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Table of Contents
Year : 2021  |  Volume : 58  |  Issue : 4  |  Page : 368-373

First detection of voltage-gated sodium channel mutations in Phlebotomus argentipes collected from Bangladesh

1 Department of Microbiology, Mymensingh Medical College, Mymensingh, Bangladesh
2 Laboratory of Molecular Immunology, Graduate School of Agricultural and Life Sciences, Univ. of Tokyo, Tokyo, Japan
3 Department of Parasitology, Ege University Faculty of Medicine, Izmir, Turkey
4 Mymensingh Medical College Hospital, Mymensingh, Bangladesh
5 Department of Medical Entomology, National Institute of Infection Diseases, Tokyo, Japan
6 Hemodialysis and Apheresis, Nephrology 107 Lab, The University of Tokyo Hospital, Tokyo, Japan

Date of Submission31-Aug-2020
Date of Acceptance14-Jul-2021
Date of Web Publication25-Mar-2022

Correspondence Address:
Chizu Sanjoba
The University of Tokyo, Graduate School of Agricultural and Life Sciences, Laboratory of Molecular Immunology, 1-1-1 yayoi, Bunkyo-ku, Tokyo 113-8657
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0972-9062.328972

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Background & objectives: Phlebotomus argentipes is the main vector of visceral leishmaniasis in Bangladesh and is controlled using deltamethrin, a synthetic pyrethroid, through indoor residual spraying (IRS). A mutation at L1014 (leucine at codon 1014) of the voltage-gated sodium channel (VGSC), known as a knockdown resistance (kdr) gene, is thought to be an important pyrethroid resistance mechanism. This study detected mutations at codon 1014, and at codons 1011, 1016, and 1020, which are kdr sites in other insects. The kdr relationship with deltamethrin resistance in P. argentipes from an IRS-targeted site in Bangladesh was also evaluated.
Methods: Sand flies were collected from Magurjora village, Mymensingh district, Bangladesh in November 2012. A WHO cone bioassay test using deltamethrin was conducted and specimens were grouped as ‘live’ or ‘dead’. After morphological identification, genomic DNA was used to genotype a partial VGSC gene from P. argentipes. The kdr/ pyrethroid resistance relationship was evaluated using Fisher’s exact test.
Results: Targeted codons were genotyped from 8 ‘live’ and 63 ‘dead’ P. argentipes. All ‘live’ specimens had mutant alleles (L1014F and L1014S) at codon 1014. The mutant allele rate was 94% for ‘live’ specimens and 55% for ‘dead’ specimens. The mutant allele survival odds were higher for the wild-type L1014L allele, and L1014F odds were lower for L1014S. There were no mutations at codons 1011, 1016, and 1020.
Interpretation & conclusion: The L1014 mutations suggested that pyrethroid resistance had appeared in Bangladesh. Further research on kdr mutations in P. argentipes is important for the appropriate IRS.

Keywords: Bangladesh; Phlebotomus argentipes; pyrethroid resistance; voltage-gated sodium channel

How to cite this article:
Sarkar SR, Kuroki A, Özbel Y, Osada Y, Omachi S, Shyamal PK, Rahman F, Kasai S, Noiri E, Matsumoto Y, Sanjoba C. First detection of voltage-gated sodium channel mutations in Phlebotomus argentipes collected from Bangladesh. J Vector Borne Dis 2021;58:368-73

How to cite this URL:
Sarkar SR, Kuroki A, Özbel Y, Osada Y, Omachi S, Shyamal PK, Rahman F, Kasai S, Noiri E, Matsumoto Y, Sanjoba C. First detection of voltage-gated sodium channel mutations in Phlebotomus argentipes collected from Bangladesh. J Vector Borne Dis [serial online] 2021 [cited 2022 May 21];58:368-73. Available from: https://www.jvbd.org/text.asp?2021/58/4/368/328972

  Introduction Top

Visceral leishmaniasis (VL) is a vector-borne disease and the most severe form of leishmaniasis. In 2012, the annual number of VL cases was estimated to be 200,000 to 400,000, and 80% of these cases were in just three countries (Bangladesh, India, and Nepal) on the Indian subcontinent (ISC)[1].

