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A novel strategy for screening mutations in the voltage-gated sodium channel gene of Aedes albopictus based on multiplex PCR-mass spectrometry minisequencing technology

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Infectious Diseases of Poverty 2023年第4期12卷 43-53页

作者： Qunzheng Mu Xin Zhao Fengfeng Li Wenyu Li Xinxin Zhou Xinchang Lun Yiguan Wang Dongdong Hua Qiyong Liu Di Xiao Fengxia Meng National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases National Institute for Communicable Disease Control and PreventionChinese Center for Disease Control and PreventionBeijing102206People’s Republic of China Weifang No.2 People’s Hospital Weifang261000ShandongPeople’s Republic of China Weifang Medical College Weifang261000ShandongPeople’s Republic of China Beijing Daxing District Center for Disease Control and Prevention Beijing102600BeijingPeople’s Republic of China

Background The current prevention and control strategy forAedes albopictus heavily relies on comprehensive management, such as environmental management and chemical control. However, the wide application of pyrethroids has facilitated the development of insecticide resistance, primarily via mutations in the voltage-gated sodium channel (VGSC) gene. This study aims to develop a novel strategy for detecting mutations in the VGSC gene inAe. albopictus using multiplex PCR-mass spectrometry (MPCR-MS) minisequencing technology.Methods We established a new strategy for detecting mutations in the VGSC gene inAe. albopictus using MPCR-MS minisequencing technology. MPCR amplification and mass probe extension (MPE) were first used, followed by single nucleotide polymorphism (SNP) typing mass spectrometry, which allows the simultaneous detection of multiple mutation sites of the VGSC gene in 96 samples ofAe. albopictus. A total of 70 wild-collectedAe. albopictus were used to evaluate the performance of the method by comparing it with other methods.Results Three target sites (1016, 1532, 1534) in the VGSC gene can be detected simultaneously by double PCR amplification combined with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, achieving a detection limit of 20 fg/μl. We applied this method to 70 wild-collectedAe. albopictus, and the obtained genotypes were consistent with the routine sequencing results, suggesting the accuracy of our method.Conclusions MPCR-MS minisequencing technology provides a sensitive and high-throughput approach toAe. albopictus VGSC gene mutation screening. Compared with conventional sequencing, this method is economical and time-saving. It is of great value for insecticide resistance surveillance in areas with a high risk of vector-borne disease.

关键词： Aedes albopictus VGSC gene Mutation Single nucleotide polymorphisms Multiplex polymerase chain reaction-mass spectrometry mini-sequencing

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Genetic structure of Aedes albopictus(Diptera:Culicidae)populations in China and relationship with the knockdown resistance mutations

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Infectious Diseases of Poverty 2023年第3期12卷 31-40页

作者： Wenqi Shan Hao Yuan Hanming Chen Haowei Dong Qiuming Zhou Feng Tao Jie Bai Huiying Chen Yajun Ma Heng Peng Department of Naval Medicine Naval Medical UniversityShanghai 200433China Department of Medical Microbiology and Parasitology College of Basic Medical SciencesNaval Medical UniversityShanghai 200433China

Background Mosquito control is needed to prevent dengue fever, which is mainly spread byAedes albopictus in China. Application of insecticides is one of the main mosquito control methods;however, this approach can fail due to the knockdown resistance (kdr) gene mutation that causes decreased sensitivity to insecticides inAe. albopictus. Thekdr mutation patterns among different regions in China differ significantly. However, the underlying mechanism and factors that influencekdr mutation remain unclear. To explore the potential influence of genetic background on the development of insecticide resistance inAe. albopictus, we analyzed the genetic structure ofAe. albopictus populations in China and its correlation with majorkdr mutations.Methods We collectedAe. albopictus from 17 sites in 11 provinces (municipalities) across China from 2016 to 2021 and extracted the genomic DNA from individual adult mosquitoes. We selected eight microsatellite loci for genotyping, and based on microsatellite scores, we estimated intraspecific genetic diversity, population structure, and effective population size. The association between the intrapopulation genetic variation and F1534 mutation rate was evaluated by the Pearson correlation coefficient.Results Based on variation analysis of the microsatellite loci of 453 mosquitoes representing 17 populations throughout China, more than 90% of the variation occurred within individuals, whereas only about 9% of the variation occurred among populations, indicating that field populations ofAe. albopictus are highly polymorphic. The northern populations tended to belong to gene pool I (BJFT 60.4%, SXXA 58.4%, SDJN 56.1%, SXYC 46.8%), the eastern populations tended to belong to pool III (SH 49.5%, JZHZ 48.1%), and the southern populations tended to belong to three different gene pools. Moreover, we observed that the greater the fixation index (FST), the lower the wild-type frequency of F1534 ofVSGC.Conclusions The degree of genetic differentiation amongAe. albopictus populations in China was

