A new knockdown resistance (kdr) mutation F1534L in Aedes aegypti associated with insecticide resistance

The control of Aedes aegypti borne-infections mainly dengue, chikungunya, yellow fever and Zika virus relies mainly on vector control measures in the absence of specific drugs or vaccines available against these infections. Emergence of insecticide resistance in Ae. aegypti may pose serious threat to the success of insecticide-based vector control programme. Here, we report the presence of multiple knockdown resistance (kdr) mutations present in an Indian Ae. aegypti population including a new mutation F1534L (not reported earlier in Ae. aegypti) which is associated with DDT and pyrethroid resistance. DNA sequencing of partial domain II, III and IV of the voltage gated sodium channel (VGSC) performed on Ae. aegypti collected from Bengaluru, India, revealed the presence of four kdr mutations, i.e., V1016G and S989P in domain II and two alternative kdr mutations F1534C and F1534L in domain III. Allele specific PCR assays (ASPCR) were developed for the detection of kdr mutations V1016G and S989P while a PCR-RFLP based strategy was adopted for the genotyping of all three known mutations in domain III (F1534L, F1534C and T1520I). Genotyping of 572 Ae. aegypti samples collected in 2014 and 2015 revealed a moderate frequency of V1016G/S989P (18.27%) and F1534L (17.48%), a relatively high frequency of F1534C (50.61%) and absence of T1520I in the population. Mutations V1016G and S989P were in complete linkage disequilibrium while they were having negative linkage disequilibrium with kdr alleles F1534C and F1534L. The new mutation F1534L showed significant protection against permethrin, deltamethrin and DDT whereas F1534C showed protection against permethrin and DDT but not against deltamethrin.

3 feed on nonhuman primates and after its domestication, Ae. aegypti has now expanded from Africa and colonized most of the pantropical world [2]. During last five decades, there have been an unprecedented emergence of epidemic arboviral diseases [2] and approximately half of the world population is under the threat of dengue [3,4]. In India, dengue and chikungunya are main arboviral infection with recent introduction of Zika virus (ZIKV). Recently there had been an outbreak of Zika infections in Jaipur city [5], where Ae. aegypti has been incriminated as Zika vector [6].
In absence of specific vaccines or drugs for the treatment of Aedes-borne infections, vector control and personal protection remain only measures to contain the spread of these arboviral infections. Use of insecticide-based control of this vector is a common approach used worldwide. Pyrethroid group of insecticides is of special interest which is being extensively used in long lasting insecticidal nets (LLIN), space spray as well as in household repellent and personal protection. Pyrethroids are preferred group of insecticides due to their low mammalian toxicity, degradability in nature and rapid knockdown effect on insect [7].
However, extensive use of pyrethroids in public health and also in agriculture sector has led to the emergence of resistance against these insecticides in many disease vectors including Ae. aegypti. Several reports of pyrethroid resistance in Ae. aegypti has been recorded from different parts of world mainly in Southeast Asia, Latin Americas and Africa, with such reports not available from India until the year 2015 [8].
Understanding the mechanisms of insecticide resistance in vector populations is crucial for an effective insecticide resistance management. One of the known mechanisms of insecticide resistance in mosquitoes against pyrethroids and DDT is knockdown resistance (kdr) which is conferred by alteration in the target site of action, i.e., the voltage gated sodium channel (VGSC) resulting from non-synonymous mutations. Several kdr mutations have been reported in Ae. aegypti in different parts of world, amongst which mutations at three loci i.e., Iso1011 (IàM/V) and Val1016 (VàG/I) in domain II and F1534 (FàC) in domain III are commonly associated with pyrethroid resistance [9][10][11][12][13][14][15][16][17][18]. Presence of such kdr mutations in the Indian subcontinent was screened in just one northern Indian population (Delhi) where we reported the presence of F1534C and a novel mutation T1520I [18]. This study reports a new mutation F1534L, co-occurring with mutations F1534C, S989P and 4 V1016G, in a southern Indian field population of Ae. aegypti. The new mutation F1534L showed positive association with DDT and pyrethroid resistance. respectively. In most of the cases, the identification of S989 and V1016 mutations were based on 1X sequencing data where forward sequence was used for identification of S989 alleles and reverse sequence was used for V1016 alleles. This was due to presence of ambiguous sequence in downstream sequence resulting from multiple indels present in intron between these two kdr locus. In course of study a total of 294 samples were sequenced for partial domain II of which 178 were homozygous wild for both residues (SS at residue S989 and VV at residue V1016), 92 were heterozygous (SP and VG) and 24 were mutant homozygous (PP and GG). Other two alternative mutations F1534C and F1534L present in domain III were due to T>C substitution on the first position of the codon, leading to Phe (TTC)àLeu (CTC) mutation, and T>G substitution on the second position of the codon leading to Phe (TTC)àCys (TGC) mutations, respectively. Out of the 27 individuals sequenced for domain III, one was homozygous wild for FF (TTC) at residue F1534, seven were homozygotes for CC (TGC), four were homozygotes for LL (CTC), four samples were heterozygotes for each of FC and FL and seven were having mixed bases at first and second position of the codon, i.e., with YKC, which could be either heterozygote for LC (CTC+TGC) or FR (TTC+CGC).

