Molecular Insight into the Knockdown Resistance (kdr) in the Voltage Gated Sodium Channel (vgsc) Gene of the Main Dengue Vector, Aedes aegypti (Diptera: Culicidae) and the Discovery of Novel Regional Specic Point Mutation A1007G in Malaysia.

Background: Characterization of the insecticide resistance mechanism imparts the society with the information on the evolutionary process involved in the adaptation of Aedes aegypti mosquito to environmental changes. Investigating the phenotypic status of the target mosquitoes, their resistance level as well as elucidating the genotypic prole provides information about the involvement of insecticide resistance mechanism, in terms of portraying the evolution of resistance in the eld, to eventually managing vector control programmes. In this current study, we investigated the quantication responses for the phenotypic and genotypic resistance of Ae. aegypti population from different states in Malaysia. Methods: We tested insecticide susceptibility status of adult Ae. aegypti from populations of States of Penang, Selangor and Kelantan (Peninsular Malaysia) against 0.25% permethrin and 0.25% pirimiphos-methyl through WHO bioassay kit. Permethrin-resistant and permethrin susceptible samples were then genotyped for domains II and III in the voltage gated sodium channel (vgsc) gene using allele specic PCR (AS-PCR) for the presence of diagnostic single nucleotide mutations. AS-PCR results were then validated in sequencing these two domains to identify any possible additional point mutations. Results: Adult WHO bioassay revealed that populations of Ae. aegypti from these three states were highly resistant towards 0.25% permethrin and 0.25% pirimiphos-methyl. Genotyping results showed that three knockdown (kdr) mutations (i.e. S989P, V1016G and F1534C) were associated with pyrethroid resistance in these populations. We also report for the rst time the presence of the A1007G mutation in Malaysian populations of Ae. aegypti. Conclusions: This study brings an insight on the occurrence and association of point mutations with insecticide resistance in Malaysian populations of Ae. aegypti. The results reveal the widespread of several kdr mutations in the eld with the consequence to compromise the use of pyrethroid insecticides in vector control programmes. Knowledge on the distribution of target site resistance throughout Malaysia is vital to ensure the success of the insecticide-based

introduced and portrays good e cacy pro le during clinical trial, there are still some restrictions which need to be solved before commercialized in large scale [16].
Various approaches have been conducted to control populations size and distribution of Ae. aegypti; most of them rely on the insecticide-based intervention targeting the immature and adult stages of this species [17]. In Malaysia, pyrethroid and organophosphate are routinely used during the vector control programmes conducted by the Ministry of Health, and also by the private pest control operators and the local communities [18,19]. Such excessive use of these insecticide classes for a long period, led to over dependence and improper usage of these insecticides to control the mosquitoes, eventually causing resistance in this vector.
Inappropriate usage and over-exposure to insecticides that share the same mode of action can lead to the selection of several types of insecticide resistance in the mosquitoes: 1) the modi cation of the mosquito cuticle, leading to reduced penetration of the insecticide into the insect's exoskeleton, 2) the presence of the single nucleotide polymorphism (SNP) resulting in the modi cation of nucleotide in the target gene and consequently changing the amino acid of the target sites in mosquito, 3) increasing in the enzymatic activity in detoxifying the insecticides; or 4) changes in the mosquito behaviour enabling them to survive in the toxic environment [20].
The two resistance mechanisms that are commonly associated with pyrethroid resistance within insects are 1) increased metabolic detoxi cation activity [21] and 2) the insensitivity of the target site such as sodium channel gene, Ace-1 gene and GABA receptor [22]. Although, organophosphate and pyrethroid have different modes of action, both insecticides target the nervous system of the insect which eventually leads to its death [23]. Several point mutations have been functionally identi ed in the vgsc gene to reduce its sensitivity by preventing the binding of the insecticide to the target gene, making the insect experience rapid nerve ring and paralysis and consequently leading to the knockdown resistance (kdr) [24,25,26,27].
In Malaysia, three common knockdown (kdr) mutations within Ae. aegypti voltage gated sodium channel gene (vgsc) are known to be associated with pyrethroid resistance. The non-synonymous mutations S989P, V1016G and F1534C correspond to a substitution of amino acid serine to proline, valine to glycine and phenylalanine to cysteine, respectively, within domains II and III. These mutations were previously shown to be widely distributed across Malaysia [28,29,30]. Other studies have reported that these mutations are widely established across Southeast Asia, including Indonesia, Thailand, Singapore, Myanmar and Vietnam [31,32,33,34,35]. Currently, a total of 13 non-synonymous mutations have been detected in the vgsc gene of pyrethroid-resistant Ae. aegypti population worldwide; ve of them (V410L, S989P, I1011M, V1016G and F1534C) have been in vitro functionally characterized in the expression system of Xenopus oocytes and reported to confer kdr resistance in Ae. aegypti [24,25,26,27,36,37,38,39,40]. Kdr mutations G923V, L982W, I1011M and V1016G were rst identi ed in 2003 which were detected in pyrethroid/DDT resistant Ae. aegypti populations [24]. In 2007, I1011V and V1016I mutations were reported in the Ae. aegypti population from Latin America [25]. Subsequently, D1763Y mutation was observed in the pyrethroid resistant population of Ae. aegypti from Taiwan in 2009 [36]. S989P and F1534C mutations were rst reported in Ae. aegypti population from Thailand in the following year [37,41]. In 2015, T1520I mutation was reported in Ae. aegypti population from India [39]. The rst kdr mutation in domain I, V410L was rst observed in the Brazilian population of Ae. aegypti in 2017 [27]. A1007G mutation was rst detected in Ae. aegypti from Vietnam in the following year [42]. Recently, F1534L mutation was reported in the pyrethroid resistant Indian Ae. aegypti populations [43].
Characterizing target site resistance mechanism is an essential key to improve the management strategies of the vector control. Hence, the aim of this study is to further elucidate the insecticide resistance mechanism associated within Malaysian Ae. aegypti population phenotypically and genotypically.

