Acta Tropica 148 (2015) 137–141
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Involvement of metabolic resistance and F1534C kdr mutation in the pyrethroid resistance mechanisms of Aedes aegypti in India R. Muthusamy, M.S. Shivakumar ∗ Molecular Entomology Laboratory, Department of Biotechnology, School of Biosciences, Periyar University, Periyar Palkalai Nagar, Salem 636011, Tamil Nadu, India
a r t i c l e
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Article history: Received 10 March 2015 Received in revised form 22 April 2015 Accepted 26 April 2015 Available online 2 May 2015 Keywords: Permethrin resistance Detoxification enzymes Aedes aegypti Target site insensitivity Synergist
a b s t r a c t Pesticide resistance poses a serious problem for worldwide mosquito control programs. Resistance to insecticides can be caused by an increased metabolic detoxification of the insecticide and/or by target site insensitivity. In the present study, we estimated the tolerance of Indian Aedes aegypti populations using adult bioassays that revealed high resistance levels of the field populations to permethrin (RR-6, 5.8 and 5.1 folds) compared to our susceptible population. Enzymatic assays revealed increased activities of glutathione S-transferase and carboxylesterase enzymes in the field populations comparatively to the susceptible population. PBO synergist assays did not confirm that cytochrome P450 monooxygenase metabolic detoxification acted as a major cause of resistance. Hence the role of target site resistance was therefore investigated. A single substitution Phe1534Cys in the voltage gated sodium channel was found in domain III, segment 6 (III-S6) of the resistance populations (allele frequency = 0.59, 0.51 and 0.47) suggesting its potential role in permethrin resistance in A. aegypti. © 2015 Published by Elsevier B.V.
1. Introduction Aedes aegypti is one of the main vectors of dengue fever in several countries such like Asia, Africa and India (WHO, 1999). During the year of 2012, 12,826 dengue cases were recorded, including 66 reported deaths in Tamil Nadu, India (NVBDCP, 2012). Control of A. aegypti populations consists primarily in eliminating the larval breeding sites by reducing standing water sources (wastes, tyres, cleaning of cement tanks, etc.) and/or treating with insecticides. For larval control, the World Health Organization Pesticide Evaluate Scheme (WHOPES) recommends ten different compounds including organophosphates and neocotinoids, Insect Growth Regulators such as chitin synthesis inhibitors, juvenile hormone analogs, and Bacillus thuringiensis var israelensis. For adult control, people rely mainly on the use of pyrethroids and organophosphates (WHOPES, 2006). In the last two decades, pyrethroid has been increasingly used as an alternative to organophosphate insecticides owing to its rapid mode of action and low toxicity to mammals (Elliott, 1977). Though, after routine use, many field mosquito populations have developed resistance to synthetic pyrethroid compounds (Jirakanjanakit et al., 2007; Chuaycharoensuk et al., 2011), pyrethroids are
∗ Corresponding author. Tel.: +91 4272345766. E-mail address: [email protected] (M.S. Shivakumar). http://dx.doi.org/10.1016/j.actatropica.2015.04.026 0001-706X/© 2015 Published by Elsevier B.V.
extensively engaged in and around households, even for pest control and mosquito protection (Ranson et al., 2011). Pyrethroid resistance in A. aegypti, as well as in other vector species, may arise through two major mechanisms. The first mechanism consists of metabolic or enzymatic resistance. In this case resistance is achieved through an increased metabolic detoxification of the insecticide and/or by target site insensitivity. They work by rapidly metabolizing and detoxifying the insecticide or by sequestration, therefore preventing the insecticide from binding its target site (Hemingway et al., 2004). The large number of enzymes present in these enzymatic families makes their study difficult but more and more studies suggested that they can play an important role in pyrethroid resistance (Feyereisen, 1995; Muthusamy et al., 2014). The second mechanism is knockdown resistance or kdr mutations, which is resulting from insecticide selection that is not overcome by metabolic inhibitors (Casida et al., 1983). This frequently consists of the single point mutation within the genes encoding protein that are targeted by insecticides. Kdr resistance is one of the foremost mechanisms of pyrethroid resistance, which target the transmembrane voltage gated sodium channel (NaV ) from the insect nervous system and responsible for reduced neuronal sensitivity to pyrethroid insecticides, is known as knockdown resistance (kdr) (Dong, 2007). The voltage-gated sodium channel is composed of four homologous domains (domain I–IV), each domains containing six hydrophobic segments (Segment 1–6) (Frank and Catterall, 2003). Because the sodium channel proteins
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are conserved among invertebrates, hence small changes are permissive without decreasing its physiological role (Ffrench-Constant et al., 1998). A series of mutations have been identified in the NaV gene of different orders of insects that affect pyrethroid susceptibility, thus being referred to as ‘knockdown resistance’ or kdr mutations (Martins and Valle, 2012). These kdr mutations may lead to conformational changes in the whole NaV channel that maintain its physiological role without permitting the insecticide action (Reilly et al., 2006). In insects, the most common occurrence of kdr mutation is the 1014 Leu/Phe amino acid substitution, followed by the Leu/Ser substitution in the same position, in Culex and Anopheles mosquitoes (Davies et al., 2007). Though several mutations have been identified in natural populations at AaNaV (Brengues et al., 2003; Rodriguez et al., 2007), only the Val1016Ile and Phe1534Cys substitutions were clearly related to the loss of pyrethroid susceptibility (Harris et al., 2010). These sites are present in the IIS6 and IIIS6 regions of the sodium channels that are known to be involved in the interaction with pyrethroids (Reilly et al., 2006). Marcombe et al. (2012) have found that elevated activity of esterase, glutathione transferase and mixed function oxidase were involved in pyrethroid resistance coupled with high frequencies of Phe1534Cys kdr mutation in Grand Cayman and Martinique populations of A. aegypti. The present study investigates the permethrin resistance mechanisms involving both detoxification enzymes and Phe1534Cys Kdr mutation in the voltage gated sodium channel gene of A. aegypti from India. 