Identification of kdr mutations in
The latter combination was ruled out as sequencing of 15 cloned PCR products, from five such samples (having sequence YKC), revealed the presence of two haplotypes, one with CTC (L) and another with TGC (C). We also observed that haplotype with F1534L mutation had a restriction site for Eco88I (5'-C↓ YCGRG-3'). Therefore, all the seven heterozygote samples with the sequence YKC were subjected to PCR-RFLP with Eco88I and all were partially cleaved indicating the presence of LC (CTC+TGC) heterozygote. DNA sequencing 5 of 12 samples for partial domain IV revealed absence of any non-synonymous mutation including D1794Y reported elsewhere [19]. Additionally, 25 samples were checked for presence of D1794Y using PCR-RFLP following Chang et al., [19] and none were found positive.

Development of PCR-based assay for genotyping of kdr alleles
For genotyping of F1534-kdr alleles, we modified PCR-RFLP developed by Kushwah et al., (2015) [18] where an additional restriction enzyme Eco88I was used for identification of new allele F1534L. For genotyping of S989-and V1016-kdr alleles, we developed allele-specific PCRs (ASPCR) for each locus.

Genotyping of kdr alleles and their linkage association
Genotyping result of kdr alleles at loci F1534, S989 and V1016 carried out on 572 field collected F0 populations are shown in Table 1

Genetic association of kdr alleles with phenotype insecticide resistance
The distribution of individuals with different kdr allele genotypes in respect to F1534, S989 and V1016 loci in dead and alive mosquitoes (F0 and F1) after exposure to 0.75% permethrin (type I pyrethroid), 0.05% deltamethrin (type II pyrethroid) and 4% DDT is shown in Supplementary Table S2. Since mutations present in domain II (S989P and V1016G) were always found in association with wild form in domain III (F1534) and have been reported to have protection with insecticides, we removed data of individual having haplotype FPG from analysis of genetic association of F1534-kdr mutations with phenotypic insecticide resistance.
Similarly, data of individual with CSV and LSV haplotypes were removed while analysing association of S989P/V1016G kdr mutations with phenotypic insecticide resistance. The analysis of genetic association of F1534-kdr alleles with phenotype insecticide resistance is shown in Table 2. Statistical analysis revealed that allele F1534L showed strong protection against permethrin (P <0.0001), moderate protection against deltamethrin (P< 0.01) and very low protection against DDT (P <0.05). Other allele 1534C showed strong protection against permethrin (P <0.0001), low protection against DDT (P <0.001) and no protection against deltamethrin. Mutations 989P and 1016G together have strong protection to permethrin and we could not establish their role against deltamethrin and DDT due to low frequency of alleles at S989 and V1016 locus.