Methods
Sampling and rearing of mosquitoes Six populations of Aedes aegypti were collected from three states in Malaysia; Penang, Selangor and Kelantan in 2017 by placing the 100 ovitraps at residential areas for ve days. The sites were selected based on the number of reported dengue cases in idengue website (https://idengue.mysa.gov.my/) which is the Malaysian national dengue database (Ministry of Health) and were routinely sprayed with insecticides particularly permethrin and pirimiphos-methyl. We selected two sites within each state (total = six sites): Sungai Dua (SD) and Balik Pulau (BP) in Penang, Alam Budiman (AB) and TUDM, Subang (TDM) in Selangor, and Pauh, Panji (PNJ) and Flat Buluh Kubu (FLT) in Kelantan. A maximum of 100 ovitraps were set up for each of the study site at a distance between 5 to 10 meters apart depending on the type of houses in the residential area. The ovitraps were randomly placed at the potential breeding sources and less exposed to direct sunlight. The traps were collected after ve days and brought back to the insectary for culturing purposes.
The eggs of Aedes mosquitoes from all the localities were hatched and upon emergence of rst instar larvae, they were fed with the larval food containing grounded cat biscuit, beef liver powder, milk powder and yeast with a ratio of 2:1:1:1. The adult mosquitoes were morphologically identi ed to the species level based on the pattern on the thorax. The adult Ae. aegypti were supplied with 10% sucrose solution. All larvae and adults were maintained at room temperature of 28 2 with a relative humidity of 75 10%. The local susceptible laboratory strain obtained from the Vector Control Research Unit (VCRU) which has never been exposed to any insecticides was used as a reference strain.

WHO adult bioassay
The adult mosquito bioassay was performed according to the World Health Organization (WHO) protocol [44]. Twenty female mosquitoes of Ae. aegypti of three to ve days old were tested against 0.25% permethrin (Type I pyrethroid) and 0.25% pirimiphos-methyl (organophosphate) for one hour and replicated ve times.. The susceptibility bioassay test was conducted in room temperature of 28 2 with a relative humidity of 75 10%. Subsequently, they were supplied with 10% sucrose solution ad libitum and maintained under insectary conditions. The number of dead and alive mosquitoes were recorded at 24 hours post exposure. After 24 hours exposure, the whole bodies of the surviving mosquitoes were transferred and kept in -80 and those of the dead samples were preserved in silica gel inside microcentrifuge tubes for kdr genotyping.

