Portal de Periódicos da CAPES

Two novel mutations in the voltage-gated sodium channel associated with knockdown resistance (kdr) in the dengue vector Aedes aegypti in Vietnam

2018; Wiley; Volume: 43; Issue: 1 Linguagem: Inglês

10.1111/jvec.12298

1948-7134

Nguyễn Thị Kim Liên, Nguyen Thi Hong Ngọc, Nguyen Thu Hien, Nguyễn Huy Hoàng, Nguyen Thi Huong Binh,

Insect Pest Control Strategies

Dengue is a rapidly spreading vector-borne disease, with approximately 390 million cases of infection worldwide and over 3.97 billion people in 128 countries at risk of the disease (Bhatt et al. 2013). Vietnam is one of the most affected countries in Southeast Asia with 50,000 cases reported in 2016 (MOH 2016). Aedes aegypti is the main dengue vector widely distributed throughout the world. It originated in Africa and is now found in all continents, including America, Asia, Australia, and Europe (Lambrechts et al. 2011, Medlock et al. 2012, Brown et al. 2014). Pyrethroids are a major class of insecticide widely used for controlling pests and disease vectors. The intensive use of pyrethroid has led to many instances of insect resistance, with primary target sites being voltage-gated sodium channels (VGSC) (Narahashi 2002, Soderlund 2012). One of the major mechanisms of pyrethroid resistance, which reduces neuronal sensitivity to this class of insecticides, is known as knockdown resistance (kdr) (Soderlund and Bloomquist 1990). Computer modeling predicts that IIS5, IIIS6 domains in voltage-gated sodium channels and the linker connecting S4 and S5 in domain II are the binding site of pyrethroid with channels, with most of the kdr mutations in these regions. Identification of kdr mutations has already led to successful development of rapid and accurate molecular methods to detect kdr-based pyrethroid resistance in field populations (Yanola et al. 2011). Such information is needed in order to design and implement suitable control interventions against this species. Mutations at three codon positions of the Voltage-Gated Sodium Channel (VGSC) gene (Ile1011Met/Val, Val1016Gly/Ile, and Phe1534Cys) have been primarily associated with both pyrethroid and DDT resistance in various Ae. aegypti populations (Brengues et al. 2003, Saavedra-Rodriguez et al. 2007, Lima et al. 2011, Martins et al. 2013, Stenhouse et al. 2013). Additionally, mutations such as Ser989Pro have also been associated with pyrethroid resistance in Ae. aegypti (Kawada et al. 2014). A high frequency of the kdr mutation 1534Cys was found in Myanmar, Malaysia, Thailand, India, and Grand Cayman (Harris et al. 2010, Yanola et al. 2011, Kawada et al. 2014, Ishak et al. 2015, Kushwah et al. 2015). Mutation 1016Gly has a high frequency in Grand Cayman, Thailand (Harris et al. 2010, Stenhouse et al. 2013) but has a low frequency in Vietnam (Kawada et al. 2009), Myanmar (Kawada et al. 2014). Otherwise, kdr mutation 1016Ile was very frequent in Latin America and its frequency was higher at places where many pyrethroids were used, such as Brazil, Martinique, and Mexico (Garcia et al. 2009, Martins et al. 2009, Marcombe et al. 2009). More new mutations associated with pyrethroid resistance have been identified in sodium channels from Ae. aegypti, consisting of Gly923Val, Leu982Thr (Brengues et al. 2003); Val1023Ile, Asp1794Tyr (Chang et al. 2009); Phe1552Cys (Yanola et al. 2010); Thr1520Ile (Kushwah et al. 2015); Ser996Pro, Val1023Gly, Phe1565Cys (Wuliandari et al. (2015); and Val 410Leu (Haddi et al. 2017). Here, we determined that the mutation in the voltage-gated sodium channel is associated with knockdown resistance to pyrethroid in Ae. aegypti populations from some provinces in Vietnam. The information about the knockdown resistance to pyrethroid will be a key for the implementation of suitable control strategies against Ae. aegypti and a reduction of the dengue burden in Vietnam. To determine the resistance level, mosquito samples were collected from the field of five provinces in Vietnam using a standard collection method. These provinces include Ha Noi, Thanh Hoa, Nghe An, Ha Tinh in the north and Khanh Hoa in the south of