Discussion
Aedes aegypti, a primary vector of dengue, yellow fever, chikungunya, Zika and other arboviral infections has attained global importance due to invasion of this species in different parts of the world from Africa. Currently, with no specific treatment or vaccine available to control transmission of these arboviral infections, vector control remains the mainstay in public health. One of the available potential methods for control of this vector is the use of 7 pyrethroid group of insecticides for space spray and personal protection measure.
Development of resistance against these insecticides is major concern for success of such application. It is therefore desirable to have knowledge on underlying mechanism(s) of insecticide resistance for an effective insecticide resistance management.
Reduced sensitivity of VGSC-the target site for DDT and pyrethroids, is one of the mechanisms of resistance in insects due to conformational changes in protein resulting from one or more mutations leading to amino acid substitution, commonly referred to as knockdown resistance (kdr). In Ae. aegypti, several such mutations are documented, of which F1534C, S989P and V1016G are widely reported kdr mutation and is known to confer resistance against DDT and pyrethroids [20,21,22]. This study reports the presence of four kdr mutations viz. S989P, V1016G, F1534C and F1534L with the latter mutation is being reported for the first time in Ae. aegypti. although its presence in Aedes albopictus has been reported [23,24,25]. The new mutation F1534L has shown significantly higher protection against permethrin (type I pyrethroid), deltamethrin (type II pyrethroid) and DDT in Ae.
aegypti. The discovery of new kdr mutations associated with insecticide resistance is of global concern as this may hamper pyrethroid-based vector control and personal protection measures.
Earlier we conducted a survey of kdr mutations on Delhi population in year 2014 [18], where we found F1534C mutation along with a novel mutation T1520I linked to F1534C but did not find the three mutations being reported in this study, i.e., S989P, V1016G and F1534L.
Similarly, a novel mutation T1520I reported in Delhi was absent from Bengaluru. To ensure that we did not skipped detection of the other three mutations (S989P, V1016G and F1534L) in Delhi population during previous study, we genotyped 184 Ae. aegypti samples collected from Delhi in August 2018. We did not find any of these three mutations in Delhi population.
Thus, there is a contrast difference in distribution of kdr alleles in two different geographical locations which are approximately 1700 km apart. In another part of India (West Bengal, eastern India), there is report of presence of three mutations, F1534C, T1520I and V1016G but the presence of S989P was not investigated in this study [26].
For monitoring of kdr mutations in field condition, we developed a highly specific PCR-RFLP-based assay for simultaneous detection of all the three mutations (total five alleles) reported in domain III-S6 of the VGSC at locus F1534 and T1520. The PCR-RFLP for the 8 identification of all five alleles is advantageous over other PCR-based methods being highly specific due to high sequence-specificity of restriction enzymes. Additionally, in this PCR-RFLP assay, unlike other assays, a single PCR is required for genotyping of all five alleles present at locus F1534 and T1520. For genotyping of domain II-S6 mutations, we developed two allele-specific PCR (ASPCR) assays, one each for S989P and V1016G mutations. Our ASPCRs for genotyping of S989P and V1016G alleles are advantageous over other available PCR assay for these mutations, as our PCR need single assay for each locus while method described by Stenhouse et al., (2013) [27] need two PCR assays for single locus. However, ASPCR being based on single base mismatch is prone to non-specific extension and high degree of optimization is required. ASPCR is sensitive to change of type of reagents and PCR thermal conditioning. Discrepancies were noticed with such PCR based genotyping of F1534 kdr-alleles in an earlier study [28].
Co-occurrence of F15134C with S989 and V1016 may be of serious concern in case all three mutations are present on same haplotype. It has been shown through the site directed mutagenesis that such combination (F1534C+S989P+V1016G) may result in a seriously high degree of resistance against permethrin and deltamethrin (1100-and 90-fold, respectively) [29]. In this study, we found that 989P, 1016G and 1534C/1534L were found together in a single mosquito but always in heterozygous condition. Phasing out of haplotypes revealed that there are just four haplotype FSV, FPG, LSV and CSV present in this population. Thus, 989P and 1016G are always found together but never with 1534C or 1534L. In such a population, a single recombination may lead to production of haplotype CPG or LPG which may have greater impact on insecticide resistance phenotype. Unlike our finding, a small proportion of mosquitoes in Myanmar have shown to have 1534C homozygotes with 1016G homozygotes (2.9%, double mutant) and with homozygous 989P+1016G+1534C (0.98%, triple mutant) suggesting presence of CPG haplotype [30]. Occurrence of such haplotype with three mutations may be selected in presence of insecticide pressure and may pose serious threat to insecticide-based vector control programme.
In this study we found 100% linkage association of 1016G with 989P [22,30,31,32]. Though, such association has been shown in several studies, the frequency of V1016G is reported higher than S989P where V1016G mutation may be found alone but S989P was always linked with V1016G [27,29,30,32]. Similar unidirectional linkage has been shown in domain III in VGSC of Ae. aegypti population, where a novel mutation T1520I (reported in Delhi, 9 India) was always found with F1534C and was suggested to be a compensatory mutation [18].
A disturbing fact we recorded in this study was the non-compliance of HWE for F1534-kdr mutations in Bengaluru. Similar departure from HWE for this locus was also noted in our previous study of Delhi-population [18] and in Grand Cayman Island [33]. It was interesting to note that another mutation T1520I that was linked to F1534C, was in compliance with HWE. While we don't have an explanation to this, probably remains this may be due to the presence of multiple VGSC gene or gene duplication as proposed by Martin et al., [34]. This will need a further investigation.