In 2005, the Kala-azar Elimination Programme (KAEP) was launched to eliminate VL from the ISC and is supported by the World Health Organization (WHO). Bangladesh, India, and Nepal participated in the program to reduce VL incidence below 1 case in every 10,000 people per year at the sub-district level[2]. The regional strategy consists of (1) early diagnosis and complete case management, (2) effective surveillance, (3) social mobilization and building partnerships, and (4) integrated vector man- agement[3]. In these countries, VL is mainly caused by the protozoan parasite Leishmania donovani[4] and the sand fly Phlebotomus argentipes is the only species known to be a vector[5],[6].

In Bangladesh, vector control targeted against P. argentipes began in 2012 in the form of indoor residual spraying (IRS)[7]. The insecticide used was deltamethrin 5WP (wettable powder), one of the most commonly used synthetic pyrethroids. Despite the reliance on pyrethroids, there have been very few studies on the pyrethroid susceptibility status of P. argentipes in Bangladesh. However, the emergence and existence of pyrethroid resistance in P. argentipes have been reported in other ISC countries. In India, one P. argentipes population in Pondicherry was resistant to deltamethrin[8], and in Nepal, P. argentipes from three endemic districts showed signs of pyrethroid resistance (alpha-cypermethrin and deltamethrin)[9].

One of the major mechanisms driving pyrethroid resistance is knockdown resistance (kdr) mutations in the para voltage-gated sodium channel (VGSC) gene. Voltage-gated sodium channels exist in nerve cells and are the target site for pyrethroids. Inhibition of in/deactivation processes in the VGSC by pyrethroids causes immediate insect paralysis and death. Amino acid mutations at specific sites in the VGSC are known to be associated with pyrethroid resistance and they are called kdr mutations. An electrophysiological study using VGSCs in Aedes aegypti showed that kdr mutations could reduce sensitivity to several types of pyrethroids[10]],[11]. Mutations at codon L1014 (leucine at codon 1014, using Musca domestica codon numbering) of the VGSC are commonly known to be kdr mutations in many insect species, such as German cockroaches, houseflies, and the Anopheles mosquito[12],[13].

The existence of L1014F/S (mutation of leucine to phenylalanine or serine) in P. argentipes sand flies was first reported from Bihar, India, in 2017[14]. The study reported that both L1014F and L1014S had a relationship with pyrethroid resistance. In this previous study, two pyrethroids (deltamethrin and alpha-cypermethrin) were researched, and survival odds for L1014F/S were higher than wild-type L1014L in both pyrethroids. Odds ratio of L1014S and L1014L were 3.47 in deltamethrin and 23.8 in alpha-cypermethrin, and odds ratio of L1014F and L1014L were 4.33 in deltamethrin and 35.7 in alpha-cypermethrin[14]. Subsequent studies also reported the existence of L1014F/S alleles and their relationship with deltamethrin resistance in P. argentipes from West Bengal, India[15]. The survival odds for L1014F/S were also higher. The odds ratio of L1014S and L1014L was 4.49 and that of L1014F and L1014L was 34.1[15].

Studies on kdr mutations in P. argentipes have never been conducted in Bangladesh. The purpose of this study was to detect mutations at codon 1014 and at codons 1011, 1016 and 1020, which are known to be kdr sites in other insects. This study also evaluated the relationship between the codon and deltamethrin resistance in P. argentipes collected from an IRS-targeted site in Bangladesh.

  Material & Methods Top

Collection of sand flies

The collection site was Magurjora village, Harirampur Union, Trishal sub-district (Upazila), Mymensingh district, Mymensingh division, Bangladesh [Figure 1]. The Mymensingh district is the most highly endemic area for VL in Bangladesh, and P. argentipes is the dominant species in this area[16]. Therefore, VL control programs are focused on this district and a first-round of IRS using 0.05% deltamethrin was conducted during the 2012 pre-mon-soon period (May–June)[7]. Sand flies for use in this study were collected using 10 CDC light traps in November, 2012. The traps were placed in and around human dwellings and cattle sheds during the night. The captured sand flies were transferred to the laboratory and released into a plexiglass cage with two sides covered by plaster of Paris. A cotton wool pad soaked with 10% sucrose was placed on the cage to provide food for the insects.
Figure 1: Locations of Bangladesh and Magurjora village (Harirampur union, Trishal sub-district, Mymensingh district, Mymensingh division; shown as a black dot) where the sandflies were collected. Geographical data was from www.gadm.org and the location details for the village were taken from its geographical coordinates (DD: 24.5212, 90.4124). Maps were produced by QGIS version 3.10.2.