Synergist bioassay
The synergism assay was conducted as a rst screening to investigate the potential role of superfamily enzyme mixed function oxidases and esterase in the insecticide metabolic detoxi cation. Piperonyl butoxide (PBO) was used before the exposure to permethrin insecticide because it is known as a common synergist for pyrethroid [45] and inhibit enzyme from the family of esterases and mixed function oxidases which are mainly associated with metabolic resistance caused by pyrethroid, organophosphate and carbamate exposure. The test was performed on the individuals from all sampling sites because the percentage mortality was less than 90% mortality from the previous WHO bioassay test. Adult female mosquitoes were exposed to 4% PBO one hour before the exposure to 0.25% permethrin. The mortality of the individual mosquitoes was recorded after 24 hours. To compare the results obtained between the two treatments, we either use only the insecticide or a combination of synergist and insecticide.

Genotyping of kdr mutations in voltage gated sodium channel (vgsc) gene in Aedes aegypti
Extraction of genomic DNA (gDNA) The gDNA of 20 dead and alive samples from all localities were extracted from legs and wings following Livak protocol [46]. The body of survived mosquitoes were kept in -80 for the quanti cation of metabolic resistance by real-time PCR. The DNA concentration and purity were measured using a Nanodrop spectrophotometer at 260 nm. The gDNA samples were stored in the -20 for the downstream application.
Detection of the V1016G mutation by using allele speci c PCR (AS-PCR) in Aedes aegypti To determine the presence of V1016G and F1534C point mutations conferring pyrethroid resistance, 20 female mosquitoes from each resistant and susceptible eld population were randomly chosen for this genotyping using AS-PCR. From a total of 100 female mosquitoes, 20 resistant individuals and 20 susceptible individuals were genotyped using AS-PCR protocol as previously described by Stenhouse et al. [32]. V1016G mutation was genotyped in 20 resistant and susceptible individuals that were exposed to permethrin previously. Each reaction used consisted of a total volume of 15 l of 1.  extension) and 72 for 2 min ( nal extension). The PCR products were loaded checked onto 3% agarose gel. The sizes of the two ampli ed fragments are 60 base pairs [bp] for valine and 80 bp for glycine.

Genotyping of F1534C by using allele speci c (AS-PCR) in Aedes aegypti
For the ampli cation of F1534C in domain III segment 6 (DIII S6), this kdr mutation was genotyped by AS-PCR as described by Yanola et al. [41]. The PCR reaction was performed in nal volume of 15l with a nal Validating polymorphism sites of the voltage gated sodium channel (vgsc) gene in Aedes aegypti by DNA sequencing Some of the AS-PCR products were sequenced in order to verify the accuracy of results obtained from the AS-PCR assay and, also, to detect any possible novel mutation. The regions of DIIS6 and DIIIS6 in the vgsc gene (where the V1016G and F1534C mutations occur) were separately ampli ed (by PCR) using the protocol described in Stenhouse et al. [32] and Yanola et al. [41]. Each reaction was performed in 20 l nal volume consisting of 1. Chromatograms were rst edited using MEGA 6 Molecular Evolutionary Genetic Analysis software, version 6.06 [47]. The sequences determined in this study have been deposited in GenBank (GenBank accession nos. MT237357-MT237435). Then, all nucleotide sequences of the haplotype for domains II and III were aligned (no indels needed). Our phylogenetic matrix comprises 46 individuals and 491 and 346 nucleotide positions for domain II and domain III respectively. We inferred the relationships among the individuals using a maximum parsimony method of phylogenetic reconstruction as implemented in PAUP* v4b10 [48]. Susceptible Musca domestica (GenBank accession number U38813.1) was selected as the outgroup to root the tree. The haplotype TCS network was built using the PopART [49,50] software to determine the correspondence/relation between haplotype and the resistance/susceptible phenotypes.

Statistical analysis
Percentage mortality at 24 hours post exposure for each of the population was used to describe the phenotype status. The classi cation of the susceptible and resistant phenotype in the tested populations were interpreted according to the WHO guidelines [44]. The tested population was considered to be susceptible if the percentage mortality ranged from 98% to 100%; mortality ranged from 90% to 97% suggests that the population has developed intermediate resistance; and the population was considered to be resistant if the percentage mortality less than 90%. Odds ratio (OR) and Fisher's exact test were conducted to compare the distribution of kdr genotypes between the alive and dead mosquitoes. Chisquare analysis was used to test the signi cant differences in percentage mortality with and without preexposure to PBO. All statistical analysis was performed using The Statistical Package for Social Science software (IBM SPSS Statistics version 24).

