Current status of insecticide resistance and its underlying mechanisms in Aedes aegypti (L.) in Punjab, Pakistan : Journal of Vector Borne Diseases

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Research Article

Current status of insecticide resistance and its underlying mechanisms in Aedes aegypti (L.) in Punjab, Pakistan

Ramzan, Hania1; Manzoor, Farkhanda1; Oneeb, Muhammad2,

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Journal of Vector Borne Diseases 60(1):p 57-64, Jan–Mar 2023. | DOI: 10.4103/0972-9062.353250
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Abstract

Aedes aegypti (Linnaeus) plays an important role as a vector of different deadly diseases particularly dengue fever. Insecticides are used as a primary tool to control Ae. aegypti. However, due to the excessive use of insecticides on agricultural, public health, and industrial levels, mosquitoes have developed resistance. In this study, the current susceptibility status of Ae. aegypti mosquitoes against different insecticides (Temephos, DDT, dieldrin, Malathion, Bendiocarb, Permethrin, Cypermethrin, and Lambda-cyhalothrin) was evaluated in district Lahore and district Muzaffargarh of Punjab, Pakistan. For this purpose, WHO bioassays and biochemical assays were performed on Ae. aegypti population from Lahore (APLa) and Aedes population from Muzaffargarh (APMg). Results of APLa and APMg showed high levels of resistance against the larvicide Temephos. Resistance against all adulticides was also observed in APLa and APMg (% mortality < 98%). The biochemical assays indicated statistically significant elevated levels of detoxification enzymes in APLa and APMg. APLa showed slightly higher levels as compared to APMg. Mosquitoes were also screened for the presence of kdr mutations. The results revealed no mutation in domain II while the presence of mutation F1534C in domain III was found in both field populations. The results showed the presence of moderate to high grade resistance against all insecticides in Ae. aegypti in district Lahore and district Muzaffargarh of Punjab, Pakistan.

INTRODUCTION

By the twentieth century, vector-borne diseases became the most serious public and animal health problem globally. Out of all disease-causing insect vectors, mosquitoes play an important role as vectors of different deadly diseases particularly in tropical and subtropical regions. Dengue fever alone is responsible for the health burden in more than 100 countries and around 3 billion humans are at risk. Aedes aegypti, an anthropophilic species, is the principal vector of dengue fever. It also transmits yellow fever, Chikungunya, and La Crosse encephalitis along with Zika Virus in Europe, Latin America, and Africa[1]. Pakistan, being a subtropical country, is a hotspot for Ae. aegypti. Pakistan has faced a number of outbreaks in different years since the last three decades. However, in 2011, dengue disease caused a major threat to public health in Punjab province and district Lahore was considered as the center of the disease. Currently, Pakistan is considered as endemic for this disease with more than 24,000 dengue cases reported from different districts of Punjab, during the last year[2,3].

At present, vaccines or drugs that could be effective against dengue or other Aedes-borne diseases are not available. A vaccine against the dengue virus (CYD-TDV) is being used in a few countries. However, it is not recommended for individuals who have never been infected by the dengue virus as it may intensify the chance of severe dengue. So, in this scenario, the reduction of Ae. aegypti population is considered to be the main defense to prevent dengue epidemics. Source reduction by eradicating breeding sites of Ae. aegypti might be an effective way to prevent the spread of the dengue vector. However, in many countries including Pakistan, ongoing strategies, to control Ae. aegypti population, rely mainly on the use of effective and safe insecticides through indoor residual spraying (IRS), space spraying and insecticide-treated nets (ITNs). The most commonly used insecticides to control mosquitoes belong to the class pyrethroids, carbamates, and organophosphates. However, pyrethroids are commonly used in public health programs in the form of IRS and ITNs[4]. So, due to over-reliance on insecticides and their excessive use, resistance in Ae. aegypti has been developed in many countries[5]. The resistance mechanisms in insects are triggered by physiological and behavioral alterations. Studies revealed that, generally, mosquitoes show resistance by three major mechanisms: reduced penetration, increased levels of enzymes, and reduced sensitivity of the target site[6]. In a study, resistance to permethrin and malathion was detected by using CDC bioassay. Levels of detoxification enzymes (acetylcholinesterases, GSTs, MFOs, p-nitro phenylacetate esterases, and esterases) and frequency of kdr mutations have been studied, previously. Similarly, several studies have shown the association of insecticide resistance with detoxifying enzymes and mutations in target sites[7,8]. kdr mutations are associated with mutations in Voltage-gated Sodium Channel (VGSC) gene and were first found in Musca domestica[9]. The most common kdr mutations found in Ae. aegypti are F1534C, S989P, and V1016G and are distributed worldwide. However, F1534C mutation is associated with the resistance to type 1 pyrethroids and DDT. It often occurs alone but can reach higher levels when combined with other kdr mutations like S989P or V1016G[10].