2. Material and methods 2.1. Mosquito population sampling and rearing A. aegypti were collected from artificial containers situated within three localities Salem (SLM), Namakkal (NKL) and Dharmapuri (DPI) between 11◦ 39 , 11◦ 13 and12◦ 08 N Latitude and 78◦ 16 , 78◦ 16 and 78◦ 13 E Longitude from TamilNadu, India in February 2012. Sampled locations consisted of detached housing, temples, and schools. Field collected populations were established between 5 and 10 larval collection sites. Mosquitoes from the same locality were pooled and maintained under a 12:12 light:dark cycle at 27 ± 1◦ C with relative humidity of 70 ± 5% in the laboratory. Females were blood-fed on chick in the mosquito rearing cages 60 × 60 cm and adults obtained from the F1 offspring were used for bioassays, biochemical and molecular studies. The susceptible mosquito colony was obtained from the National Centre for Disease Control (NCDC) Mettupalayam and used for all experiments. 2.2. Preparation of piperonyl butoxide (PBO) impregnated papers Piperonyl butoxide (PBO; 2-(2-butoxyethoxy) ethyl 6propylpiperonyl ether, Bangalore, India) impregnated filter papers were prepared with as described by Herath and Davidson (1981) with a ratio of 1:5 (permethrin:PBO) (Kumar et al., 1991). A similar procedure was applied for the control except that the paper was treated with acetone alone. 2.3. WHO adult bioassays The adult bioassays were carried out according to the WHO standard test protocol (World Health Organization, 1981). Fifteen sucrose fed 2–3 day old female mosquitoes per replicate were used for this bioassay. This test was divided in to three different bioassays with similar test procedure using different types of impregnate papers. The first bioassay comprised of mosquitoes only exposed to impregnated papers with diagnostic dosage of permethrin (0.75%) recommended by WHO. In the second bioassay, mosquitoes were
exposed to PBO impregnated papers alone. In the third bioassay consisted of mosquitoes were exposed to PBO before being exposed to permethrin (0.75%). As a control, mosquitoes were exposed to papers impregnated with acetone only. All mosquitoes were exposed to the diagnostic dosage of permethrin and/or PBO for 1 h. Cumulative mortality counts were recorded every minute during the exposure time. After the exposure period, the mosquitoes were held for a 24-h recovery period before the mortality was recorded again. Sucrose solution was provided for the mosquitoes. If the control mortality was between 5% and 20%, the percentage of mortality was corrected by Abbott’s (1925) formula. LT50 was calculated using probit analysis (Finney, 1977). Resistance ratios of the all field populations of A. aegypti were calculated using the following formula: resistance ratio (RR) = LT50 of the field population/LT50 of the laboratory Sus population. For the synergistic effect of PBO, the following formula was used: synergistic factor (SR) = LT50 of the permethrin/LT50 of the PBO + permethrin. All survivors were collected and immediately used for the measurement of enzyme activities. 2.4. Biochemical assays Carboxylesterases (ESTs), glutathione S-transferases (GSTs), mixed function oxidase (MFOs) and acetylcholinesterase (AChE) activities were determined from thirty individual 3 days-old F1 female mosquitoes according to the methods described by Brogdon et al. (1989) and Penilla et al. (1998). The substrate utilized in each assay included ␣- and  naphthyl acetate for ␣-Est and -Est respectively, CDNB (1-chloro 2,4-dinitrobenzene) for GSTs, TMBZ (3,3 ,5,5 -tetramethyl-benzidine dihydrochloride) for MFO. Total protein quantification of mosquito homogenates was performed as per the method of Lowry et al. (1951) with bovine serum albumin as the standard protein in order to normalize enzyme activity levels by protein content. The enzyme activities in the field population and susceptible populations were compared using Kruskal–Wallis test using Prism Graph Pad software (Version 6.0). 2.5. A. aegypti sodium channel partial sequencing Total Genomic DNA was extracted from individual female mosquitoes from each locality and susceptible population by using a HiPurATM insect DNA purification kit (Himedia), as recommended by manufacturer. The polymerase chain reactions (PCR) were carried out to amplify a region of 350 bp of the exon 31 of the voltage-gated sodium channel, as described by Harris et al. (2010), which encode domain III, subunit 6. This region allows detection of Phe1534Cys kdr mutation AaEx31P (F-5 TCGCGGGAGGTAAGTTATTG-3 and AaEx31Q R-5 GTTGATGTGCGATGGAAATG-3 ). The PCR was carried in a 20 L reaction volume, which contains 10 L of 2× PCR Taq Mix [(SRL-Sisco Research Laboratories PVT. LTD.) (20 mM Tris–HCl (pH 8.0), 100 mM KCl, 3 mM MgCl2 , 400 M dNTP, 0.1 U/L Taq DNA Polymerase)] 4 L of 0.4 M each forward and reverse primers, 4 L of nuclease free water and finally 2 L of total genomic DNA extracted from a single mosquito as template. Cycling conditions were as follows: Initial denaturation of 95 ◦ C for 4 min followed by 30 cycles of 94 ◦ C for 60 s, 62 ◦ C for 30 s, and 72 ◦ C for 1 min, then a final elongation at 72 ◦ C for 7 min. The PCR products were visualized by gel electrophoresis and then sequenced in both directions using the ABI 3730 Applied Biosystem. The partial sequence of A. aegypti sodium channel gene were compared with those of other sodium channel sequences deposited in GenBank using the “BLAST-N” tools available on the National Center for Biotechnology Information (NCBI) website. The aminoacid sequence of A. aegypti partial sodium channel was deduced from the corresponding sequence using the translation tool at
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Table 1 Susceptibility of adult Aedes aegypti mosquitoes exposed to permethrin alone and piperonyl butoxide (PBO) + permethrin. Exposure
Permethrin
Population
LT50 (min) 95%CL
Mortality (%)
RR
Permethrin + PBO (synergist) LT50 (min) 95%CL
Mortality (%)
SR
DPI SLM NKL Sus
55.45 (53.23–56.00) 54.43 (51.45–56.57) 47.37 (45.35–49.32) 9.23 (8.12–11.34)
48.5 48.3 50.7 92.2
6.0 5.8 5.1 –
26.45 (25.22–27.25) 23.55 (22.24–24.13) 22.40 (23.23–24.43) 05.55 (07.32–09.93)
59.2 62.7 68.0 97.5
2.0 2.3 2.1 –
PBO = piperonyl butoxide (synergist); RR = resistance ratio; SR = synergistic ratio; CL = confidence limit (95%).
