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Year : 2018  |  Volume : 36  |  Issue : 3  |  Page : 435--438

Bioassay and molecular study for detection of insecticide resistance dengue causing mosquito vectors

Monika Soni1, Chandrakanta Bhattacharya1, Jitendra Sharma2, Prafulla Dutta1,  
1 Division of Entomology and Filariasis, Regional Medical Research Centre (ICMR), Dibrugarh, Assam, India
2 District Surveillance Unit, Office of the Joint Director of Health Services, Lakhimpur, Assam, India

Correspondence Address:
Ms. Monika Soni
DST, Women Scientist-A, Regional Medical Research Centre (ICMR), Dibrugarh, Assam
India

Abstract

Nowadays, dengue infection creates a major problem across the country. The vector species carrying dengue infection has progressively started to developed resistance against most of the currently used insecticides. Hence, a study was carried out in dengue-endemic areas of Assam and Arunachal Pradesh to find the current situation of insecticide susceptibility status of dengue vectors. Based on the previous history of dengue incidence, Aedes mosquitoes were collected from Dibrugarh, Kamrup, Sivasagar, Tezpur and Tinsukia districts in Assam and Pasighat district in Arunachal Pradesh to test the insecticide resistance status through bioassay and molecular methods. The WHO standard bioassay test kits were used to detect insecticide susceptibility status among dengue vectors. In molecular study, allele-specific polymerase chain reaction (PCR) method was done for the detection of mutations in paratype voltage-gated sodium channel gene of Aedes aegypti and Aedes albopictus mosquitoes. In bioassay method, 100% A. aegypti mosquitoes were found to be resistant towards dichlorodiphenyltrichloroethane (DDT), 8% towards pyrethroid and 4% towards malathion. Similarly, 92% A. albopictus mosquitoes have shown resistance competency towards DDT, 12% towards pyrethroid and 8% towards malathion. In allele-specific PCR methods, V1016G heterozygous mutations were detected from the field collected A. aegypti and A. albopictus mosquitoes of Tinsukia, Dibrugarh and Sivasagar district. Similarly, F1534C heterozygous mutations were observed from A. aegypti mosquitoes of Tezpur, Tinsukia and Sivasagar district and A. albopictus mosquitoes of Tinsukia, Dibrugarh and Sivasagar district. From the study, it was concluded that the Aedes mosquitoes have progressively started to developed resistance towards commonly used insecticides.

How to cite this article:
Soni M, Bhattacharya C, Sharma J, Dutta P. Bioassay and molecular study for detection of insecticide resistance dengue causing mosquito vectors.Indian J Med Microbiol 2018;36:435-438

How to cite this URL:
Soni M, Bhattacharya C, Sharma J, Dutta P. Bioassay and molecular study for detection of insecticide resistance dengue causing mosquito vectors. Indian J Med Microbiol [serial online] 2018 [cited 2019 Dec 5 ];36:435-438
Available from: http://www.ijmm.org/text.asp?2018/36/3/435/245379

Full Text

 Introduction



Dengue is a mosquito borne viral disease. Nowadays it is spreading in different states of India. The Northeast (NE) states of India are most vulnerable for dengue virus infection due to huge diversity of mosquito species, favorable environmental situation for mosquito proliferation, presence of large forest fringe areas, hygiene as well as flooding. The presence of huge diversity of mosquito vectors are posing a serious threat in a community by transmitting viruses, bacteria and other causal organisms from infected person to a healthy individual. To reduce the incidence of these mosquito borne diseases, many control policies are undertaken in dengue reporting areas of India. Among them, the vector control strategies are gaining more importance as vectors are the carriers responsible for transmitting dengue virus from infected person to a healthy individual. These vector control strategies include the use of several classes of insecticides for combating against most of the mosquito vectors carrying viral and parasitic pathogens. These insecticides have different ability to repel and kill different mosquito species. However, the uses of insecticides are limited up to certain extents as the mosquito species have started to developed resistance capability towards most of the insecticides used so far in public health programme and due to which the insecticide-based control programmes meant for the prevention of mosquito is not as much beneficial as it expected.[1],[2]

