江西省口岸区域白纹伊蚊特异性病毒Aedes albopictus anphevirus基因组序列研究

目的昆虫特异性病毒在白纹伊蚊中与致病性传染病病原体如登革病毒、基孔肯雅病毒和寨卡病毒等的传播和复制有着密切联系,本文对其基因组序列进行研究,为研发新型虫媒病毒防治策略提供理论基础。方法本研究利用二代测序技术,对江西省3个口岸区域(南昌昌北机场、九江城西港和赣州陆港)采集的野外白纹伊蚊种群携带的昆虫特异性病毒进行高通量测序。结果3个白纹伊蚊种群中广泛存在昆虫特异性病毒Aedes albopictus anphevirus(AealbAV)。利用MEGAHIT软件对这些病毒reads进行组装,得到2株AealbAV全基因组序列,与美国株MW147277.1高度同源(相似度均大于98%),基因登录号分别为九江株OR715784,赣州株OR729834,南昌株仅拼接出部分序列。结论AealbAV在江西白纹伊蚊种群中普遍存在,其全基因组研究为口岸区域利用昆虫特异性病毒进行白纹伊蚊综合防治提供了理论基础。

Larvicidal efficacy of crude and fractionated extracts of Dracaena loureiri Gagnep against Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, and Anopheles minimus mosquito vectors

Objective:To evaluate the larvicidal efficacy of crude and fractionated extracts of Dracaena loureiri endocarp against Aedes aegypti,Aedes albopictus,Culex quinquefasciatus,and Anopheles minimus mosquitos.Methods:Larvicidal activity was tested according to World Health Organization standard protocol.The third-stage larvae of each mosquito species were exposed to various concentrations of Dracaena loureiri crude extract and six groups of Dracaena loureiri fractionated extracts(RC-DT 009-014).Larval mortality rates were observed after 24 h and48 h of exposure.Then,a computerized probit analysis of the mortality data was performed to determine lethal concentration 50(LC_(50))and lethal concentration 90 values.Results:Anopheles minimus larvae(24-h LC_(50)77.88 mg/L)had the highest susceptibility to crude extract,whereas others(Aedes aegypti,24-h LC_(50)224.73 mg/L;Aedes albopictus,24-h LC_(50)261.75 mg/L;and Culex quinquefasciatus,24-h LC_(50)282.86 mg/L)were significantly less susceptible.The most effective groups of fractionated extracts were RC-DT 012 and RC-DT 013.The mosquito species most susceptible to fractionated extracts was Culex quinquefasciatus,with 24-h LC_(50)values of 0.66 and 0.94 mg/L for RC-DT 012 and RC-DT 013,respectively.Conclusions:The larvicidal activity of fractionated extracts is more effective than that of crude extract against all tested mosquito species.For the most effective alternative larvicide,purification and a phytochemical constituent analysis must be performed.

Larvicidal activities of hydro-ethanolic extracts of three Cameroonian medicinal plants against Aedes albopictus

Objective: To investigate the larvicidal activity of Syzygium guineense(Myrtaceae)(S. guineense), Monodora myristica and Zanthoxylum heitzii(Rutaceae)(Z. heitzii) extracts against Aedes albopictus(Ae. albopictus).Methods: The larvicidal activity of the hydro-ethanolic extracts from these plant species was assessed at three different concentrations(50, 100 and 200 mg/L) on first-instar of Ae. albopictus larvae in comparison with untreated controls. Mortality rate was recorded daily for a period of 12 days. The values of LC_(50) and lethal time killing 50% of the tested individuals(LT_(50)) were calculated using the log-probit analysis.Results: The root extract of S. guineense exhibited the best activity with 100% mortality after 8 days of treatment at 200 mg/L, followed by the fruit extract of Z. heitzii with83.33% mortality at the same concentration. Nonetheless, larvae were most susceptible to the fruit extract of Z. heitzii both in terms of LC_(50)(39.89 mg/L) and LT_(50)(145.68 h). A statistically significant difference between the control and the group treated at 200 mg/L was noticed in all the extracts.Conclusions: The present study shows that the hydro-ethanolic extracts of S. guineense,Monodora myristica and Z. heitzii tested have significant larvicidal activity. These preliminary results are of great interest and some of these plant species can be proposed for the formulation of new bioinsecticides to control Ae. albopictus populations.