the ExPASy Proteomics website (http://www.expasy.org/tools/dna. html). Further for genotyping tests Hardy Weinberg equilibrium was performed using Genepop version 4.2. 2.6. Kdr genotyping An amino acid substitution, F1534C, was detected in the sequenced regions of the sodium channel of A. aegypti from DPI, SLM and NKL populations. For this assay, two internal allele-specific primers AaEx31 F: 5 -CCTCTACTTTGTGTTCTTCATCATCTT-3 and AaEx31 R: 5 -GCGTGAAGAACGACCCGC-3 were used. These primers flanking within the region of 350 bp primer sequences and the PCR was performed as described above. Approximately 100 mosquitoes were used for genotyping test. 3. Results and discussion The LT50 values for the three field populations were 55.45 min from the DPI, 54.43 min from the SLM and 47.37 min from NKL compared to the laboratory population 9.23. Among the field populations, DPI and SLM were slightly more resistant compared to NKL (RR 5.1). Furthermore, the resistance ratios for the
A
C
permethrin exposed populations ranged from 5.8 to 6.0 folds, respectively (Table 1). These results suggested that A. aegypti from field populations were resistant to permethrin. Exposure to PBO synergist, prior to permethrin enhanced the effectiveness of permethrin by reducing the LT50 values by 2–2.3 times compared to the LT50 values of permethrin alone exposed mosquitoes. Indicating a significant role of P450 in the resistance of adults to permethrin. Similar results were observed by WanNorafikah et al. (2010) where they reported that the range of RR for permethrin-exposed A. aegypti from Taman Melati, Vista Angkasa and Desa Tasik field strains were 4.47, 4.35 and 4.28 folds respectively. Selvi et al. (2006) reported 0.5–1.7 fold RR for permethrin selected A. aegypti strains. Compared to other studies, our work showed resistance ratios 1.5–4 folds higher with permethrin suggesting an increase of resistance within the last few years. Moreover the lower LT50 values recorded for all PBO + permethrinexposed population suggested that the permethrin resistance in these mosquitoes could be linked with an increase activity of mixed function oxidases. The measurement of global detoxification enzyme activities showed a higher level of alpha-esterases in DPI, SLM and NKL compared to the susceptible population (1.5-fold, 1.0-fold and
B
D
Fig. 1. Comparison of detoxification enzymes activities between the permethrin resistant and susceptible population (A) ␣-esterase and -esterase (B) activities were measured with the naphthyl acetate method and expressed as mol ␣- or -naphthol produced/mg protein/min (±SD). (C) GST activities were measured with the CDNB and expressed as mol of conjugated CDNB/mg protein/min (±SD). (D) P450 activities were measured with the TMBZ and expressed as TMBZ/mg protein/min (±SD). Statistical comparison of enzyme activities between the field and susceptible population were performed with a Kruskal–Wallis test (* p < 0.05).
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Cys
CGTCCTCGATCCTTCCAGGTGGGAAAGCAGCCGATTCGCGAGACCAACATCTACATGTAC 60 R P R S F Q V G K Q P I R E T N I Y M Y
Phe
CGTCCTCGATCCTTCCAGGTGGGAAAGCAGCCGATTCGCGAGACCAACATCTACATGTAC 120 R P R S F Q V G K Q P I R E T N I Y M Y
Cys
CTCTACTTTGTGTTCTTCATCATCTGCGGGTCGTTCTTCACGCTGAATCTGTTCATCGGT 120 L Y F V F F I I C G S F F T L N L F I G
Phe
CTCTACTTTGTGTTCTTCATCATCTTCGGGTCGTTCTTCACGCTGAATCTGTTCATCGGT 180 L Y F V F F I I F G S F F T L N L F I G
Cys
GTCATCATCGACAACTTCAACGAGCAGAAGAAGAAAGCCGGTGGCTCACTGGAAATGTTC 180 V I I D N F N E Q K K K A G G S L E M F
Phe
GTCATCATCGACAACTTCAACGAGCAGAAGAAGAAAGCCGGTGGCTCACTGGAAATGTTC 240 V I I D N F N E Q K K K A G G S L E M F
Cys
ATGACGGAGGATCAGAAAAAGTACTACAACGCCATGAAAAAGATGGGCTCGAAGAAGCCG 240 M T E D Q K K Y Y N A M K K M G S K K P
Phe
ATGACGGAGGATCAGAAAAAGTACTACAACGCCATGAAAAAGATGGGCTCGAAGAAGCCG 300 M T E D Q K K Y Y N A M K K M G S K K P
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Cys
CTGAAAGCTATTCCACGGCCTAGGGTAAGGCATTTCCATCGCACATCAAC 290 L K A I P R P R V R H F H R T S
Phe
CTGAAAGCTATTCCACGGCCTAGGGTAAGGCATTTCCATCGCACATCAAC 350 L K A I P R P R V R H F H R T S
||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Fig. 2. Partial sequence of the Aedes aegypti sodium channel from susceptible population (1534Phe), compared with the field populations (1534Cys). The amino acid translation is given below the sequence data and the mutation leading to Phe1534Cys (TTC-TGC) substitution is highlighted in black color.
