Assessment of insecticide resistance in primary dengue vector, Aedes aegypti (Linn.) from Northern Districts of West Bengal, India

Accepted Manuscript Title: Assessment of Insecticide Resistance in Primary Dengue Vector, Aedes aegypti (Linn.) From Northern Districts of West Bengal Authors: Minu Bharati, Dhiraj Saha PII: DOI: Reference:

S0001-706X(18)30221-3 https://doi.org/10.1016/j.actatropica.2018.07.004 ACTROP 4709

To appear in:

Acta Tropica

Received date: Revised date: Accepted date:

21-2-2018 10-5-2018 4-7-2018

Please cite this article as: Bharati M, Saha D, Assessment of Insecticide Resistance in Primary Dengue Vector, Aedes aegypti (Linn.) From Northern Districts of West Bengal, Acta Tropica (2018), https://doi.org/10.1016/j.actatropica.2018.07.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Title: Assessment of Insecticide Resistance in Primary Dengue Vector, Aedes aegypti (Linn.) From Northern Districts of West Bengal

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Authors: Minu Bharati and Dhiraj Saha*

Affiliation: Insect Biochemistry and Molecular Biology Laboratory,

Department of Zoology, University of North Bengal, Raja Rammohunpur, P.O. North

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Bengal University, Siliguri – 734013, District – Darjeeling, West Bengal, India.

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Graphical abstract

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Highlights:

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First report of insecticide susceptibility status against four major groups of insecticide .in Ae. aegypti from dengue endemic districts of northern West Bengal, India. Widespread insecticide resistance along with underlying role of metabolic detoxification in development of resistance against insecticide in dengue vector were recorded from this region of India. This study also reports first ever Permethrin and Propoxur resistance in wild populations of Ae. aegypti from north east India.

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Abstract:

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Aedes mosquitoes are the major vectors transmitting several arboviral diseases such as dengue,

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zika and chikungunya worldwide. Northern districts of West Bengal is home to several

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epidemics vectored by mosquito including dengue infections, proper control of which depends

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on efficient vector control. However the onset of insecticide resistance has resulted in failure of vector control approaches. This study was carried out to unveil the level of insecticide resistance

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prevailing among the primary dengue vector in this dengue endemic region of India. It was

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observed that, field caught populations of Ae. aegypti were moderately to severely resistant to

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majority of the insecticide classes tested, i.e. Organochlorine (DDT), Organophosphates (temephos, malathion), Synthetic Pyrethroids (deltamethrin, lambdacyhalothrin and permethrin)

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and carbamate (propoxur). In majority of the populations, metabolic detoxification seemed to play the underlying role behind the development of insecticide resistance.This study seems to be

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the first report revealing the pattern of insecticide resistance in Ae. aegypti from Northern West Bengal. Efficient disease management in this region can only be achieved through proper insecticide resistance management. This study may help the concerned authorities in the formulation of an effective vector control strategy throughout this region incorporating the knowledge gained through this study. 2

Keywords: Aedes aegypti, Insecticide resistance, Insecticide detoxifying enzyme, Dengue, vector control

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1. Introduction: Mosquitoes pose a major human health threat due to its key role in transmission of diseases such as dengue, malaria, chikungunya, Japanese encephalitis, filariasis etc. Dengue, chikungunya and yellow fever collectively infect 50-100 million people every year, with over 2.5 billion people

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living in disease endemic areas1. Aedes mosquitoes namely, Ae. aegypti and Ae. albopictus have

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been long known for transmission of dengue virus (DENV) and chikungunya virus (CHIKV)2.

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As an impact of globalisation, both the mosquitoes have expanded their geographic distribution

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to many previously unexplored areas3. Since its first proved outbreak in 1963, most of the Indian states have now become endemic for dengue along with the recent appearance of a novel dengue

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serotype in the subcontinent4-5.

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In India, around 1,57,220 cases of dengue infections causing 250 deaths occurred in 20176. In

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West Bengal, 10,697 people were infected with dengue and 19 deaths were reported till October 20176. West Bengal, the most densely populated state of India is highly endemic to most of the

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mosquito transmitted diseases particularly dengue and chikungunya. The presence of large vegetation cover, high rainfall, poor sanitation practices and improper garbage disposal strategies

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along with a large population presents the ideal ambience for the growth cycle of Aedes mosquitoes and its transmitted disesases in northern part of West Bengal7. As no drug or vaccine is available for the treatment of dengue/ Dengue haemorrhagic fever (DHF), the sole method of prevention of disease spread is through the control of vector 3

mosquitoes i.e. Aedes mosquitoes8. The Local vector control program aims to follow a long term strategy for prevention and control of dengue in India through integrated vector management (for transmission reduction) by conducting entomological surveillance including larval surveys,

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following anti larval measures such as source reduction, use of larvicides and larvivouros fish, environment management etc and following anti adult measures through either Indoor residual

spray by 2% pyrethrum extract or fogging by 5% malathion during disease outbreaks along with personal protection measures such as, use of long lasting insecticide treated nets and other mosquito control tools 6.

