Yajun Ma

The Hyrcanus Group of Anopheles (Anopheles) in China (Diptera: Culicidae): Species Discrimination and Phylogenetic Relationships Inferred by Ribosomal DNA Internal Transcribed Spacer 2 Sequences

Abstract The Hyrcanus group comprises many closely related species with wide distributions in the Oriental and Palaearctic regions. The sequences of the second internal transcribed spacer (ITS2) of ribosomal DNA were determined for 12 species in China—An. crawfordi, An. hyrcanus, An. junlianensis, An. kunmingensis, An. kweiyangensis, An. lesteri, An. liangshanensis, An. peditaeniatus, An. pullus, An. sinensis, and two unknown species within the group. The length of the ITS2 ranged from 436 bp in An. hyrcanus to 469 bp in An. crawfordi, with GC contents of 44.9–46.8%. Intraspecific variation was found in three species (An. junlianensis, An. liangshanensis, and An. pullus) at the level of 0.0–0.4%, whereas interspecific differences ranged from 1.6% between An. liangshanensis and An. kunmingensis to 50.8% between An. peditaeniatus and sp. 1. The ITS2 comparisons revealed two unknown species, verified the valid species status for An. kunmingensis, and found An. pullus in China. We agree that An. anthropophagus is a junior synonym of An. lesteri. The validation of An. junlianensis awaits recognition of the molecular identity of the entity identified as An. yatsushiroensis. The ITS2 divergences were used for inferring phylogenetic relationships among 12 species in China. The estimation revealed close relationships among An. liangshanesis, An. kunmingensis, An. kweiyangensis, An. lesteri, and An. sinensis. Our study emphasizes the need for the molecular identity of the species members in integrated studies in systematics, bionomics, and population genetics for the Hyrcanus group.

Population Structure of the Malaria Vector Anopheles sinensis (Diptera: Culicidae) in China: Two Gene Pools Inferred by Microsatellites

Background Anopheles sinensis is a competent malaria vector in China. An understanding of vector population structure is important to the vector-based malaria control programs. However, there is no adequate data of A. sinensis population genetics available yet. Methodology/Principal Findings This study used 5 microsatellite loci to estimate population genetic diversity, genetic differentiation and demographic history of A. sinensis from 14 representative localities in China. All 5 microsatellite loci were highly polymorphic across populations, with high allelic richness and heterozygosity. Hardy–Weinberg disequilibrium was found in 12 populations associated with heterozygote deficits, which was likely caused by the presence of null allele and the Wahlund effect. Bayesian clustering analysis revealed two gene pools, grouping samples into two population clusters; one includes six and the other includes eight populations. Out of 14 samples, six samples were mixed with individuals from both gene pools, indicating the coexistence of two genetic units in the areas sampled. The overall differentiation between two genetic pools was moderate (F ST = 0.156). Pairwise differentiation between populations were lower within clusters (F ST = 0.008–0.028 in cluster I and F ST = 0.004–0.048 in cluster II) than between clusters (F ST = 0.120–0.201). A reduced gene flow (Nm = 1–1.7) was detected between clusters. No evidence of isolation by distance was detected among populations neither within nor between the two clusters. There are differences in effective population size (Ne = 14.3-infinite) across sampled populations. Conclusions/Significance Two genetic pools with moderate genetic differentiation were identified in the A. sinensis populations in China. The population divergence was not correlated with geographic distance or barrier in the range. Variable effective population size and other demographic effects of historical population perturbations could be the factors affecting the population differentiation. The structured populations may limit the migration of genes under pressures/selections, such as insecticides and immune genes against malaria.

Mitochondrial genetic differentiation across populations of the malaria vector Anopheles lesteri from China (Diptera: Culicidae)

BackgroundAnopheles lesteri is a primary vector of Plasmodium spp. in central China. A complete understanding of vector population structure and the processes responsible for the differentiation is important to the vector-based malaria control programmes and for identifying heterogeneity in disease transmission as a result of discrete vector populations. There is no adequate An. lesteri population genetic data available.MethodsPolymorphism of sequence variations in mitochondrial COII and Cytb genes were assessed to explore the level of genetic variability and differentiation among six populations of An. lesteri from China.ResultsThere were 30 (4.37%) and 21 (5.33%) polymorphic sites for mtDNA-COII and Cytb gene, respectively. Totally 31 COII and 30 Cytb haplotypes were obtained. The range of FST values was from 0.101 to 0.655 by mtDNA-COII, and 0.029 to 0.231 by Cytb gene. The analysis of molecular variance (AMOVA) showed that the percentage of variation within populations (65.83%, 88.48%) was greater than that among populations (34.17%, 11.52%) using both genes. The Tajimas D and Fus Fs values were all negative, except Tajimas D values of YN and HNB populations, which suggest a large number of low-frequency mutations in populations and the populations were in expansion proceeding.ConclusionsLevels of genetic variation within An. lesteri populations were higher than among them. While these results may suggest considerable levels of gene flow, other explanations, such as the effect of historical population perturbations can also be hypothesized.