Usually, the insecticides binds with the sodium channels and causing prolonged opening of sodium channels which resulted in repetitive firing or nerve conduction blocking, and ultimately, it leads to paralysis and death of the insect. However, an in-depth knowledge of resistance mechanisms is necessary to guide insecticide use in vector control programmes. For detection of insecticide resistance in mosquito vectors, the molecular methods are gaining more importance out of several diagnostic tools. The molecular studies over the past decades have identified several polymorphisms in the paratype voltage-gated sodium channel (VGSC) gene, and these are associated with the resistance against pyrethroid and dichlorodiphenyltrichloroethane (DDT), also known as knockdown resistance (kdr). Several earlier reports indicated that insecticide resistance dengue vectors are predominant in the Southern part of India.[3],[4] However, the information regarding the insecticide resistance gene polymorphism of dengue vectors, such as Aedes (Stegomyia) aegypti and Aedes (Stegomyia) albopictus widespread in the North Eastern region of India, are not available. Hence, the present study aims to explore the allelic distribution of VGSC gene mutations among dengue vectors collected from selected dengue reporting areas in NE states.

 Materials and Methods



Study area

The study areas were selected based on the previous history having high prevalence rate of dengue infection. Our study includes five highly endemic districts, i.e., Dibrugarh, Kamrup, Sivasagar, Tezpur and Tinsukia in Assam. Furthermore, our study has covered the entire Pasighat district of Arunachal Pradesh which shared inter-state boundary with Assam [Figure 1]. All the collection sites were from urban settings. The study was carried out in the Regional Medical Research Centre (ICMR), Dibrugarh, which is the apex research laboratory of North Eastern part of India in the field of biomedical science.{Figure 1}

Mosquito collection sites

To fulfil the objective of the study, Aedes aegypti and Aedes albopictus mosquitoes were searched in discarded tyres, plastic containers and other waste containers holding stagnant water and collected using BG-sentinel trap. The collected mosquitoes were kept in separate cages according to different locations. Later on, the collected mosquitoes were separated based on species identification tool, namely stereoscope. To get sufficient mosquitoes of equal age for bioassay method F2 generations were used along with the molecular study.

Adult bioassay

The insecticide susceptibility test was done by using the WHO standard procedures. Different insecticide groups, such as organochlorine (DDT-4%), organophosphate (malathion-5%) and pyrethroids (permethrin and deltamethrin-0.05%), were used against 3–5-day-old females populations collected from different locations. The observed mortality rate was calculated by using the following formula:

Observed mortality = total number of dead mosquitoes in four tubes/total sample sizes in four tubes × 100

Average control mortality rate below 5% (in two tubes) was considered for validation of the test.

Knockdown resistance genotyping

Thirty-five individual female mosquitoes from each location site were genotyped for both 1016 and 1534 positions. Total genomic DNA was extracted using Qiagen blood and tissue kit (Qiagen, Inc., Hilden, Germany) following manufacturer's instructions. Allele-specific polymerase chain reaction (PCR) for Val/Gly and Phe/Cys was carried out using primers described from previous work.[5]

Polymerase chain reaction amplification of the fragment of voltage-gated sodium channel gene

Allele-specific PCR method was done for the detection of V1016G mutation in the VGSC gene of A. aegypti and A. albopictus by using the primers as mentioned in [Table 1]. For detection of valine/glycine at 1016 codon position, 3 μl of mosquito DNA was added in a 10 μl of reaction volume containing 5 μl of master mix, 0.25 μl of forward primer, 0.125 μl of reverse primer and 1.625 μl of nuclease-free water. The product size for valine and glycine was 60 and 80 bp, respectively. The cycling condition involves 35 cycles with initial denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s and final extension at 72°C for 2 min.{Table 1}

For detection of phenylalanine/cysteine at 1534 codon in the VGSC gene, 3 μl of mosquito DNA was added in a 10 μl of reaction volume containing 5 μl of master mix, 0.5 μl of each forward and reverse primer and 1 μl of nuclease-free water. The product size for phenylalanine and cysteine was 88 bp and 110 bp, respectively. The thermal profile was initial denaturation at 95°C for 2 min, denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s and final extension at 72°C for 2 min. All the amplifications were performed in a thermal cycler (Gene Amp® PCR System 9700 and Veriti, Applied Biosystems, Foster City, CA, USA). Products were visualised on 4% gel to obtain band of expected size.