0.8-fold with p < 0.05). For beta esterases, although all three resistant populations showed higher activities, only DPI is higher (0.8-fold with p < 0.05). The same profile is shown for GST activities (2.0-fold with p < 0.05 respectively). Finally, the three field populations did not show any significant increased MFO activities compared to the susceptible one (Fig. 1). However biochemical assays only provide an overall estimate of MFO enzymatic activities and there might be a chance of other P450s may involve in this resistance mechanism. Previous reports of Djouaka et al. (2008) revealed that though the reduced P450 enzymatic activity in Anopheles gambiae was found, microarray analysis suggest that a multiple P450 genes were involved in pyrethroid resistance mechanism from Southern Benin and Nigeria. Elevated GST levels have also been frequently associated with insect resistance to pyrethroid insecticides (Enayati et al., 2005; Lumjuan et al., 2005). Our biochemical data also suggest the role GSTs in insecticide resistance in the DPI population. Sequencing of the voltage-gated sodium channel identified single amino acid substitutions in DPI, SLM and NKL populations compared with the susceptible population (Fig. 2). The substitution at codon 1534 (Phe to Cys kdr polymorphic site) where a single base pair substitution changes the codon from TTC to TGC resulting in a phenylalanine to cysteine substitution in domain III, subunit 6 (numbering of residues is based on the reference sequence from Musca domestica Williamson et al. (1996) and exon assignment is based on the Chang et al. (2009) annotation of the A. aegypti sodium channel gene). As the Phe1534Cys kdr mutation was detected in the field populations with 321 bp fragments, the sequence were submitted to GenBank (Accession numbers: KM519597 KM519598). Similar results were found in Linss et al. (2014) study were he found only single kdr mutation at the site of 1534 leading to phenylalanine to cysteine replacement revealed pyrethroid resistance in A. aegypti Brazilian natural populations. Substitutions in an alternative phenylalanine residue in IIIS6, F1538, have been associated with pyrethroid resistance in the southern cattle tick, Boophilus microplus (He et al., 1999) and the two-spotted spider
Table 2 Genotypes and resistance allele frequencies of three populations of Aedes aegypti for the F1534C mutationsa Populations
DPI SLM NKL
1534 F/F
F/C
C/C
Freq C
P value
10 7 7
65 40 35
52 30 27
0.59 0.51 0.47
0.653 0.557 0.004
a Tests for Hardy Weinberg equilibrium were applied to the data and the P values are shown. The final row shows the combined analysis for all three populations.
mite, Tetranychus urticae (Tsagkarakou et al., 2009). Additionally, a gene duplication event was recently described in the AaNaV of natural populations and in a laboratory strain selected for pyrethroid resistance (Martins et al., 2013). Although there are at least seven different mutations described in the AaNaV , only that corresponding to the 1534 positions is clearly related to resistance; which is placed in a domain of the sodium channel that interacts directly with the pyrethroid molecule (Du et al., 2013). The tetraplex PCR assay was used to detect F1534C frequency in field populations. Thirty mosquitoes from each area (DPI, SLM and NKL) were genotyped at F1534C loci. The frequency of the 1534C allele was 0.59, 0.51 and 0.47 respectively (Table 2). Previous study of Yanola et al. (2011) revealed that the presence of 1534C mutation with allele frequency of 0.77 could be associated with permethrin resistance in A. aegypti Thailand populations. 4. Conclusions In conclusion, permethrin resistance grows fast and it means that new strategies are needed before having the resistance completely filed in the populations. Hence care should be taken while permethrin used in the field control of Aedes mosquito populations. It is not surprising that pyrethroids are the most employed insecticide worldwide for use in nets, curtains and households (Zaim et al., 2000). Although many novel control strategies are
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being tested in the field, insecticides will particularly play an important role for vector control program. Hence the knowledge about pyrethroid metabolic resistance and commonly occurred kdr mutations in natural populations are crucial for preventing the effectiveness of pyrethroid compound as a sustainable tool against A. aegypti. Conflict of interest We have no conflict of interest. Acknowledgements We acknowledge the financial support received from DST-SERB Young Scientist Program, New Delhi, India. (Ref. No. SR/FT/LS23/2011). Authors’ also thank Biotechnology Department, Periyar University, TamilNadu, India for their support to carry out this work. The authors thank Dr. Rodolphe Poupardin Biology Centre AS CR, v. v. i., Institute of Entomology, Branisovska 31, CZ-370 05 Ceske Budejovice, Czech Republic for editing the English manuscript. References Abbott, W.S., 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 18, 265–267. Brengues, C., Hawkes, N.J., Chandre, F., McCarroll, L., Duchon, S., Guillet, P., Manguin, S., Morgan, J.C., Hemingway, J., 2003. Pyrethroid and DDT cross-resistance in Aedes aegypti is correlated with novel mutations in the voltage-gated sodium channel gene. Med. Vet. Entomol. 17, 87–94. Brogdon, W.G., 1989. Biochemical resistance detection: an alternative to bioassay. Parasitol. Today 5, 56–60. Casida, J.E., Gammon, D.W., Glickman, A.H., Lawrence, L.J., 1983. Mechanisms of selective action of pyrethroid insecticides. Annu. Rev. Pharmacol. Toxicol. 23, 413–438. Chang, C., Shen, W.K., Wang, T.T., Lin, Y.H., Hsu, E.L., Dai, S.M., 2009. A novel amino acid substitution in a voltage-gated sodium channel is associated with knockdown resistance to permethrin in Aedes aegypti. Insect Biochem. Mol. Biol. 39, 272–278. Chuaycharoensuk, T., Juntarajumnong, W., Boonyuan, W., Bangs, M.J., Akratanakul, P., Thammapalo, S., Jirakanjanakit, N., Tanasinchayakul, S., Chareonviriyaphap, T., 2011. Frequency of pyrethroid resistance in Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in Thailand. J. Vector Ecol. 36, 204–212. Davies, T.G., Field, L.M., Usherwood, P.N., Williamson, M.S., 2007. A comparative study of voltage-gated sodium channels in the Insecta: implications for pyrethroid resistance in Anopheline and other Neopteran species. Insect Mol. Biol. 16, 361–375. Djouaka, R.F., Bakare, A.A., Coulibaly, O.N., Akogbeto, M.C., Ranson, H., Hemingway, J., Strode, C., 2008. Expression of the cytochrome P450s, CYP6P3 and CYP6M2 are significantly elevated in multiple pyrethroid resistant populations of Anopheles gambiae s.s. from Southern Benin and Nigeria. BMC Genomics 9, 538. Dong, K., 2007. Insect sodium channels and insecticide resistance. Invert. Neurosci. 7, 17–30. Du, Y., Nomura, Y., Satar, G., Hu, Z., Nauen, R., He, S.Y., Zhorov, B.S., Dong, K., 2013. Molecular evidence for dual pyrethroid-receptor sites on a mosquito sodium channel. Proc. Natl. Acad. Sci. U.S.A. 110, 11785–11790. Elliott, M.,1977. Synthetic pyrethroids. In: “Synthetic Pyrethroids” ACS Symposium Series 42. ACS, Washington, DC, pp. 1–28. Enayati, A.A., Ranson, H., Hemingway, J., 2005. Insect glutathione transferases and insecticide resistance. Insect Mol. Biol. 14 (1), 3–8. Feyereisen, R., 1995. Molecular biology of insecticide resistance. Toxicol. Lett. 82–83, 83–90. Ffrench-Constant, R.H., Pittendrigh, B., Vaughan, A., Anthony, N., 1998. Why are there so few resistance-associated mutations in insecticide target genes. Philos. Trans. R. Soc. Lond. B: Biol. Sci. 353, 1685–1693. Finney, D.J., 1977. Probit Analysis: A Statistical Treatment of the Sigmoid Response Curve. Cambridge University Press, London, NY/Melbourne, pp. 333. Frank, H.Y., Catterall, W.A., 2003. Overview of the voltage-gated sodium channel family. Genome Biol. 4, 207. Harris, A.F., Rajatileka, S., Ranson, H., 2010. Pyrethroid resistance in Aedes aegypti from Grand Cayman. Am. J. Trop. Med. Hyg. 83, 277–284. He, H.Q., Chen, A.C., Davey, R.B., Ivie, G.W., George, J.E., 1999. Identification of a point mutation in the para-type sodium channel gene from a pyrethroid-resistant cattle tick. Biochem. Biophys. Res. Commun. 261, 558–651. Hemingway, J., Hawkes, N.J., McCarroll, L., Ranson, H., 2004. The molecular basis of insecticide resistance in mosquitoes. Insect Biochem. Mol. Biol. 34, 653–665.