关键词： Aedes albopictus Population structure Microsatellite Knockdown resistance China

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Climate change and Aedes albopictus risks in China:current impact and future projection

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Infectious Diseases of Poverty 2023年第2期12卷 123-124页

作者： Hongmei Liu Xiaodan Huang Xiuxia Guo Peng Cheng Haifang Wang Lijuan Liu Chuanhui Zang Chongxing Zhang Xuejun Wang Guofa Zhou Maoqing Gong Shandong Institute of Parasitic Diseases Shandong First Medical University and Shandong Academy of Medical Sciences Jining People’s Republic of China Program in Public Health University of California Irvine USA Shandong Center for Disease Control and Prevention Jinan China

Background Future distribution of dengue risk is usually predicted based on predicted climate changes using general circulation models(GCMs).However,it is difficult to validate the GCM results and assess the uncertainty of the predictions.The observed changes in climate may be very different from the GCM results.We aim to utilize trends in observed climate dynamics to predict future risks of Aedes albopictus in China.Methods We collected Ae.albopictus surveillance data and observed climate records from 80 meteorological stations from 1970 to 2021.We analyzed the trends in climate change in China and made predictions on future climate for the years 2050 and 2080 based on trend analyses.We analyzed the relationship between climatic variables and the prevalence of Ae.albopictus in different months/seasons.We built a classification tree model(based on the average of 999 runs of classification and regression tree analyses)to predict the monthly/seasonal Ae.albopictus distribution based on the average climate from 1970 to 2000 and assessed the contributions of different climatic variables to the Ae.albopictus distribution.Using these models,we projected the future distributions of Ae.albopictus for 2050 and 2080.Results The study included Ae.albopictus surveillance from 259 sites in China found that winter to early spring(November–February)temperatures were strongly correlated with Ae.albopictus prevalence(prediction accuracy ranges 93.0–98.8%)—the higher the temperature the higher the prevalence,while precipitation in summer(June–September)was important predictor for Ae.albopictus prevalence.The machine learning tree models predicted the current prevalence of Ae.albopictus with high levels of agreement(accuracy>90%and Kappa agreement>80%for all 12 months).Overall,winter temperature contributed the most to Ae.albopictus distribution,followed by summer precipitation.An increase in temperature was observed from 1970 to 2021 in most places in China,and annual change rates varied substantially from-0.22℃/year to 0.58℃/year

关键词： Aedes albopictus Observed climate change Projected future climate Observed risks distribution Projected future risk distribution

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Modelling the probability of presence of aedes aegypti and Aedes albopictus in Iran until 2070

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Asian Pacific Journal of Tropical Medicine 2023年第1期16卷 16-25页

作者： Mohammad Mehdi Sedaghat Faramarz Bozorg Omid Mohammad Karimi Sajjad Haghi Ahmad Ali Hanafi-Bojd Department of Vector Biology&Control School of Public HealthTehran University of Medical SciencesTehranIran Department of GIS Faculty of Geodesy and Geomatics EngineeringK.N.Toosi University of TechnologyTehranIran Zoonoses Research Center Tehran University of Medical SciencesTehranIran

Objective:To determine the suitable ecological habitats of aedes(Ae.)aegypti and Ae.albopictus in Iran due to climate change by the 2070s.Methods:All data relating to the spatial distribution of Ae.aegypti and Ae.albopictus worldwide,which indicated the geographical coordinates of the collection sites of these mosquitoes,were extracted from online scientific websites and entered into an Excel file.The effect of climatic and environmental variables on these mosquitoes was evaluated using the MaxEnt model in the current and future climatic conditions in the 2030s,2050s,and 2070s.Results:The most suitable areas for the establishment of Ae.aegypti are located in the southern and northern coastal areas of Iran,based on the model outputs.The modelling result for suitable ecological niches of Ae.albopictus shows that in the current climatic conditions,the southern half of Iran from east to west,and parts of the northern coasts are prone to the presence of this species.In the future,some regions,such as Gilan and Golestan provinces,will have more potential to exist/establish Ae.albopictus.Also,according to the different climate change scenarios,suitable habitats for this species will gradually change to the northwest and west of the country.The temperature of the wettest season of the year(Bio8)and average annual temperature(Bio1)were the most effective factors in predicting the model for Ae.aegypti and Ae.albopictus,respectively.Conclusions:It is required to focus on entomological studies using different collection methods in the vulnerable areas of Iran.The future modelling results can also be used for long-term planning to prevent the entry and establishment of these invasive aedes vectors in the country.

