Characterization and establishment of a reference deltamethrin and cypermethrin resistant tick line (IVRI-IV) of Rhipicephalus (Boophilus) microplus

Accepted Manuscript Characterization and establishment of a reference deltamethrin and cypermethrin resistant tick line (IVRI-IV) of Rhipicephalus (Boophilus) microplus

Srikant Ghosh, Snehil Gupta, KG Ajith Kumar, Anil Kumar Sharma, Sachin Kumar, Gaurav Nagar, Rinesh Kumar, Souvik Paul, Ashutosh Fular, Gajanan Chigure, Abhijit Nandi, HV Manjunathachar, Aquil Mohammad, MR Verma, BC Saravanan, Debdatta Ray PII: DOI: Reference:

S0048-3575(17)30084-6 doi: 10.1016/j.pestbp.2017.03.002 YPEST 4040

To appear in:

Pesticide Biochemistry and Physiology

Received date: Revised date: Accepted date:

11 June 2016 31 January 2017 1 March 2017

Please cite this article as: Srikant Ghosh, Snehil Gupta, KG Ajith Kumar, Anil Kumar Sharma, Sachin Kumar, Gaurav Nagar, Rinesh Kumar, Souvik Paul, Ashutosh Fular, Gajanan Chigure, Abhijit Nandi, HV Manjunathachar, Aquil Mohammad, MR Verma, BC Saravanan, Debdatta Ray , Characterization and establishment of a reference deltamethrin and cypermethrin resistant tick line (IVRI-IV) of Rhipicephalus (Boophilus) microplus. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Ypest(2017), doi: 10.1016/j.pestbp.2017.03.002

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ACCEPTED MANUSCRIPT Characterization and establishment of a reference deltamethrin and cypermethrin resistant tick line (IVRI-IV) of Rhipicephalus (Boophilus) microplus Srikant Ghosh*, Snehil Gupta, Ajith Kumar KG, Anil Kumar Sharma, Sachin Kumar, Gaurav

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Nagar, Rinesh Kumar, Souvik Paul, Ashutosh Fular, Gajanan Chigure, Abhijit Nandi, HV

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Manjunathachar, Aquil Mohammad, MR Verma1, BC Saravanan and Debdatta Ray

Entomology Laboratory, Division of Parasitology, ICAR-Indian Veterinary Research Institute,

Division of Economics and statistics, ICAR-Indian Veterinary Research Institute, Izatnagar –

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1

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Izatnagar – 243122 (U.P.), India

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243122 (U.P.), India

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*Correspondence: [email protected]

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ACCEPTED MANUSCRIPT ABSTRACT The problem of ticks and tick borne diseases is a global threat and growing reports of resistance to commonly used insecticides further aggravated the condition and demands for country specific

resistance monitoring tools and possible solutions of the problem.

Establishment of standard reference is prerequisite for development of monitoring tools. For

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studying possible role of different mechanisms involved in development of resistance in

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Rhipicephalus (Boophilus) microplus population and to develop newer drug to manage the

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problem of resistance, a deltamethrin exposed and selected tick colony, referred to as IVRI-IV, was characterized using reference susceptible IVRI-I tick line as control. The RF values of

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IVRI-IV ticks against deltamethrin, cypermethrin and diazinon were determined as 194.0, 26.6, 2.86, respectively, against adults . The esterase enzyme ratios of 2.60 and 5.83 was observed

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using α-naphthyl and β-naphthyl acetate while glutathione S–transferase (GST) ratio was 3.77. Comparative analysis of IVRI-I and IVRI-IV carboxylesterase gene sequences revealed 13 reported for the first time. The C190A

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synonymous and 5 non synonymous mutations,

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mutation in the domain II S4-5 linker region of sodium channel gene leading to leucine to isoleucine (L64I) amino acid substitution was also detected in the IVRI-IV population. In the

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present study, monitorable indicators for the maintenance of the reference IVRI-IV colony, the

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first established deltamethrin and cypermethrin resistant tick line of India, were identified. Keywords: Rhipicephalus (Boophilus) microplus, pyrethroid resistant, sodium channel gene mutation, CES gene mutation, enzyme alteration

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ACCEPTED MANUSCRIPT 1. Introduction Ticks are obligate hematophagous arthropods parasitize every class of vertebrates in almost every region of the world [1]. In India, Rhipicephalus (Boophilus) microplus has been reported from almost all states except Andaman and Nicobar islands, Manipur and is responsible for transmission of Babesia bigemina and Anaplasma marginale in cattle [2,3] and the cost of

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controlling ticks and tick borne diseases has been estimated at the tune of 498.7 million USD per

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annum [4]. The R.(B.) microplus has been ranked 6th amongst the resistant arthropods and

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resistance to almost every chemicals has been reported from different parts of the world including India [5,6].