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Deltamethrin bioassay

The susceptibility test was conducted according to the WHO cone bioassay protocol with modification[17]. K-Othrine WG250 (deltamethrin 25%w/w, Bayer AG) was diluted with water at a given concentration and sprayed onto plywood boards (15 × 15 cm) from a height of 50 cm using a spray gun connected to an air pump. The application rate was 40 mL/m2 and the boards were weighed immediately after spraying to confirm the treatment amount. Boards with an error of 10% or more were discarded. In the sand fly control group, the plywood boards were treated with the same amount of water rather than deltamethrin. Approximately 15–30 field-collected sand flies were introduced into the WHO plastic cones and exposed for 30 min. Each test was repeated three times. After exposure, the sand flies were transferred into plastic cups and provided with 10% sucrose-in-water on cotton wool and held for 24 h at 25 °C ± 2°C and 80% ± 10% relative humidity. The mortality rate was recorded at 5, 10, 30, 60 min, and then at 2, 3, 4, 5, 6, 12 h. At 24 h, all specimens were removed from the plastic cups and divided into ‘live’ and ‘dead’ groups. They were labeled and individually stored in 99.9% ethanol at room temperature. The percent corrected mortality was calculated using Abbott’s formula[18]:

Percent corrected mortality = {(observed % mortality) – (% control mortality)} / (100% control mortality) X 100.

Identification of sand flies

After the bioassay, the head and abdominal segments of the tested specimens were cut from the body and mounted on labeled slides in Swan solution for morphological identification. The remaining body was transferred to 99.9% ethanol for DNA extraction. Morphological identification was conducted using published keys and descriptions[19],[20], which were based on the following distinguishing features: cibarium, pharynx, and spermathecae in females and genitalia, coxite, and style in males. All specimens were observed using BX51 and SZ61 microscopes (Olympus Co., Japan) and recorded using a DP73 microscope camera (Olympus Co., Japan).

DNA isolation and genotyping of VGSC codons

The DNA was individually extracted from the P. argentipes in the test group and the methods used were different for the ‘live’ and ‘dead’ groups. The DNA of a ‘live’ specimen was extracted using MagExtractor™ -Genome- (Toyobo Co., Ltd., Japan). Before the extraction, each specimen was homogenized using a pipette tip and incubated in a 10 μL mixture containing 1 μL proteinase K (Sigma, USA), 0.5% SDS, 2 mM CaCl2, 8 mM Tris-HCl, and 8 mM EDTA at 50 °C for 16 h. The DNA of a ‘dead’ specimen was extracted by alkali extraction. Zirconia beads (2.3 mm) (BMS, Japan) and 10 μL of 0.2 M NaOH were added to each specimen. Then the samples were homogenized using TissueLyser II (QIAGEN, Germany) at 30 Hz for 3 min. After 10 min incubation at 70 °C, a 40 μL mixture containing 22.5 mM Tris-HCl and 0.3125 mM EDTA was added to neutralize the sample. All extracted DNA samples were stored at -20°C for further study.

The PCR amplification was conducted separately in a 25 μL mixture containing 12.5 μL of 2 × PCR buffer for KOD FX (Toyobo Co., Ltd., Japan), 5 μL of 2 mM dNTPs, 0.75 μL of 10 μM forward and reverse primers, 0.5 μL of KOD-FX DNA polymerase (Toyobo Co., Ltd., Japan), and 1 μL of genomic DNA. The thermocycle conditions consisted of an initial denaturation step of 5 min at 95 °C followed by 38 cycles at 96°C for 30 sec, 56°C for 30 sec, and 68°C for 30 sec, with a final extension step at 68°C for 5 min. The primers used were Vssc8F (5’ -AATGTGGGATTGCATGCTGG-3’) and Vssc1bR (5’ -CGTATCATTGTCTGCAGTTGGT-3’), as previously described[14].