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Pyrethroid resistance in African anopheline mosquitoes: what are the implications for malaria control. Trends Parasitol. 27 (2), 91–98. Reilly, A.O., Khambay, B.P., Williamson, M.S., Field, L.M., Wallace, B.A., Davies, T.G., 2006. Modelling insecticide-binding sites in the voltage-gated sodium channel. Biochem. J. 396, 255–263. Rodriguez, K.S., Marquez, L.U., Rajatileka, S., Moulton, M., Flores, A.E., Salas, I.F., Bisset, J., Rodriguez, M., Mccall, P.J., Donnelly, M.J., Ranson, H., Hemingway, J., Black, W.C., 2007. A mutation in the voltage-gated sodium channel gene associated with pyrethroid resistance in Latin American Aedes aegypti. Insect Mol. Biol. 16, 785–798. Selvi, S., Edah, M.A., Nazni, W.A., Lee, H.L., Azahari, A.H., 2006. The development of resistance and susceptibility of Aedes aegypti larvae and adult mosquitoes against selection pressure to malathion, permethrin and temephos insecticides and its cross-resistance relationship against propoxur. Malays. J. Sci. 25, 1–13. Tsagkarakou, A., Van Leeuwen, T., Khajehali, J., Ilias, A., Grispou, M., Williamson, M.S., Tirry, L., Vontas, J., 2009. Identification of pyrethroid resistance associated mutations in the para sodium channel of the two-spotted spider mite Tetranychus urticae (Acari: Tetranychidae). Insect Mol. Biol. 18, 583–593. Wan-Norafikah, O., Nazni, W.A., Lee, H., Zainol-Ariffin, P., Sofian-Azirun, M., 2010. Permethrin resistance in Aedes aegypti (Linnaeus) collected from Kuala Lumpur, Malaysia. J. Asia-Pac. Entomol. 13, 175–182. WHOPES, 2006. Pesticides and Their Application for the Control of Vectors and Pests of Public Health Importance (WHO/CDS/NTD/WHOPES/GCDPP/2006.1). World Health Organization, Geneva. Williamson, M.S., Martinez-Torres, D., Hick, C.A., Devonshire, A.L., 1996. Identification of mutations in the housefly para-type sodium channel gene associated with knockdown resistance (kdr) to pyrethroid insecticides. Mol. Gen. Genet. 252, 51–60. World Health Organization, 1981. 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Involvement of metabolic resistance and F1534C kdr mutation in the pyrethroid resistance mechanisms of Aedes aegypti in India R. Muthusamy, M.S. Shivakumar ∗ Molecular Entomology Laboratory, Department of Biotechnology, School of Biosciences, Periyar University, Periyar Palkalai Nagar, Salem 636011, Tamil Nadu, India
a r t i c l e
i n f o
Article history: Received 10 March 2015 Received in revised form 22 April 2015 Accepted 26 April 2015 Available online 2 May 2015 Keywords: Permethrin resistance Detoxification enzymes Aedes aegypti Target site insensitivity Synergist
a b s t r a c t Pesticide resistance poses a serious problem for worldwide mosquito control programs. Resistance to insecticides can be caused by an increased metabolic detoxification of the insecticide and/or by target site insensitivity. In the present study, we estimated the tolerance of Indian Aedes aegypti populations using adult bioassays that revealed high resistance levels of the field populations to permethrin (RR-6, 5.8 and 5.1 folds) compared to our susceptible population. Enzymatic assays revealed increased activities of glutathione S-transferase and carboxylesterase enzymes in the field populations comparatively to the susceptible population. PBO synergist assays did not confirm that cytochrome P450 monooxygenase metabolic detoxification acted as a major cause of resistance. Hence the role of target site resistance was therefore investigated. A single substitution Phe1534Cys in the voltage gated sodium channel was found in domain III, segment 6 (III-S6) of the resistance populations (allele frequency = 0.59, 0.51 and 0.47) suggesting its potential role in permethrin resistance in A. aegypti. © 2015 Published by Elsevier B.V.