Sequencing

To validate the above allelic-specific PCR method, amplified products of expected size were purified by using QIAGEN gel extraction kit and sent for sequencing to a commercial company. After sequencing, the PCR products were edited manually in BioEdit software, submitted in GenBank and analysed in Mega5 and DnaSP version v. 5.10.01 for the detection of point mutations.

 Results and Discussion



In insecticide susceptibility test, 100% A. aegypti mosquitoes were found to be resistance towards DDT. However, only 8% A. aegypti mosquitoes have shown resistance towards pyrethroid, and 4% A. aegypti mosquitoes were found to be resistance against malathion. Similarly, 92% A. albopictus mosquitoes have shown resistance competency towards DDT, 12% towards pyrethroid and 8% towards malathion. As per the epidemiological investigation records, DDT spraying and fogging were done in all those mosquito collection areas at frequent interval by the employees of state government in previous time. Hence, the mosquitoes prevalent in those areas are basically exposed towards DDT and build resistance competency towards the insecticide. This epidemiological record was found comparable with our insecticide susceptibility test results. Apart from this, previous studies conducted in different parts of India have reported varying degree of resistance towards DDT and pyrethroid.[6],[7],[8],[9],[10],[11]

The VGSC gene of individual A. aegypti and A. albopictus mosquitoes was amplified through allele-specific PCR method to test the presence of V1016G and F1534C mutations. In allele-specific PCR analyses, valine to glycine heterozygous mutations (V1016G) associated bands were detected from the field collected A. aegypti and A. albopictus mosquitoes of Tinsukia, Dibrugarh and Sivasagar district [Figure 2]. Similarly, phenylalanine to cysteine heterozygous mutations (F1534C) related bands were detected from A. aegypti mosquitoes of Tezpur, Tinsukia and Sivasagar district as well as A. albopictus mosquitoes of Tinsukia, Dibrugarh and Sivasagar district. The presence of heterozygous mutations in the concerned gene position indicated that the Aedes mosquitoes have progressively started to developed resistance capability towards DDT, pyrethroid, malathion and deltamethrin. Recently, the authors have found high-frequency mutations at codon F1534C among A. aegypti mosquitoes collected from Delhi, which has been associated with resistance against DDT and deltamethrin.[12] However, as per few reports in other part of the country, it has been documented that deltamethrin and malathion insecticides are still effective against A. albopictus vectors due to the absence of important knockdown resistance mutations as well as higher susceptibility level detected in bioassay.[13] It is interesting to note from the present study that there is no V1016G polymorphism from the mosquitoes collected from Arunachal Pradesh region. From this finding, we can conclude that the commonly used insecticides are still susceptible against mosquito vectors (A. aegypti) in Pasighat area of Arunachal Pradesh. Hence, Government of Arunachal Pradesh has to focus on large-scale use of insecticides to reduce the numbers of Aedes mosquitoes.{Figure 2}

In mutation analysis of sequencing products, no such mutations were detected in any of the analysed mosquito samples. However, the presence of heterozygous mutations observed in allele-specific PCR revealed that the mosquito population has progressively started to develop mutations in the concerned position of VGSC gene. Hence, such mosquito population has a chance to develop mutations at specific codon 1016 and 1534 of VGSC (in both loci) gene in near future as one of its allele has already shown mutation.

The presence of such heterozygous mutant Aedes mosquito population may create a challenge in near future. Hence, the government has to focus on reducing such mosquito population by using recently developed larval control methods such as using larvivorous fish, genetically engineered mosquito population, etc. In the same time, general public should become aware about the transmission of dengue and how to get rid from such incidence.

 Conclusion



From this study, it is concluded that the Aedes mosquitoes prevalent in Assam have progressively started to developed resistance capability towards currently used insecticides. In near future, these may bring an indication of major dengue and chikungunya outbreak in NE states of India. For prevention of an impending outbreak in near future, time-to-time bioassay and molecular study to test the insecticide susceptibility status in dengue reporting areas is very much essential.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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