Mosquito collection
Immatures (eggs, larvae and pupae) Ae. aegypti were collected from domestic and peridomestic breeding sites from Basavangudi area of Bengaluru city (77° 56-57′ E, 12° 92-95′ N) during years 2014 and 2015. Oral informed consent was obtained from the owners of the houses for collection of immature at residential premises. Immature were reared in the laboratory till the emergence into adult (F0). In addition, F1 progenies were also obtained. To get F1 progenies, mosquitoes were fed on blood through artificial membrane (Parafilm®) and eggs were obtained after 72 hrs of blood feeding. Eggs were allowed to hatch in water and larvae were reared in enamel trays with a supplementation of fish-food till pupation. Pupae were removed from the tray and placed in a bowl containing water inside an insect cage (measuring 30 cm X30 cm X 30 cm) for their emergence into adult. Adults were fed on 10% glucose soaked on cotton pad. Insectary was maintained at a temperature of 27±°C, relative humidity (RH) 60-70% and photoperiod of 14h:10h (light:dark) ratio.

Exposure of insecticide to mosquitoes (Bio-assay)
Two-to four-days old sugar fed adult Ae. aegypti female mosquitoes (F0, and F1), were exposed to 0.05% deltamethrin-, 0.75% permethrin-or 4% DDT-impregnated papers (supplied by WHO collaborative centre, Vector Control Research, Universiti Sains, Malaysia) for one-hour following WHO's standard insecticide-susceptibility test guidelines [35]. Following exposure of insecticide, they were transferred to recovery tubes and mortalities were recorded after 24 hours of recovery. Individual dead and alive mosquitoes were kept in 1.5 mL micro centrifuge tubes with a piece of silica gel for DNA isolation and at -20 °C. All bioassays were carried out in a laboratory maintained at 25°C and RH 60-70%.

DNA isolation and sequencing
DNA from individual mosquitoes was isolated following the method described by Livak et al., [36] after removing 1/3rd of the posterior abdomen that carries spermatheca (to avoid contamination of sperm from mating male partner) and stored at 4°C. Some of the mosquitoes were sequenced for domain II, III and IV of the VGSC. Primers used for amplification of domain II, III and IV are shown in Supplementary Table 3

Cloning and sequencing
It was not possible to identify the correct amino acid codon through DNA sequencing in samples which were found heterozygous for two nucleotide base positions i.e., first and second codon of the F1534 residue. We therefore cloned and sequenced five such heterozygous samples to identify the correct codon. PCR product was amplified using primers AekdrF and AekdrR and high fidelity taq DNA polymerase (Phusion® High-Fidelity DNA Polymerase) purified using QIAquick PCR Purification Kit (Qiagen Inc.) and cloned in 11 pGem-T Easy Vector Systems (Promega Corporation) as per vendor's protocol. Competent E. coli cells were transformed with the recombinant DNA and grown on LB-Agar plate.
Positive clones were selected by blue/white screening and amplified using universal primers SP6 and T7. The PCR product of individual clone was sequenced at Macrogen, South Korea, using primers SP6 and T7. Sequences were aligned using Mega5 [37].

Genotyping of domain II kdr alleles (V1016G and S989P)
For genotyping of kdr alleles at residue S989 and V1016, separate allele-specific PCR assays were designed. The list of primers used for ASPCR are shown in Supplementary Table 3.
The PCR conditions for both PCR were identical. The PCR was carried out using Thermo

Genotyping of domain III kdr alleles (T1520I, F1534C and F1534L)
Earlier we reported a PCR-RFLP for the genotyping T1520I and F1534C, where a single PCR product amplified using primers AekdrF and AekdrR were subjected to RFLP using two restriction enzymes BsaBI and SsiI respectively [9]. In order to include RFLP for identification of new mutation F1534L, we searched for F1534L-specific restriction enzyme site using an online tool available at http: //insilico.ehu.es/restriction/two_seq. We found a unique restriction site Eco88I in the DNA sequence with F1534L and therefore for genotyping of all kdr alleles present in domain III, PCR-RFLP method was modified. In modified procedure additional restriction-digestion was performed in a separate tube with Eco88I in addition to BsaBI and SsiI. Additional restriction-digestion reaction mixture (20 μl) contained 5 μl of PCR-amplified product, 2 units of Eco88I enzyme and 1X buffer (Thermo Fisher scientific). This was incubated for 4 hours or overnight at 37°C following electrophoretic run on 2.5% agarose gel. The RFLP product and visualized in gel documentation system. The criteria for scoring of F1534 alleles were modified which are presented in Table 3, while the criterion for scoring of T1520 alleles remain unchanged.

Statistical analysis:
Hardy-Weinberg equilibrium (HWE) of kdr-alleles in a population was tested using Fisher's exact test. Phasing of haplotypes and estimation of their frequencies were performed using Arlequin 3.5 software [38]. Association of kdr genotypes with phenotype resistance was tested using co-dominant model and test of significance was estimated using Fisher's exact test and odds ratio (OR).
The Wright's inbreeding coefficient was calculated using formula F = (He-Ho)/He, where 'He' is expected heterozygosity and 'Ho' is observed heterozygosity.