In Pakistan, despite the large-scale insecticide usage, no systematic study has been done so far to monitor the levels of insecticide resistance, due to the lack of expertise and updated knowledge of vectors’ epidemiology, biology, and susceptibility status to insecticides. Insecticide resistance in mosquitoes has been reported in Pakistan[11]. The data regarding the involvement of metabolic resistance mechanisms along with knockdown resistance (kdr) in insecticide resistance in Ae. aegypti is scarce, especially in the arid zone. Therefore, the present study was designed to determine the insecticide resistance status of the field populations of Ae. aegypti mosquitoes, from district Lahore and district Muzaffargarh, on both enzymatic and molecular levels. This study investigated all main mechanisms involved in insecticide resistance in Ae. aegypti from the arid agro-ecological zone of Pakistan for the first time. This study will enhance the knowledge about insecticide resistance in Ae. aegypti of arid and semi-arid agro-ecological zones of Pakistan. Thus, it will contribute to vector control monitoring programs that would eventually decrease the Aedes-borne diseases burden in Pakistan.

MATERIAL & METHODS

Study area

The current study was conducted in two districts of Punjab, Pakistan, i.e., Lahore (31.34° N and 74.22° E) and Muzaffargarh (30.40 °N, 71.20 °E) [Figure 1]. District Lahore is bounded on the North and West by district Sheikhupura and on the South by district Kasur. River Ravi flows on the northern side of Lahore. In the East, it shares an international border with India. The total area of the city is 1772 km2. It is located 217 m above sea level. District Muzaffargarh is located at the elevation of 123 meters above sea level across the Chenab River. It is occupying about 8435 km2 of area. It is surrounded by districts Multan and Khanewal in the East, Layyah in the North and Rahimyar Khan and Bahawalpur in the South.

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Figure 1:
Map of Pakistan where study areas i.e., Lahore and Muzaffargarh are magnified. This map is generated on open-source software QGIS (version 2.18.9).

Sample collection and rearing

Mosquitoes were collected from different urban and rural areas of both districts according to the convenient sampling technique from September 2018 to March 2020. From district Lahore, they were collected from Allama Iqbal town, Marghazar Colony, Model Town, Samanabad, Taj Garh, Nathoke, Manhala, and Dial. While, areas of Ganesh Wah Canal, Khan pur, Muradabad, Hayatabad and Khurshidabad from district Muzaffargarh were selected for sample collection [Figure 1]. Adult Ae. aegypti mosquitoes were collected by using the hand catch method. For this purpose, mosquitoes resting on different surfaces were collected by using both mouth (WHO) and mechanical aspirators. CDC sweeper (CDC, USA) was also used in areas where mosquitoes were found in rich density. Larvae of Ae. aegypti mosquitoes were collected by using a dipper.

Adult mosquitoes were pooled and placed in pre-labeled cages covered with damp cotton and provided with a 10% sucrose solution. The collected mosquitoes were identified under CO2 anesthesia under a stereomicroscope by using a morphological identification key. Mosquitoes were reared to obtain F1 generation to get maximum growth synchronization for bioassays and to detect metabolic and target-site resistance. Mosquito populations from Lahore and Muzaffargarh were referred to as APLa and APMg, respectively.

Insecticide susceptible lab strain was used as a control for different tests. This lab strain was originally collected from district Lahore in the 1970s and has undergone continuous rearing at NIMRT institute, Lahore. This strain was then shifted to the Entomology lab, Department of Parasitology, University of Veterinary and Animal Sciences, Lahore in 2008 and has been reared there since then by following standard mosquito-rearing methods before the experiments[12].