1. Introduction Aedes aegypti is one of the main vectors of dengue fever in several countries such like Asia, Africa and India (WHO, 1999). During the year of 2012, 12,826 dengue cases were recorded, including 66 reported deaths in Tamil Nadu, India (NVBDCP, 2012). Control of A. aegypti populations consists primarily in eliminating the larval breeding sites by reducing standing water sources (wastes, tyres, cleaning of cement tanks, etc.) and/or treating with insecticides. For larval control, the World Health Organization Pesticide Evaluate Scheme (WHOPES) recommends ten different compounds including organophosphates and neocotinoids, Insect Growth Regulators such as chitin synthesis inhibitors, juvenile hormone analogs, and Bacillus thuringiensis var israelensis. For adult control, people rely mainly on the use of pyrethroids and organophosphates (WHOPES, 2006). In the last two decades, pyrethroid has been increasingly used as an alternative to organophosphate insecticides owing to its rapid mode of action and low toxicity to mammals (Elliott, 1977). Though, after routine use, many field mosquito populations have developed resistance to synthetic pyrethroid compounds (Jirakanjanakit et al., 2007; Chuaycharoensuk et al., 2011), pyrethroids are
∗ Corresponding author. Tel.: +91 4272345766. E-mail address: [email protected] (M.S. Shivakumar). http://dx.doi.org/10.1016/j.actatropica.2015.04.026 0001-706X/© 2015 Published by Elsevier B.V.
extensively engaged in and around households, even for pest control and mosquito protection (Ranson et al., 2011). Pyrethroid resistance in A. aegypti, as well as in other vector species, may arise through two major mechanisms. The first mechanism consists of metabolic or enzymatic resistance. In this case resistance is achieved through an increased metabolic detoxification of the insecticide and/or by target site insensitivity. They work by rapidly metabolizing and detoxifying the insecticide or by sequestration, therefore preventing the insecticide from binding its target site (Hemingway et al., 2004). The large number of enzymes present in these enzymatic families makes their study difficult but more and more studies suggested that they can play an important role in pyrethroid resistance (Feyereisen, 1995; Muthusamy et al., 2014). The second mechanism is knockdown resistance or kdr mutations, which is resulting from insecticide selection that is not overcome by metabolic inhibitors (Casida et al., 1983). This frequently consists of the single point mutation within the genes encoding protein that are targeted by insecticides. Kdr resistance is one of the foremost mechanisms of pyrethroid resistance, which target the transmembrane voltage gated sodium channel (NaV ) from the insect nervous system and responsible for reduced neuronal sensitivity to pyrethroid insecticides, is known as knockdown resistance (kdr) (Dong, 2007). The voltage-gated sodium channel is composed of four homologous domains (domain I–IV), each domains containing six hydrophobic segments (Segment 1–6) (Frank and Catterall, 2003). Because the sodium channel proteins
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are conserved among invertebrates, hence small changes are permissive without decreasing its physiological role (Ffrench-Constant et al., 1998). A series of mutations have been identified in the NaV gene of different orders of insects that affect pyrethroid susceptibility, thus being referred to as ‘knockdown resistance’ or kdr mutations (Martins and Valle, 2012). These kdr mutations may lead to conformational changes in the whole NaV channel that maintain its physiological role without permitting the insecticide action (Reilly et al., 2006). In insects, the most common occurrence of kdr mutation is the 1014 Leu/Phe amino acid substitution, followed by the Leu/Ser substitution in the same position, in Culex and Anopheles mosquitoes (Davies et al., 2007). Though several mutations have been identified in natural populations at AaNaV (Brengues et al., 2003; Rodriguez et al., 2007), only the Val1016Ile and Phe1534Cys substitutions were clearly related to the loss of pyrethroid susceptibility (Harris et al., 2010). These sites are present in the IIS6 and IIIS6 regions of the sodium channels that are known to be involved in the interaction with pyrethroids (Reilly et al., 2006). Marcombe et al. (2012) have found that elevated activity of esterase, glutathione transferase and mixed function oxidase were involved in pyrethroid resistance coupled with high frequencies of Phe1534Cys kdr mutation in Grand Cayman and Martinique populations of A. aegypti. The present study investigates the permethrin resistance mechanisms involving both detoxification enzymes and Phe1534Cys Kdr mutation in the voltage gated sodium channel gene of A. aegypti from India. 2. Material and methods 2.1. Mosquito population sampling and rearing A. aegypti were collected from artificial containers situated within three localities Salem (SLM), Namakkal (NKL) and Dharmapuri (DPI) between 11◦ 39 , 11◦ 13 and12◦ 08 N Latitude and 78◦ 16 , 78◦ 16 and 78◦ 13 E Longitude from TamilNadu, India in February 2012. Sampled locations consisted of detached housing, temples, and schools. Field collected populations were established between 5 and 10 larval collection sites. Mosquitoes from the same locality were pooled and maintained under a 12:12 light:dark cycle at 27 ± 1◦ C with relative humidity of 70 ± 5% in the laboratory. Females were blood-fed on chick in the mosquito rearing cages 60 × 60 cm and adults obtained from the F1 offspring were used for bioassays, biochemical and molecular studies. The susceptible mosquito colony was obtained from the National Centre for Disease Control (NCDC) Mettupalayam and used for all experiments. 2.2. Preparation of piperonyl butoxide (PBO) impregnated papers Piperonyl butoxide (PBO; 2-(2-butoxyethoxy) ethyl 6propylpiperonyl ether, Bangalore, India) impregnated filter papers were prepared with as described by Herath and Davidson (1981) with a ratio of 1:5 (permethrin:PBO) (Kumar et al., 1991). A similar procedure was applied for the control except that the paper was treated with acetone alone. 2.3. WHO adult bioassays The adult bioassays were carried out according to the WHO standard test protocol (World Health Organization, 1981). Fifteen sucrose fed 2–3 day old female mosquitoes per replicate were used for this bioassay. This test was divided in to three different bioassays with similar test procedure using different types of impregnate papers. The first bioassay comprised of mosquitoes only exposed to impregnated papers with diagnostic dosage of permethrin (0.75%) recommended by WHO. In the second bioassay, mosquitoes were
