J Arthropod-Borne Dis, September 2018, 12(3): 310–320 MA Gorouhi et al.: Biochemical Basis of … 310 http://jad.tums.ac.ir Published Online: September 30, 2018 Original Article Biochemical Basis of Cyfluthrin and DDT Resistance in Anopheles stephensi (Diptera: Culicidae) in Malarious Area of Iran Mohammad Amin Gorouhi 1, 2, *Mohammad Ali Oshaghi 2, *Hassan Vatandoost 2, 3, Ahmad Ali Enayati 4, Mohamad Reza Abai 2, 3, Mohsen Karami 5, Yaser Salim-Abadi 6, Ahmad Ali Hanafi-Bojd 2, 3, Abbas Aghaei-Afshar 1, Azim Paksa 2, Fatemeh Nikpour 2, 3 1Department of Vector Biology and Control, School of Health, Kerman University of Medical Sciences, Kerman, Iran 2Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 3Department of Chemical Pollutants and Pesticides, Institute for Environmental Research, Tehran University of Medical Sciences, Tehran, Iran 4Department of Medical Entomology, Mazandaran University of Medical Sciences, Sari, Iran 5Infectious Diseases and Tropical Medicine, Babol University of Medical Sciences, Babol, Iran 6Department of Health Services and Health Promotion, School of Health, Rafsanjan University of Medical Sciences, Rafsanjan, Iran (Received 12 Apr 2018; accepted 24 Sep 2018) Abstract Background: Anopheles stephensi is a key urban malaria vector in the Indian subcontinent and Middle East includ- ing south and southeast of Iran. Wide application of insecticides resulted in resistance of this species to various insec- ticides in these regions. This study was conducted to reveal the role of metabolic mechanisms in the development of resistance in An. stephensi to DDT and cyfluthrin. Methods: Field mosquito specimens were collected from Chabahar Seaport, southeast corner of Iran, in 2015. Insec- ticide susceptibility and enzyme assays were conducted as recommended by WHO. Results: Mean enzyme ratios were 3.95 and 3.04 for α- esterases and 2.40 and 1.97 for β- esterases in the DDT and cyfluthrin- resistant populations correspondingly compared with the susceptible strain. The GSTs enzyme mean ac- tivity ratios were 5.07 and 2.55 in the DDT and cyfluthrin- resistant populations compared with the susceptible beech strain. The cytochrome p450s enzyme ratios were 1.11 and 1.28 in the DDT and cyfluthrin- resistant populations respectively compared with the susceptible beech strain. Conclusion: Metabolic mechanisms play a crucial role in the development of DDT and cyfluthrin resistance in An. stephensi, therefore, further evaluation of the mechanisms involved as well as implementation of proper insecticide resistance management strategies are recommended. Keywords: Anopheles stephensi, Insecticide, Resistance mechanisms, Malaria Introduction Malaria is still a major public health prob- lem in southeast corner of Iran (1). There are seven Anopheles species as malaria vectors in Iran including An. stephensi, An. culcifa- cies s.l., An. maculipennis s.l., An. sacharovi, An. superpectus s.l., An. dthali, and An. flu- viatilis s.l.. Anopheles stephensi is the most important malaria vector in southern region of the country (2-10). Application of chemical insecticides is one of the most important interventions for malaria control, used in Iran during past dec- ades. Different groups of insecticides includ- ing organochlorines (DDT, dieldrin and BHC), organophosphates (pirimiphos-methyl and mal- athion), carbamate (propoxur) and pyrethroids (lambdacyhalothrin and delthamethrin) in dif- *Corresponding authors: Dr Mohammad Ali Oshaghi, E-mail: moshaghi@sina.tums.ac.ir, Dr Hassan Vatandoost, E-mail: hvatandoost1@yahoo.com, vatando@tums.ac.ir http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2018, 12(3): 310–320 MA Gorouhi et al.: Biochemical Basis of … 311 http://jad.tums.ac.ir Published Online: September 30, 2018 ferent forms of application such as indoor re- sidual spraying (IRS) and insecticide-treated nets (ITNS) for adult mosquito control and or- ganophosphates for larviciding were used in malarious areas of the country (11-13). Iran has embarked on the malaria elimina- tion program since 2007 relying on application of chemical insecticides specially pyrethroid compounds for malaria vector control (14). Anopheles stephensi is resistant to sever- al insecticides including DDT, dieldrin, and malathion (12, 15-19). The first indication of pyrethroid resistance was reported from Chaba- har Seaport, southeast of Iran in 2012 (13). Moreover, there are many reports on