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Reference resistant homogenous tick colonies are the essential biological material required

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to study the mechanism of development of resistance and to formulate newer tick management strategy for field situation. In recent years, resistance reports have gained paramount attention amongst the tick researchers and drug manufacturers, but little attention has been paid towards

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development of country specific reference tick line for generation of country specific base line data [7,8].

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For the last several years, a number of reference R. (B.) microplus strains susceptible and resistant to SP compounds are being maintained by CFTRL (Texas), CSIRO (Australia),

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UFGRS (Brazil) etc. for generation of country specific tools for resistance monitoring [9]. In India, specific susceptible lines viz., IVRI-I, R. (B.) microplus and IVRI-II, Hyalomma anatolicum strains were characterised and registered in the national registration system (NBAII/IVRI/BM/1/1998 and NBAII/IVRI/HA/1/1998). However, as such no reference resistance tick lines were available to study the mechanism of development of resistance. In the present study, an attempt was made to characterise and establish a reference resistant tick line to support the resistance studies.

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ACCEPTED MANUSCRIPT Development of resistance against cypermethrin, and deltamethrin had been reported from various parts of India and considered to be a serious issue in livestock development programme [10,11, 12]. To unravel the factors associated with development of SP resistance, a deltamethrin exposed and selected tick colony is maintained since 2009 in the Entomology laboratory of the institute, as IVRI-IV. Attempts have also been made to develop diazinon and multi drug

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resistant R. (B.) microplus lines in the entomology laboratory, IVRI by providing continuous selection pressure of acaricides for the last five to six years but characterization is awaited. The

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present study was undertaken to characterize the repeatedly deltamethrin exposed tick colony,

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IVRI-IV, to establish it as reference material for tick research and to enrich the registry of

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characterized arthropods of importance.

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2. Material and Methods 2.1. Acaricides

grade cypermethrin, deltamethrin,

and diazinon were procured from

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Technical

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AccuStandard® Inc. / Sigma-Aldrich, U.S.A., and were used for the preparation of stock solutions in methanol/acetone. For dose-dependent bioassay, different working concentrations

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(in multiples of DC generated using reference susceptible IVRI-I line) were prepared from the stock solution in distilled water. Commercialised insecticide preparations were avoided to

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maintain repeatability of the results and to avoid additional effect of synergist or any proprietary additives used by manufacturers.

2.2. Animals For maintaining ticks, weaned crossbred (Bos taurus male x B. indicus female) male calves were reared in tick proof animal houses of the division of Parasitology, Indian Veterinary Research Institute and fed with calf starter, milk, concentrate mixture, wheat bran and water ad

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ACCEPTED MANUSCRIPT libitum. Two animals were used alternatively and after 2-3 cycles of release of larvae for feeding, the animal was given rest for 1-2 months and alternatively second animal was used for the recovery of sufficient number of engorged ticks for experimentation. The calves were reared as per the guidelines of statutory Indian body, “Committee for the Purpose of Control and

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Supervision on Experimentation on Animals” (CPCSEA).

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2.3. Reference susceptible IVRI-I line

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The homogenous colony of acaricide susceptible IVRI-I line of R. (B.) microplus (National strain registration no. NBAII/IVRI/BM/1/1998) was maintained at 28°C and 85 ± 5% RH in the

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Entomology Laboratory, Division of Parasitology since 1998. On an average 8-10 hrs light

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period was maintained for rearing of ticks in incubator. The genetic homogeneity (99.8%) in the colony was confirmed by PCR amplification and sequence analysis of mitochondrial 16S rDNA [13]. This tick line was adapted in the laboratory and is continuously being reared for the last

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several years. The discriminating concentrations (DC) of chemical acaricides used for resistance characterization were determined by adult immersion test (AIT) and larval packet test (LPT) using the reference line [14,15]. For biochemical and molecular characterization, larvae and

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2.4. Tick colony

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adults were stored in -80°C.

