Prevalence and molecular characterization of Wolbachia in field-collected Aedes albopictus, Anopheles sinensis, Armigeres subalbatus, Culex pipiens and Cx. tritaeniorhynchus in China

Wolbachia are maternally transmitted intracellular bacteria that can naturally and artificially infect arthropods and nematodes. Recently, they were applied to control the spread of mosquito-borne pathogens by causing cytoplasmic incompatibility (CI) between germ cells of females and males. The ability of Wolbachia to induce CI is based on the prevalence and polymorphism of Wolbachia in natural populations of mosquitoes. In this study, we screened the natural infection level and diversity of Wolbachia in field-collected mosquitoes from 25 provinces of China based on partial sequence of Wolbachia surface protein (wsp) gene and multilocus sequence typing (MLST). Among the samples, 2489 mosquitoes were captured from 24 provinces between July and September, 2014 and the remaining 1025 mosquitoes were collected month-by-month in Yangzhou, Jiangsu province between September 2013 and August 2014. Our results showed that the presence of Wolbachia was observed in mosquitoes of Aedes albopictus (97.1%, 331/341), Armigeres subalbatus (95.8%, 481/502), Culex pipiens (87.0%, 1525/1752), Cx. tritaeniorhynchus (17.1%, 14/82), but not Anopheles sinensis (n = 88). Phylogenetic analysis indicated that high polymorphism of wsp and MLST loci was observed in Ae. albopictus mosquitoes, while no or low polymorphisms were in Ar. subalbatus and Cx. pipiens mosquitoes. A total of 12 unique mutations of deduced amino acid were identified in the wsp sequences obtained in this study, including four mutations in Wolbachia supergroup A and eight mutations in supergroup B. This study revealed the prevalence and polymorphism of Wolbachia in mosquitoes in large-scale regions of China and will provide some useful information when performing Wolbachia-based mosquito biocontrol strategies in China.

Identification and phylogenetic analysis of voltage-gated sodium channel haplotypes in the malaria vector Anopheles sinensis using a high-throughput amplicon sequencing approach

Background Anopheles sinensis is a dominant vector for malaria transmission in Asian countries. Voltage-gated sodium channel (VGSC) mutation-mediated knock-down resistance (kdr) has developed in many A. sinensis populations because of intensive and long-term use of pyrethroids. Our previous study showed that multiple mutations at position 1014 of the VGSC were heterogeneously distributed in A. sinensis populations across Sichuan, China. Methods To understand resistance genotypes at the haplotype level and reconstruct the phylogenetic relationship of VGSC haplotypes, a cost-effective next-generation sequencing (NGS)-based amplicon sequencing approach was established to clarify haplotypes containing codon 1014 of the VGSC gene from a total of 446 adults collected in 12 locations of Sichuan, China. Results Nineteen (19) haplotypes were identified, including 11 wild 1014L, 6 resistance 1014F, and 2 resistance 1014C haplotypes. We found that resistance haplotypes of A. sinensis VGSC were widely distributed at frequencies ranging from 3.67 to 92.61%. The frequencies of the 1014C haplotype in the southeast of Sichuan (Luzhou, Guangan, and Suining) were relatively higher than those in other sampling locations. Phylogenetic analyses support that kdr-type mutation at position 1014 is not singly originated and resistance 1014C haplotypes evolve from TTT-encoding 1014F. Conclusions A cost-effective next-generation sequencing (NGS)-based amplicon sequencing approach has been established in this study. The data revealed the patchy distribution of VGSC resistance haplotypes with overall high frequencies in Sichuan, China. Phylogenetic analyses support multiple origins and sequential evolution (1014L → 1014F → 1014C) for kdr-type mutations in A. sinensis. Graphical abstract

Polymorphism and Geographical Distribution of vgsc and ace-1 Gene in Anopheles Sinensis Field Populations in Guizhou Province, China