WHO adult bioassay
The diagnostic dosage for Aedes mosquito was used for permethrin and the tentative dosage for Anopheles was used for pirimiphos-methyl. WHO bioassays on the eld strains revealed phenotypic resistance in populations from Selangor, Penang and Kelantan with percentage mortality less than 90% after exposure towards permethrin and pirimiphos-methyl ( Fig. 1). Aedes aegypti from all six populations were highly resistant towards both insecticides with percentage mortality, after 24 hours exposure, varying from 0% to only 18% and 3% to 58% for 0.25% permethrin and 0.25% pirimiphos-methyl, respectively. Full susceptibility was observed in Ae. aegypti from the VCRU laboratory strain, after the exposure towards both insecticides ( Fig. 1).
High resistance towards permethrin was observed in all six populations. The lowest percentage of mortality was observed in the populations from Pauh, Panji (PNJ) and Flat Buluh Kubu (FLT), Kelantan with 0% and 1% mortality, respectively, indicating that these two populations developed very strong resistance and the need of additional assessment regarding the genetic mechanisms and distribution of such resistance. Aedes aegypti from TUDM, Subang (TDM) shows the highest mortality compared to other populations with 18%, although such percentage of mortality is still quite low. Widespread resistant towards 0.25% pirimiphos-methyl was also observed in all populations with the highest resistance level recorded in Alam Budiman, Selangor (AB) with 3% mortality. Meanwhile, the population from Balik Pulau, Penang (BP) exhibited the highest mortality against pirimiphos-methyl with 58% mortality.

Synergism assay with piperonyl butoxide (PBO)
Percentage mortality of all populations except Pauh, Panji, (PNJ) after subjected to one-hour preexposure to 4% PBO before being exposed to permethrin, displays an increase in the mortality ranging from 2% to 69% (Fig. 1). Pre-exposure to synergist showed an increase in mortality for populations from TUDM, Subang (TDM) and Balik Pulau (BP) with percentage mortality of 29% and 69% respectively after being exposed to permethrinThese two populations showed a major increase of percentage mortality compared to the other populations, in addition, Ae. aegypti from Balik Pulau (BP) showed a signi cant increase in susceptibility towards permethrin (X 2 = 7.244, df= 1, P = 0.007). No impact of pre-exposure to synergist PBO was observed in the population from Pauh, Panji (Kelantan) with 0% mortality before and after exposure to synergist.  (Table 1).
At codon 1016, a polymorphism was detected in populations from Penang and Selangor (but not in those of Kelantan). A mutation from valine to glycine at codon position 1016 was identi ed within these populations. A mutation of wildtype GTA to mutant allele GGA was detected from those populations with allelic frequency ranging from 5% to 55% (Table 2). Among 167 samples genotyped, a total of 46 samples were partially sequenced in domain II to valid the results obtained from AS-PCR for point mutation V1016G. Eighteen samples showed discrepancy where heterozygous allele (V/G) (from AS-PCR) turned out to be homozygous wild type allele (V/V) after Sanger sequencing. This might be due to the presence of two consecutive alternative mutations in domain II segment 6 leading to genotyping error, hence resulting in the false positive results [28]. In addition, the ampli cation of the non-speci c band happens due to the mismatch of the single base in the gene, hence it is unable to prevent the non-speci c ampli cation during the PCR extension [51].
A novel non-synonymous substitution has been discovered at codon position 1007, a mutation from alanine (GCC) to glycine (GGC), happens due to changes of nucleotide C to G at position 1007 in the sequence. Our results show that only permethrin resistant samples from Pauh, Panji (PNJ) and Flat Buluh Kubu (FLT) have this novel amino acid substitution, with the percentage of 1007G allele were 85% and 90%, respectively. Interestingly, the populations that possess this mutation will not co-occur with other point mutations either S989P or V1016G in domain II and could only be found in samples from Kelantan state. Our results indicate this point mutation alone in domain II might be responsible in conferring the high resistance in the phenotypes since there is no other point mutation in domain II that coexist after genotyping using direct sequencing (Fig. 2). Result from the direct sequencing reveals populations from Kelantan that possess this novel mutation will coexist with another point mutation in domain III which is the F1534C mutation (Fig. 2, Fig. 3). All samples from these localities were a mixture of heterozygous and homozygous for the double substitution mutations. These populations were either heterozygous or homozygous mutant to codon 1007 and shares another amino acid change at codon 1534 and are homozygous mutant at this position (Additional le: Table S3). This might explain why the population from Kelantan were highly resistant towards permethrin.
In domain III, a change from phenylalanine (TTC) to cysteine (TTG) at codon 1534 was detected in all six populations with allelic frequency ranging from 0.028 to 0.975 and populations from Kelantan showing the highest allele frequency for the permethrin-resistant samples; more than 90%. We also recorded that the mutant allele, 1534C are common in the susceptible samples from Penang and TUDM, Subang (TDM) ( Table 3, Additional le: Fig. S1).