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Mosquito control intervention makes the heavy use of insecticide at both household as well as

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higher levels. But due to uncontrolled and indiscriminate use of these insecticides, both the target

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as well as non target species have evolved strategies to resist the actions of those chemicals in

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their body. Different mechanisms interrupting the chemicals to manifest their planned actions is

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known as Insecticide resistance9. Insecticide resistance results in the failure of mosquito control

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programmes to achieve their planned targets, thereby increasing the risk of DENV infection even after insecticide spray during severe disease outbreaks9.

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Resistance to insecticides can be achieved by an array of modifications within a mosquito,

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including behavioural alteration, physiological modifications in cuticle (thickened cuticle) reducing the insecticide penetration, biochemical changes in the activity of major insecticide

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detoxifying enzymes or structural modification within the target site of the insecticide thereby blocking the insecticide binding and subsequent action10. All the above mentioned mechanisms have been noted to occur in field populations of Aedes mosquitoes in response to a varying degree of insecticide resistance throughout the world9.

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Mosquitoes have developed insecticide resistance both as a direct effect of insecticides targeted on them as well as an indirect exposure of insecticide sprayed on agricultural field7, 11-12. Reduced susceptibilities of Aedes species to insecticides have been linked to both over activity of

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detoxifying enzymes as well as altered target site in field populations. Over expression or gene amplification of enzyme classes, Carboxylesterases (CCEs), Glutathione S-transferases (GSTs) and Cytochrome P450s (CYP450) or Mixed Function Oxidases (MFOs) have been reported to

confer insecticide resistance in many populations of insecticide resistant Ae. aegypti and Ae. albopictus population worldwide1,13. Through advanced studies incorporating transcriptome

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studies and Detox chip analysis, all the three above mentioned enzyme classes namely, CYP450s,

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CCEs, GSTs have been implicated in conferring insecticide resistance against insecticides. Also,

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many point mutations conferring target site alteration in voltage gated sodium channel gene and

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acetylcholinisterase (AchE) gene have been identified in Ae. aegypti mosquitoes1,13. Knockdown

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resistance (kdr) mutations are widespread in different Aedes population and have been shown to

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provide varying degrees of selective advantage over pyrethroid and organochlorine insecticide pressure in many populations of Aedes aegypti1,14.

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Dengue prevention is highly dependent on effective vector control alone. The major obstacle in

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vector control is presented by insecticide resistance. Variation in insecticide resistance patterns have important implications for vector control, especially when the vector control is aimed for a

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wide geographical area. So, the information regarding the prevailing status of insecticide resistance and the underlying mechanisms of resistance in the field/wild populations of Aedes mosquitoes may help in planning efficient mosquito control strategies. The present study reveals the prevailing degree of insecticide resistance along with the involved mechanism in action throughout five dengue endemic districts of West Bengal. The overall trend presented here may 5

be helpful in devising approaches to manage insecticide resistance during vector control programmes. 2. Materials and methods:

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2.1 Selection of sampling districts: Five different sampling districts were selected based on the dengue prevalence in northern part of West Bengal, namely, Alipurduar, Jalpaiguri, Darjeeling, Coochbehar and North Dinajpur. The geographical coordinates and other relevant biotic and abiotic factors of the sampling sites are recorded in Table 1.

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2.2 Collection of larva and adult mosquitoes: The selected sampling sites (Figure 1) were

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screened for the larva and adult Aedes mosquitoes. Mosquito larvae were collected from different

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field habitats such as discarded automobile tyres, earthen pots, artificial containers, water holding

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tanks, discarded buckets, aloevera plantations, tree holes, pots etc. The larva once initially

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identified as Aedes were then collected and transferred to plastic containers and brought to the

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laboratory. The sampling was done during March to October 2017, pre-monsoon, monsoon and post-monsoon seasons and the details of total collection (sampling site and season wise) is

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provided in Table 1.

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2.3 Rearing of field caught population of mosquitoes: In the laboratory, the larvae were identified upto subspecies level following standard identification keys15-16. All the collected

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mosquitoes were identified to be Aedes aegypti aegypti. The field collected larvae (F0) were then reared at temperature 252⁰C and 70-80% relative humidity. The rearing was done based on the standard method7for successive generations. The larvae were reared to F1 generation upto adults to ensure the homogeneity of the field collected populations. The emerged adults were cross checked with adult identification keys16. The F1 adults were used for bioassays and detoxifying enzyme 6

activity studies. The field caught populations were allowed to rear for successive generations without any exposure to insecticides in the laboratory maintaining the same physical factors as mentioned earlier and provided with anesthesised rat as a source of blood for the females in each

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generation. The tenth generation larvae and adults were taken as control/ susceptible population (SP).