Diversity of bacteriome associated with Phlebotomus chinensis (Diptera: Psychodidae) sand flies in two wild populations from China

Sand fly Phlebotomus chinensis is a primary vector of transmission of visceral leishmaniasis in China. The sand flies have adapted to various ecological niches in distinct ecosystems. Characterization of the microbial structure and function will greatly facilitate the understanding of the sand fly ecology, which would provide critical information for developing intervention strategy for sand fly control. In this study we compared the bacterial composition between two populations of Ph. chinensis from Henan and Sichuan, China. The phylotypes were taxonomically assigned to 29 genera of 19 families in 9 classes of 5 phyla. The core bacteria include Pseudomonas and enterobacteria, both are shared in the sand flies in the two regions. Interestingly, the endosymbionts Wolbachia and Rickettsia were detected only in Henan, while the Rickettsiella and Diplorickettsia only in Sichuan. The intracellular bacteria Rickettsia, Rickettsiella and Diplorickettsia were reported for the first time in sand flies. The influence of sex and feeding status on the microbial structure was also detected in the two populations. The findings suggest that the ecological diversity of sand fly in Sichuan and Henan may contribute to shaping the structure of associated microbiota. The structural classification paves the way to function characterization of the sand fly associated microbiome.

Historical survey of the kdr mutations in the populations of Anopheles sinensis in China in 1996–2014

BackgroundAnopheles sinensis has become an important malaria vector in China. The long-term extensive utilization of pyrethroids for ITNs and IRS for mosquito control in the last three decades has resulted in the occurrence of resistant An. sinensis populations in many regions. Knockdown resistance (kdr), caused by point mutations in the VGSC gene, is one of the mechanisms that confer resistance to DDT and pyrethroids. Recently, several investigations revealed the kdr occurrence in some An. sinensis populations, however, no kdr data were available earlier than 2009. A survey tracking the dynamics of the kdr mutations in past decades would provide invaluable information to understand how the kdr alleles spread in mosquito populations temporally and spatially.MethodsA survey was conducted on the kdr alleles at condon 1014 of the VGSC gene and their distributions in 733 specimens of An. sinensis and 232 specimens of the other eight member species of the Anopheles hyrcanus group that were collected from 17 provinces in China in 1996–2014.ResultsA total of three kdr alleles, TTT (F), TTG (F) and TGT (C) were detected, and TGT (C) and TTT (F) were already present in the specimens from Jiangsu and Shandong as early as 1997. The TTT (F) was the most frequent mutant allele, and largely distributed in central China, namely Shandong, Jiangsu, Anhui, Henan, Shanghai, Jiangxi and Hubei. When data were analysed in three time intervals, 1996–2001, 2005–2009, 2010–2014, the prevalence of kdr alleles increased progressively over time in the populations in central China. In contrast, the kdr alleles were less frequent in the samples from other regions, especially in Yunnan and Hainan, despite the documented presence of pyrethroid resistant populations in those regions. Interestingly, no mutant alleles were detected in all 232 specimens of eight other species in the An. hyrcanus group.ConclusionThe survey revealed that the kdr occurrence and accumulation in the An. sinensis populations were more frequent in central China than in the other regions, suggesting that the kdr mutations may contribute significantly to the pyrethroid resistance in the mosquitoes in central China.

Genetic differentiation between sandfly populations of Phlebotomus chinensis and Phlebotomus sichuanensis (Diptera: Psychodidae) in China inferred by microsatellites

BackgroundPhlebotomus chinensis is a primary vector of visceral leishmaniasis; it occurs in various biotopes with a large geographical distribution, ranging from Yangtze River to northeast China. Phlebotomus sichuanensis, a species closely related to P. chinensis in high altitude regions, has a long term disputation on its taxonomic status. Both species occur in the current epidemic regions and are responsible for the transmission of leishmaniasis. Population genetic analysis will help to understand the population structure and infer the relationship for morphologically indistinguishable cryptic species. In this study, microsatellite markers were used for studying the genetic differentiation between P. chinensis and P. sichuanensis.MethodsSandflies were collected in 6 representative localities in China in 2005-2009. Ten microsatellite loci were used to estimate population genetic diversity. The intra-population genetic diversity, genetic differentiation and effective population size were estimated.ResultsAll 10 microsatellite loci were highly polymorphic across populations, with high allelic richness and heterozygosity. Hardy-Weinberg disequilibrium was found in 23 out of 60 (38.33%) comparisons associated with heterozygote deficits, which was likely caused by the presence of null allele and the Wahlund effect. Bayesian clustering analysis revealed three clusters. The cluster I included almost all specimens in the sample SCD collected at high altitude habitats in Sichuan. The other two clusters were shared by the remaining 5 populations, SCJ in Sichuan, GSZ in Gansu, SXL and SXX in Shaanxi and HNS in Henan. The diversity among these 5 populations was low (FST = -0.003-0.090) and no isolation by distance was detected. AMOVA analysis suggested that the variations were largely derived from individuals within populations and among individuals. Consistently, the analysis of ribosomal DNA second internal transcribed spacer (ITS2) sequence uncovered three types of variants, which corresponded with the three gene pools revealed by microsatellites.ConclusionsThe data suggested that the SCD population carried a distinct gene pool, which was differentiated from the other populations. The high altitude ecological habitats, distinctive ITS2 and herein divergence inferred by microsatellite loci support the species status of P. sichuanensis. The P. chinensis populations did not have a significant divergence from each another.