Insecticides used

World Health Organization (WHO) recommended diagnostic dosages of different insecticides for adults i.e., DDT (4%), Dieldrin (4%), Malathion (5%), Bendiocarb (0.1%), Permethrin (0.75%), Deltamethrin (0.05%), and Lambda-cyhalothrin (0.05%) and concentrations of Temephos for larval bioassay were used to determine the susceptibility status of mosquitoes. These insecticides were obtained from WHO Collaborator Centre, The Vector Control and Research Unit (VCRU) at Universiti Sains, Penang, Malaysia (USM).

Insecticide susceptibility tests

Larval bioassay

Dose-Response bioassay was conducted according to WHO standard protocol against larvicides[13]. Different concentrations of Temephos i.e., 1.25 mg/l, 6.25 mg/l, 31.25 mg/l, and 156.25 mg/l were tested against the mosquito larvae of Ae. aegypti from both districts, separately. For each concentration, L3 or L4 larvae (n=100±10), in four replicates of about 25 larvae/cup, were transferred to small disposable cups.. For the control group (n=25±5), alcohol was added instead of insecticide. The results were calculated according to WHO criteria.

Adult bioassay

Adult bioassay was done by following the WHO susceptibility kit protocol. Four replicates of 3–5 days old, non-blood-fed mosquitoes (n=25±5) were introduced into holding tubes to acclimatize for 30 min. They were then transferred to tubes with insecticide-impregnated papers. After 1 h of exposure, mosquitoes were transferred back to holding tubes for 24 h at 25±5 °C and 80% humidity. For the control group, mosquitoes (n=25±5) were exposed to insecticides free papers. Susceptibility status was evaluated according to WHO recommended criteria.

Biochemical assays

Enzyme-based metabolic mechanisms involved in producing insecticide-resistant mosquitoes were detected by using biochemical methods. Alterations in enzymatic levels of AChE, esterases, GSTs, and cytochrome P450 were calculated by following WHO protocols[14]. Nonblood fed, 3–5 days old, individual females from APLa (n=29) and APMg (n=23) were used in each assay.

DNA Extraction and amplification of VGSC

The genomic region of Voltage-Gated Sodium Channels comprising domains II and III was investigated to evaluate kdr mutations, found in pyrethroid-resistant mosquitoes. Genomic DNA was extracted from Female Ae. aegypti (n=42) of both APLa and APMg by using the manual protocol of the “Absolute-Ethanol” method, with few modifications[15]. Lower abdominal segments were removed to avoid any amplification of DNA present in their spermathecae. Quantification of purified DNA was done by using NanoDrop Spectrophotometer (ThermoFisher ScientificTM). The extracted pellets of DNA were stored at -20°C till further use.

PCR approach was employed to amplify the voltage-gated sodium channel (VGSC) gene of mosquitoes by using published primers[16,17]. The optimized thermocycling conditions for domain II were as follows: Initial denaturation at 94°C for 5 min, 35 cycles each of 94°C for 30 sec, 62°C for 45 sec, and 72°C for 30 sec, followed by a final step at 72°C for 10 min. While the PCR conditions for domain III were as follows: Initial denaturation at 95°C for 5 min, 35 cycles each of 94°C for 30 sec, 59°C for 30 sec, and 72°C for 30 sec, followed by a final step at 72°C for 10 min. The amplified products were visualized on 1% agarose gel and bands for domain II (473 bp) and domain III (350 bp) were excised and purified by using the GeneAll ExpinTM gel extraction kit. Purified amplicons were quantified by using NanoDrop, spectrophotometer. These purified amplicons from both APLa and APMg were then submitted for Sanger sequencing (n=11).

Statistical analysis

The statistical analysis was done by using Microsoft Excel, GraphPad Prism, and Ldp Line Software. The resistance status of the tested population was evaluated as follows: mortality rate >98% is susceptible population; mortality rate <98% is resistant population. Abbott’s formula was used to correct the percentage mortality when mortality in the control was between 5% and 20%. Other tests include ANOVA, Kruskal-Wallis H Test, and Mann-Whitney U Test.