resistance of this species to different insecticide groups including pyrethroids from Iran neighboring countries including Pakistan, Afghanistan, the Indian subcontinent as well as the Middle East countries (14, 15, 18, 20-25). Due to the importance of pyrethroids in malaria control program and the slow process of development of new insecticide compounds, monitoring and management of insecticide re- sistance are necessary (26, 27). Metabolic and target site insensitivity are two common re- sistant mechanisms in insects. In metabolic resistance, alteration in the levels or activities of detoxification enzymes such as esterases, glutathione S-transferases (GSTs), and cyto- chrome P450s may occur (24, 28). In target site insensitivity, mutations in the sodium chan- nel, acetylcholinesterase and GABA receptor genes occur (29). Therefore, determination of resistance mechanisms in An. stephensi is es- sential for proper management of insecticide resistance through vector control interventions. The aim of this study was to determine the possible involvement of enzymes groups’ in DDT and pyrethroid insecticides resistance functioning in An. stephensi, the main malar- ia vector in southeast of Iran. Materials and Methods Mosquito collection and rearing Anopheles stephensi larvae were collected from larval habitats using the standard dipper from Chabahar Seaport (25°25ˊN, 60°45ˊE) Sistan and Baluchestan Province, southeast of Iran (Fig. 1) during Apr to June 2015. The larvae specimens were transported in cool box- es to insectary of the Medical Entomology and Vector Control Department, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran and reared to adult stage under standard condition at 25 °C, 80% relative hu- midity with a 12h day/night lighting cycle. The adult mosquito specimens were identified to species level using the identification key (30). Moreover, a pyrethroid susceptible strain (Beech strain) originated in India in 1940 and kept in the insectary without being exposed to insecticides used as a control in all experiments. Adult susceptibility tests and selection Six different insecticide impregnated pa- pers including DDT 4%, lambdacyhalothrin 0.05%, deltamethrin 0.05%, cyfluthrin 0.15%, permethrin 0.75%, and etofenprox 0.5% sup- plied by WHO were used for evaluating the susceptibility status of An. stephensi popula- tions from Chabahar. Two or three days old adult female mosquitos that were kept on 10% aqueous sucrose solution were used for sus- ceptibility test procedure according to the WHO method (27). Then the mosquito populations with the lowest mortality rates were subjected to selection pressure of the two insecticides in the laboratory. The mosquito populations were exposed to the two insecti- cides in two separate lines over 18 and 19 generations throughout four and five selection phases. For both insecticides, mortality rate was calculated in different times and regres- sion lines were plotted in each generation us- ing Microsoft Excel (ver. 2013). A subset (40 specimens for each sample) of resistant and susceptible mosquito populations were placed in a 1.7ml tubes and kept in freezer (-80 °C). These frozen specimens were then transported in a cold chain to the Pesticide Biochemistry http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2018, 12(3): 310–320 MA Gorouhi et al.: Biochemical Basis of … 312 http://jad.tums.ac.ir Published Online: September 30, 2018 Laboratory of Medical Entomology Depart- ment, School of Public Health, Mazandaran University of Medical Sciences, Sari, Iran for further biochemical assays. Biochemical assays Biochemical tests were performed accord- ing to the method described by Hemingway (1998) (31). The enzyme activities/contents of P450s, glutathione S-transferases (GSTs), and esterases using corresponding fresh buffer solutions were quantified. Frozen adult mos- quito specimens were individually put in wells of flat-bottomed 96-well microtiter plate and manually homogenized using a steel pestle in 250µL cold distilled water at 4 °C. The plate was spun at 3000rpm for 20min in a Beck- man Coulter (Beckman Inc., USA) centrifuge at 4 °C and the supernatant was used as the source of enzymes in reaction mixtures. In each biochemical assay, blank replications (all component of the reaction mixture except for the enzyme source) were provided. Prepara- tion of all reaction mixtures was carried out on ice (31). Total Protein assay