2.4 Insecticide source:Larvicide, namely temephos andadult insecticide impregnated papers

(malathion, deltamethrin, lambda cyhalothrin, DDT, Propoxur and permethrin) were purchased

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from Vector control unit, Universiti sains Malaysia.

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2.5 Larval bioassay: The susceptibility of Ae. aegypti larvae against temephos (an

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organophosphate) was tested following the WHO guidelines17. Two discriminating doses were

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selected: 1. WHO recommended dose (0.020 mg/L) and 2. India government recommended dose (0.0125 mg/L). Thirty (30) late third instar or early fourth instar larvae of each population (field

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caught and laboratory reared susceptible population) were exposed to test cups containing 0.0200

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mg/L and a 0.0125 mg/L solution of Temephos in water. The bioassay was done in triplicate with

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one set of control (using 1 ml of solvent) in laboratory conditions. Mortality percentage was recorded after 24 hours of exposure to temephos. Larvae were considered dead or moribund if

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they failed to evoke any response when touched17.

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For the determination of lethal concentrations (LC50 and LC90) of temephos, around 25 larvae from each population (including susceptible) were transferred to plastic cups containing different concentration of Temephos18. Four replicates for each concentration and 4-6 serially selected concentrations in the range of the insecticide causing 10% - 90% mortality (0.0001 - 0.01 mg/L) were set to determine LC50 and LC90 values. Control sets were also run containing only pure

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solvent (i.e. ethanol) in water. Larval mortality was recorded after 24 hours of insecticide exposure and the criteria for discriminating between dead and live larvae remained the same.

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2.6 Adult bioassay: Adult bioassays were also performed following WHO guidelines19. Thirty (30) non blood-fed adults were exposed to insecticide impregnated papers with WHO

recommended diagnostic dose of insecticide (5% malathion, 0.05% deltamethrin and 0.05%

lambda cyhalothrin, 4% DDT, 0.1% Propoxur and 0.75% permethrin) placed in tubes for 1 hour.

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After one hour, the mosquitoes were transferred to another tube that carried cotton balls soaked in

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10% glucose solution. The whole set was maintained at laboratory conditions for 24 hours.

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Mortality was recorded 24 hours post-exposure. For control, mosquitoes were place in tubes

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containing papers impregnated with silicone oil and acetone. The whole set was performed along

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with three replicates. Mortality percentages were calculated as the mean of four set of assays.

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2.7 Insecticide detoxifying enzymes’ activity: Single adult Ae. aegypti were homogenized in 100 μL of 0.1M sodium phosphate buffer (pH 7.2)

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with a teflon micro-pestle in a 1.5 mL centrifuge tube. The pestle was washed with another100 μL

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of 0.1M sodium phosphate buffer (pH 7.2) making the whole solution 200 μL. The homogenate was centrifuged at 12,000 rpm (revolutions per minute) for 15 minutes in a centrifuge (Sigma

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3K30, Sigma,U.K.)7. The supernatant was stored at -20⁰C and was used within 3-4 days as enzyme source for detoxifying enzyme activity assays. For each biochemical test, a minimum of thirty individuals were assayed. A duplicate set was also run for each enzyme assay.

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2.7.1 Non-specific esterase (carboxylesterase) assay: The activity of carboxylesterases (CCE) hydrolyzing α- and β- napthyl acetate as substrate were assayed according to standard WHO guidelines20 with minor modifications for using in microplate7. Fast blue B salt was used as the

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staining agent. Absorbance was recorded at 540 nm using microplate reader (Biotek ELx800, USA). Standard curves of α- and β- napthol were prepared and the CCE activity were estimated. 2.7.2 CYP450 assay: The activity of CYP450 was also measured according to standard WHO guidelines20 using 3,3',5,5'-Tetramethyl benzidine (TMBZ) as a substrate and H2O2 as the

peroxidising agent. Absorbance was recorded at 630nm using the microplate reader. A standard

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curve for the heme peroxidase activity was prepared using different concentrations of cytochrome

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c (0.0025 nM to 0.0200 nM) for horse heart type VI (Sigma Aldrich). The total CYP450 was

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expressed as CYP450equivalent units (EUs) in mg protein.

2.7.3 Glutathione S-transferase (GST) assay: GST activity was assessed following the WHO

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protocol20. 10 μL of homogenate was mixed with 200 μL of CDNB/GSH (working solution) in

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wells of microtitre plate. Blanks were set with 10 μL of distilled water along with 200 μL of working solution. The plate was read continuously for 5 minutes at 340 nm and the GST activity

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(μM mg protein-1 min-1) was calculated.

