J Arthropod-Borne Dis, December 2016, 10(4): 493–500 MA Gorouhi et al.: Current Susceptibility … 493 http://jad.tums.ac.ir Published Online: October 04, 2016 Original Article Current Susceptibility Status of Anopheles stephensi (Diptera: Culicidae) to Different Imagicides in a Malarious Area, Southeastern of Iran Mohammad Amin Gorouhi 1, *Hassan Vatandoost 1, *Mohammad Ali Oshaghi 1, Ahmad Raeisi 2, Ahmad Ali Enayati 3, Hossein Mirhendi 4, Ahmad Ali Hanafi-Bojd 1, Mohammad Reza Abai 1, Yaser Salim-Abadi 1,5, Fatemeh Rafi 1 1Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2National Malaria Control Department, CDC, Ministry of Health and Medical Education, Iran 3School of Public Health and Health Sciences Research Centre, Mazandaran University of Medical Sciences, Sari, Iran 4Department of Parasitology and Mycology, School of Public Health, Tehran University of Medical Sci- ences, Tehran, Iran 5School of Health, Rafsanjan University of Medical Sciences, Rafsanjan, Iran (Received 22 Nov 2014; accepted 10 Agu 2015) Abstract Background: Anopheles mosquitoes are an important group of arthropods due to their role in transmission of ma- laria. The present study was conducted for determination of susceptibility status of Anopheles stephensi to different imagicides collected from malarious area in Chabahar city, Iran. Methods: In the present study seven insecticides including: DDT 4%, lambdacyhalothrin 0.05%, deltamethrin 0.05%, permethrin 0.75%, cyfluthrin 0.15% and etofenprox 0.5% were tested based on WHO method. Regression line was plotted for each insecticide using mortality of different exposure times. Bioassay data were analyzed using Probit software and the lethal time for 50% and 90% mortality (LT50 and LT90) values were calculated. Results: The susceptibility levels of field strain of An. stephensi to the discriminative dose of different imagicides were determined 100, 98, 96, 89, 82 and 62% for etofenprox, permethrin, deltamethrin, lambdacyhalothrin, cyfluth- rin and DDT, respectively. Our finding indicated that An. stephensi is resistant to DDT, lambdacyhalothrin and cyfluthrin, and susceptible to etofenprox and permethrin and candidate of resistant to deltamethrin based on WHO criteria. Conclusion: Our findings indicated that An. stephensi is resistant to DDT and some pyrethroid insecticides which can be developed due to application of insecticides in health and agriculture. These results can provide a clue for future chemical control program in the study area. Keywords: Susceptibility test, Anopheles stephensi, Chabahar, Pyrethroid resistance Introduction Mosquitoes as a big group of arthropods play an important role in transmission of many diseases to human such as malaria, filariasis, yellow fever, dengue fever (Horsfall 1955, Tabachnick 1991, Service 2003, Azari- Hamidian 2011). Some species of Anopheles mosquitoes are vectors of malaria in differ- ent parts of the world. For example, Anoph- eles stephensi Liston (Diptera: Culicidae) is the main malaria vector in Eastern Medi- terranean region and south of Asia continent (Zahar 1974,Vatandoost et al. 2006). In Iran there are some species of malaria vectors including: An. stephensi, An. dthali, An. cu- licifacies, An. fluviatilis, An. superpictus s.l., An. sacharovi, An. maculipennis Complex (Naddaf et al. 2003, Azari-Hamidian 2011, Mehravaran et al. 2011, Oshaghi et al. 2011). *Corresponding author: Dr Hassan Vatandoost, E-mail: hvatandoost1@yahoo.com, vatando@tums.ac.ir, Dr Moham- mad Ali Oshaghi, E-mail: moshaghi@tums.ac.ir J Arthropod-Borne Dis, December 2016, 10(4): 493–500 MA Gorouhi et al.: Current Susceptibility … 494 http://jad.tums.ac.ir Published Online: October 04, 2016 Before initiating of national malaria control program in 1957, malaria cases were report- ed from most parts of Iran, Since then, due to implementing of many continuous in- terventions, malaria confined to south east- ern parts of the country including Sistan va Baluchestan, Hormozgan and southern parts of Kerman Provinces (Edrissian 2006, Vatan- doost et al. 2011). For controlling of malaria, vector control is one the most important ap- proach which focuses on chemical control of mosquitoes. Up to now different group of insecticides including: organochlorines (DDT, dieldrin and BHC), organophosphates (pi- rimiphos-methyl, malathion), carbamates (propoxur) and pyrethroids (lambdacyhalo- thrin, delthamethrin) in different forms of application such as Indoor Residual Spray- ing (IRS), Insecticide Treated Nets (ITNS) for adult stage and some organophosphates for larviciding were used in malariaous area of Iran (Salim Abadi et al. 2010, Hanafi- Bojd et al. 2012, Vatandoost and Hanafi- Bojd 2012). Resistance of Anopheles spp to DDT and pyrethroid insecticides were re- ported from different countries around the world like China, Turkey, India, some coun- tries of Africa and Latin America (Kasap et al. 2000, Hargreaves et al. 2003, Syafruddin et al. 2010, Lol et al. 2013, Soltani et al. 2013, Chang et al. 2014). In Iran many re- searches have evaluated susceptibility status of malaria vectors against different insec- ticides (Vatandoost and Hanafi-Bojd 2005, Hanafi-Bojd et al. 2006, Vatandoost et al. 2006, Vatandoost et al. 2011, Vatandoost and Hanafi-Bojd 2012). Approximately