Vietnam. The larvae were collected from natural habitats and reared to adults in the insectary. All specimens were morphologically identified to species. Bioassays were carried out according to the WHO procedure (WHO 2016) using two to five-day-old F2 generations of Ae. aegypti with four replicates of 25 mosquitoes per tube. The insecticides tested were: 0.75% Permethrin (type I of Pyrethroid), 0.05% Deltamethrin (Type II of Pyrethroid), 0.05% Lambda-Cyhalothrin (Pyrethroid), 4% DDT (Organochlorine), 1% Propoxur (Carbamate), and 5% Malathion (Organophosphate). Resistance status was classified according to WHO (2016) criteria: resistance for 98% mortality. All these resistant mosquitoes were genotyped for kdr mutations by direct sequencing. The Bora susceptible strain that is susceptible to all insecticide classes was used in this study as a control. The adult mosquitoes were collected with sterile single-use vessels and placed into sterile test tubes. They were then immediately put on dry ice and stored at −20° C until they could be processed. Forelegs of individual mosquitoes were transferred to a sterile 1.5 ml micro-centrifuge tube, 50 μl of extraction buffer added (5 M NaCl, 1mM Tris-HCl pH 8.6, 0.5 mM EDTA, pH 8.0), and were carefully crushed with the help of a sterile plastic pestle. The homogenate was vortexed briefly followed by short spinning in a micro-centrifuge, incubated in a water bath maintained at 65° C for 30 min and finally centrifuged at 8,000 rpm for 2 min. Seven μl of Kac 8 M were added and the tube was kept at −20° C for 30 min, then centrifuged 12,000 rpm for 20 min and the supernatant transferred into a new tube. Afterwards, 100 μl of cold ethanol was added and the tube centrifuged at 12,000 rpm for 20 min. The supernatant was then removed and the precipitate dissolved in 50 μl of TE (10 mM Tris HCl pH 8.0, 1 mM EDTA pH 8.0) buffer. To identify potential kdr mutations, two fragments of the coding region of the VGSC gene spanning exon 19 to exon 31 (covering the 989, 1011, 1016, and 1534 coding positions) were amplified from DNA samples and directly sequenced (ten for each province). The PCR was carried out using 10 pmol of each primer (Kawada et al. 2014) and 20 ng of DNA as template in 25 μl reactions containing 1X Dream Taq buffer, 0.2 mM dNTPs, 1.5 mM MgCl2, and 1U Dream Taq (Thermo, U.S.A.). The cycle conditions were 95° C for 1 min and 35 cycles of 95° C for 10 s, 55° C for 30 s and 72° C for 30 s, followed by a final extension step of 72° C for 10 min. The PCR amplification was carried out on an Eppendorf Mastercycler EP gradient (USA Scientific, Inc). The PCR products were purified using the Qiaquick PCR purification kit (Qiagen, Germany) and sequenced directly using an ABI PRISM 3500 (U.S.A.). The sequences were aligned and compared to the reference sequences in GenBank (EU399179 for Bora strain) using BioEdit software version 7.0.9.0 to detect the mutations. The percentage of mosquito mortality after one h exposure showed that the Bora strain was sensitive to all types of insecticide. However, insecticide resistance in mosquito populations that were collected from the field was very different. All the mosquito populations were sensitive to the organophosphate (except the strain from Ha Tinh province) but strongly resistant to organochlorine (DDT), carbamate, and lambda-cyhalothrin (Table 1). For deltamethrin, 24 h mortalities were 22%, 37%, and 92% in Ha Noi, Khanh Hoa, and Nghe An populations, respectively, but were 100% in Thanh Hoa and Ha Tinh. For permethrin, the mortality rate ranged from 2% in Ha Noi to 7% in Khanh Hoa and up to 100% in Thanh Hoa, Nghe An, and Ha Tinh. These results suggested the resistance to lambda-cyhalothrin, deltamethrin, and permethrin was present. Ha Noi, Khanh Hoa populations can be characterized as highly resistant, whereas Nghe An, Thanh Hoa, and Ha Tinh populations have a lower level of resistance (Figure 1). Similar results were observed in Ae. aegypti from Punjab, Pakistan (Mohsin et al. 2016). Resistance to DDT, deltamethrin, and permethrin in most adult mosquitoes has been reported in