Background: Vector control has been a key strategy in malaria prevention and elimination for decades. However, insecticide resistance is becoming a serious threat to vector control. Anopheles sinensis is one of the important transmission vectors for malaria in Guizhou Province, China. However, little is known on insecticide resistance status and related mechanism. In this study, the diversity and frequency of the major insecticide resistance associated genes such as voltage-gated sodium channel (vgsc) and acetylcholinesterase-1 ( ace-1 ) genes that encoded the target proteins of Pyrethroids and OPs were investigated in field populations.Methods: Adult mosquitoes were collected from 12 sampling sites across Guizhou by lamp trapping. Female An. sinensis were identified by morphological and molecular identification. Genomic DNA was extracted to amplify vgsc and ace-1 gene fragments. PCR products were sequenced bi-directly. Mutations of vgsc gene at locus 1014 and that of ace-1 at locus 119 were analyzed using MEGA 7.1 software, and the frequencies of mutations were calculated respectively.Results: 5 kdr mutation alleles at the locus 1014 of vgsc gene as a result of three amino acid replacements ( namely 1014F/C/S) in 548 samples of 12 An. sinensis populations. The total frequency of kdr mutation alleles was 27.4%, of which the TTT/C (F) allele had a highest mutation frequency of 22.5%. The top three mutation genotypes were from XiShui, TongZhi and DeJiang populations collected in north Guizhou. There were three alleles at locus 119 in ace-1 gene with 49.47% of GGC/G, 0.17% of GGT/G and 50.36% of AGC/S. The 100% frequency of mutation genotypes (GS, SS) was found in CeHeng, LuoDian and SanDu populations gathered in southwest Guizhou.Conclusion: A diverse genetic mutations of vgsc and ace-1 genes are found in An. sinensis in Guizhou. There are a significant geographical heterogeneities of allele frequency among different populations in Guizhou. A high frequency of kdr mutation (>44 %) in north Guizhou. The 119S mutation of ace-1 gene is present at a high frequency in most An. sinensis populations in Guizhou, especially in the previously highly endemic malaria regions. These findings suggest continued monitoring of the genotypic diversity of insecticides resistance genes may assist to formulate a region-customized resistance management strategies.

Metagenomic sequencing reveals viral abundance and diversity in mosquitoes from the Shaanxi-Gansu-Ningxia region, China

Background Mosquitoes host and transmit numerous arthropod-borne viruses (arboviruses) that cause disease in both humans and animals. Effective surveillance of virome profiles in mosquitoes is vital to the prevention and control of mosquito-borne diseases in northwestern China, where epidemics occur frequently. Methods Mosquitoes were collected in the Shaanxi-Gansu-Ningxia region (Shaanxi Province, Gansu Province, and Ningxia Hui Autonomous Region) of China from June to August 2019. Morphological methods were used for taxonomic identification of mosquito species. High-throughput sequencing and metagenomic analysis were used to characterize mosquito viromes. Results A total of 22,959 mosquitoes were collected, including Culex pipiens (45.7%), Culex tritaeniorhynchus (40.6%), Anopheles sinensis (8.4%), Aedes (5.2%), and Armigeres subalbatus (0.1%). In total, 3,014,183 (0.95% of clean reads) viral sequences were identified and assigned to 116 viral species (including pathogens such as Japanese encephalitis virus and Getah virus) in 31 viral families, including Flaviviridae, Togaviridae, Phasmaviridae, Phenuiviridae, and some unclassified viruses. Mosquitoes collected in July (86 species in 26 families) showed greater viral diversity than those from June and August. Culex pipiens (69 species in 25 families) and Culex tritaeniorhynchus (73 species in 24 families) carried more viral species than Anopheles sinensis (50 species in 19 families) or Aedes (38 species in 20 families) mosquitoes. Conclusion Viral diversity and abundance were affected by mosquito species and collection time. The present study elucidates the virome compositions of various mosquito species in northwestern China, improving the understanding of virus transmission dynamics for comparison with those of disease outbreaks.

From malaria elimination to post‐elimination: a 10-year surveillance data study in Shanghai

Background The aim of this study was to investigate and analyse the characteristics of malaria in Shanghai from 2010 to 2019 and to provide suggestions for areas with a similar elimination process in China in order to prompt development of strategies and interventions in the post-elimination stage. Methods This was a cross-sectional study exploring the malaria characteristics during 2010–2019 in Shanghai, China. Malaria data from the Infectious Diseases Information Reporting Management System (IDIRMS) between 2010 and 2012 and data from the Parasitic Diseases Information Reporting Management System (PDIRMS) between 2013 and 2019 were combined for analysis in this study. Results From 2010 to 2019, a total of 436 malaria cases were reported in Shanghai. Among them, 415 (95.18%) were imported from abroad, 19 (4.36%) were domestically acquired from other provinces, 1 (0.23%) case was caused by blood transfusion, and 1 (0.23%) had a long incubation. Only Plasmodium vivax was found in domestically indigenous cases; Plasmodium falciparum accounted for the largest proportion of imported cases. Domestically acquired cases were only reported in 2010–2011 and 88% occurred in June to September; no significant seasonal difference was observed for imported cases over the 10 years. No local transmission has occurred in Shanghai since 2012. The median interval from fever onset to diagnosis was 3 days. Between 2010 and 2019, among 308 foci, 33 were classified as potential transmission and dispersed in suburb areas (Minhang, Baoshan, Jiading, Pudong, Jinshan, Songjiang, Qingpu, Fengxian, and Chongming). Only Anopheles sinensis was present and the proportion of Anopheles sinensis in different species of mosquitoes under surveillance in Shanghai decreased from 2011 to 2019. Conclusions Shanghai faces the challenge of malaria re-establishment caused by imported malaria in the post-elimination stage. Therefore, risk investigation and assessment should be carried out, and receptivity and susceptibility should be assessed for every point of focus. Training should be continued to strengthen facility staff capability, and multisectoral coordination and cooperation need to be conducted efficiently to maintain malaria elimination in Shanghai.