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2.7.4 Total protein content: Total protein of each individual of Ae. aegypti was determined

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according to standard WHO guidelines20 to cancel out any size differences among individuals and for the correct expression of enzyme activity. 2.8 Electrophoretic analysis of α- and β-esterases: Native Polyacrylamide Gel Electrophoresis (PAGE) of different Ae. aegypti populations i.e. field collected as well as from laboratory reared control populations were carried out in 8% 9

polyacrylamide gels using equal amount of protein for each populations in tris-glycine (pH 8.3) buffer system at 120V for 4-5 hours at 40C. The gels were stained for CCE isozymes21 and the Rf (Retardation factor) values of CCE isozyme bands were determined according to the standard

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method. 2.9 Calculation:

In the bioassays, control mortalities were below 5%, so no calculation of corrected mortality was needed. LC50, LC90 and LC99 were estimated at 95% confidence interval by putting log dose

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against probit in SPSS 16.0 software and the obtained linear regression coefficient (r2) was used to

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assess the linearity of the data set. Double the extrapolated value of LC99 was taken as the

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recommended discriminating dose of the insecticide for that specific region/area. The population

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with mortality percentages when > 98 is said to be susceptible, 80-97 is assessed as resistance not confirmed (=unconfirmed resistance= incipient status) and <80 as resistant17.

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3.Result:

3.1 Larval bioassays: Most of the tested populations were completely susceptible to both the

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concentrations of temephos. However, one population, i.e. NDP was found to have reduced mortality percentages 91.8% (for WHO recommended dose)and 79.1 % (for NVBDCP

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recommended dose) in the range of incipient resistance andresistance respectively. The LC50 value ranged from 9.4 x 10-5ppm to 2.0 x 10-3 ppm, i.e. 1.64 times to 35.08 times the LC50 value

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of SP. The highest value of LC50 and LC99 was noted for NDP population. The recommended Temephos dosage for all the studied districts were also evaluated, that ranged from 5.2 x 10-1 ppm to 6.4 x 10-4ppm.

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3.2 Adult bioassays: Through the study of adult bioassays, it was noted that widespread resistance was prevalent among the tested populations against an array of insecticides (Figure 2). For malathion, four of the populations showed >98% mortality after 24 hours, though two of the

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populations, i.e. APD and DAR had mortality percentages below 98% (Table 3). None of the tested populations were completely susceptible to DDT. All field populations exhibited moderate to severe resistance with mortality percentages ranging from 47.9% to 72.0%. Against type II

pyrethroids (deltamethrin and lambdacyhalothrin), none of the tested populations were found to be totally resistant though three of the populations i.e. APD, JPG, NDP were found to possess

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unconfirmed resistance (or incipient resistance status). Through the result of permethrin

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bioassay, it was found that all the tested populations were resistant or incipiently resistant against

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permethrin with mortality percentages as low as 60% to 83.3%. Similar pattern was noted for the

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only carbamate insecticide used in this study, i.e. propoxur, mortality percentages were noted to

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range from 50 % to 97.2%.

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3.3 Biochemical enzyme assays:The activity of major detoxifying enzyme groups were varying among different field caught populations of Ae. aegypti (Table 4). The activity of α-CCEs ranged

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from 0.231 to 0.672 µmoles mg protein-1 min-1, i.e. 1.07 to 3.11 times that of the SP. Similarly, the activity of β-CCEs were 1.11 to 2.46 times the activity level of SP. For both α- and β-CCEs,

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the highest level of activity was noted for NDP population, i.e. 0.672 and 0.414µmoles mg

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protein-1 min-1 respectively. The level of CYP450 monoxygenase activity were similar throughout the tested mosquito populations, ranging from 0.047 to 0.063 nmoles mg protein-1 min-1 . The highest level of activity was noted for JPG population, i.e. 1.53 times higher than SP. The GSTsactivity ranged from 0.33 to 0.43 GSH-CDNB conjugate μM mg protein-1 min-1 amongst the tested mosquito populations. 11

3.4 Qualitative analysis through native PAGE: Through the study of native PAGE, it was found that altogether five different bands were found for CCEs reacting with α-napthyl acetate among the tested populations. Only one population, i.e. NDP was found to express all the five

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bands with their Rf value ranging from 0.62 to 0.97, whereas the remaining showed the presence of a single band with Rf value 0.95to 0.96 differing in its intensity among the tested populations (Table 5, Figure 3). Similarly, for CCEs reacting with β-napthyl acetate, a total of three distinct bands were observed among the tested populations. All the three observed bands were expressed by NDP population with Rf value ranging from 0.60 to 0.97 whereas all other populations

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expressed a single band with Rf value 0.96 to 0.97 (Table 6). However, the intensity of the bands

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were varying among different populations (Table 6, Figure 4).