In order to minimize the error due to dif- ferent size and protein contents of mosquitos and homogenizing process, total protein com- ponent of each specimen was measured using Bradford method in triplicate by adding 300 µL of Bio-Rad solution (diluted with distilled water by 1:4) to 10µL of the homogenate. Af- ter 5min incubation at room temperature, the absorbance was measured at 570nm in a Bi- otek ELX808 Ultra Microplate Reader (Bio- tek Inc, USA) (31). The value was changed into product concentration using a bovine serum albumin standard curve obtained with the same reagents and method. Cytochrome P450s assay This test quantifies the amount of hem con- taining protein in the specimens. In each well, the reaction cocktail comprised of 20µl of the mosquito homogenate in duplicate, 80µL of 0.0625M potassium phosphate buffer PH 7.2, 200µL of 3, 3’, 5, 5’ tetramethyl benzi- dine (TMB) solution (0.01g TMB dissolved in 5ml methanol plus 15ml of 0.25M sodium acetate buffer pH 5.0) and 25µL of 3% hy- drogen peroxide. After 2h incubation in room temperature, the absorbance was measured at 450nm. The protein contents were described as correspondent units of cytochrome (EUC) P450sec/mg protein corrected for the known hem content of P450s and cytochrome C us- ing a standard curve of purified cytochrome C (31). Glutathione S-transferase assay The reaction mixture contained 200µL of reduced glutathione plus 1-coloro-2, 4-dini- trobenzene (CDNB) added to 10µL of the mos- quito homogenate in duplicate. The increase in absorbance was measured at 340nm for 5 min. The amount of conjugate produced/min/ mg protein (mM) using the extinction coef- ficient of CDNB corrected for the path length of the solution in the microplate well was reported as enzyme activity (31). General esterase assay In this assay, the activity of α-esterase and β-esterase with the alfa and beta-naphthyl ac- etate as universal substrates were measured. To a reaction mixture of 200µL of alpha or beta-naphthyl acetate solution was added to 20µL of mosquito homogenate in duplicate. After 30min incubation at room temperature, 50µL of fast blue solution was added to each mixture. Plates were incubated at room tem- perature for another 5min and then absorb- ance was recorded at 570nm (19). The opti- cal densities (OD) of solutions were convert- ed to product concentration as µM of product formed/min/mg protein using standard curves of ODs for known concentrations of the prod- ucts α- or β –naphthol (31). In each biochemical assays, four blank rep- licates were set using the same materials of http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2018, 12(3): 310–320 MA Gorouhi et al.: Biochemical Basis of … 313 http://jad.tums.ac.ir Published Online: September 30, 2018 each assay except for distilled water added in- stead of the mosquito homogenate. The ODs of the wells containing mosquito homogenates were adjusted by deducting with the average ODs of the blank replicates. Data conversion and analyses The activity/contents of the enzymes were measured and used for further analysis by Microsoft Excel. The data then were trans- formed into the actual enzyme activity val- ues using standard curves. Mean values of the enzyme activities of all populations were compared using ANOVA in conjunction with the Tukey’s statistical test using SPSS ver. 19 software (Chicago, IL, USA) (P< 0.05). En- zyme ratios (ER) were computed by dividing the mean activities of each resistant popula- tion with those of the Beech susceptible strain (24). Results Selection process Susceptibility tests showed that An. ste- phensi Chabahar strain was susceptible to permethrin and etofenprox, resistant candi- date to deltamethrin and resistant to DDT, cyfluthrin, and lambda cyhalothrin. This strain showed the highest resistance to DDT 4% and cyfluthrin 0.15% respectively among the insecticides tested (Fig. 2). Populations with resistance ratio (RR) to Cyfluthrin of 11.6 and to DDT of 2.05 RR in comparison with the susceptible strain were chosen for insecticide selection process. This process con- tinued for four and five phases throughout 18 and 19 generations respectively to achieve resistance ratio of 28.75 for the population exposed to DDT (R1) and 6.8 for the popula- tion exposed to cyfluthrin (R2). Biochemical assays Activities of α- and ß-esterases, glutathione- S-transferase (GST) and the contents of cy- tochrome P450s were tested for the