Cloning and sequence analysis of β-actin gene from Aedes albopictus (Diptera: Culicidae)

Objective： To obtain the complete β-actin gene from Aedes albopictus. Methods： Total RNA was extracted from C6/36 cells. Degenerate primers were designed based on the β-actin sequences of An. gambiae, Ae. aegypti, Cx. pipiens pallens and D. melanogaster. By RT-PCR, the product was amplified, purified, cloned into the pGT vector and sequenced. The β-actin sequence was aligned and phylogenetically analyzed by the BLAST program and the CLUSTAL W program. Results： A sequence of 1132 bp including an open reading frame of 1131 bp was obtained （GenBank DQ657949）. The deduced protein had 376 amino acids. Aligned to SWISS-PROT, it exhibited a high level of identity with β-actins from Anopheles, Drosophila and Culex at the amino acid sequence level. Phylogenetic analysis indicated that Ae. albopictus β-acfin was much more homologous with invertebrate β- actin than with vertebrate β-actin. Conclusion： The gene may be used as the internal control in the experiments of Ae. albopictus.

Larvicidal activity of Xenorhabdus and Photorhabdus bacteria against Aedes aegypti and Aedes albopictus

Objective: To evaluate the efficacy of symbiotic bacteria, Xenorhabdus indica, Xenorhabdus stockiae, Photorhabdus luminescens subsp. akhurstii and Photorhabdus luminescens subsp. hainanensis as a larvicide against Aedes aegypti and Aedes albopictus. Methods: Larvae(L3-L4) of Aedes aegypti and Aedes albopictus were given 2 m L of a suspension 107-108 CFU/m L of each symbiotic bacterium. Distilled water and Escherichia coli ATCC襅25922 were used as the control. The mortality rate of the larval mosquitoes was observed at 24, 48, 72 and 96 h. The experiment was performed in triplicates. Results: The larvae of both Aedes species started to die at 24 h exposure. Aedes aegypti showed the highest mortality rate(87%-99%), 96 h after exposure to Xenorhabdus stockiae(b NBP22.2_TH). The mortality rate of Aedes albopictus was between 82% and 96% at 96 h after exposure to Xenorhabdus indica(b KK26.2_TH). Low effectiveness of distilled water and Escherichia coli ATCC襅25922 were observed in both Aedes larvae, with a mortality rate of 2% to 12%. Conclusions: The study confirms the oral toxicity of Xenorhabdus and Photorhabdus bacteria against Aedes spp. Xenorhabdus stockiae and Xenorhabdus indica may be an alternative agent for control Aedes spp. This is basic information for further study on the mechanism of action on Aedes larvae or application to control mosquito larvae in the community.

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

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.

First identification of kdr allele F1534S in VGSC gene and its association with resistance to pyrethroid insecticides in Aedes albopictus populations from Haikou City,Hainan Island,China