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4. Discussion:

The study revealed the level of insecticide resistance prevailing in the wild populations of Aedes

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aegypti in five dengue endemic districts of sub-Himalayan West Bengal. Majority of the studied

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larval Ae. aegypti populations were found to be susceptible to temephos except one population i.e. NDP population. In NDP population, moderate resistance (91.8% mortality) against WHO

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recommended dosage of temephos (0.02 ppm) and severe resistance (79% mortality) against

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Indian government (0.0125 ppm) recommended dosage was recorded (Table 2). The NDP mosquito population were collected from national highways and surrounding habitable areas,

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which points on the danger of possessing this kind of insecticide resistance. In a similar type of study in northeastern province of India, three out of the four studied field populations of Ae. aegypti were found to possess similar degree of resistance as found in present study22. The occurrence of increased temephos resistance in NDP thus imparts light on the level of insecticide selection pressure prevailing in this population23. Such an intense insecticide selection pressure 12

could have been achieved due to routine use of temephos for control of both malaria and dengue vector by Government6 and non- government pest and vector control agencies owing to its ease of availability and low cost in Indian market. Since, temephos is the widest used larvicide in

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India6, development of resistance against this larvicide may have serious implications in dengue prevention efforts24.

Generally, the major detoxification enzyme groups associated with resistance against

organophosphate are mostly CCEs25-26. Through biochemical assays of enzyme activity, the

involvement of CCEs behind temephos resistance was recorded as the activity of α- and β- CCEs

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were significantly enhanced in NDP population compared to other tested populations (Table 4).

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Similar associations between CCEs and development of resistance against temephos have also

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been noticed worldwide in both artificially selected populations27 as well as field collected

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populations of Ae. aegypti26,28. Similar interpretations were also made from the study of

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qualitative analysis of CCEs through the study of native PAGE. NDP population was found to

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express numerous deeply stained bands, i.e. five for α-CCEs and three for β-CCEs (Table 5, Table 6). It has been shown in different populations of Ae. aegypti that presence of isozyme

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variations of CCEs correspond to resistance against temephos29-30 and other Organophosphates31. Also, the greater intensity of the band in NDP population may correspond to over expression of

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the corresponding isozyme of CCE32, thus resulting in the resistance found in our study. Similar

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associations between isozyme overexpression and insecticide resistance have also been observed in Ae. albopictus in Indian state of Tamilnadu33. Resistance against one more Organophosphate insecticide i.e. malathion was assessed in this study among different field populations of Ae. aegypti. Here, two of the six tested populations, namely, APD and DAR were revealed to have moderate and possible resistance respectively 13

against malathion. Both the districts, Darjeeling and Alipurduar are characterized by having intense tea plantations, on which the much of their economy is dependent34. Moreover, the close vicinity of Tea plantations to human habitats in these districts indicate a possibility of indirect

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exposure to agriculturally sprayed malathion in Aedes mosquitoes by contaminating their breeding habitats. The pattern of resistance observed in APD and DAR population of Ae. aegypti may thus be pertained to the heavy use of this insecticide in agricultural field throughout India.

Similar cases of insecticide resistance in mosquitoes as a result of indirect exposure to malathion from agricultural sector have also been reported through different wild populations worldwide35.

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Resistance against malathion have also been linked to the over-activity of CCE enzymes in

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different field populations of Ae. aegypti37. In this study, though the quantitative levels of α- and

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β-CCEs were noted to be significantly higher in the resistant populations than the susceptible

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ones (with the exception of NDP), the observations of qualitative study of CCEs seem to provide

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a clearer insight into the observed resistance phenomenon (Table 5, Table 6). In APD, single but deeply stained bands were observed for both α- and β- CCEs, similarly for DAR also, moderately

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darker bands were observed having similar Rf values, i.e. 0.96 (Figure 3-4, Table 5-6). Since the intensity of bands in native page provides an indication of over-expression of specific

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isozymes32, it may be interpreted that the over-expressed CCE isozymes (through higher

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detoxification) may contribute towards the observed resistance against malathion. Variable patterns of resistance were observed in this study amongst different field populations of Ae. aegypti against two of the tested OPs, i.e. temephos and malathion. Such an observation may be explained by the fact that resistance against malathion, which is an open chain OP has been

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shown to be provided by different carboxylesterase mechanism with no relation, i.e. cross resistance to temephos38. The widespread use of DDT for malaria vector control throughout the world since the mid 20th

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century has forced the strong selection of DDT resistant population of Ae. aegypti throughout different corners of world1,39-41. In this study, all field populations of Ae .aegypti were found to be resistant to DDT (Table 3), with mortality percentages ranging from as low as 47.9% to

72.0% and 98.2 % for SP (Table 3). Generally detoxification of DDT is conferred either by

metabolic enzymes (GSTs and partly due to P450s or CCEs)42 or due to kdr mutation43 or by a

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combination of both14. There was no significant variation in the level of GST activity among the

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tested populations which may explain the similar levels of DDT resistance recorded throughout

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different sites (Table 4). The trend of DDT resistance observed in this region has been found to

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be similar as observed in nearby regions of India in Aedes mosquitoes44.