exposed to PBO impregnated papers alone. In the third bioassay consisted of mosquitoes were exposed to PBO before being exposed to permethrin (0.75%). As a control, mosquitoes were exposed to papers impregnated with acetone only. All mosquitoes were exposed to the diagnostic dosage of permethrin and/or PBO for 1 h. Cumulative mortality counts were recorded every minute during the exposure time. After the exposure period, the mosquitoes were held for a 24-h recovery period before the mortality was recorded again. Sucrose solution was provided for the mosquitoes. If the control mortality was between 5% and 20%, the percentage of mortality was corrected by Abbott’s (1925) formula. LT50 was calculated using probit analysis (Finney, 1977). Resistance ratios of the all field populations of A. aegypti were calculated using the following formula: resistance ratio (RR) = LT50 of the field population/LT50 of the laboratory Sus population. For the synergistic effect of PBO, the following formula was used: synergistic factor (SR) = LT50 of the permethrin/LT50 of the PBO + permethrin. All survivors were collected and immediately used for the measurement of enzyme activities. 2.4. Biochemical assays Carboxylesterases (ESTs), glutathione S-transferases (GSTs), mixed function oxidase (MFOs) and acetylcholinesterase (AChE) activities were determined from thirty individual 3 days-old F1 female mosquitoes according to the methods described by Brogdon et al. (1989) and Penilla et al. (1998). The substrate utilized in each assay included ␣- and  naphthyl acetate for ␣-Est and -Est respectively, CDNB (1-chloro 2,4-dinitrobenzene) for GSTs, TMBZ (3,3 ,5,5 -tetramethyl-benzidine dihydrochloride) for MFO. Total protein quantification of mosquito homogenates was performed as per the method of Lowry et al. (1951) with bovine serum albumin as the standard protein in order to normalize enzyme activity levels by protein content. The enzyme activities in the field population and susceptible populations were compared using Kruskal–Wallis test using Prism Graph Pad software (Version 6.0). 2.5. A. aegypti sodium channel partial sequencing Total Genomic DNA was extracted from individual female mosquitoes from each locality and susceptible population by using a HiPurATM insect DNA purification kit (Himedia), as recommended by manufacturer. The polymerase chain reactions (PCR) were carried out to amplify a region of 350 bp of the exon 31 of the voltage-gated sodium channel, as described by Harris et al. (2010), which encode domain III, subunit 6. This region allows detection of Phe1534Cys kdr mutation AaEx31P (F-5 TCGCGGGAGGTAAGTTATTG-3 and AaEx31Q R-5 GTTGATGTGCGATGGAAATG-3 ). The PCR was carried in a 20 L reaction volume, which contains 10 L of 2× PCR Taq Mix [(SRL-Sisco Research Laboratories PVT. LTD.) (20 mM Tris–HCl (pH 8.0), 100 mM KCl, 3 mM MgCl2 , 400 M dNTP, 0.1 U/L Taq DNA Polymerase)] 4 L of 0.4 M each forward and reverse primers, 4 L of nuclease free water and finally 2 L of total genomic DNA extracted from a single mosquito as template. Cycling conditions were as follows: Initial denaturation of 95 ◦ C for 4 min followed by 30 cycles of 94 ◦ C for 60 s, 62 ◦ C for 30 s, and 72 ◦ C for 1 min, then a final elongation at 72 ◦ C for 7 min. The PCR products were visualized by gel electrophoresis and then sequenced in both directions using the ABI 3730 Applied Biosystem. The partial sequence of A. aegypti sodium channel gene were compared with those of other sodium channel sequences deposited in GenBank using the “BLAST-N” tools available on the National Center for Biotechnology Information (NCBI) website. The aminoacid sequence of A. aegypti partial sodium channel was deduced from the corresponding sequence using the translation tool at
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Table 1 Susceptibility of adult Aedes aegypti mosquitoes exposed to permethrin alone and piperonyl butoxide (PBO) + permethrin. Exposure
Permethrin
Population
LT50 (min) 95%CL
Mortality (%)
RR
Permethrin + PBO (synergist) LT50 (min) 95%CL
Mortality (%)
SR
DPI SLM NKL Sus
55.45 (53.23–56.00) 54.43 (51.45–56.57) 47.37 (45.35–49.32) 9.23 (8.12–11.34)
48.5 48.3 50.7 92.2
6.0 5.8 5.1 –
26.45 (25.22–27.25) 23.55 (22.24–24.13) 22.40 (23.23–24.43) 05.55 (07.32–09.93)
59.2 62.7 68.0 97.5
2.0 2.3 2.1 –
PBO = piperonyl butoxide (synergist); RR = resistance ratio; SR = synergistic ratio; CL = confidence limit (95%).
the ExPASy Proteomics website (http://www.expasy.org/tools/dna. html). Further for genotyping tests Hardy Weinberg equilibrium was performed using Genepop version 4.2. 2.6. Kdr genotyping An amino acid substitution, F1534C, was detected in the sequenced regions of the sodium channel of A. aegypti from DPI, SLM and NKL populations. For this assay, two internal allele-specific primers AaEx31 F: 5 -CCTCTACTTTGTGTTCTTCATCATCTT-3 and AaEx31 R: 5 -GCGTGAAGAACGACCCGC-3 were used. These primers flanking within the region of 350 bp primer sequences and the PCR was performed as described above. Approximately 100 mosquitoes were used for genotyping test. 3. Results and discussion The LT50 values for the three field populations were 55.45 min from the DPI, 54.43 min from the SLM and 47.37 min from NKL compared to the laboratory population 9.23. Among the field populations, DPI and SLM were slightly more resistant compared to NKL (RR 5.1). Furthermore, the resistance ratios for the
A
C
permethrin exposed populations ranged from 5.8 to 6.0 folds, respectively (Table 1). These results suggested that A. aegypti from field populations were resistant to permethrin. Exposure to PBO synergist, prior to permethrin enhanced the effectiveness of permethrin by reducing the LT50 values by 2–2.3 times compared to the LT50 values of permethrin alone exposed mosquitoes. Indicating a significant role of P450 in the resistance of adults to permethrin. Similar results were observed by WanNorafikah et al. (2010) where they reported that the range of RR for permethrin-exposed A. aegypti from Taman Melati, Vista Angkasa and Desa Tasik field strains were 4.47, 4.35 and 4.28 folds respectively. Selvi et al. (2006) reported 0.5–1.7 fold RR for permethrin selected A. aegypti strains. Compared to other studies, our work showed resistance ratios 1.5–4 folds higher with permethrin suggesting an increase of resistance within the last few years. Moreover the lower LT50 values recorded for all PBO + permethrinexposed population suggested that the permethrin resistance in these mosquitoes could be linked with an increase activity of mixed function oxidases. The measurement of global detoxification enzyme activities showed a higher level of alpha-esterases in DPI, SLM and NKL compared to the susceptible population (1.5-fold, 1.0-fold and
B
D
Fig. 1. Comparison of detoxification enzymes activities between the permethrin resistant and susceptible population (A) ␣-esterase and -esterase (B) activities were measured with the naphthyl acetate method and expressed as mol ␣- or -naphthol produced/mg protein/min (±SD). (C) GST activities were measured with the CDNB and expressed as mol of conjugated CDNB/mg protein/min (±SD). (D) P450 activities were measured with the TMBZ and expressed as TMBZ/mg protein/min (±SD). Statistical comparison of enzyme activities between the field and susceptible population were performed with a Kruskal–Wallis test (* p < 0.05).
