Mechanism Study Of Pyrethroid Resistance In Aedes Albopictus

Currently,dengue is the most rapidly spreading vector-borne disease in the world with 2.5 billion people at risk and approximately 500,000 people developing severe dengue annually.The increasing negative impact of dengue on humans is partly associated with rapid expansions of its vectors,Aedes aegypti and Aedes albopictus mosquitoes(Ae.albopictus).Besides dengue,Ae.aegypti and Ae.albopictus mosquitoes are efficient vectors of Chikungunya,and other arboviruses as well as dog heartworm and filarial nematodes.Today,Ae.albopictus is ranked as one of the world’s 100 most invasive species4,making it an increasingly important vector world-wide.In the past 30-40 years,this species has spread to every continent except Antarctica from its native home range in East Asia and was responsible for recent dengue and Chikungunya outbreaks in Hawaii,Mauritius,Gabon,Madagascar and Europe.Traditionally,Ae.albopictus mosquitoes live at the edge of forests and breed in natural habitats and are therefore considered rural vectors.However,this species has adapted to urban environments and has become the most important disease vector in urban areas in southern China,where dengue represents a serious public health problem and Southern Europe,including Italy and France,where the first endogenous transmissions of Chikungunya and dengue in Europe occurred.Ever since the first outbreak in Foshan in 1978,major and minor Dengue outbreaks has happened in Guangdong,with a more frequent currencies in recent years,such as an outbreak in May 2002 with a occurrence of 1422 confirmed cases in Guangzhou.In 2014,south China and Southeast Asia experienced an outbreak of history.There are 45,171 confirmed cases discovered in Guangdong,with 37,354 confirmed cases in Guangzhou and 6 cases of death.There are more than 4 billion of people each year infected with Dengue virus.The epidemic of Dengue fever is causing severe damage to health to human being and sometimes cause major public health incidence.The aggressive dispersal and thus world-wide invasiveness in recent years,in addition to increased vector competence to Chikungunya viruses,have proved high public health impact of Ae.albopictus.For instance,global spread of Ae.albopictus is linked to Zika virus outbreaks in urban areas of central Africa,Asia and the Pacific.At present,there are neither effective vaccines nor therapeutic treatments targeted for viruses vectored by Ae,albopictus,especially when early symptom of Dengue fever is very similar to flu,thus easily misdiagnosed.Once the case is confirmed,a large number of patiens has already been infected with the virus and are difficult to quarantine.The difficulty in diagnosing and lack of vaccine makes treating vector population control the only option to limit disease transmission.Current vector control strategies primarily rely on the source reduction of larval breeding sites and use of insecticides targeting the larval and adult stages,using various chemical compounds such as pyrethroids,organophosphates and carbamates or other biological compounds such as Bacillus thuringiensis sub.israeliensis toxins(Bti).Treatment of public areas with high population density of Ae.albopictus with adulticides is a recommended protective measure and pyrethroids are the most commonly used adulticides.Also,these same chemical compounds are used for agricultural practices.Increased use of insecticides for public health vector control and for agricultural pest control has imposed tremendous selection pressure on mosquito populations for increased resistance.Insecticide resistance has been documented or emerging in Ae.albopictus populations from China,Pakistan,Malaysia,Thailand,Central Africa and Haiti.Pyrethroid resistance was also reported in Ae.albopictus in the US in two strains,one from Alabama and another from Florida.Insecticide resistance levels of Ae.albopictus populations from Europe are currently not available.The establishment and wide-spread insecticide resistance has significantly hindered control programs.Despite increased resistance of mosquitoes to insecticides,chemical controls remain the most important method for Ae.albopictus due to the lack of other efficient and cost-effective alternatives.Accordingly,vector insecticide resistance is an outstanding issue in the control and prevention of vector-borne diseases worldwide.Among the four major synthetic insecticides groups,pyrethroids are the most widely used adulticide due to its low mammalian toxicity and its rapid knockdown effect.It has been intensively used for space spray to control Aedes mosquitoes and dengue transmission.Extensive and prolonged use of pyrethroids imposes selection pressure on Ae.albopictus populations and eventually increases the potential of resistance.The Insecticide Resistance Action Committee(IRAC)practically defines insecticide resistance as "the selection of a heritable characteristic in an