关键词： aedes aegypti Aedes albopictus Dengue fever Chikungunya Ecological Niche Modeling Climate change

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Alternative splicing patterns of doublesex reveal a missing link between Nix and doublesex in the sex determination cascade of Aedes albopictus

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Insect Science 2021年第6期28卷 1601-1620页

作者： Binbin Jin Yijie Zhao Yunqiao Dong Peiwen Liu Yan Sun Xiaocong Li Xin Zhang Xiao-Guang Chen Jinbao Gu Department of Pathogen Biology Guangdong Provincial Key Laboratory of Tropical Disease ResearchSchool of Public HealthSouthern Medical UniversityGuangzhou510515China Reproductive Medical Centre of Guangdong Women and Children Hospital Guangzhou511442China

Sexual development in insects is regulated by a complicated hierarchical cascade of sex determination.The primary signals are diverse,whereas the central nexus doublesex(dsx)gene is relatively conserved within the pathway.aedes(Stegomyia)albopictus is an important vector with an extensive worldwide distribution.We previously reported that Ae.albopictus dsx(Aalbdsx)yields one male-(Aalbdsx^(M))and three female-specific isoforms(Aalbdsx^(F1-3));however,the spatiotemporal expression profiles and mechanisms regulating sex-specific alternative splicing require further investigation.In this study,we demonstrated that the Aalbdsx^(M) messenger RNA(mRNA)represents the default pattern when analyzed in human foreskin fibroblasts and HeLa cells.We combined reverse transcription polymerase chain reaction with RNA immunoprecipitation using specific antibodies against tagged Ae.albopictus male-determining factor AalNix and confirmed that AalNix indirectly regulates dsx pre-mRNA and regulates its alternative splicing.During the early embryo stage(0-2 and 4-8 h),maternal dsx^(F) and default splicing dsx^(M) were detected in both sexes;the expression of dsx^(M) then decreased until sufficient AalNix transcripts accumulated in male embryos at 20-24 h.These findings suggest that one or more potential dsx splicing enhancers can shift dsx^(M) to dsx^(F) in both sexes;however,the presence of Nix influences the function of this unknown splicing enhancer and ultimately leads to the formation of dsx^(M) in males.Finally,our results provide important insight into the regulatory mechanism of dsx alternative splicing in the mosquito.

关键词： Aedes albopictus alternative splicing Doublesex\mosquito control sex determination

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Integrated analysis of miRNAs and transcriptomes in Aedes albopictus midgut reveals the differential expression profiles of immune-related genes during dengue virus serotype-2 infection

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Insect Science 2016年第3期23卷 377-385页

作者： Yan-Xia Liu Fen-Xiang Li Zhuan-Zhuan Liu Zhi-Rong Jia Yan-He Zhou Hao Zhang Hui Yan Xian-Qiang Zhou Xiao-Guang Chen Key Laboratory of Prevention and Control of Emerging Infectious Diseases of Guangdong Higher Education Institutes Department ofPathogen Biology School of Public Health and Tropical Medicine Southern Medical University Guangzhou Laboratory of EmergingInfectious Diseases and Division of Laboratory Medicine Zhujiang Hospital Southern Medical University Guangzhou BGI-Shenzhen Shenzhen Guangdong Province China

Mosquito microRNAs （miRNAs） are involved in host-virus interaction, and have been reported to be altered by dengue virus （DENV） infection in Aedes albopictus （Diptera： Culicidae）. However, little is known about the molecular mechanisms of Aedes albopictus midgut--the first organ to interact with DENV--involved in its resistance to DENV. Here we used high-throughput sequencing to characterize miRNA and messenger RNA （mRNA） expression patterns in Aedes albopictus midgut in response to dengue virus serotype 2. A total of three miRNAs and 777 mRNAs were identified to be differentially expressed upon DENV infection. For the mRNAs, we identified 198 immune-related genes and 31 of them were differentially expressed. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses also showed that the differentially expressed immune-related genes were involved in immune response. Then the differential expression patterns of six immune-related genes and three miRNAs were confirmed by real-time reverse transcription polymerase chain reaction. Furthermore, seven known miRNA-mRNA interaction pairs were identified by aligning our two datasets. These analyses of miRNA and mRNA transcriptomes provide valuable information for uncovering the DENV response genes and provide a basis for future study of the resistance mechanisms in Aedes albopictus midgut.