R1 and R2 An. stphensi populations are summarized in Table 1 and Fig. 3. The cytochrome P450s enzyme ratios were 1.11 and 1.28 fold in the DDT and cyfluthrin- resistant populations com- pared with the susceptible strain. Although the median activities of the resistant population were 2.2 and 2.7 times more than the suscep- tible one, however, the mean activity/content of P450 enzymes in the resistant and the sus- ceptible strains was not significant (P< 0.05). The enzyme ratios for esterases with α-naph- thyl acetate were 3.95 and 3.04 and with β- naphthyl acetate were 2.4 and 1.97 in the DDT and cyfluthrin- resistant populations corre- spondingly compared with the susceptible strain. The GSTs enzyme ratios were 5.07 and 2.55 in the DDT and cyfluthrin- resistant pop- ulations compared with the susceptible Beech strain. The enzyme ratios for both the esterases and GST enzymes were higher in the DDT- resistant population than the cyfluthrin- re- sistant population (Fig. 3). Statistical analy- sis showed that the activity/content of the two esterases and GST enzymes of the selected populations and the beech susceptible strain were significantly different (P< 0.05) (Fig. 3). Table 1. Details of enzyme activities and enzyme ratios (ER) measured in Anopheles stephensi resistant populations from southeastern Iran. Beech, susceptible, R1, DDT resistant, and R2, the Cyfluthrin resistant population Enzyme Population N Median Mean±SE Enzyme Ratio P450 Beech 53 1.014e-005 2.365e-005±4.899e-006 1 R1 58 2.183e-005 2.626e-005±2.035e-006 1.11 R2 69 2.753e-005 3.050e-005±2.319e-006 1.28 GST Beech 71 0.03485 0.03856±0.002610 1 R1 66 0.15330 0.19590±0.01373 5.07 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2018, 12(3): 310–320 MA Gorouhi et al.: Biochemical Basis of … 314 http://jad.tums.ac.ir Published Online: September 30, 2018 R2 69 0.08683 0.09857±0.006261 2.55 α-eseterase Beech 80 0.0002753 0.0002825±1.307e-005 1 R1 80 0.001022 0.001116±6.949e-005 3.95 R2 80 0.0007664 0.0008604±5.001e-005 3.04 β-eseterase Beech 80 0.0003146 0.0003397±1.588e-005 1 R1 80 0.0007474 0.0008167±5.603e-005 2.4 R2 79 0.0006188 0.0006718±3.299e-005 1.97 Fig. 1. The map of the study area in Chabahar Seaport, Southeast of Iran Fig. 2. Mortality rate (mean and SEM) of Anopheles stephensi Chabahar strain to six insecticides Table 1. Continued … http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2018, 12(3): 310–320 MA Gorouhi et al.: Biochemical Basis of … 315 http://jad.tums.ac.ir Published Online: September 30, 2018 Beech R 1 R 2 0.0 0.2 0.4 0.6 G S T e n z y m e a c ti v it y Beech R 1 R 2 0.000 0.001 0.002 0.003 0.004  -e s tr a s e a c ti v it y Beech R 1 R 2 0.000 0.001 0.002 0.003  -e s ta s e a c ti v it y Beech R 1 R 2 0.00000 0.00002 0.00004 0.00006 0.00008 0.00010 P 4 5 0 a c ti v it y Fig. 3. Mean and SEM activity profiles of P450, GST and α- and ß-Esterase enzymes in the DDT resistant (R1), the Cyfluthrin resistant (R2), and the susceptible Beech-strain of Anopheles stephensi Discussion This study revealed that An. stephensi from Chabahar District, southeast of Iran is resistant to pyrethroids including cyfluthrin and lamb- da cyhalothrin, DDT and tolerant to deltame- thrin. Therefore, far various modes of resistance including modification or overexpression of detoxification enzymes, target site insensitiv- ity, as well as behavioral adaptations have been developed and documented in insects (32, 33). Resistance to insecticides might be due to in- creased monitoring, misuse of insecticides, ge- ographical extension of resistance, and new re- sistance genes (34) leading to decrease in the effectiveness of vector control programs. Re- sistance to pyrethroids in An. stephensi has been reported in several countries in the Eastern Mediterranean Region, notably Afghanistan, and Oman (12, 15, 16, 24, 34, 35). In addition, there are reports on DDT resistance in Yem- en (34, 35), DDT and pyrethroid resistant in Anopheles mosquito of Iran (13, 15). There have been reports of resistance to three of the four insecticides classes in An. stephensi mosquitoes in Afghanistan (24, 34). Resistances to all four classes of insecticide have been reported in An. stephensi from So- malia and Sudan, including widespread re- sistance to DDT and an increasing frequency of resistance to pyrethroids (34, 36). Unfor- tunately, most of the new resistance reports are to pyrethroid compounds that are the on- ly