insect population that results in the repeated failure of an insecticide product to provide the intended level of control when used as recommended".An essential step in resistance management is the detection and surveillance of insecticide resistance.Pyrethroid resistance detection in Ae.albopictus has been based on the traditional bioassay detection methods.The bioassay method is too crude for routine field use because of several major limitations.First,it requires a large number of field-caught mosquitoes,but variation in the age and physiological conditions of field-caught mosquitoes is too large and affects the consistence of bioassay results.Second,standardization of any bioassay method is often difficult across sentinel sites because different sites have different temperatures,humidity and testing conditions,which significantly confound the bioassay results.Larval bioassays require collecting a large number of larvae from the field and comparing the susceptibility status with respect to a laboratory strain,which is often difficult.Thus,sensitive and reliable molecular or biochemical methods for resistance diagnosis and surveillance are highly desirable.The understanding of the mechanisms of insecticide resistance is essential for developing reliable resistance monitoring tools.Recognized mechanisms of insecticide resistance in mosquito vectors include two major categories:1)mutations in target sites that affect the affinity with which insecticides bind(target site resistance)and 2)metabolic detoxification of insecticides before they reach their targets(metabolic resistance).For the first mechanism,the voltage-gated sodium channel,encoded by the gene VGSC is the target site of pyrethroids.Acetylcholinesterase(AChE),encoded by the Acel gene,is the target site for organophosphates and carbamates.A small number of amino acid residues are consistently mutated in the VGSC and Acel genes across a wide range of mosquito species studied in relation to resistance,which allowed the development of gene amplification-based techniques for resistance diagnosis.For the second mechanism,detoxification enzymes such as esterases,P450 mono-oxigenases and glutation-S transferases play important roles.In addition to these two resistance mechanisms,other mechanisms have been proposed,including the thickening of cuticle and modification in the digestive tract lining that may prevent or reduce insecticide penetration and absorption(penetration resistance)Insecticide resistance in Ae.albopictus populations has been monitored since the early 2000s using bioassay methods and these studies have documented that insecticide resistance is developing in this species.However the state-of-the-art techniques have not been applied to the studies of insecticide resistance.Analyses of resistance in Ae.albopictus has been focusing on knockdown resistance(kdr)induced by mutations in the VGSC gene.However,the relationship between mutations in the VGSC gene and the resistance phenotype is often not linear.This is not surprising because other resistance mechanisms such as metabolic resistance may play an important role in resistance and/or different resistance mechanisms may be present simultaneously(multiple resistance).New genomic approaches are available to achieve a holistic understanding of insecticide resistance mechanisms.One very valuable technology is the RNA-seq methodology which allows the sequencing of millions of short fragments,in one experiment.RNA-seq methods are becoming the technique of choice for transcriptome analyses due to the following advantages:1)they can be performed without a priori knowledge of the genome sequence;2)the resolution is at the single nucleotide level,thus avoiding the problem of cross-hybridization to similar sequences;3)they provide absolute rather than relative gene expression measurements and reproducibility is high;4)the methods provide both measurements of expression levels and coding sequence information.The RNA-seq technology has already been applied in insects as Drosophila melanogaster and congeneric species to study tissue-specific expression or to compare the transcriptome of different states and tissues.RNA-seq has also been applied to disease vectors,leading to de novo transcriptome assemblies in An.funestus and Ae.aegypti and expression profiles at specific life stages and tissues.In many insect species,kdr L1014F mutation is the most well-known mutation related to pyrethroids and DDT resistance.In Ae,aegypti and Ae.albopictus,mutations occur in different codons of the VGSC gene.Previously described kdr mutations were V1016G,V1016I and F1534C in Ae.aegypti.For Ae.albopictus,F1534C was first reported in Singapore in 2011,and the same mutation was subsequently found in Malaysia as well as in the United States but with a different allele(F1534L).Along with the emergence and spread of kdr mutations,recent studies have demonstrated pyrethroid resistance in Ae.albopictus adults from