Association between kdr mutation at domain II and III with pyrethroid resistance
To assess the correlation with the resistance phenotype, a total of 167 resistant and susceptible mosquitoes from all populations were then genotyped at domains II and III. To ascertain the impact of kdr mutations at different codons; 989, 1007, 1016 and 1534 of the vgsc gene towards pyrethroid resistance, the S989P, A1007G, V1016G and F1534C mutations were analysed separately for their associations with the permethrin resistance.
The S989P mutation in domain II is not signi cantly associated with the pyrethroid resistance in all populations (Fisher's exact test, P = 0.146) and we presume the populations were not at Hardy-Weinberg equilibrium due to low samples size tested for genotyping at codon 989 (  Table 2). In most of the localities, the differences in the allelic frequencies between alive and dead mosquitoes were not signi cantly correlated (P = 0.429). More heterozygote mosquitoes survived after the exposure towards permethrin (Additional le: Fig. S1).
We cannot determine the correlation between novel mutation, A1007G with the permethrin resistance in population from Kelantan due to the low number of susceptible samples obtained after WHO bioassay.
Despite that, we found an extremely high 1007G allele frequency ranging from 85% to 90% (Table 4). This probably explained why the mortality of the population from Kelantan were exceptionally low ( Table 4).  Table S2). Triple-locus wild type homozygote, S989+V1016+F1534 (Type 1) and quadruple-locus wild type homozygote, S989+A1007+V1016+F1534 (Type 10) was found in four susceptible samples from Selangor and one susceptible sample from Kelantan respectively. Most of the locus genotyped were a combination of two to three amino acid substitution. We noticed triple-loci kdr genotypes ( Seven haplotypes were identi ed with ve haplotypes present in domain II and two haplotypes was observed in domain III. These haplotype variations produced four amino acid substitution. In the coding region of domain II, we found ve polymorphic sites resulting in three non-synonymous changes (S989P, A1007G and V1016G mutations) and two synonymous changes. Meanwhile in the coding region of domain III, one polymorphic site at codon 1534 could be observed leading to the two haplotypes created in this vgsc fragment. In general, the vgsc gene exhibits low polymorphism level for all six populations in Malaysia with a low number of the mutational steps between the haplotypes in domain II and III as shown in the TCS network (Fig. 4). From the TCS network analysis in domain II, four resistant haplotypes were observed with one singleton haplotype 4 (H4) and haplotype 5 (H5) which is a combination of the new mutation and synonymous change were detected in Kelantan samples only.
There are ve different haplotypes with two major haplotype that are established from all six Ae. aegypti populations in Malaysia (Fig. 4A). Interestingly, in haplotype 4 (H4) and haplotype 5 (H5), we found a novel regional mutation in domain II segment 6 which comprised of resistant samples from Kelantan state only and consisted of both homozygous and heterozygous A1007G mutation. These samples from Pauh, Panji and Flat Bulu Kubu were homozygous susceptible for S989P and V1016G mutations.
There are only two haplotypes that were found to be associated with the F1534C mutation in domain III of the vgsc gene. In domain III, haplotype 1 (H1) was associated with F1534C mutation revealing that all resistant samples from six populations were homozygous resistant for F1534C mutation (Fig. 4B).
There is no signi cant difference in the Tajima's D estimation in the vgsc fragment for both domain II and III demonstrating a low number of the polymorphisms in the vgsc within those populations ( Table 5). The presence of the predominant haplotypes in both domain II and III gave us an idea that there is selection pressure in the vgsc gene fragment in both domain II and III which is in agreement with the existence of the kdr mutation in this Malaysian population of Ae. aegypti. Maximum parsimony phylogenetic tree analysis of the vgsc gene display an association between the pyrethroid resistance and the single nucleotide polymorphism of domain II and III in the vgsc gene respectively (Additional le: Fig. S2A, Fig.  S2B). The existence of polymorphism in exon 15 to 16 domain II and exon 23 to 25 of domain III of vgsc gene potentially correlated with the permethrin resistance. Reconstruction of the maximum parsimony tree revealed the haplotype pattern within those domains clustered according to the phenotype of the mosquito samples.