Bangkok, Thailand (Komalamisra et al. 2011). The high resistance to DDT and reduction in susceptibility to deltamethrin, lambda-cyhalothrin, and propoxur have been reported in Ae. aegypti from Senegal and Cape Verde Archipelago (Dia et al. 2012). Resistance to DDT and deltamethrin was also observed in Ae. aegypti in the Central African Republic (Ngoagouni et al. 2016). Insecticide (diagnostic dose) Ha Noi strain Thanh Hoa strain Nghe An strain Ha Tinh strain Khanh Hoa strain Mortality (%) Resistance status Mortality (%) Resistance status Mortality (%) Resistance status Mortality (%) Resistance status Mortality (%) Resistance status DDT (4%) 1 R 10 R 20 R 57 R 0 R Propoxur (1%) 40 R 66 R 80 R 84 R 42 R Malathion (5%) 100 S 100 S 100 S 87 R 100 S Deltamethrin (0.05%) 22 R 100 S 92 R 100 S 37 R Lambda-Cyhalothrin (0.05%) 3 R 95 R 85 R 88 R 21 R Permethrin (0.75%) 2 R 100 S 100 S 100 S 7 R Interestingly, no mutation was detected at the 989, 1011, 1016, and 1534 positions in VGSC protein, but two novel amino acid substitution mutations, Ala1007Gly and Phe1558Cys (GenBank accession number: MG257775-MG257780), were detected in the VGSC protein in Khanh Hoa, Nghe An, and Ha Noi populations that had been phenotyped for resistance to pyrethroid (Figure 2). The results demonstrated the presence of knockdown resistance at the above provinces but none in Thanh Hoa and Ha Tinh populations. In Kawada et al. (2009), Ile1011Met/Val and Leu1014Phe mutations were not found in Ae. aegypti from provinces in the south of Vietnam. Val1016Cys mutation was only detected in two heterozygous individuals from Quang Ngai province. Mutation Phe1269Cys was discovered in Quang Tri, Thua Thien – Hue, Quang Nam, Quang Ngai, Binh Dinh, and Khanh Hoa provinces in the middle and south of Vietnam but none in Ae. aegypti from the north. Stenhouse et al. (2013) indicated that kdr mutations were not enough to explain the resistance level in some Ae. aegypti populations. Thus, there may have been other mechanisms, such as detoxification enzymes, playing a major role in the resistance to these cases. In addition, the difference in genotypes is influenced by geographic factors and the types of chemicals that are used. This has been displayed in several previous studies. Yanola et al. (2010) reported that Phe1534Cys was related to modification of type I pyrethroid and not to type II. Lima et al. (2011) showed that Ile1011Met was related to cypermethrin resistance (type II) in Ceara, Brazil. While Val1016Ile was associated with resistance to type I and type II of pyrethroid (Brengues et al. 2003), Val1016Gly mutation appears to be the most important mutation in Ae. aegypti strains in southern China (Li et al. 2015). Ser989Pro has always been reported in relation with Val1016Gly and reduces the channel sensitivity to deltamethrin (type II of pyrethroid) (Hirata et al. 2014, Ishak et al. 2015) and permethrin (type I of pyrethroid) in Indonesia (Hamid et al. 2017). Kawada et al. (2014) also reported that Ile1011Met and Leu1014Phe mutations were not found in Myanmar. In contrast, Val1016Gly and Ser989Pro mutations were widely distributed in Myanmar with high frequencies (84.4% and 78.8%, respectively) (Kawada et al. 2014). However, Ser989Pro and Phe1534Cys mutations were not detected in resistant strains in Taiwan and Malaysia (Chang et al. 2009, Ishak et al. 2017) or in Indonesia populations (Hamid et al. 2017). In conclusion, all five populations in our study were resistant to pyrethroid. Ae. aegypti from Ha Noi, Khanh Hoa could be characterized as highly resistant, whereas Nghe An, Thanh Hoa, and Ha Tinh had a lower level of resistance. We also detected two novel mutations in VGSC protein, Ala1007Gly (in Khanh Hoa) and Phe1558Cys (in Ha Noi, Nghe An, Khanh Hoa). However, these changes were not found in Thanh Hoa and Ha Tinh populations. The difference in genotype proved that studies to determine pyrethroid resistance mechanisms of Ae. aegypti are important for better control. This study was supported by the National Foundation for Science and Technology Development, Vietnam (NAFOSTED, grant no. 106-NN.02–2015.17).