Background:Aedes albopictus is distributed widely in China,as a primary vector of Dengue fever and Chikungunya fever in south of China.Chemical insecticide control is one of the integrated programmes to prevent mosquito-borne diseases.Long-term applications of pyrethroids have resulted in the development of resistance in Ae.albopictus populations in China.However,the susceptibility of Ae.albopictus to pyrethroids in Hainan Island was unclear.Knockdown resistance(kdr),caused by point mutations in the VGSC gene,is one of the mechanisms that confer resistance to DDT and pyrethroids.This study was to investigate the resistance level of Ae.albopictus populations in Haikou City to three pyrethroid insecticides,and elucidate the relationship between the resistant phenotype and kdr mutations.Methods:The Aedes albopictus samples were collected in Xinbu Island(XI),Longtang Town(LT),Shishan Town(ST),Baishamen Park(BP),and Flower Market(FM)from Haikou City,Hainan Island,China.The larval susceptibility to deltamethrin,permethrin and beta-cypermethrin was tested by larval bioassays,and adult susceptibility to deltamethrin and DDT was determined by adult bioassays.The degree of resistance was determined by resistance ratio value(RR50>3)for larvae and by mortality for adult.The kdr alleles at codon 1534 of the VGSC gene were genotyped.The relationship between kdr genotypes and resistant phenotypes was analyzed by Chi-square test.Results:Out of five populations,assessed by larval bioassays,XI was susceptible to deltamethrin and permethrin;LT was susceptible to permethrin and beta-cypermethrin;and ST was susceptible to permithrin.FM and BP both were resistant to all of the three pyrethroids,and FM showed the highest degree of resistance,with RR50 values from 65.17 to 436.36.A total of 493 individuals from the larval bioassays were genotyped for kdr alleles.Five alleles were detected,including two wildtype alleles,TTC(F)(67.04%)and TTT(F)(0.41%),and three mutant alleles,TGC(C)(0.30%),TCC(S)(31.54%)and TTG(L)(0.71%).There was a clear correlation between mutant alleles(or F1534S)and resistant phenotypes(P<0.01).Conclusion:Two novel kdr mutant alleles F1534S and F1534L were detected in the pyrethroid resistant populations of Ae.albopictus in Haikou Hainan,China.For the first time,the mutant F1534S was associated with pyrethroid resistance in Ae.albopictus.

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

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(OR<1),while F1534S mutation was positively correlated with deltamethrin and permethrin resistance(OR>1).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.

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

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 among sites,with the largest increase in temperature occurring from February to April(an annual increase of 1.4–4.7℃ in monthly mean,0.6–4.0℃ in monthly minimum,and 1.3–4.3℃ in monthly maximum temperature)and the smallest in November and December.Temperature increases were lower in the tropics/subtropics(1.5–2.3℃ from February–April)compared to the high-latitude areas(2.6–4.6℃ from February–April).The projected temperatures in 2050 and 2080 by this study were approximately 1–1.5℃ higher than those projected by GCMs.The estimated current Ae.albopictus risk distribution had a northern boundary of north-central China and the southern edge of northeastern China,with a risk period of June–September.The projected future Ae.albopictus risks in 2050 and 2080 cover nearly all of China,with an expanded risk period of April–October.The current at-risk population was estimated to be 960 million and the future at-risk population was projected to be 1.2 billion.Conclusions The magnitude of climate change in China is likely to surpass GCM predictions.Future dengue risks will expand to cover nearly all of China if current climate trends continue.

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

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.

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

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.

Photoperiodic diapause in a subtropical population of Aedes albopictus in Guangzhou,China:optimized fieldlaboratory-based study and statistical models for comprehensive characterization

Background:Aedes albopictus is among the 100 most invasive species worldwide and poses a major risk to public health.Photoperiodic diapause provides a crucial ecological basis for the adaptation of this species to adverse environments.Ae.albopictus is the vital vector transmitting dengue virus in Guangzhou,but its diapause activities herein remain obscure.Methods:In the laboratory,yeast powder and food slurry were compared for a proper diapause determination method,and the critical photoperiod(CPP)was tested at illumination times of 11,11.5,12,12.5,13,and 13.5 h.A 4-parameter logistic(4PL)regression model was selected to estimate the CPP.In the field,the seasonal dynamics of the Ae.albopictus population,egg diapause,and hatching of overwintering eggs were investigated monthly,weekly,and daily,respectively.A distributed lag non-linear model(DLNM)was used to assess the associations of diapause with meteorological factors.Results:In the laboratory,both the wild population and the Foshan strain of Ae.albopictus were induced to diapause at an incidence greater than 80%,and no significant difference(P>0.1)was observed between the two methods for identifying diapause.The CPP of this population was estimated to be 12.312 h of light.In the field,all of the indexes of the wild population were at the lowest levels from December to February,and the Route Index was the first to increase in March.Diapause incidence displayed pronounced seasonal dynamics.It was estimated that the day lengths of 12.111 h at week2016,43 and 12.373 h at week2017,41 contributed to diapause in 50%of the eggs.Day length was estimated to be the main meteorological factor related to diapause.Conclusions:Photoperiodic diapause of Ae.albopictus in Guangzhou of China was confirmed and comprehensively elucidated in both the laboratory and the field.Diapause eggs are the main form for overwintering and begin to hatch in large quantities in March in Guangzhou.Furthermore,this study also established an optimized investigation system and statistical models for the study of Ae.albopictus diapause.These findings will contribute to the prevention and control of Ae.albopictus and mosquito-borne diseases.