insecticides used for long lasting insecti- cide nets (LLINs). This study showed that biochemical mech- anisms are driving the resistance in this field population. This was shown by measuring the activities of the enzymes which could be re- sponsible for the insecticide resistance in An. stephensi from Chabahar. The differences be- tween activities of three enzyme groups in- cluding alpha and beta esterases, and GSTs in the Chabahar population were higher than those of the susceptible Beech strain, esterases and GSTs could all be involved in insecticide resistance in this population. Our result showed that in order α-esterase, β-esterase and GST enzyme have played the highest role in re- sistance to the DDT resistant populations (Ta- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2018, 12(3): 310–320 MA Gorouhi et al.: Biochemical Basis of … 316 http://jad.tums.ac.ir Published Online: September 30, 2018 ble 1). This order was α-esterase, GST enzyme, and then β-esterase for the cyfluthrin resistant population. Accordingly, almost similar situ- ation has been reported in the field populations of An. stephensi in Afghanistan, a neighbor- ing country sharing border line with Chabahar (24). Esterases and cytochrome P450s are in- volved in pyrethroid resistance in An. stephensi (21, 37), An. gambiae (38), An. albimanus (39, 40), and An. minimus (41). Moreover, esteras- es are involved in organophosphate (OP) re- sistance with cross-resistance to pyrethroids (21, 40, 42, 43). Rising enzyme activities in many insects have been reported including mosquitoes which are resistant to various in- secticides from different parts of the world (17, 28, 42, 44-47). As biochemical mecha- nisms are involved in insecticide resistance in the Chabahar population, using of synergists in formulation of pyrethroid insecticides should be evaluated. Different forms of resistance mechanisms have been reported in different species of Anopheles so that in some species only met- abolic resistance has currently been reported. For example in the study on An. funestus s.s in Uganda just enzymatic resistant have been reported (48), whereas both metabolic and tar- get-site insensitivity have been found as re- sistant mechanisms in An. gambiae s.s. in Af- rica (48, 49). In this study the target-site insensitivity (kdr) mutations as a potential resistance mech- anism in the An. stephensi populations were not examined. These mutations have been shown in An. stephensi from Afghanistan (24) and might be present in Chabahar population. Therefore this molecular assay is highly rec- ommended to test the presence of kdr resistance mechanism in this population. Conclusion The An. stephensi Chahbahar population is becoming resistant to deltamethrin. This in- secticide is currently used in malaria elimina- tion program against malaria vectors including An. stephensi in the region. Although this in- secticide may still be useful to combat An. stephensi in the area, surveillance of the sus- ceptibility of populations by bioassay as well as biochemical and molecular assays are recom- mended to prevent building up of deltamethrin resistance levels. Insecticide resistance man- agement strategies are also recommended to suspend or to slow the rate of resistance devel- opment to deltamethrin in Chahbahar District. This study showed enzyme elevation and enzymatic resistance in the resistant popula- tion. Therefore conducting biochemical assays along with bioassay can be helpful for moni- toring and management of resistant phenom- ena. Biochemical assays can be involved in routine malaria program for better monitoring and management of resistance in vector pop- ulations. Moreover, using other insecticides with different mode of action can be helpful for vector resistant management. The main resistance mechanism in An. ste- phensi from the study area is metabolic and dif- ferent enzyme groups play various roles in the resistance. Therefore, continuous surveillance of the susceptibility of populations and mon- itoring of insecticide resistance in the malar- ia vectors is crucial for successful control measures in Iran. Acknowledgements The authors are grateful for kind collabo- ration of the Department of Medical Ento- mology of Mazandaran University of Medi- cal Sciences, Iran. This study was financially supported by Tehran University of Medical Sciences, grant Code 22675. 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