A survey of insecticide resistance-conferring mutations in multiple targets in Anopheles sinensis populations across Sichuan, China

Background Sichuan province is located in the southwest of China, and was previously a malaria-endemic region. Although no indigenous malaria case has been reported since 2011, the number of imported cases is on the rise. Insecticide-based vector control has played a central role in the prevention of malaria epidemics. However, the efficacy of this strategy is gravely challenged by the development of insecticide resistance. Regular monitoring of insecticide resistance is essential to inform evidence-based vector control. Unfortunately, almost no information is currently available on the status of insecticide resistance and associated mechanisms in Anopheles sinensis, the dominant malaria vector in Sichuan. In this study, efforts were invested in detecting the presence and frequency of insecticide resistance-associated mutations in three genes that encode target proteins of several classes of commonly used insecticides. Methods A total of 446 adults of An. sinensis, collected from 12 locations across Sichuan province of China, were inspected for resistance-conferring mutations in three genes that respectively encode acetylcholinesterase (AChE), voltage-gated sodium channel (VGSC), and GABA receptor (RDL) by DNA Sanger sequencing. Results The G119S mutation in AChE was detected at high frequencies (0.40–0.73). The predominant ace-1 genotype was GGC/AGC (119GS) heterozygotes. Diverse variations at codon 1014 were found in VGSC, leading to three different amino acid substitutions (L1014F/C/S). The 1014F was the predominant resistance allele and was distributed in all 12 populations at varying frequencies from 0.03 to 0.86. The A296S mutation in RDL was frequently present in Sichuan, with 296SS accounting for more than 80% of individuals in six of the 12 populations. Notably, in samples collected from Chengdu (DJY) and Deyang (DYMZ), almost 30% of individuals were found to be resistant homozygotes for all three targets. Conclusions Resistance-related mutations in three target proteins of the four main classes of insecticides were prevalent in most populations. This survey reveals a worrisome situation of multiple resistance genotypes in Sichuan malaria vector. The data strengthen the need for regular monitoring of insecticide resistance and establishing a region-customized vector intervention strategy.

Association of Anopheles sinensis average abundance and climate factors: Use of mosquito surveillance data in Goyang, Korea

Malaria is a vector-borne disease transmitted by Anopheline mosquitoes. In Korea, Plasmodium vivax malaria is an endemic disease and the main vector is Anopheles sinensis. Plasmodium vivax malaria is common in the northwestern part of South Korea, including in the city of Goyang in regions near the demilitarized zone. This study aimed to identify the best time-series model for predicting mosquito average abundance in Goyang, Korea. Mosquito data were obtained from the Mosquito Surveillance Program of the Goyang Ilsanseogu Public Health Center for the period 2008–2012. Black light traps were set up periodically in a park, a senior community center, and a village community center, public health center, drainage pumping station, cactus research center, restaurant near forest, in which many activities occur at night. In total, 9,512 female mosquitoes were collected at 12 permanent trapping sites during the mosquito season in the study period. Weekly An. sinensis average abundance was positively correlated with minimum grass temperature (r = 0.694, p < 0.001), precipitation (r = 0.326, p = 0.001). The results showed that seasonal autoregressive integrated moving average (SARIMA) (1,0,0)(0,0,1)21 with minimum grass temperature variable at time lag0 weeks and the precipitation variable at time lag1 weeks provided that best model of mosquito average abundance. The multivariate model accounted for about 54.1% of the mosquito average abundance variation. Time-series analysis of mosquito average abundance and climate factors provided basic information for predicting the occurrence of malaria mosquitoes.