Research Article: Prevalence of kdr mutations and insecticide susceptibility among natural population of Aedes aegypti in West Bengal

Date Published: April 15, 2019

Publisher: Public Library of Science

Author(s): Pabitra Saha, Moytrey Chatterjee, Sudeep Ballav, Akash Chowdhury, Nandita Basu, Ardhendu Kumar Maji, Luciano Andrade Moreira.


Aedes albopictus and Aedes aegypti are the major vectors of arboviral diseases. As effective vaccines are not available for most of the arboviral diseases, vector control by using insecticides play the key role to reduce the disease transmission. The emergence and spread of resistance to different classes of insecticides by the vectors is a major obstacle to control the disease transmission. Information about vector susceptibility to different insecticides and their mechanisms are very important for formulating proper vector control measures. The present study was designed to assess the susceptibility of Ae. aegypti against three different classes of adulticides, one larvicidal agent available and polymorphisms in the voltage-gated sodium channel (VGSC) gene related to insecticide resistance.

Immature stages of Ae. aegypti were collected from three dengue endemic municipal areas of West Bengal and reared in the laboratory. Larvae and adults (F1 progeny) were used for insecticide bioassay as per WHO protocols. Knock down resistance gene (kdr) mutations were assessed by direct sequencing of PCR products.

The Ae. aegypti population was found to be susceptible to type II pyrethroids and malathion but highly resistant to DDT. A high rate of polymorphisms in the VGSC gene was observed among the collected mosquitoes. A double mutant V1016G + F1534C was found to be associated with DDT resistance but neither V1016G nor F1534C alone showed the same association. Association between the kdr mutations and the susceptibility status of pyrethroids could not be established due to very small sample size. A low to moderate level of resistance was noticed against temephos among the larval population based on WHO criteria.

The replacement of DDT by type II pyrethroids for the management of dengue vectors is an appropriate decision taken by the national program which is supported by the findings of a higher level of resistance to DDT. Persistence of polymorphisms in the VGSC gene might be an indication of emergence of resistance against pyrethroid insecticides that should be monitored at a regular interval. Attempts should be made to determine the effectiveness of other larvicides for replacement of temephos if needed in future. Along with the chemical insecticides different biological vector control methods as well as biopesticides should also be used in vector control programmes.

Partial Text

Mosquito-borne arboviral diseases like dengue, chikungunya, yellow fever and Zika are major public health problem with more than 4 million disability adjusted life years globally [1, 2]. The major causes behind emergence and spread of arboviral diseases are demographic changes, massive urbanization, population movement, trade, transport and lack of effective vector control strategies which favour the world-wide distribution of these viruses and vector mosquitoes [3, 4, 5, 6, 7]. During the last decade a higher level of mortality and morbidity has been observed due to dengue and Zika virus infection [8]. Both these diseases are mainly transmitted by Aedes albopictus and Ae. aegypti mosquitoes [9, 10]. The spread of the vectors was amplified during the Second World War due to rapid human movement and transportation leading to dengue epidemic [6]. After the war, rapid urbanization led to rapid spread of dengue and hyper-endemicity with multiple serotypes in most South East Asian countries, with severe forms of the disease [11]. Urban and sub-urban colonization comes with new man-made breeding sites for mosquitoes such as regular water containers, disposed water-holding vessels, waste disposal areas, small containers, and discarded tyres all that may help Ae. albopictus and Ae. aegypti to thrive and multiply [4, 12, 13]. Ae. albopictus and Ae. aegypti are potential vectors for dengue epidemics as they breed preferentially in artificial containers [14, 15, 16, 6]. To date no effective anti-viral agent is recommended against arboviruses including dengue virus. A vaccine against dengue, Dengvaxia® (CYDTDV), has been licensed since 2015, but the overall efficacy of trials has been about 60% and it has not been used on a large scale [17]. Recently the World Health Organization (WHO) does not recommend wide spread vaccination with Dengvaxia® as it increases the rate of dengue haemorrhagic fever in sero-negative individuals [18]. Effective vector control plays the key role for reducing transmission of arboviruses worldwide and is the essential component of the WHO strategy for the prevention, control, and elimination of Neglected Tropical Diseases [19]. However, the emergence and spread of insecticide resistance in vector mosquitoes is becoming a major obstacle to reaching the goals set by WHO. Resistance to different classes of insecticides have been recorded among both the Aedes vector species in different parts of the World [20]. The worldwide insecticide resistance network supported by the World Health Organisation is established to track insecticide resistance among the vectors of arboviruses and to evaluate the potential for deployment of alternative vector control interventions [21]. Four mechanisms have been found to be associated with insecticide resistance-metabolic enzyme-based resistance, reduced target site sensitivity due to mutations in target genes, reduced penetration of insecticide due to thickening of the cuticles and behavioural changes [22]. The first two mechanisms are studied extensively [23, 24, 25, 26] but the role of cuticular penetration has not been well explained [20]. Increased production of three metabolic enzymes i.e. cytochrome P450 monooxygenases (P450s), esterases and glutathione S-transferases are principally associated with insecticide resistance [22, 27]. Resistance due to target site insensitivity is associated with mutations at the VGSC gene, commonly referred to as knockdown resistance (kdr). The VGSC mutations modify the target site of insecticide so that insecticide does not bind and cause the prolonged opening of the sodium channel resulting in rapid paralysis of the insects [28].

Ae. aegypti is highly anthropophilic, aggressive day biter with peak activities during early morning and late afternoon. They prefer to feed indoors and rest outside in close proximity to their breeding sites [16]. It is very difficult to control the adult mosquitoes through IRS (indoor residual spray) due to typical feeding and resting behaviour. Use of insecticides as space sprays using thermal fogging and ultra-low volume application are the choice of methods for controlling the Aedes population. In general, management of breeding sites with effective larvicides plays an important role for this purpose. The selection of effective insecticidal agents (larvicide and adulticide) is very important. In the present study, we attempted to determine the susceptibility status of Ae. aegypti in three different classes of insecticides; DDT (organochlorine), deltamethrin and alpha-cypermethrin (type II pyrethroid), malathion (organophosphate) as adulticide, temephos as larvicide and polymorphisms in VGSC gene among the mosquitoes collected from three different districts of West Bengal.

From the present study it was evident that the Ae. aegypti populations from each of the study areas were susceptible to the currently used pyrethroid i.e. 0.05% alpha cypermethrin and also to 0.05% deltamethrin, but highly resistant to DDT. However, presence of a high level of polymorphisms in VGSC gene may be an indication of emerging pyrethroid resistance. So, the susceptibility of used pyrethroid and polymorphisms in target genes should be monitored at regular intervals to detect the emergence of pyrethroid resistance among the Ae. aegypti population. As malathion is highly sensitive, it might be an alternative in near future if needed. A low to moderate level of resistance to temephos among larval populations was also noticed. Further study is required to observe the larval susceptibility to other larvicides to replace temephos for proper management of Ae. aegypti. Along with the chemical insecticides different biological vector control methods as well as biopesticides should also be used in vector control programmes.