关键词： Aedes albopictus DENV host-pathogen interaction midgut miRNA mRNA

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Effects of Overwintering on the Survival and Vector Competence of Aedes albopictus in the Urban Life Cycle of Dengue Virus in Guangzhou,China

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Virologica Sinica 2021年第4期36卷 755-761页

作者： Yue Chen Ronghua Chen Jianrong Gao Chunyuan Li Jun Liu Zhijian Zhou Ruiwen Ren Center for Disease Prevention and Control of the Southern Theatre Command of the PLA Guangzhou510507China Guangdong Arbovirus Diseases Emergency Technology Research Center Guangzhou510507China

The Pearl River Delta,where Aedes albopictus(Ae.albopictus)is the only vector for dengue transmission,has exhibited one of the highest dengue burdens in southern China in recent decades.However,whether dengue virus(DENV)can overwinter in Ae.albopictus in the Pearl River Delta has not been determined to date.In this study,300 field-derived Ae.albopictus mosquitoes from Guangzhou that were infected with the predominant endemic DENV-1 strain were investigated under simulated urban balcony environment from October 16,2016,to June 16,2017.The vertical transmission of DENV in the infected overwintering Ae.albopictus was analyzed.The DENV infected overwintering mosquitoes were evaluated for viral load at nine-time points using reverse transcription-quantitative PCR.The vector competence of the infected overwintering Ae.albopictus was also investigated by using suckling mice.Adult mosquitoes and larvae were found during the observation period.The vertical transmission of DENV-1 was documented.The DENV-1-positive rates between overwintering males and females had no difference.The proportion of DENV-1-positive overwintering mosquitoes decreased over time and had no difference beyond three months after the experiment.Overwintering mosquitoes can spread DENV-1 to hosts.No engorged mosquitoes at an ambient temperature below 15℃were observed.The ratio of engorged mosquitoes was positively correlated with the ambient temperature ranging from 15 to 30℃.Our results demonstrated that DENV can overwinter in Ae.albopictus in the Pearl River Delta,Ae.albopictus is the competent vector for DENV,and maintain autochthonous dengue outbreaks in the Pearl River Delta through vertical transmission.

关键词： Dengue virus(DENV) Aedes albopictus Overwintering Vertical transmission Subtropical region

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Mosquito densovirus signifcantly reduces the vector susceptibility to dengue virus serotype 2 in Aedes albopictus mosquitoes(Diptera:Culicidae)

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Infectious Diseases of Poverty 2023年第3期12卷 91-92页

作者： Ling Kong Jie Xiao Lu Yang Yuan Sui Duoquan Wang Shaoqiang Chen Peiwen Liu Xiao-Guang Chen Jinbao Gu Guangdong Provincial Key Laboratory of Tropical Disease Research Depart-ment of Pathogen BiologySchool of Public HealthSouthern Medical Uni-versityGuangzhou 510515GuangdongChina Brown School Washington UniversitySt.LouisMO 63130USA National Institute of Parasitic Diseases Chinese Center for Disease Control and PreventionShanghai 200025China Shenzhen Aiming Pest Control Operation Service Company Limited Shenz-henGuangdongChina