The quality and quantity of the PCR products were analyzed using MultiNa (Shimadzu, Japan). The products were then purified using ExoSAP-IT (Thermo Fisher Scientific, USA) and sequenced using PCR primers, and the sequence data were read using a 3130xl Genetic Analyzer (Applied Biosystems, USA).

The partial sequence included codons 1011, 1014, 1016, and 1020 of the para VGSC gene. The mutations were analyzed using BioEdit Sequence Alignment Editor v7.0.5 and the codon numbering was based on the house fly para VGSC (Musca domestica, GenBank accession no. X96668).

Data analysis

Fisher’s exact test was used to assess the relationship between allele frequencies at codon 1014 and the ‘live’ or ‘dead’ phenotypes. R software version 3.6.1 was used for the calculations. The level of significance was P < 0.05.

  Results Top

Deltamethrin bioassay of field-collected sand flies and morphological identification

The results of the insecticide susceptibility tests are shown in [Figure 2]. The sand fly mortality recorded at 24 h after 30 min exposure was 87.9%. The morphological identification results after the bioassay showed that 79.8% (75/94) of the sand flies tested were P. argentipes. The remaining sand flies were all identified as Sergentomyia spp. Eight out of nine sand flies in the ‘live’ group and 67 out of 85 sand flies in the ‘dead’ group were identified as P. argentipes. The number of female/male sand flies was 8/1 and 46/39 in the ‘live’ and ‘dead’ groups, respectively. One female sand fly was identified as Sergentomyia spp. within the ‘live’ group.
Figure 2: Mortality rates over time for the collected Phlebotomus sandflies after exposure to deltamethrin.

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Prevalence of VGSC mutations and their relationship with resistance

The targeted codons were successfully genotyped from 71 P. argentipes specimens in the test group. These consisted of 8 ‘live’ (7 females and 1 male) and 63 ‘dead’ (29 females and 34 males) specimens. At codon 1014, a wild-type (TTA) allele and three different types of mutant alleles (TTT, TTC, and TCA) were detected. Two mutant codons (TTT and TTC) replace the wild-type leucine with phenylalanine (L1014F) and the other mutant codon (TCA) replaces leucine with serine (L1014S). All the ‘live’ specimens had mutant alleles and the frequency was 94% (15/16), which was higher than for the ‘dead’ specimens (55%, 69/126) [Table 1]. The genotype frequencies showed that the leucine (Leu, wild-type) homozygotes were all dead and only 2% (1/44) of the specimens whose genotypes contained at least one Leu allele were alive [Table 2]. Out of a total of six genotypes, only three genotypes (Leu/Ser, Ser/Ser, and Phe/Ser; Ser: serine, Phe: phenylalanine) had ‘live’ specimens and the percentage number of mutant homozygote genotypes that were alive (Ser/Ser (31%), Phe/Ser (33%)) was higher than for the heterozygote Leu/Ser genotype (4%). In males, the mutant allele frequencies for ‘live’ and ‘dead’ specimens were 100% (2/2) and 56% (38/68), respectively. For females, it was 93% (13/14) and 53% (31/58) for ‘live’ and ‘dead’ specimens, respectively.
Table 1: Allele frequencies at codon 1014 and relationship with deltamethrin susceptibility.

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Table 2: Genotype frequencies at codon 1014 and its relationship with deltamethrin susceptibility

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Fisher’s exact test showed that the survival odds for mutant alleles were higher than for the wild-type L1014L allele. A comparison of the two mutant alleles showed that the L1014F odds ratio for survival (OR) values (OR = 8.29, P = 0.0671) were lower than for L1014S (OR = 13.7, P = 0.00212) [Table 1]. No mutations were found at codons 1011, 1016, and 1020. At these codons, all the genotypes were wild-type homozygotes (Ile/Ile at 1011, Val/Val at 1016, and Phe/Phe at 1020; Ile: isoleucine, Val: valine) [Table 3].
Table 3: Genotype frequencies at codon 1011, 1016 and 1020

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  Discussion Top

This study reported the first detection of L1014F/S mutations in P. argentipes from Bangladesh. The statistical analysis showed that the mutant alleles had a relationship with survival in the deltamethrin bioassay, and they are considered to be a deltamethrin resistance mechanism. There were no significant differences in the frequency of mutant alleles in the ‘live’ and ‘dead ‘groups between the sexes. The higher survival odds for L1014S than for L1014F were the opposite to previous reports from India[14],[15]. An electrophysiological study using Drosophila VGSCs and data from Anopheles gambiae also suggested that L1014F had a stronger effect on pyrethroid resistance than L1014S[21],[22]. These results suggest that further research is needed to assess the effect of each allele.