The colony of R. (B.) microplus is continuously maintained since 2009 exerting repeated selection pressure of deltamethrin in each generation of adults. The ticks were originally collected from Danapur village of Patna district in Bihar, India. To maintain the resistance level, ticks of each generation was selected after exposure to particular concentration of deltamethrin. The resistance factor of the line was calculated at every five generations employing laboratory standardized AIT and LPT procedures. The 40th generation of the colony which developed high rate of survivability to exposed dose of deltamethrin were selected for characterization. 5

ACCEPTED MANUSCRIPT 2.5. Bioassays The AIT was performed as described by Benavides et al. [16] with minor modifications [10,17]. To determine LC50 and LC95 values of chemical acaricides, the dose-response assay using reference susceptible IVRI-I tick line was conducted using serial dilution prepared from the stock solution. The resistance factor (RF) of IVRI-IV ticks was estimated by dividing the

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LC50 value of respective chemical acaricide against IVRI-IV ticks with LC50 against susceptible

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ticks [17]. The LPT was conducted as per FAO guidelines with minor modifications and RF was

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calculated [18]. Based on the RF value, resistance level of IVRI-IV is categorised [10]. The probit analysis and chi square test of dose-response data of IVRI-IV ticks against deltamethrin,

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2.6.1. Esterases

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2.6. Biochemical characterization

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cypermethrin and diazinon were performed using SAS 9.3 statistical software.

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Esterase activity based on substrate – product reaction was determined according to the method of Hemingway and Brogdon [19] with laboratory standardized modifications. Forty

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larvae of 10-14 days old stored at -80°C were homogenized with 150 µl of distilled water followed by centrifugation at 1100xg at 4°C for 15 min and then supernatant was collected for

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enzyme assay. A 30 mM stock solution of α and β naphthyl acetate was prepared in acetone and stored separately at 40 C. The working solution of α and β naphthyl acetate was prepared fresh by adding 100 µl of 30 mM α or β naphthyl acetate in 9.9 ml of 0.02 M phosphate buffer, pH 7.2. The fast blue stain solution was also made fresh by adding 15 mg fast blue in 1.5 ml distilled water and 3.5 ml of 5% SDS in 0.1 M sodium phosphate buffer (pH 7.2). In a 96 well microtitre plate, 20 µl of the homogenate was added to adjacent wells in duplicate with 150 µl of either α or β naphthyl acetate working solution, respectively. In the control blank wells, 20 µl of distilled water was added in place of tick homogenate. The reaction mixtures were incubated at 6

ACCEPTED MANUSCRIPT room temperature for 15 min, 50 µl of fast blue solution was then added to each well and the plates were incubated at room temperature for 5 min to allow the colour formation to occur and the absorbance was measured at 570 nm in an ELISA reader. The amount of naphthol produced was divided by the number of minutes the substrate and homogenate were incubated before the stain was added. The total protein concentration of each sample was determined according to

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dye-binding method of Bradford using a microtiter plate (Axygen, USA). Standard bovine serum albumin (2mg/ml; Sigma aldrich) was used for the preparation of protein standard curve.

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The data were subjected to linear regression analysis. The absorbance was measured at 595 nm

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and the mean concentration of protein was determined. The amount of naphthol produced per

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minute obtained as above was again divided by the protein value. Esterase activities were

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reported as µmoles of product formed/min/mg protein [20].