Discussion
The development of insecticide resistance worldwide has become worrisome problem since a few decades ago [10]. Malaysia is one of the countries that is confronted with this scenario [52]. The present study reveals that the susceptibility status and the distribution of the kdr allele in the Malaysian main dengue vector, Aedes aegypti in Malaysia. This study focuses on the signi cant role of target site mutation in conferring pyrethroid resistance in this species.

Resistance pro les of Aedes aegypti
Currently, pyrethroid and organophosphate are the major classes of insecticides that are widely used to eliminate the Aedes mosquitoes during the dengue outbreak and these classes of insecticides have been routinely switched by the Ministry of Health Malaysia throughout the control programmes. In addition, the pest control industry also has been using the same classes of insecticides for the same purpose. This action has probably resulted in the selection pressure within the natural populations due to the high exposure towards the insecticides during the control measures and hence have the ability in adapting to the changes of the harsh environment [53]. High occurrence of permethrin resistance in Ae. aegypti is frequently reported in many countries from Southeast Asia including Singapore, Indonesia, Cambodia, Laos, Thailand, Myanmar and Vietnam [31,34,35,54,55,56,57,58,59]. The increasing occurrence of the pyrethroid resistance is alarming to governments and private sectors involved in vector management.
The overall low percentage mortality observed in all six populations against 0.25% permethrin and 0.25% pirimiphos-methyl indicated that they showed high resistance towards these insecticides and suggests these insecticides might not be effective for the control of these mosquito populations. Resistance ratio (RR) cannot be determined in this study because there were no knocked down mosquitoes during the onehour exposure towards permethrin and pirimiphos-methyl and this probably re ects that the populations are highly resistant towards both insecticides. Results of the susceptibility bioassay revealed that Ae. aegypti populations from Penang, Kelantan and Selangor exhibit high resistance towards the insecticides from the class of pyrethroid (Type I: permethrin) and organophosphate (pirimiphos-methyl) with the percentage mortality after 24-hour exposure less than 90% con rming these populations are resistant as shown in previous studies recently [28,29,30]. However, our results contradicted a study conducted in 2011 revealing that Ae. aegypti populations from Penang were at that time fully susceptible towards pirimiphos-methyl and malathion [60]. One explanation could be that the Penang populations recently developed resistance against these insecticides compromising the application of this insecticide during vector control programmes. Phenotypic pyrethroid resistance is still increasing in Malaysian population of Ae. aegypti as a consequence of the wide usage of pyrethroid for decades. To understand the genetic basis of resistance mechanisms and their distribution in Malaysia, those populations were subjected to additional investigation.
In Malaysia, dengue vector control programme highly relies on the use of chemical insecticides [61]. Application of chemical insecticides during thermal fogging and space spray to control adult Ae. aegypti has been conducted for more than a decade [18]. Generally, pyrethroid and organophosphate insecticides have been widely used for the insecticidal treatment against the primary vector for dengue, Ae. aegypti. To date, permethrin and pirimiphos-methyl have been used simultaneously to control adult Ae. aegypti during the vector control programme by the public health authorities [62]. Previously, in the early 1970, malathion was rst used in fogging to control against adult mosquito vectors. However, its usage discontinued and was replaced by permethrin and deltamethrin in 1998 [52]. These type I and type II pyrethroids have continued to be used until today. Not only that, pyrethroid is also used in the household insecticide products such as liquid vaporizer, aerosol, mosquito coils and mosquito mats [61]. These products have formulations containing active ingredients of meto uthrin, tetramethrin, d-allethrin, trans uthrin, d-phenothrin, s-bioallethrin, cyphenothrin, deltamethrin, d-trans allethrin, prallethrin and permethrin [61,62,63]. The extensive usage of household insecticide products may exert selection pressure in the target mosquito population and thus might cause cross-resistance in these pyrethroid insecticides.
Apart from controlling adult Ae. aegypti, targeting at the larval stage using larvicidal treatment such as temephos also has been conducted as a part of vector control. Temephos has been widely used to control mosquito larvae since 1973. As a consequence, effective long term usage of temephos has been compromised due to the development of resistance towards this particular insecticide [28,64]. Therefore, surveillance on the phenotypic status of Malaysian Ae. aegypti across all the other states in Malaysia as well as their genotypic frequencies between the kdr alleles is crucial in the vector control programme and should be monitored over the time. This could provide conclusive evidence in deciding which vector control strategies should be implemented to improve the management of the control programme.
Development of resistance towards pyrethroid insecticide in Malaysia is notably observed since the year 2001 and it is gradually increasing yearly [52]. Although the authorities practice insecticide rotation, the use of insecticides from the same class and having the same mode of action causes the resistance problem to not be solved. In addition, metabolic cross-resistance could possibly occur between pyrethroid and organophosphate insecticides as a results of insecticide rotation [65]. In particular, insecticide rotation for permethrin and pirimiphos-methyl have been adopted during the control programme as one of the strategies in the insecticide resistance management (IRM) in Malaysia. Hence, further investigation is needed to con rm the involvement of metabolic cross-resistance in Malaysian population of Ae. aegypti. It is important to note that we did not carry out intensity assay in addition to the 2 diagnostic dose which was recommended in WHO guideline [66] due to insu cient mosquito samples. Future studies such as dose-response study and intensity bioassay should be conducted in local populations to determine the strength of the resistance level among these populations, thus can assist in managing insecticide resistance.
Resistance is usually the combination of two or more mechanisms. Pyrethroid resistance in Ae. aegypti is often associated with target site resistance and metabolic resistance [21,22]. al. [30] found the presence of those mutations in the populations from Selangor associated with pyrethroid and DDT resistance. Our present study is in line with the reported point mutations which is associated with pyrethroid resistance in the Malaysian population of Ae. aegypti.
In addition, we unexpectedly discovered a potential novel substitution mutation, A1007G from Kelantan population, which occurs as a single mutation in domain II without the association of other mutations, S989P and V1016G, but co-occur with the point mutation in domain III, F1534C. We observed a signi cant correlation between V1016G and F1534C genotypes with the pyrethroid resistance in several localities within these states in Malaysia which shows that those genotypes could possibly attribute towards the pyrethroid type II resistance. In addition, high frequency of the kdr mutations; S989P, A1007G, V1016G and F1534C was detected amongst the six populations in Malaysia, demonstrating target site resistance is partially associated with the resistance towards permethrin. The widespread of these point mutations of S989P, V1016G and F1534C in the kdr gene of the Malaysian population Ae. aegypti might explain the large-scale usage of the insecticide from the class of pyrethroid which had been extensively used by the government and the private sector to control the population of mosquito vectors and especially Aedes species in Malaysia.