The wild-type L1014L frequency was relatively high (41%), which suggested that selection for kdr alleles had not yet progressed at the collection point. Continuous IRS from 2012 has possibly increased the frequency of L1014F/S alleles at the IRS-targeted sites in Bangladesh.

The L1014S ratio was higher than for L1014F, which was similar to the results reported in Indian studies[14],[15]. During the early stage of selection, the ratio is affected by the previous frequency before IRS. Therefore, the L1014S frequency could have been higher at the beginning. The ratio is also affected by the fitness cost of each allele. These results suggest that further observations are needed to predict mutant allele transitions.

An analysis of the Leu allele frequency (45%) and the Leu/* genotype frequency (68%) showed that their genotype frequencies in the ‘dead’ group were relatively high. This may be due to the recessive or incompletely recessive status of the kdr gene[23],[24]. In addition, 20/63 of the kdr homozygote specimens were in the ‘dead’ group. This could be because the specimens had died naturally or resistance mechanisms other than kdr (such as detoxification enzymes and reduced cuticle permeability) are present. It is possible that two or more resistance mechanisms are needed to survive.

This study revealed that there were no mutations at codons 1011, 1016, and 1020. In the previous studies, the genotypes at codons 1011, 1016, and 1020 were also all wild-type homozygotes[14],[15]. The relationship between the mutations at theses codons and pyrethroid resistance in sand flies needs to be researched continuously.

IRS plays an important role in the prevention and elimination of VL. One month after the IRS, the density of P. argentipes in households decreased by 22.5% in Bangladesh[7]. Therefore, development of pyrethroid resistance in P. argentipes can be a serious problem. In India, DDT has been replaced by pyrethroids from 2015 because Phlebotomus in VL-endemic areas are highly resistant to DDT[25]. The existence of the mutant alleles in P. argentipes suggested that pyrethroid resistance had developed in Bangladesh. An appropriate IRS strategy is necessary to sustainably eliminate VL and it is applying in the endemic areas of Bangladesh for a long time in the scope of the Kala-azar Elimination Programme launched in 2005 together with Nepal and India[2]. This program is now in the consolidation phase and IRS using pyrethroids is still important for decreasing VL cases. If the populations of P. argentipes with insecticide resistance increase, the number of resistant individuals in the new generations will increase, and then serious problems may occur in VL control and the efforts spent for a successfully implemented program may be wasted or repeated. Therefore, further research on kdr mutations in local P. argentipes populations is becoming increasingly important, and research on alternative insecticides to pyrethroids may need to be considered. In addition to this, we would like to emphasize that regular monitoring of P. argentipes population size and the implementation of conventional insecticide resistance testing by local authorities is essential.

  Conclusion Top

This study is the first to detect L1014F/S mutations of VGSCs in P. argentipes from Bangladesh. The analysis showed that both L1014F and L1014S had a relationship with deltamethrin resistance. Further studies on the mechanisms driving pyrethroid resistance are needed so that the appropriate IRS method can be applied.

Conflict of interest: None

  Acknowledgements Top

The authors appreciate the support given by Mr. Md. Abdus Salam, Trishal Health Complex, and Ms. Sohana Asma during the sandfly collection period in Magurjora village. The authors also appreciate the WHO cone bioassay technical support provided by Mr. Kazunori Ohashi. This study was part of a project entitled Research and Development of Prevention and Diagnosis for Neglected Tropical Diseases, which is supported by JST/JICA and the Science and Technology Research Partnership for Sustainable Development.

  References Top

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  [Figure 1], [Figure 2]

  [Table 1], [Table 2], [Table 3]


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