2.6.2 Glutathione S- transferase (GST)

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The GST assay was performed according to Habig et al. [20] with some modifications. Forty 10-14 day old larvae stored at -80°C were homogenized in mortar with 150 µl of distilled water followed by centrifugation at 1100xg at 4°C for 15 min and then supernatant was collected

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for GST assay. The working solution for the assay was prepared by adding 2.5 ml of 10 mM

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reduced glutathione (GSH) in phosphate buffer (pH 6.5) with 125 µl of 63 mM chloroditrobenzene (CDNB). In a 96 wells microtitre plate 150 µl working solution was added with10 µl of larval homogenate in duplicate adjacent wells. All the reagents were freshly prepared and used within 2 hrs. In the control blank well, 10 µl water was added in lieu of tick homogenate. The plates were incubated for 20 min in dark and absorbance was read at 340 nm. The concentration of GST was calculated by Lambert-Beer’s law A = CL Where, A: Absorbance at 340 nm after 20 min; 7

ACCEPTED MANUSCRIPT Extinction coefficient of product (3-(2-chloro-4-nitrophenyl)-glutathione)=4.39mM-1; L: Path length, which in this case was 6 mm; C: Concentration For calculating the specific activity of GST, the GST concentration of the homogenate was

Therefore, GST activity in the homogenate was calculated as

Optical Density X 1000

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GST (mM/mg/min) =

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divided by total protein as calculated by Bradford assay.

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4.39 mM-1 X 6 mm X total protein in 10 µl homogenate X 20 min For each sample duplicate wells were used and data obtained were analyzed by one way

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ANOVA.

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2.7. Molecular Characterization

Total RNA from randomly selected adult female ticks (n=10 for each) of IVRI-I and IVRI-

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IV was extracted using Trizol® reagent (Thermo Scientific, USA). The integrity of RNA was

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checked by gel electrophoresis and the concentration was determined in NanoDrop spectrophotometer and cDNA was synthesized using oligo dT primer. The complete cds of CES

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gene (1635 bp) was amplified using primers, CES F 5’ATGGCGGTGAAAGCAGCTGTG3’ and CES R 5’GCGATTTCCCTTAGAAGAGTGACTT3’custom designed from accession no.

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AF182283. The 50µl PCR reaction was set up containing 5 µl 10x Dream Taq buffer; 5 µl cDNA (1:5 dilution, 100ng/ µl); 2 µl 25mM MgCl2; 1 µM each primer; 0.4mM dNTPs and 0.5 µl Dream Taq polymerase (5U/ µl) (Thermo Scientific, USA) and cycling conditions were 95°C for 3 minutes, 40 cycles of 95°C for 30 sec, 63°C for 30 sec, 72°C for 2 minutes and finally 72°C for 10 minutes. The amplified gene was cloned using pTZ57R/T vector and E. coli DH5α cells. Five positive clones, each from a different RNA sample were outsourced to Delhi University DNA sequencing facility for double stranded sequencing. To screen for mutations in the CES gene, the forward and reverse sequence data of IVRI-IV and IVRI-I were aligned, 8

ACCEPTED MANUSCRIPT analysed and compared using Clustal W in laser gene software (DNA Star Inc., Madison, USA) and BTI software (Gene Tool lite, USA). The C190A mutation in S4-5 linker region of sodium channel gene has been detected earlier in the IVRI-IV tick population and regularly monitored at each generation. In the current study, 40th generation was screened for the presence of C190A mutation. The PCR amplification,

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cloning and sequencing was performed following the protocol of Kumar Rinesh et al. (2013)

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[21].

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3. Results

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3.1. Bioassay

The results obtained in AIT and LPT based bioassays against pyrethroid resistant tick

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line-IV as compared to the susceptible IVRI-I line of R.(B.) microplus revealed the RF of 194.0 and 67.46 against deltamethrin, respectively, while 26.6 and 7.67 against cypermethrin with

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respect to their LC50 estimates. However, the line also showed slight resistance to diazinon in

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AIT bioassay showing RF of 2.86 while larvae were found sensitive. The results of chi square test (χ2) did not indicate significant difference between observed and expected response of ticks

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treated with cypermethrin, deltamethrin and diazinon using AIT and LPT bioassay (Table 1). The observed chi square values for AIT against these chemicals were ranged from 0.4607 to

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0.7766 and in case of LPT the values were ranging from 2.809 to 9.582 and were insignificant (P>0.05). The observed level of significance was 0.088 to 0.8584 (P>0.05). The chi square test of the data revealed the goodness of fit data in probit model and also showed the homogeneity of the IVRI-IV line (Table 1).