A video clip of the biting midge Culicoides anophelis ingesting blood from an engorged Anopheles mosquito in Hainan, China.

BackgroundBiting midges are hematophagus ectoparasites of insects, humans and other animals. Culicoides (Trithicoides) anophelis Edwards1922 is a predator of engorged mosquitoes.FindingsIn a field trip of wild mosquito collections, C. anophelis was found on two Anopheles mosquitoes. One mosquito with a midge clinging onto its abdomen was caught on video demonstrating the act of the midge taking blood from the engorged mosquito Anopheles vagus. The midge C. anophelis has a broad host range. Documented in the literature, the midge has been found in various mosquito species in the genera Anopheles, Culex, Aedes and Armigeres.ConclusionsA video clip was presented demonstrating a midge taking blood from an engorged mosquito. The host promiscuity of C. anophelis raises a concern about its potential as a mechanic or biological vector to spread viruses among mosquito populations.

Erratum to: Genetic diversity and population structure of the primary malaria vector Anopheles sinensis (Diptera: Culicidae) in China inferred by cox1 gene

Author details WHO Collaborating Center for Tropical Diseases, Key Laboratory of Parasite and Vector Biology, Ministry of Public Health, National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai 200025, China. Department of Tropical Infectious Disease, Second Military Medical University, Shanghai 200433, China. Second Military Medical University Press, Shanghai 200433, China.

A multiplex microfluidic loop-mediated isothermal amplification array for detection of malaria-related parasites and vectors

Malaria infection poses a great threaten to public health even nowadays. The conventional diagnosis tools of malaria parasites and vectors require systematic training for the observers accompanied by the low throughput. In this study, a new detection system, i.e., multiplex microfluidic loop-mediated isothermal amplification (mμLAMP) array, was developed to provide a convenient, rapid and economical detection system for malaria diagnosis. A microfluidic-based detection chip was designed and developed, targeting the conserved gene of four Anopheles and two Plasmodium species responsible for most of the malaria cases occurred in China. The DNA preparation of Anopheles and Plasmodium samples was realized by using a newly-developed DNA extraction method. For this mμLAMP array system, the detection limit was determined to be 1pg of targeting DNA with high sensitivity (>95%) and specificity (100%). Further, the accuracy of such mμLAMP analysis was evaluated by the analysis of 48 Anopheles mosquito samples, of which 30 were termed to be target Anopheles, displaying high consistency with that by morphological analysis. In conclusion, the mμLAMP detection system was proved to be a visible, sensitive, specific and high-throughput diagnostic tool. Considering the portable manipulation of such detection system, our studies shed light on its potential application of malaria surveillance on the spot.

Structural divergence of chromosomes between malaria vectors Anopheles lesteri and Anopheles sinensis

BackgroundAnopheles lesteri and Anopheles sinensis are two major malaria vectors in China and Southeast Asia. They are dramatically different in terms of geographical distribution, host preference, resting habitats, and other traits associated with ecological adaptation and malaria transmission. Both species belong to the Anopheles hyrcanus group, but the extent of genetic differences between them is not well understood. To provide an effective way to differentiate between species and to find useful markers for population genetics studies, we performed a comparative cytogenetic analysis of these two malaria vectors.ResultsPresented here is a standard cytogenetic map for An. lesteri, and a comparative analysis of chromosome structure and gene order between An. lesteri and An. sinensis. Our results demonstrate that much of the gene order on chromosomes X and 2 was reshuffled between the two species. However, the banding pattern and the gene order on chromosome 3 appeared to be conserved. We also found two new polymorphic inversions, 2Lc and 3Rb, in An. lesteri, and we mapped the breakpoints of these two inversions on polytene chromosomes.ConclusionsOur results demonstrate the extent of structural divergence of chromosomes between An. lesteri and An. sinensis, and provide a new taxonomic cytogenetic tool to distinguish between these two species. Polymorphic inversions of An. lesteri could serve as markers for studies of the population structure and ecological adaptations of this major malaria vector.