competence and insecticide resistance, making it crucial for accurate species identification. Conventional species identification conducted using individual genotyping (or barcoding) based on species-specific molecular markers requires extensive time commitment and financial resources.ResultsA population-based quantitative sequencing (QS) protocol developed in this study provided a rapid estimate of species composition ratios among pooled mosquitoes as a cost-effective alternative to individual genotyping. This can be accomplished by using species- or group-specific nucleotide sequences of the mitochondrial cytochrome C oxidase subunit I (COI) and the ribosomal RNA internal transcribed spacer 2 (ITS2) region as species identification alleles in a two-step prediction protocol. Standard genomic DNA fragments of COI and ITS2 genes were amplified from each Anopheles species using group-specific universal primer sets. Following sequencing of the COI or ITS2 amplicons generated from sets of standard DNA mixtures, equations were generated via linear regression to predict species-specific nucleotide sequence frequencies at different positions. Species composition ratios between An. sineroides, An. pullus and An. lesteri were estimated from QS of the COI amplicons based on the mC.260A, mC.122C and mC.525C alleles at the first step, followed by the prediction of species composition ratios between An. sinensis, An. kleini and An. belenrae based on QS of the ITS2 amplicons using the rI.370G and rI.389T alleles. The COI copy number was not significantly different between species, suggesting the reliability of COI-based prediction. In contrast, ITS2 showed a slightly but significantly higher copy number in An. belenrae, requiring an adjustment of its predicted composition ratio. A blind test proved that predicted species composition ratios either from pooled DNA specimens or pooled mosquito specimens were not statistically different from the actual values, demonstrating that the QS-based prediction is accurate and reliable.ConclusionsThis two-step prediction protocol will facilitate rapid estimation of the species composition ratios in field-collected Anopheles Hyrcanus Group populations and is particularly useful for studying the vector ecology of Anopheles population and epidemiology of malaria.

Project description: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, suggesting that the species was in the process of differentiation. 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 Anopheles hyrcanus group members based on ITS2 rDNA.

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Molecular phylogeny of Anopheles hyrcanus group members based on ITS2 rDNA.

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