A novel mutation in Malaysian Aedes aegypti population
The mutation A1007G was found in the population from Kelantan with a high frequency was observed (f 85%). Direct neurophysiological analysis has not yet been proven, and this step is crucial in seeking evidence whether this new mutation is responsible in causing the pyrethroid resistance within the permethrin-resistant population. We hypothesize this particular mutation play a similar function as the other kdr mutations since this mutation is located within one of the four speci c amino acid residues in the P-region segments, DIIS6 [67]. Changes of nucleotide C to G at position 1007 in domain II leading to the amino acid substitution from alanine to glycine presumably give rise to the alteration of the target site in the sodium channel, hence reducing its sensitivity towards permethrin. This is further justi ed by the results from synergist assay that showed PBO might be considered as a partially ineffective synergist.
Modi cation in the insect voltage gated sodium channel which normally regulates sodium ion within the gene, makes the channel less functional, hence delaying the closing of the channel [68,69]. The previous studies from other research groups are able to detect other regional mutations in the Asian continent.
First report in Vietnam found point mutation L982W in domain II is associated with the DDT and pyrethroid cross resistance in the resistant population of Ae. aegypti [24]. In 2009, a research group from Taiwan concluded that a high resistant fold in their permethrin resistant strain is conferred by the association of a novel mutation, D1763Y with the other substitution mutation, V1016G [36]. These coexisting mutations conceivably causes synergist effect in the knockdown resistance towards permethrin. The sensitivity of the vgsc gene towards permethrin resistant is reduced by 190-fold. The presence of the point mutation, T1520I and F1534L in the domain III of the kdr gene in the Indian Ae. aegypti population might be partly responsible towards the pyrethroid resistance [39,43]. These new mutations have yet to be functionally con rmed in the oocytes' system. Similarly, our study also has a limitation in validating this new mutation. Due to facilities constraint, we are unable to conduct the functional validation of our potential novel substitution mutation, A1007G express in the Xenopus oocytes. By conducting the experiment, we might possibly know the conformational occurrence of this new mutation in conferring pyrethroid resistance. However, high occurrence of the 1007G allele frequency together with the 1534C allele in that populations provide us an information that the co-occurrence of this new mutation with F1534C could be one of the contributing factors in the pyrethroid resistance in the Kelantan population.
To our current understanding, Lien et al. [42] discovered this new mutation in pyrethroid resistant population from Vietnam which is in line with our study. However, they did not further elucidate the role of this particular mutation. An attempt was made by a research group from United States of America to investigate this mutation in the American population of Ae. aegypti [70]. Unfortunately, this mutation could not be detected from any samples, suggesting kdr allele A1007G, is a part of other geographic mutations similar to V410L [40], G923V, L982W [24], I1011V/M [25], T1520I [39], F1534L [43], D1763Y [36].
Low genetic diversity was observed in the vgsc gene fragment spanning point mutation S989P, A1007G, V1016G and F1534C from all localities, indicate these genes are under selection pressure which might support that knockdown resistance play a role in the pyrethroid resistance within Malaysian population of A. aegypti. This selection might be due to low polymorphism of the vgsc gene fragment with low number of mutational steps between haplotypes. The similar pattern was also observed in the previous report by