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Amongst the studied population, except one population i.e. COB was found to possess reduced

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susceptibility for all tested pyrethroid insecticide. Moreover, none of the tested populations were found to be completely resistant to deltamethrin or lambdacyhalothrin (though incipient

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resistance was widespread). Whereas, all the population were found to have reduced mortality

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percentages, i.e. below susceptibility range for permethrin, the sole type I pyrethroid tested. Pyrethroid susceptible populations of Ae. aegypti have been reported from other parts of India44. This is first report of reduced susceptibilities to pyrethroids, resistance against permethrin and

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higher levels of incipient resistance against deltamethrin and lambdacyhalothrin in Ae. aegypti from this region.

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Two types of mechanisms have generally been linked to resistance against pyrethroid insecticides in Ae. aegypti, i.e. metabolic detoxification by CYP450 (in majority of cases), GSTs and CCEs or due to the presence of different kdr mutations, i.e. target site insensitivity1,41,46-47.

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Through the study of detoxification enzymes activity, it was found that CYP450 activity was significantly higher in most of the field populations in comparison to susceptible population. Moreover, the significantly higher level of CYP450 activity in APD, JPG, DAR and NDP

population indicates a possibility of involvement of CYP450 enzyme in the tested resistant (/

incipiently resistant) field populations of Ae. aegypti. The activity of α- and β- CCEs may also

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play a minor role in conferring resistance against pyrethroid insecticides in APD and NDP

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population as has been reported in some other Ae. aegypti populations24. However, the presence

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of widespread resistance against DDT throughout the study area brings into focus the vital role

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of prevailing kdr mutations in providing resistance against pyrethroid insecticides40,46-47 as

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evident in COB population (resistant to both DDT and permethrin).

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Since Ae. aegypti is an anthropophilic mosquito, the reduced susceptibilities against pyrethroid insecticides observed in this study may be either due to direct exposure to household mosquito

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repellent techniques, such as coils, fumigants, creams22 or indirect exposure to agricultural pyrethroids. Pyrethroids from agricultural sources have been shown to contaminate the mosquito

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breeding site and thereby beginning the onset of insecticide resistance in mosquito population7,11. The fact that both DDT and insecticides of pyrethroid origin share their target site of action,

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increases the chances of pyrethroid resistance in field populations of Ae. aegypti. Amongst the populations tested for resistance against propoxur, the only carbamate insecticide in this study, two field populations( JPG and DAR) were found to be resistant, whereas remaining (APD and COB) were found to possess unconfirmed resistance against propoxur (Table 3). Very 16

few studies have reported resistance against propoxur in field strains of Ae. aegypti42. Resistance against propoxur has generally been associated with detoxification through CCEs48 and rarely through CYP450 and GSTs41 or through altered AchE, i.e. ace mutations42. In this study no

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correlation could be established between resistance against propoxur and biochemical activity of CCEs, CYP450 or GSTs. Since AChE is the shared target of both OP and carbamate insecticide, the presence of altered AChE generally results in cross resistance to both OP and carbamates,

which may be the scenario for DAR and APD population (cross resistance between malathion and propoxur) but not in JPG and COB population. So, it may be concluded that mutation in

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acetylcholinisterase may have some role in resistance against malathion and propoxur in DAR

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and APD population, but some other mechanism may be the underlying reason behind severe

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propoxur resistance in JPG Population. In India, propoxur is not used in mosquito control

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programmes6, yet the presence of widespread resistance in Ae. aegypti against it may be due to the indirect exposure through household insect repellent tools which contain upto 2 % propoxur

5. Conclusion:

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(such as No-P spray for bed bugs and termites, Baygon spray etc).

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This study seems to be the first study revealing the prevailing level of resistance against a

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multitude of insecticides in Ae. aegypti from this region. The endemicity of this region for majority of vector borne diseases and associated vector control approaches may be the reason for

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the widespread resistance recorded against different insecticides. There is an urgent need to generate knowledge on the molecular mechanisms involved behind development/maintenance of resistance in Ae. aegypti mosquitoes, so that an effective vector control strategy (incorporating insecticide resistance management approaches) could be designed and followed. Since none of the insecticides could result in complete susceptibility, yet, temephos seems to be the most 17

preferred choice of insecticide among the six tested insecticides in majority of the district except North Dinajpur. In India the introduction of Insect Growth Regulators (IGRs) and neonicotinoids in vector control may help achieve efficient mosquito control. Also, more studies should be

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directed to map the insecticide resistant status throughout different regions of India and to formulate site specific insecticide choice and dosage.