Ethical statement: Not applicable

RESULTS

Larval bioassay

WHO recommended dose-response assay was performed to evaluate the susceptibility status of larvae of Ae. aegypti against insecticide Temephos by determining the lethal concentrations (LCs) and resistant ratios (RR). For APLa, LC50 and LC90 values were 14.65 mg/l and 207.39 mg/l, respectively. LC50 and LC90 values, for APMg, were 17.74 mg/l and 480.89 mg/l, respectively. High levels of resistance were found in both populations (Table 1).

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Table 1:
LC50 and LC90 values and resistance ratio (RR) of Ae. aegypti larval strain against temephos

Adult susceptibility test

Diagnostic doses of 7 adulticides were used against both field populations. Mortality was corrected using Abbott’s formula, where needed. Laboratory strain was used as the positive control. Both populations tested showed low to high levels of resistance against all insecticides.

Resistance ratio based on KDT (RRKDT50) of APLa was highest for Malathion in both APLa (13.99) and APMg (7.77). The lowest RRKDT50 value for APLa was against Bendiocarb (3.67) and for APMg, it was against Lambda-cyhalothrin (2.16) (Table 2).

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Table 2:
KDT50 and KDT90 values and resistance ratio (RR) of Ae. aegypti adults of APLa and APMg

Biochemical assays for insecticide resistance

The AChE inhibition activity was significantly higher in both APLa and APMg as compared to the lab strain (Kruskal-Wallis test=51.42, d.f.=2, P <0.0001). The mean of propoxur inhibition (%) of APLa was 79% (95% CI = 71.85-86.16) and for APMg, it was 70.76% (95% CI = 64.06-77.46) [Figure 2]A. The results of GST activity showed elevated levels in APLa and APMg as compared to lab strain (Kruskal-Wallis test= 63.88, d.f.=2, P= <0.0001). The overall mean activity of GST was 0.4903 conjugated GSH/min/mg protein (95% CI = 0.4759-0.5047) [Figure 2]B. The mean values of a-esterase activity in Ae. aegypti were calculated for APLA is 3.327 (95% CI= 3.040-3.614) and APMg is 1.934 (95% CI= 1.616-2.253) mM β-naphthol/min/mg protein, respectively. Both field populations were compared with lab strain by using Kruskal-Wallis analysis (test= 62.52, d.f. = 2, P= <0.0001) [Figure 2]C. Similarly, the mean values of α-esterase of both field populations were also analyzed. The mean values for APLa and APMg were 4.417 (95% CI= 4.606-6.228) and 4.312 (95% CI= 3.653-4.970) mM β-naphthol/min/mg protein, respectively. Results showed a significant difference between both APLa and APMg when compared to lab strain (Kruskal-Wallis test= 51.77, d.f. = 2, P= <0.0001) [Figure 2]D. Furthermore, Both Ae. aegypti field populations showed higher levels of cytochrome P450 as compared to the lab strain (Kruskal-Wallis test=59.98, d.f. = 2, P= <0.0001). The mean MFOs activity in Lahore population was 0.3270 equivalent units of P450/mg protein (95% CI= 0.3088-0.3451) and 0.2479 equivalent units of P450/mg protein (95% CI= 0.2294-0.2664) in Muzaffargarh population. The mean activity of cytochrome P450 in both APLa and APMg was 0.292 equivalent units of P450/mg protein (95% CI= 0.2753-0.3087) [Figure 2]E.

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Figure 2:
Detoxification enzyme activities in Ae. aegypti from district Lahore and Muzaffargarh in comparison with susceptible lab strain. A. AChE, B. α-esterases, C. β-esterases, D. GSTs and E. Cytochrome P450.

Detection of potential kdr mutations

A total of 11 Aedes aegypti mosquitoes from both districts were genotyped to detect kdr mutations. The sequences of exonic regions of domains II and III of VGSC were obtained after Sanger sequencing. No amino-acid mutations were detected in the exons of domain II in both APLa and APMg. However, the presence of a mutation at position 1534 in domain III was observed in a few samples (these positions are presented based on the amino acid sequences of the VGSC gene of a variant of the housefly, GenBank accession No. AAB-47604 and AAB-47605). From APLa, F1534C mutation was detected in 3 samples. APMg showed F1534C mutation in 2 samples [Figure 3]. This F1534C mutation occurs due to the replacement of the second base of codon TTC to TGC (phenylalanine to cysteine).