3.2. Biochemical characterization 3.2.1. Esterase activity 9

ACCEPTED MANUSCRIPT The mean specific activity of esterases was found to be 0.687 ± 0.0499 nmoles α-napthol formed/min/mg protein and 1.292 ± 0.0845 nmoles β-napthol formed/min/mg protein using α and β-napthyl acetate , respectively, for reference susceptible IVRI-I tick. In comparison, IVRIIV showed immediate colour transition with high mean esterase activity of 1.811±0.132 nmoles α-napthol formed/min/mg protein and 6.862 ± 0.420 nmoles β-napthol formed/min/mg protein

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indicating 2.60 fold and 5.83 folds elevations. It was observed that esterase activity using β-

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napthyl acetate is a better monitoring indicator than use of α- napthyl acetate (Table 2).

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3.2.2. Glutathione S-transferase (GST) activity

The specific activity of GST in case of IVRI-I was found to be 0.32±0.02 mmole/min/mg

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protein, however, a significant (p<0.001) fold increase of 3.77 in GST activity (1.23±0.41

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mmole/min/mg protein) was recorded in IVRI-IV with rapid production of CDNB-GSH conjugate (Table 2).

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3.3. Molecular characterization

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The in silico analysis of full length sequence of IVRI-IV CES (accession no. KT215345) gene detected 18 nucleic acid substitutions of which 13 were found to be synonymous and 5

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were non-synonymous. Among these, 3 substitutions (A848G, A1175G and C1376T) were found consistently in IVRI- IV tick population (Table 3) which resulted in three amino acid

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substitutions viz. E283G, E392G and P459L (Table 4). The C190A mutation in the sodium channel gene was also detected in the 25th generation of IVRI-IV tick colony. 4. Discussion Globally, for generation of scientifically repeatable data, few tick research laboratories established reference characterized tick lines and generated significant data. The reference lines were exploited to establish DC of various acaricides; standardization of various bioassay viz. AIT, LPT, LIT, larval tarsal test [22,23], larval immersion microassay [24]; vaccine trials, for 10

ACCEPTED MANUSCRIPT comparative analysis of herbal and chemical acaricides efficacy [25]; mechanism of action of drugs as well as to study the mechanism of development of resistance which may be biochemical [26] or molecular [27,28].

The use of OP and SP compounds forms the backbone of arthropod management in tropical

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and sub-tropical countries. However, due to indiscriminate use of these acaricides, ticks have

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developed resistance to most of the introduced acaricides. During the last few years, after

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determination of DC (at IVRI laboratory) of commonly used acaricides, resistance has been reported comparatively at a faster pace and the subject has been reviewed recently [6]. However,

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the knowledge on mechanisms of development of resistance and the possible solution is very limited. The present study was focused to characterize a resistant tick population and to correlate

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the possible role of over-expression of functional enzymes and changes in the targeted genes

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with development of resistance.

Previously, reference tick lines were characterized using resistance monitoring tools like AIT, LPT and by native PAGE profiling for esterase. For instances, following characterization

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of San Felipe, Corrales and Coatzacoalcos strains, resistance ratio (RR) of 1840, 6900 and 250,

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respectively, were reported against permethrin, and 1.4, 1.3 and 3.6 against coumaphos, respectively [26]. The RR of 250 against permethrin in Coatzacoalcos strain was reported with a higher esterase activity on native PAGE system [29]. The Brazilian Santa Luiza strain was reported to be highly resistant to permethrin (RR 93.0) and to amitraz (RR 188.0) along with domain II sodium channel mutation [30]. The Pasqueria strain was found to be resistant to both diazinon and coumaphos [31]. In the present study, RF of the IVRI-IV tick against deltamethrin, cypermethrin and diazinon was determined as 194.0, 26.0 and 2.86, respectively, although the ticks were repeatedly selected after treatment with different dosages of deltamethrin only.

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Mainly three enzymes viz., esterase, monooxygenase and glutathione S-transferase are reported to be involved in metabolic detoxification and often correlated with development of acaricide resistance [32]. Significant increase in esterase, GST and monooxygenase activity in field resistant R. bursa population collected from difference provinces of Iran was reported but

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failed to correlate the higher enzyme activity with resistance due to non-availability of reference strain for comparison [8]. In the present study, GST activity in IVRI-IV were 3.77 (p<0.001)

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folds higher than IVRI-I line, however, the higher activity cannot be correlated directly with

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resistance because of high sensitivity of reaction conditions, a small variation can mislead the

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results. Previously, no role of GST in conferring resistance in Corrales, san Felipe and Coatzacoalcos strains was reported [33]. In the same context, GST activity in field ticks

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collected from Punjab and Bihar states of India was found to be elevated but could not be

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correlated with development of resistance [23,34].