India which is a part of global mega biodiversity region should be screened critically for the

presence of compounds of botanical origin with potency for mosquitocidal or mosquito repellent activity. Discovery of novel insecticide groups with novel target sites should be targeted which

U

may result in delayed onset of resistance against those insecticides. Transposable elements

N

favouring the duplication of detoxifying enzymes in Ae.aegypti (and hence resulting in

A

insecticide resistance) should be paid immense attention and ways to combat it should be

M

searched49. Critical screening of Aedes breeding habitats and their destruction along with study

D

of other techniques such as sterile male mosquito, new tools for efficient mechanical control of

TE

mosquito, prospect of Wolbachhia in dengue prevention should be aimed to reduce the dependencies on insecticides of chemical nature for mosquito control. This study may help the

EP

concerned authorities immensely in the formulation of an effective resistant management strategy for dengue vector control throughout the region incorporating the knowledge gained

CC

through this study.

A

6. Acknowledgements: The authors express their sincere thanks to the Head, Department of Zoology, University of North Bengal, for providing laboratory facilities and funds from the departmental budget allocation. The authors also express their sincere appreciation to those scientists and authors upon whose concepts, hypotheses and scientific contributions the present work has been formulated, experimented and results discussed. Thanks are also expressed to the 18

University of North Bengal for providing uninterrupted LAN (Local Area Network) that has helped immensely in searching and collecting related information. The first author expresses sincere gratitude to University Grants Commission (UGC) for providing financial assistance

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throughout this work through award letter Sr. no. 2121430414, Ref no.: 21/12/2014 (ii) EU-V, Dated 03/06/2015.

U

Conflict of interest: The authors declare that they have no conflict of interest.

N

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Figure 1: Map of the sampling districts 26

120

80 60 40 20 0 APD

JPG

DAR

Sampling Sites Deltamethrin

Lambdacyhalothrin

Permethrin

U

DDT

NDP

SP

Propoxur

N

Malathion

SC RI PT

Mortality percentages

100

A

CC

EP

TE

D

M

A

Figure 2: Insecticide susceptibility status against six adulticides among wild Ae. aegypti populations rom northern districts of West Bengal

*APD: Alipurduar, COB: Coochbehar, JPG: Jalpaiguri, DAR: Darjeeling, NDP: North Dinajpur, SP: Susceptible population Figure 3: Qualitative estimation of α-CCEs in different field caught Ae. aegypti populations

27

SC RI PT U N A

M

*APD: Alipurduar, COB: Coochbehar, JPG: Jalpaiguri, DAR: Darjeeling, NDP: North Dinajpur, SP: Susceptible population

TE

D

Figure 4: Qualitative estimation of β-CCEs in different field caught Ae. aegypti populations

Population abbreviated name

CC

Districts

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Table 1: Details of the Sampling Districts:

Disease endemicity

Last season of dengue outbreak

APD

26.69° N 89.47° E

Dengue, Malaria, JE

2017

Coochbehar COB

26.34° N 89.46° E

Dengue, Malaria, JE, Filariasis

2017

A

Alipurduar

Geographical coordinates

28

Total numbers of mosquito (larvae and pupae) sampled Pr M-1354 M-1207 Po M-1095 Pr M-1412 M-1126 Po M-1114

Mosquito Generation used in Experiments

F1

F1

JPG

26.52° N 88.73° E

Dengue, Malaria, JE, AES

2017

Pr M-1371 M-1048 Po M-991

F1

Darjeeling

DAR

26.71° N 88.43° E

Dengue, Malaria, JE, AES

2017

Pr M-1243 M-1197 Po M-1013

F1

North Dinajpur

NDP

26.27° N 88.20° E

2017

Pr M- 1821 M-915 Po M-888

F1

Susceptible population

SP

--

Dengue, Malaria, JE, AES --

--

--

F10

A

N

U

SC RI PT

Jalpaiguri

M

Table 2: Susecptibility to temephos in larval Ae. aegypti collected from districts of northern Bengal Mortality %age

LC50 dose (ppm) ±S.E.

LC99 dose (ppm) ±S.E.

Recommended

(0.02ppm)

(0.0125ppm)

(95%C.I.)