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

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 DENV-2 is suppressed in MDVinfected Ae.albopictus,thus reducing vector susceptibility to DENV-2.Our study supports the hypothesis that MDVs may contribute to reducing transmission of DENV and provides an alternative strategy for mosquito-transmitted disease control.

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

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.

Seasonality modeling of the distribution of Aedes albopictus in China based on climatic and environmental suitability

Background:Aedes albopictus is a highly invasive mosquito species and a major vector of numerous viral pathogens.Many recent dengue fever outbreaks in China have been caused solely by the vector.Mapping of the potential distribution ranges of Ae.albopictus is crucial for epidemic preparedness and the monitoring of vector populations for disease control.Climate is a key factor influencing the distribution of the species.Despite field studies indicating seasonal population variations,very little modeling work has been done to analyze how environmental conditions influence the seasonality of Ae.albopictus.The aim of the present study was to develop a model based on available observations,climatic and environmental data,and machine learning methods for the prediction of the potential seasonal ranges of Ae.albopictus in China.Methods:We collected comprehensive up-to-date surveillance data in China,particularly records from the northern distribution margin of Ae.albopictus.All records were assigned long-term(1970–2000)climatic data averages based on the WorldClim 2.0 data set.Machine learning regression tree models were developed using a 10-fold crossvalidation method to predict the potential seasonal(or monthly)distribution ranges of Ae.albopictus in China at high resolution based on environmental conditions.The models were assessed based on sensitivity,specificity,and accuracy,using area under curve(AUC).WorldClim 2.0 and climatic and environmental data were used to produce environmental conduciveness(probability)prediction surfaces.Predicted probabilities were generated based on the averages of the 10 models.Results:During 1998–2017,Ae.albopictus was observed at 200 out of the 242 localities surveyed.In addition,at least 15 new Ae.albopictus occurrence sites lay outside the potential ranges that have been predicted using models previously.The average accuracy was 98.4%(97.1–99.5%),and the average AUC was 99.1%(95.6–99.9%).The predicted Ae.albopictus distribution in winter(December–February)was limited to a small subtropical-tropical area of China,and Ae.albopictus was predicted to occur in northern China only during the short summer season(usually June–September).The predicted distribution areas in summer could reach northeastern China bordering Russia and the eastern part of the Qinghai-Tibet Plateau in southwestern China.Ae.albopictus could remain active in expansive areas from central to southern China in October and November.Conclusions:Climate and environmental conditions are key factors influencing the seasonal distribution of Ae.albopictus in China.The areas predicted to potentially host Ae.albopictus seasonally in the present study could reach northeastern China and the eastern slope of the Qinghai-Tibet Plateau.Our results present new evidence and suggest the expansion of systematic vector population monitoring activities and regular re-assessment of epidemic risk potential.