Role of metabolic resistance in Aedes aegypti
A major increase of the susceptibility in Balik Pulau, Penang (BP) and a slight increase of the susceptibility in the other locations after the pre-exposure towards PBO suggest that metabolic resistance could be involved in conferring permethrin resistance in the Malaysian population of Ae. aegypti. PBO is widely known as the most frequent synergist used with the combination of the pyrethroid insecticide in controlling the resistant mosquitoes [72]. In general, synergist like PBO can act as an enzyme inhibitor in the metabolic enzyme defence system and acts by binding the PBO metabolites to the enzyme from superfamily group of monooxygenases P45O and non-speci c esterases, hence, resulting in the detoxi cation of enzyme to oxidize. Thus, the effectiveness of the pyrethroid will increase against the pyrethroid resistant mosquitoes [45]. In the present study, the pre-exposure towards PBO might be considered as an ineffective synergist against the resistant Ae. aegypti mosquitoes as it is unable to restore full susceptibility after the exposure towards permethrin. To note, no mortality was observed in the population from Pauh, Panji (Kelantan) after the pre-exposure to PBO, suggesting that target site resistance might play a major role in this population. There might be other possible involvement of the resistance mechanism such as modi cation of the mosquitoes' cuticle within the Malaysian population, as the enhancement of the metabolic enzyme system can lead to the reduced penetration of the insecticides towards the cuticular insects [73,74].

Conclusions
By elucidating the resistance mechanism involved in the Malaysian strain of Aedes aegypti, we can gure out the geographical distribution of the mutations involved alongside with their frequencies. The present study re ects the high occurrence of the reported mutations and the arising of new point mutations in the vgsc gene within the Malaysian permethrin-resistant strain. Henceforth, surveillance and monitoring of these mutations in the vgsc gene should be conducted regularly in order to detect any possible involvement of new point mutations and also the frequency level. This act can instigate an insightfuldecision making factor on the proper usage of insecticide against the target vector which is a part of the integrated resistance management that enables the authorities to control the widespread of the resistance within the target mosquito population in Malaysia. Understanding the resistance mechanism involved in the mosquito population will enlighten the authorities to have better planning in the management of the vector control program.  "-" No individuals died during adult bioassay, thus the genotype and the allelic frequency cannot be determined. N/A indicates that it could not be determined because no individual died for susceptible phenotype. suggests a high risk for the association between phenotype and genotype. Table 2 Results on the association of V1016G allele with the insecticide resistance phenotype.

Declarations
"-" No individuals died during adult bioassay, thus the genotype and the allelic frequency cannot be determined. N/A indicates that it could not be determined because no individual died for susceptible phenotype. suggests a high risk for the association between phenotype and genotype. "-" No individuals died during adult bioassay, thus the genotype and the allelic frequency cannot be determined. N/A indicates that it could not be determined because no individual died for susceptible phenotype. suggests a high risk for the association between phenotype and genotype. Table 4 Association of potential novel mutation A1007G allele with the insecticide resistance phenotype.
"-" No individuals died during adult bioassay, thus the genotype and the allelic frequency cannot be determined. N/A indicates that it could not be determined because no individual died for susceptible phenotype. suggests a high risk for the association between phenotype and genotype.