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Figure 3:
Sequences of domain III of Ae. aegypti aligned in BioEdit software showing mutation at position 1534. The nucleotide sequence at 122 is showing the mutation (TTC to TGC).

DISCUSSION

In the absence of effective vaccines and drugs, the vector control program is the only viable method in controlling the Aedes-borne diseases. In Pakistan the available empirical data on insecticide resistance in mosquitoes is quite scarce, however, there have been few studies regarding the semi-arid zone, while no studies regarding the arid zone are available[18]. Lahore falls in a semi-arid agro-ecological zone with an abundance of rice, wheat, and maize fields in the vicinity. District Lahore, being the second most populated city of Pakistan and the provincial capital of Punjab has faced many dengue outbreaks in the last decade. Muzaffargarh district being in an arid agro-ecological zone is abundant in mango, rice, and wheat fields. District Muzaffargarh lies in southern Punjab where notably a few numbers of dengue cases have been reported in the last few years. Owing to its humid environmental condition, Lahore presents ideal breeding conditions for Ae. aegypti while the drier environment of Muzaffargarh provides unfeasible breeding conditions in comparison. The disproportionate division of urban and rural areas in both districts is another contributing factor in the breeding of Ae. aegypti. As the proliferation of Ae. aegypti thrives in the urban environment, therefore, Lahore with its abundant urban areas provides an ideal breeding ground for these mosquitoes.

Moreover, the abundant use of insecticides in crop fields of both districts resulted in insecticide resistance in Ae. aegypti which make these regions ideal places for mosquitoes to breed. As per the Directorate General of Pest Warning and Quality Control of Pesticides, Punjab, the insecticides used in agriculture belong to all four main classes. Insecticides from these classes have previously been used for controlling malaria which later provided a solution to control dengue as well[19]. So, this study was designed to determine the current status of insecticide resistance in Ae. aegypti mosquitoes in district Lahore and district Muzaffargarh.

In the present study, both APLa and APMg indicated low to high resistance to all adulticides tested. Recently pyrethroids are being widely used for IRS, LLITNs, and fogging against mosquitoes. The extensive use of pyrethroids in crop fields has resulted in insecticide resistance in different crop pests and mosquitoes. Pyrethroids such as deltamethrin and permethrin are also commonly used in Urban Pest Management Programs which might be another reason for pyrethroid resistance in Ae. aegypti[18]. The results of this study also indicated low to high levels of resistance in APLa and APMg Ae. aegypti mosquitoes. The results of two previous studies conducted in a semi-arid zone showed resistance against bifenthrin and deltamethrin in Aedes mosquitoes[20,21]. So, it can be predicted that the current status of pyrethroid resistance could be because of cross-resistance due to the wide use of DDT or the increased selection pressure of pyrethroids due to their use on a domestic and agricultural scale. Therefore, it could be assumed that pyrethroid-based mosquito control programs are under threat.

In mosquito control programs, after pyrethroids, organophosphates and carbamates are the commonly used class of insecticides. Based on this suggestion, the present study has employed malathion and bendiocarb to determine their susceptibility status in Ae. aegypti. A previous study suggested susceptibility to malathion and bendiocarb in Ae. aegypti[22], however, the results of the present study indicated a high resistance value against these insecticides in both APLa and APMg. The possible cause of these contradictory results could be the use of agrochemicals in these selected areas. It could also be due to the seasonal variations. Henceforth, a complete surveillance plan should be implemented in Pakistan to determine the precise status of malathion and bendiocarb in Ae. aegypti.

Although many countries have banned the use of organochlorines, however, the empirical data on the reversal of resistance has encouraged many countries to use DDT for IRS following the WHO guidelines. The slow biodegradation of DDT in the environment and its accumulation in animal tissues suggest that it has a pervasive nature[23]. Even though DDT is no longer used in Pakistan, the high levels of DDT resistance in both APLa and APMg could be explained by keeping this factor in mind that it could be because of residue from the use of DDT in previous mosquito control programs or agricultural pest control programs.