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Cys
CGTCCTCGATCCTTCCAGGTGGGAAAGCAGCCGATTCGCGAGACCAACATCTACATGTAC 60 R P R S F Q V G K Q P I R E T N I Y M Y
Phe
CGTCCTCGATCCTTCCAGGTGGGAAAGCAGCCGATTCGCGAGACCAACATCTACATGTAC 120 R P R S F Q V G K Q P I R E T N I Y M Y
Cys
CTCTACTTTGTGTTCTTCATCATCTGCGGGTCGTTCTTCACGCTGAATCTGTTCATCGGT 120 L Y F V F F I I C G S F F T L N L F I G
Phe
CTCTACTTTGTGTTCTTCATCATCTTCGGGTCGTTCTTCACGCTGAATCTGTTCATCGGT 180 L Y F V F F I I F G S F F T L N L F I G
Cys
GTCATCATCGACAACTTCAACGAGCAGAAGAAGAAAGCCGGTGGCTCACTGGAAATGTTC 180 V I I D N F N E Q K K K A G G S L E M F
Phe
GTCATCATCGACAACTTCAACGAGCAGAAGAAGAAAGCCGGTGGCTCACTGGAAATGTTC 240 V I I D N F N E Q K K K A G G S L E M F
Cys
ATGACGGAGGATCAGAAAAAGTACTACAACGCCATGAAAAAGATGGGCTCGAAGAAGCCG 240 M T E D Q K K Y Y N A M K K M G S K K P
Phe
ATGACGGAGGATCAGAAAAAGTACTACAACGCCATGAAAAAGATGGGCTCGAAGAAGCCG 300 M T E D Q K K Y Y N A M K K M G S K K P
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Cys
CTGAAAGCTATTCCACGGCCTAGGGTAAGGCATTTCCATCGCACATCAAC 290 L K A I P R P R V R H F H R T S
Phe
CTGAAAGCTATTCCACGGCCTAGGGTAAGGCATTTCCATCGCACATCAAC 350 L K A I P R P R V R H F H R T S
||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Fig. 2. Partial sequence of the Aedes aegypti sodium channel from susceptible population (1534Phe), compared with the field populations (1534Cys). The amino acid translation is given below the sequence data and the mutation leading to Phe1534Cys (TTC-TGC) substitution is highlighted in black color.
0.8-fold with p < 0.05). For beta esterases, although all three resistant populations showed higher activities, only DPI is higher (0.8-fold with p < 0.05). The same profile is shown for GST activities (2.0-fold with p < 0.05 respectively). Finally, the three field populations did not show any significant increased MFO activities compared to the susceptible one (Fig. 1). However biochemical assays only provide an overall estimate of MFO enzymatic activities and there might be a chance of other P450s may involve in this resistance mechanism. Previous reports of Djouaka et al. (2008) revealed that though the reduced P450 enzymatic activity in Anopheles gambiae was found, microarray analysis suggest that a multiple P450 genes were involved in pyrethroid resistance mechanism from Southern Benin and Nigeria. Elevated GST levels have also been frequently associated with insect resistance to pyrethroid insecticides (Enayati et al., 2005; Lumjuan et al., 2005). Our biochemical data also suggest the role GSTs in insecticide resistance in the DPI population. Sequencing of the voltage-gated sodium channel identified single amino acid substitutions in DPI, SLM and NKL populations compared with the susceptible population (Fig. 2). The substitution at codon 1534 (Phe to Cys kdr polymorphic site) where a single base pair substitution changes the codon from TTC to TGC resulting in a phenylalanine to cysteine substitution in domain III, subunit 6 (numbering of residues is based on the reference sequence from Musca domestica Williamson et al. (1996) and exon assignment is based on the Chang et al. (2009) annotation of the A. aegypti sodium channel gene). As the Phe1534Cys kdr mutation was detected in the field populations with 321 bp fragments, the sequence were submitted to GenBank (Accession numbers: KM519597 KM519598). Similar results were found in Linss et al. (2014) study were he found only single kdr mutation at the site of 1534 leading to phenylalanine to cysteine replacement revealed pyrethroid resistance in A. aegypti Brazilian natural populations. Substitutions in an alternative phenylalanine residue in IIIS6, F1538, have been associated with pyrethroid resistance in the southern cattle tick, Boophilus microplus (He et al., 1999) and the two-spotted spider
Table 2 Genotypes and resistance allele frequencies of three populations of Aedes aegypti for the F1534C mutationsa Populations
DPI SLM NKL
1534 F/F
F/C
C/C
Freq C
P value
10 7 7
65 40 35
52 30 27
0.59 0.51 0.47
0.653 0.557 0.004
a Tests for Hardy Weinberg equilibrium were applied to the data and the P values are shown. The final row shows the combined analysis for all three populations.
mite, Tetranychus urticae (Tsagkarakou et al., 2009). Additionally, a gene duplication event was recently described in the AaNaV of natural populations and in a laboratory strain selected for pyrethroid resistance (Martins et al., 2013). Although there are at least seven different mutations described in the AaNaV , only that corresponding to the 1534 positions is clearly related to resistance; which is placed in a domain of the sodium channel that interacts directly with the pyrethroid molecule (Du et al., 2013). The tetraplex PCR assay was used to detect F1534C frequency in field populations. Thirty mosquitoes from each area (DPI, SLM and NKL) were genotyped at F1534C loci. The frequency of the 1534C allele was 0.59, 0.51 and 0.47 respectively (Table 2). Previous study of Yanola et al. (2011) revealed that the presence of 1534C mutation with allele frequency of 0.77 could be associated with permethrin resistance in A. aegypti Thailand populations. 4. Conclusions In conclusion, permethrin resistance grows fast and it means that new strategies are needed before having the resistance completely filed in the populations. Hence care should be taken while permethrin used in the field control of Aedes mosquito populations. It is not surprising that pyrethroids are the most employed insecticide worldwide for use in nets, curtains and households (Zaim et al., 2000). Although many novel control strategies are
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