different parts of southeast Asia such as Malaysia and Thailand and from Central Africa.Previous studies examined kdr mutations in limited number of populations,systematic examination of kdr mutations in Ae.albopictus populations from a broader geographic range would provide important information on kdr mutation distribution and potential risk of resistance spread.RNA interference(RNAi)is a biological process in which RNA molecules inhibit gene expression,typically by causing the destruction of specific mRNA molecules.RNA interference(RNAi)is by far one of the most valualbe research tools both in cell culture and in living organisms because synthetic dsRNA or siRNA introduced into cells can selectively and robustly induce suppression of specific genes of interest.Next,this cleaved message is targeted for degradation,which ultimately results in the loss of protein expression.Our study used siRNA to interrupt target gene expression inside mosquito body by either micro injection or oralfeeding of siRNA nano particles,to investigate the function of 3 candidate gene in both susceptible and resistant lab strain Ae.albopictus mosquitoes.Such studies provided fundamental basis for future resistance passway study.3 D protein silicone modeling and simulation can assist protein structure stability or functional analysis.Through Alignment modeling,our research use Ae.albopictus P450 amino acid sequences of intreast for BLAST in SWISSPORT,followed by SWISS-MODEL to construct 3D protein model based on know protein structure CYP3A4.After model construction,we mapped the mutations on the the model,and check the residual locations for possible functional theories of the mutation,before we test the SNPs in resistant and susceptible populations.Pyrethroid is the insecticide being promoted for mosquito vector control in China,and world-wide,our proposed research will contribute to mosquito control in China and other countries by providing better tools for resistance surveillance.Objective of the research:Aedes albopictus is a major dengue and Chikungunya vector and highly invasive.In the absence of effective treatment for the arbovirus infections,vector control is the only viable option.Pyrethroids are the most widely used insecticide for vector control programs due to low mammalian toxicity and rapid knockdown action.Extensive and prolonged use of pyrethroids imposes selection pressure on mosquito populations and eventually increases the potential of resistance.Monitoring pyrethroid resistance and investigate pyrethroid resistance.Our goal is to systematically examine the spatial distribution of kdr mutations in 12 Ae.albopictus populations worldwide and establish the association between resistance phynotype and mutation genotype;to investigate possible protential candidate genes positivelly related to resistance and protential SNP biomarkers for resistance monitering by the method of NA4-sequencing of resistant and susceptible wild Ae.albopictus mosquitoes,analysis of the transcription data as well as functional verification.Such work will lay fundatin to future insecticide resistance study and development of resistance mornitoring biomarker.Methods:A total of 597 Ae.albopictus individuals from 12 populations across Asia,Africa,America and Europe were examined for mutations in the voltage-gated sodium channel gene.Three domains for a total of 1,107 bp were sequenced for every individual.Two populations from southern China were examined for pyrethroid resistance using the World Health Organization standard tube bioassay,and the association between kdr mutations and phenotypic resistance was tested.The total RNA was extracted from 12 wild resistant and 12 wild susceptible 3-5 days old adult Ae.albopictus after WHO standard bioassay.Libraries were constructed and developed for each single mosquito before sequenced by hiseq system.The gene assembly,gene annotation,differentially expressed genes(DEGs)analysis and qPCR verification for selected genes was performed.In addition,the molecular markers screening for SNP was also performed in this study.Two RNA interference methads were adopted in this study,siRNA targeting 3 candidate genes:Cyp6a8,and 2 genes without annotation:Unknown gene 1 and Unknown gene 2 were designed and either injected into adult lab strain mosquito body or orally fed through siRNA nano particles at larvae stage to study the target genes expression level at adult stage.When verified that the genes can be significantly knockeddow,resistant lab strain Ae.albopictus mosquitoes were targeted using the 2 methods,to monitor the gene expression level at adult stage.After injection or oralfeeding of the siRNA,the adult mosquitoes were also applied for WHO resistance assay to examin the resistance level to check the resistance level change.Statistically check the significance of SNPs in the RNA-seq data and pick the 9 SNPs that were related to P450 super gene families.By bioinformatic analysis using online tool