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Majority of the synthetic pyrethroids are ester compounds, and high esterase activity has been often correlated with the development of resistance [35,36,37]. In the present study, a

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significantly elevated esterase activity of 2.60 and 5.83 fold (p<0.001) in IVRI-IV ticks suggested the possible role of esterases in pyrethroid resistance development. In the quantitative

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assay of esterases, visual interpretation is possible which persist for a comparatively longer period of time and thus proposed to be a suitable monitorable indicator for maintenance of the tick line in the laboratory. Previously, several tick researchers exploited native PAGE profiling of esterase as a monitorable indicator of resistance in reference tick line. An elevated activity of CzEST-9 esterase in Coatzacoalcos strain was noticed [30] and higher activity of CzEST-9 has been exploited as monitorable indicator by various tick researchers while maintaining tick lines in their laboratories [27,29]. An elevated activity of EST-1, an acetylcholinesterase, was reported and linked with resistance in Brazilian population [36]. In a similar fashion, higher 12

ACCEPTED MANUSCRIPT activity of EST-1, in IVRI-IV tick was found to be a monitorable indicator for maintenance of the tick line in the laboratory[37].

Sodium channel is the prime target of synthetic pyrethroids. Mutations in the sodium channel gene have been reported in various arthropods of agriculture and veterinary importance.

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The C190A mutation in the domain II S4-5 linker region of sodium channel resulting in an

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amino acid substitution from leucine in the susceptible isolate to an isoleucine (L64I) in the

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resistant isolate had been reported from Australian ticks [28]. Similar mutation has been identified in the IVRI-IV consistently and thus serves as one of the monitoring indicators for the

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routine maintenance of the tick line in laboratory condition. A G1120A mutation was identified in CES gene which generates a EcoRI site in gene sequence[38]. However, no such mutation

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was recorded in IVRI-IV and in the field isolates collected from three Indian states [39]. On the contrary, in this study three novel mutations (A848G, A1175G and C1376T) resulting in amino

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acid substitutions namely E283G, E392G and P459L were recorded (accession no. KT215345)

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while analyzing the full length sequence of carboxylesterase gene in IVRI-IV population. Previously, mutations in carboxylesterase gene linked with insecticide resistance have been

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identified in blowfly [40] and in Anisopteromalus calandrae [41].

Finally, following characters were identified as monitorable indicators for maintenance of IVRI-IV line in the laboratory. These are: high resistance to deltamethrin (RF= 194.0), moderate to cypermethrin (RF=26.60) and mild to diazinon (RF=2.86); high esterase activity; uninhibited EST-1 esterase in the presence of specific inhibitors and sodium channel mutation at S4-5 linker region of domain II.

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ACCEPTED MANUSCRIPT Although the problem of acaricide resistance was witnessed long back but few reference lines were established and maintained globally [9]. The present tick line has recently been registered in the national registration system (NBAIR-IVRI-BM-4-2009) and will be used as reference for tick research. As the RF of the IVRI-IV line against SP compounds is very high it can be presumed that drugs to be developed in future, if found effective against IVRI-IV line

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may contribute significantly in the management of SP resistant field ticks.

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5. Conclusion

A characterized deltamethrin and cypermethrin resistant R. (B.) microplus tick line, IVRI-IV

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has been established as reference tick line for tick research. Monitorable indicators were

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identified for continuous maintenance of the line in the laboratory. Acknowledgements

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The authors are grateful to the Indian Council of Agricultural Research, New Delhi for

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funding through the World Bank funded National Agricultural Innovation Project No. [NAIP/Comp-4/C2066/2008-09] and National Fund for Basic Strategic and Frontier Application

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Research in Agriculture Project No. [NFBSFARA/BSA-4004/2013-14].