Contrasting resistance patterns to type Ⅰ and Ⅱ pyrethroids in two major arbovirus vectors Aedes aegypti and Aedes albopictus in the Republic of the Congo, Central Africa

Background:In the Republic of Congo,with two massive outbreaks of chikungunya observed this decade,little is known about the insecticide resistance profile of the two major arbovirus vectors Aedes aegypti and Aedes albopictus.Here,we established the resistance profile of both species to insecticides and explored the resistance mechanisms to help Congo to better prepare for future outbreaks.Methods:Immature stages o f Ae.aegypti and Ae.albopictus were sampled in May 2017 in eight cities of the Republic of the Congo and reared to adult stage.Larval and adult bioassays,and synergist(piperonyl butoxide[PBO])assays were carried out according to WHO guidelines.F1534C mutation was genotyped in field collected adults in both species and the polymorphism of the sodium channel gene assessed in Ae.oegypti.Results:All tested populations were susceptible to temephos after larval bioassays.A high resistance level was observed to 4%DDT in both species countrywide(21.9-88.3% mortality).All but one population(Ae.oegypti from Ngo)exhibited resistance to type I pyrethroid,permethrin,but showed a full susceptibility to type II pyrethroid(deltamethrin)in almost all locations.Resistance was also reported to 1% propoxur in Ae.aegypti likewise in two Ae.albopictus populations(Owando and Ouesso),and the remaining were fully susceptible.All populations of both species were fully susceptible to 1%fenitrothion.A full recovery of susceptibility was observed in Ae.oegypti and Ae.albopictus when pre-exposed to PBO and then to propoxur and permethrin respectively.The F1534C kdr mutation was not detected in either species.The high genetic variability of the portion of sodium channel spanning the F1534C in Ae.aegypti further supported that knockdown resistance probably play no role in the permethrin resistance.Conclusions:Our study showed that both Aedes species were susceptible to organophosphates(temephos and fenitrothion),while for other insecticide classes tested the profile of resistance vary according to the population origin.These findings could help to implement better and efficient strategies to control these species in the Congo in the advent of future arbovirus outbreaks.

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

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 low. These populations were divided into three gene pools, in which the northern and eastern pools are relatively homogeneous, while the southern gene pool is heterogeneous. The possible correlation between its genetic variations andkdr mutations is also noteworthy.

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

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.

Screening for larvicidal activity of ethanolic and aqueous extracts of selected plants against Aedes aegypti and Aedes albopictus larvae

Objective:To screen for larvicidal activity of aqueous and ethanolic extracts(95%ethanol)from Selaginella elmeri,Christella dentata,Elatostema sinnatum,Curculigo capitulata,Euphorbia hirta,Murraya koenigii(M.koenigii),Alpinia speciosa,Cymbopogon citratus,Eucalyptus globulus(E.globulus),Jatropha curcas(J.curcas),Psidium guajava,Gliricidia sepium,Ixora coccinea and Capsicum frutescens(C.frutescens)against Aedes aegypti(A.aegypti)and Aedes albopictus(A.albopictus)3rd instar larvae.Methods:Ethanolic and aqueous extracts were screened for larvicidal activity by exposing the A.aegypti and A.albopictus 3rd instar larvae(15 larvae per trial,triplicates)for 48 h,counting the mortalities every 24 h.Additionally,phytochemical screening for flavonoids,tannins,alkaloids,anthraquinones,anthrones,coumarins,indoles and steroids were performed on active extracts using spray tests.Results:Against A.aegypti,the three most active extracts were C.frutescens ethanolic(100%after 24 and 48 h),J.curcas ethanolic(84.44%after 24 h and 88.89%after 48 h)and M.koenigii ethanolic(53.33%after 24 h and 71.11%after 48 h).On the other hand,against A.albopictus,the three most active extracts were C.frutescens ethanolic(93.33%after 24 h and 100%after 48 h),J.curcas ethanolic(77.78%after 24 h and 82.22%after 48 h)and E.globulus ethanolic(64.44%after 24 h and 73.33%after 48 h).Phytochemical screening was also performed on the active extracts,revealing alkaloids,tannins,indoles and steroids.Conclusios:The results demonstrate the larvicidal activities of ethanolic extracts of Cymbopogon citratus,Euphorbia hirta,Ixora coccinea,Gliricidia sepium,M.koenigii,E.globulus,J.curcas and C.frutescens against A.aegypti and A.albopictus 3rd instar larvae.These could be used as potential larvicidal agents for the control of these mosquitoes.