SWISS-MODLE and SWISS-PORT,to predice Ae.albopictus P450 protein model,afterwards,the known crystallized structure CYP3A4 protein was use to do the mapping through PyMol to mimic the binding of deltamethrin molecule to the protein model,and to search the mutation residuals’ protential function on the protein model.Also the SNPs frequencies in resistance and susceptible wild Ae.albopictus populations were detected in 27-62 individulas to verify the mutation rate.Results:A total of 29 synonymous mutations were found across domain Ⅱ,Ⅲ and Ⅳ of the VGSC gene.Non-synonymous mutations in two codons of the VGSC gene were detected in 5 populations from 4 countries.A novel mutation at 1532 codon(I1532T)was found in Rome,Italy with a frequency of 19.7%.The second novel mutation at codon 1534(F1534S)was southern China and Florida,USA with a frequency ranging from 9.5-22.6%.The WHO insecticide susceptibility bioassay found 90.1%and 96.1%mortality in the two populations from southern China,suggesting resistance and probable resistance.Positive association between kdr mutations with deltamethrin resistance was established in these two populations.is essential to effective management of resistance.The voltage-gated sodium channel gene is the target site of pyrethroids,and mutations in this gene result in knockdown resistance(kdr).Pyrethroid resistant transcription data were established through RNA-seq in Ae.albopictus mosquitoes,we detected 11090 pyrethroid resistance related transcripts,out of which 742 are statistically significant,and they were generated with Gene Ontology(GO)analysis.Furthermore,a total of 1418 single nucleotide polymorphisms(SNPs)were identified.Target gene expression level were both knocked down in microinjection group and orfeeding siRNA nano particle group,the knocked down levels were around 50-60%.Lab resistant Ae.albopictus strain were changed from resistant to susceptible after RNA interance by both methods.The WHO resistance asay indicated that 24 h mortality changed from 90.5%to 100%.Of the 2 unknown genes studied,the unknown gene 1 was not expressed in larvae stage.Nine SNPs in 8 P450 genes were mapped to know protein structure and used for functional prediction.During population detection of the 9 SNPs,5 of them were significantly correlated to pyrethroid resistance.Two of the mutation,Arg226Serand Cys212Ser are located on the F/G loop,which allows the P450 enzymes to open up to allow substrate to enter the activie site,which is followed by closing around the substrate to allow for catalytic chemistry to occur before releasing the substrate by opening once again.Leu282Arg residues is located near the protein redox partner binding surface,the location may case an effect on the secondary partner for FMN redox.Pro 175Gln is known for posing a positive impact on the stability of the P450 enzyme.Other than thses 5 SNPs,2 of the genes failded to amplify and 2 SNPs the Odds ratio were not significant.Conclusions:1 Two novel kdr mutations,I1532T and F1534S were found in Ae.albopictus.This is the first report of I1532T mutations in Italy and F1534S mutation in China and US.Significant association between kdr mutation and protection from deltamethrin raised the possibility that kdr mutation may be a viable biomarker for pyrethroid resistance surveillance in Ae.albopictus.2 Chinese Ae.albopictus in the south region Guangzhou and Shenzhen have developed resistance toward pyrethroid in adult,which calls for a dosage control and insecticide types rotation in the mosquito vector control movement,which could protentially reduced the evolving speed of resistance in Ae.albopictus.It’s necessary to evaluate the classes,effective dosage and efficacy of pyrethroids used in the vactor control actions,so as to get rid of the potential threat to achieving of eliminating mosquitoes.3 kdr mutation at 1534 codon is positively related to pyrethroid resistance in Chinese wild Ae.albopictus populations.This could be a protential biomarker for resistance monitor in Ae.albopictus.4 RNA-seq was first used to construct pyrethroid resistance transcription data by single library preparation of resistant and susceptible Ae.albopictus mosquitoes.And DEGs candidate gene were selected and functionally verified for its positive coorelation to pyrethroid resistance.5 Cyp6a8 and 2 other unannotated genes were functionally verified to be positively related to pyrethroid resistance in Ae.albopictus.Of the 2 unknown genes,the unknown gene 1 is only expressed in adult mosquitos thus may be regulating only in mosquito adult stage instead of through larvae and adult.6 9 SNPs related to 8 P450 genes were for the first time,mapped to protein structure,for mutation residue detection.And these mutations were verified through gene sequencing in wild resistant and suscdeptible Ae.albopictus mosquitoes.Out of the 5 significant SNPs verified,4 of the mutations were given theratical explanations regarding the function of the mutations.