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ACCEPTED MANUSCRIPT newly developed phyto-formulations against field collected and resistant tick lines of Rhipicephalus (Boophilus) microplus, J. Parasit. Dis. 40 (2016) 1590-1596. [26] L.D. Foil, P. Coleman, M. Eisler, H. Fragoso-Sanchez, Z. Garcia-Vazquez, F.D. Guerrero, N.N. Jonsson, I.G. Langstaff, A.Y. Li, N. Machila, R.J. Miller, J. Morton, J.H. Pruett, S. Torr, Factors that influence the prevalence of acaricide resistance and tick-borne diseases,

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ACCEPTED MANUSCRIPT Table 1.Comparative dose-response data of IVRI-IV and IVRI-I ticks treated with deltamethrin, cypermethrin and diazinon using LPT and AIT bioassay

ay LPT

Diazinon

Deltamethrin

des Cypermethrin

Acarici Bioass

AIT

LPT

AIT

LPT

AIT

LC50 (CL) (ppm)

LC95 (CL) (ppm)

IVRI-IV

IVRI-I

IVRI-IV

IVRI-I

1858.84

242.4

7468.91

350.7

(1721.1-1791.5)

(241.2–243.6)

(6383.7-8738.6)

(347.2–354.2)

3692.45

138.5

9378.90

349.1

(3516.6-3877.1)

(134.5–142.6)

(8449.4-10410.6)

(323.2–377.0)

796.1

11.8

1668.70

35.5

(765.5-827.9)

(11.6–12.0)

(1545.1-1802.2)

(34.1–36.9)

2600.0

13.4

6686.00

29.6

(2476.2-2730)

(12.4–14.5)

(6023.4-7421.5)

(27.7–31.7)

242.57

499.7

1481.84

636.6

(220.4-266.7)

(497.7–501.7)

(1214.6-1807.8)

(630.9–642.3)

1067.93

372.0

3458.54

635.2

(341.3–405.3)

(3033.8-3942.7)

(582.75–692.37)

(998.04-1142.6)

C C

T P E

D E

P value

T P

RF

9.582

0.088

7.67

0.4607

0.7943

26.60

2.809

0.422

67.46

0.7626

0.8584

194.0

4.577

0.206

0.48

0.7766

0.6782

2.86

I R

C S U

N A

M

χ2

A

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Table 2. Comparative analysis of enzyme activities in IVRI-I and IVRI-IV lines of R.(B.) microplus using optimum number of larvae and standardized reaction time.

α-napthol Tick lines

(µmoles/min/mg

C S U

(µmoles/min/mg protein) IVRI-I

0.687 ± 0.05

IVRI-IV

1.811 ± 0.13

D E 2.60c

Enzyme Ratio

protein)

GST (mM/mg/min)

1.292 ± 0.08

0.32±0.02

6.862 ± 0.42

1.23±0.41

5.83c

3.77c

N A

M

T P

I R

β-napthol

T P E a

significant at p<0.01; c significant at p<0.001

C C

A

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Table 3. Results of analysis of nucleotide substitution in complete cds (1635 bp) of carboxylesterase gene

Site

78*

93*

171*

175

207*

237*

243*

306*

345*

366*

386

417*#

IVRI-I

T

C

C

C

G

G

C

C

C

C

C

A

IVRI-IV

C

T

T

G

A

A

G

T

T

T

T

G

T P

420*

C S U

I R C T

603* 612* 848# 1175#

1376#

C

C

A

A

C

T

T

G

G

T

N A

*synonymous mutation, # consistently found in all clones.

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M

T P E

C C

A

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Table 4. Amino acid substitutions detected at different sites of carboxylesterase gene.

Tick Line Sites

283

392

459

IVRI-I

E

E

P

IVRI-IV

G

N A

L

D E

T P

Carboxylesterase gene

I R

C S U

G

M

T P E

C C

A

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Graphical abstract

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ACCEPTED MANUSCRIPT Highlights



Established a deltamethrin (RF= 194.0) and cypermethrin (RF = 26.6) resistant reference R. (B.) microplus tick line as IVRI- IV. Three amino acids substitutions in CES and mutation in domain II S4-5 linker region of sodium channel genes were recorded.

Higher activity of esterase and glutathione S-transferase was observed in the

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

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

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IVRI- IV line.

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