Background Dengue virus(DENV)is a major public health threat,with Aedes albopictus being the confrmed vector responsible for dengue epidemics in Guangzhou,China.Mosquito densoviruses(MDVs)are pathogenic mosquitospecifc viruses,and a novel MDV was previously isolated from Ae.albopictus in Guangzhou.This study aims to determine the prevalence of MDVs in wild Ae.albopictus populations and investigate their potential interactions with DENV and impact on vector susceptibility for DENV.Methods The prevalence of MDV in wild mosquitoes in China was investigated using open access sequencing data and PCR detection in Ae.albopictus in Guangzhou.The viral infection rate and titers in MDV-persistent C6/36 cells were evaluated at 12,24,48,72,96,and 120 h post infection(hpi)by indirect immunofuorescence assay(IFA)and real time quantitative PCR(RT-qPCR).The midgut infection rate(MIR),dissemination rate(DR),and salivary gland infection rate(SGIR)in various tissues of MDV-infected mosquitoes were detected and quantifed at 0,5,10,and 15 days post infection(dpi)by RT-PCR and RT-qPCR.The chi-square test evaluated dengue virus serotype 2(DENV-2)and aedes aegypti densovirus(AaeDV)infection rates and related indices in mosquitoes,while Tukey’s LSD and t-tests compared viral titers in C6/36 cells and tissues over time.Results The results revealed a relatively wide distribution of MDVs in aedes,Culex,and Anopheles mosquitoes in China and an over 68%positive rate.In vitro,signifcant reductions in DENV-2 titers in supernatant at 120 hpi,and an appar‑ent decrease in DENV-2-positive cells at 96 and 120 hpi were observed.In vivo,DENV-2 in the ovaries and salivary glands was frst detected at 10 dpi in both monoinfected and superinfected Ae.albopictus females,while MDV super‑infection with DENV-2 suppressed the salivary gland infection rate at 15 dpi.DENV-2 titer in the ovary and salivary glands of Ae.albopictus was reduced in superinfected mosquitoes at 15 dpi.Conclusions MDVs is widespread in natural mosquito populations,and replication of DEN

关键词： DENV-2 Mosquito densovirus Superinfection Aedes albopictus Vector susceptibility

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Isolation, identification, and phylogenetic analysis of a dengue virus strain from Aedes albopictus collected in Mawei town in Guizhou Province, China

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Chinese Medical Journal 2004年第12期17卷 1847-1849页

作者：左丽舒莉萍 Department of Immunology Guiyang Medical College Guiyang 550004 China

Dengue virus (DEN), a single-stranded RNA virus of the family Flaviviridae, is transmitted from one infected person or animal to another by the mosquitos Aedes albopictus and aedes aegypti. There are four serotypes of the dengue virus (serotypes 1-4). The virus is responsible for dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). In the recent years, the incidence rate of DF/DHF has been increasing around the world, particularly in tropical and subtropical zones. Dengue is the most significant arthropod-borne viral disease affecting public health.1

关键词： Aedes albopictus dengue virus

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Correlation between adult pyrethroid resistance and knockdown resistance(kdr)mutations in Aedes albopictus(Diptera:Culicidae)field populations in China

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Infectious Diseases of Poverty 2018年第1期7卷 870-878页

作者： Jing-Peng Gao Han-Ming Chen Hua Shi Heng Peng Ya-Jun Ma Department of Tropical Infectious Diseases Second Military Medical UniversityShanghai 200433China Institute of Disease Control and Prevention People’s Liberation Army of ChinaBeijing 100071China Department of Medical Microbiology and Parasitology College of Basic Medical SciencesSecond Military Medical UniversityShanghai 200433China

Background:Arboviral disease transmitted by Aedes albopictus such as dengue fever is an important threat to human health.Pyrethroid resistance raises a great challenge for mosquito control.A systematic assessment of Ae.albopictus resistance status in China is urgently needed,and the study of correlation between pyrethroid resistance and knockdown resistance(kdr)mutations would provide information to guide the control of the Ae.albopictus vector.Methods:Five field populations of Ae.albopictus were collected from Jinan(JN),Hangzhou(HZ),Baoshan(BS),Yangpu(YP)and Haikou(HK),China in 2017.Insecticide-impregnated papers were prepared with four pyrethroid chemicals,deltamethrin,permethrin,beta-cypermethrin and lambda-cyhalothrin.The susceptibility of Ae.albopictus to pyrethroids was tested by the WHO tube assay.Kdr mutations were identified by PCR and sequencing.Moreover,the correlation analysis between kdr alleles and pyrethroid resistance was performed.Results:All five populations of Ae.albopictus showed resistance to four pyrethroid insecticides.One kdr mutant allele at codon 1532 and three at 1534 were detected with frequency of 5.33%(I1532T),44.20%(F1534S),1.83%(F1534 L)and 0.87%(F1534C),respectively.Both 1532 and 1534 mutation mosquitoes were found in the BS and YP populations.Allele I1532T was negatively correlated with deltamethrin resistance phenotype(OR1).Conclusions:The five field populations of Ae.albopictus adults were all resistant to deltamethrin,permethrin,betacypermethrin and lambda-cyhalothrin.Mutant F1534S was clearly associated with pyrethroid resistance phenotype in Ae.albopictus and this could be developed as a molecular marker to monitor the pyrethroid resistance problem in China.

关键词： Aedes albopictus Pyrethroid Insecticide resistance kdr mutation WHO tube bioassay

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