(95%C.I.)

dosage

1.71x 10-4  1.7 x 10-5

2.9 x 10-3  1.9x 10-4

5.8 x 10-3

EP

(ppm)

100

9.4 x 10-5  1.1 x 10-6

3.2 x 10-4  1.6 x 10-5

6.4 x 10-4

CC

TE

D

Mortality %age

100

5.3 x 10-4  3.2 x 10-5

8.1 x 10-3  1.2 x 10-4

1.6 x 10-2

100

100

3.1 X 10-4  2.1 x 10-5

4.3 X 10-3  3.9 x 10-4

8.6 x 10-3

A

Sites

NDP

91.8

79.1

2.0 x 10-3  1.2 x 10-4

2.6 x 10-1  5.4 x 10-2

5.2 x 10-1

SP

100

100

5.7 x 10-5  1.1 x 10-6

1.1 x 10-4  2.9 x 10-5

--

APD

100

COB

100

JPG

100

DAR

100

29

Table 3: Insecticide susceptibility status of adult Ae. aegypti against six adulticides. Sites

Malathion

DDT

Deltamethrin

Lambdacyhal

Permethrin

Propoxur

(%age)  S.E.

(%age)  S.E.

(%age)  S.E.

othrin

(%age)  S.E.

(%age)  S.E.

55.4  1.42

89.2  0.81

80.9  0.76

83.3  0.88

91.3  0.71

(n=102)

(n=92)

(n=93)

(n=105)

(n=132)

(n=92)

70.0 1.02

100.0 0.00

100.0 0.00

76.5  0.99

97.2  0.72

(n=92)

(n=90)

(n=90)

(n=97)

(n=98)

(n=108)

99.0 0.33

72.01.15

91.90.56

81.50.33

64.5 1.41

77.2 1.21

(n=101)

(n=100)

(n=99)

(n=103)

(n=124)

(n=101)

47.9 1.41

100.00.00

60.01.03

50.01.33

(n=95)

(n=98)

(n=92)

(n=92)

(n=90)

(n=98)

100.0  0.00

56.6 1.32

90.3 1.01

85.0 1.13

#

#

(n=91)

(n=90)

(n=93)

(n=99)

100.00.00

98.20.77

SP

(n=93)

TE

NDP

D

DAR 92.6 0.89

N

JPG

(n=92)

100.00.00

A

COB 100.0 0.00

U

72.5  1.26

M

APD

SC RI PT

(%age)  S.E.

100.00.00

100.00.00

100.00.00

100.00.00

(n=95)

(n=101)

(n=90)

(n=97)

CC

EP

#enough sample not available for performing bioassay.

Table 4: Activities of major detoxifying enzymes in different field caught populations of Ae.

A

aegypti Sites

APD

α-CCEs

β-CCEs

CYP450Monoxygenase

GSTs

(µmoles mg protein-1

(µmoles mg protein-1

(nmoles mg protein-1 min-1)

(μMmg protein-1 min-1)  S.E.

min-1 )  S.E.

min-1 )  S.E.

 S.E.

0.322  0.007b*

0.231  0.004b

0.058  0.0017b 30

0.37  0.002a

0.187  0.002b

0.047  0.0006a

0.33  0.003a

0.276  0.003b

0.201  0.006b

0.063  0.0021b

0.43  0.007a

DAR 0.367  0.009b

0.211  0.008b

0.061  0.0019b

0.39  0.004a

NDP

0.672  0.018c

0.414  0.009c

0.052  0.0011b

0.34  0.006a

SP

0.216  0.003a

0.168  0.004a

0.041  0.0007a

JPG

SC RI PT

COB 0.231  0.004a

0.31  0.004a

*Within columns, means followed by the same letter do not differ significantly (P=0.05) in Tukey’s multiple comparison test (HSDa).

Band number

Intensity

APD

α-Est V

+++

COB

α-Est V

JPG

α-Est V

+

M

0.95

SLG

α-Est V

++

0.95

NDP

α-Est I

TE

+++

0.62

α-Est II

+++

0.68

α-Est III

+++

0.73

α-Est IV

+++

0.82

α-Est V

++

0.97

α-Est V

+

0.95

A

D

EP SP

+

Rf value

N

Sites

CC A

U

Table 5: Details of electrophoregram of α-CCE isozymes

0.96 0.95

+++ represents severe, ++ represents moderate and + represents low expression of isozymes of CCEs.

31

Table 6: Details of electrophoregram of β-CCE isozymes Band name

Intensity

Rf value

APD

β-Est III

+++

0.96

COB β-Est III

+

0.96

JPG

β-Est III

+

0.97

SLG

β-Est III

++

0.96

NDP

β-Est I

+++

0.62

β-Est II

+++

0.80

β-Est III

++

0.97

β-Est III

+

U N

SP

SC RI PT

Sites

0.96

A

CC

EP

TE

D

M

A

+++ represents severe, ++ represents moderate and + represents low expression of isozymes of CCEs.

32