The enzymes which are related to the metabolic detoxification of insecticides are esterases, MFOs, and GSTs and increased levels of these enzymes are responsible to increase the resistance of insects against insecticides. In Pakistan, there is limited data available on the metabolic mechanism of mosquitoes as only a few studies focus on the elevated levels of detoxifying enzymes in mosquitoes and only a single study has been conducted on Ae. aegypti till date[24]. The results of the present study illustrate that the enzymatic levels of APLa and APMg were higher as compared to the lab strain. The elevated cytochrome P450 levels indicated higher resistance levels in both field populations of Ae. aegypti. According to the results of the current study, APLa showed slightly higher levels of cytochrome P450 as compared to APMg. In a study conducted in Sri Lanka, high levels of GST were found in DDT and pyrethroid-resistant Ae. aegypti and Ae. albopictus mosquitoes[25]. In another study conducted in Laos, altered levels of cytochrome P450 monooxygenases were detected in adults of Ae. aegypti confirming the presence of pyrethroid resistance[26]. Increased levels of AChE inhibition suggested its association with malathion resistance in the field population of Ae. aegypti. Similarly, results of α both α - and β - esterases revealed the presence of resistance against bendiocarb. Higher levels of AChE and esterases have also been reported in a previous study[27]. This study suggested that the presence of mixed enzyme alterations in Ae. aegypti indicates the occurrence of multiple resistance.

Mutation F1534C in domain III (exon 31) of the VGSC gene was detected in APLa and APMg in this study. While, no mutations on any position like 989, 1011, 1014, and 1016 in exons 20 and 21 of domain II were found. Out of all mutations, S989F, F1534C, and V1016G are the most commonly found kdr mutations. These mutations, alone or in combination, are linked with resistance against pyrethroids and DDT. The limited data available on Ae. aegypti in Pakistan have indicated the presence of F1534C mutation in larvae of Ae. aegypti and adults of Ae. aegypti and Ae. albopictus[10]. The previously mentioned studies have only employed the population of Ae. aegypti in the semi-arid agro-ecological zone while the present study detected the kdr mutations from both semi-arid and arid agro-ecological zones. kdr mutations in the VGSC gene have been reported in Ae. aegypti from different Asian countries[28,29]. Elucidation of kdr mutations especially mutations at point 1534 in Ae. aegypti would offer a valuable understanding of dengue hemorrhagic fever epidemiology and provide beneficial information for vector control strategies.

This study also employed larval bioassays which exhibited high resistance in Ae. aegypti in both APLa and APMg. Previously, Ae. aegypti has shown high resistance against temephos in several districts of Punjab[30,31]. It might be due to the fact that in Pakistan, only temephos has been used to control Ae. aegypti and other mosquitoes’ larvae since 1969 till date[32]. However, a recent study has suggested low levels of temephos resistance in Ae. aegypti[10]. These low levels of temephos resistance could be nominal because temephos is still being used in fields. Therefore, the decline in temephos resistance in Ae. aegypti is a long-term process that may take several years to occur in fields without any selection pressure thus it is of paramount importance to conduct more studies on temephos resistance in Ae. aegypti to understand this phenomenon precisely.

CONCLUSION

The present study highlighted biochemical and genetic mechanisms of mosquitoes conferring insecticide resistance in two districts of Punjab, Pakistan viz., Lahore and Muzaffargarh which indicated the presence of resistance in both agro-ecological zones. Based on the results of this study, it is recommended that extensive research should be conducted to identify the detoxification genes in resistant mosquitoes, accurately. Also, scientific studies identifying different mutations in field populations of mosquitoes should be performed. There is a need to monitor insecticide regulations with surveillance data on resistance in mosquitoes. Moreover, the insecticide industry should produce reversible or irreversible inhibitors for metabolic enzymes.

Conflict of interest:

None

Acknowledgements

The authors acknowledge the help of Mr. Waqas Ahmad Gondal in the collection of mosquitoes. The authors are also grateful to the owners of farms for their collaboration for mosquito collection during this study.

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Keywords:

Aedes aegypti; Agro-ecological zones; Metabolic resistance; kdr mutations

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