in all previous conducted studies on An. ste- phensi in Iran, resistance to DDT and sus- ceptibility to pyrethroids have been reported, but in 2012 first indication of resistance to pyrethroid compounds was reported from south eastern parts of the country (Vatan- doost and Hanafi-Bojd 2012). Resistances to DDT, mainly in the adult stage of An. ste- phensi, have been widely distributed in Middle-East and Indian subcontinent caus- ing operational problems for control pro- grams (WHO 1985, WHO 1992). This study aims to monitor susceptibility status of main malaria vector, An. stephensi, to some in- secticides in Chabahar City, Sistan va Ba- luchestan Province, Iran. Materials and Methods Study area This study was performed in Chabahar seaport (25o 25/ N, 60 o 45/ E), Sistan va Ba- luchestan Province of Iran during April to June 2013 (Fig. 1). Mosquito sampling and rearing Collected larvae from the study area were transferred to the insectary for rearing under standard conditions (Temperature= 25–29o C, photo-period=12:12 Hours (light: Dark) and Humidity=50–70%). Emerged adult mosquitoes were fed with 10% aqueous su- crose solution. Adult susceptibility test Adult susceptibility tests were carried out according to the current World Health Organization method (WHO 2013). For each insecticide mortality rate in various times also were calculated and then regression line to each insecticide plotted using Microsoft Excel (version. 2013). Insecticide impregnated papers The following insecticides impregnated papers were supplied according to WHO Test procedure including: DDT 4%, lamb- dacyhalothrin 0.05%, deltamethrin 0.05%, permethrin 0.75%, cyfluthrin 0.15% and etofenprox 0.5%. Mineral oil, and silicon oil impregnated papers were used for organo- chlorine insecticides and pyrethroids as con- trol, respectively (WHO 1981, WHO 2013). J Arthropod-Borne Dis, December 2016, 10(4): 493–500 MA Gorouhi et al.: Current Susceptibility … 495 http://jad.tums.ac.ir Published Online: October 04, 2016 Statistical analysis Results were analyzed by using of Probit program (Finney 1971). In case of mortality, when the control mortality was between 5% to 20% it was corrected by Abbott’s formula (Abbott 1925). Error bars for each mortality were calculated based on statistical method at α=5%. The lethal Time for 50% and 90% mortality (LT50 and LT90) values and their 95% confidence interval also Probit regres- sion line parameters were determined with Finney method and then the regression line of all Insecticides were plotted using Mi crosoft Excel (version. 2013). Results The results of susceptibility test for each insecticides are shown in tables 1,2. Mortal- ity rate and lethal Time for 50% mortality (LT50) of different insecticides were calcu- lated. Our finding indicated that Etofenprox, Deltamethrin, Lambdacyhalothrin, Perme- thrin, Cyfluthrin and DDT have the lowest to highest LT50 value respectively (Fig. 2). Fig. 1. The map of Chabahar City representing rural districts, Sistan and Baluchistan Province (Study area), Iran Table 1. Probit regression line parameters of Anopheles stephensi exposed to different insecticides Insecticide A B±SE LT50 , 95% C.I. (Second) LT90, 95% C.I. (Second) X2(df) P value Etofenprox 0.05% -2.68 1.33± 0.14 75 626 5.11 (3) >0.05104 957 138 1749 Permethrin 0.75% -5.61 2.22±0.21 277 984 2.89 (2) >0.05335 1266 401 1775 Cyfluthrin0.15% -3.79 1.3±0.12 656 5121 1.97(4) >0.05812 7805 1010 14160 J Arthropod-Borne Dis, December 2016, 10(4): 493–500 MA Gorouhi et al.: Current Susceptibility … 496 http://jad.tums.ac.ir Published Online: October 04, 2016 Lambdacyhalothrin 0.05% -3.25 1.36±0.12 185 1414 0.04(2) >0.05246 2146 324 3791 Deltamethrin 0.05% -2.69 1.22±0.15 101 1194 0.95(2) >0.05159 1785 221 3277 DDT 4% -4.47 2.5±0.33 2820 7560 6.99(3) >0.053240 10200 3840 16560 A= y-intercept, B= the slope of the line, SE= Standard error, CI= confidence interval, x2= heterogeneity about the regression line, df= degree of freedom, P> 0.05 =represent no het- erogeneity in the population of tested mosquitos. Table 2. Mortality rate and susceptibility status of Anopheles stephensi exposed to different insecticides Chabahar, southeastern Iran, 2013 Insecticide MR±EB* Resistance status** Deltamethrin 96±3.8 RC Lmbdacyhalothrin 89±2.8 R Cyfluthrin 82±3.5 R Permethrin 98±1 S Etofenprox 100 S DDT 62±4.8 R control - *Mortality Rate±Error Bar **R Resistance, RC Resistant Candidate, T Tolerance, S Susceptible Fig. 2. Regression lines of Anopheles stephensi exposed to different insecticides (field population), 2013 Table 1. Continued… J Arthropod-Borne Dis, December 2016, 10(4): 493–500 MA Gorouhi et al.: Current Susceptibility … 497 http://jad.tums.ac.ir Published Online: October 04, 2016 Discussion In the current study seven insecticides including: DDT 4%, lambdacyhalothrin 0.05%, deltamethrin 0.05%, permethrin 0.75%, cyfluthrin 0.15% and etofenprox 0.5% were used to determine susceptibility status of An. stephensi collected from Chabahar City. Based on WHO criteria that suggested (98– 100% mortality indicates susceptibility, 90– 97% mortality indicates resistance candidate (more investigation is needed) and less than 90% mortality suggests resistance (WHO 2013). Results indicated that species is re- sistant to DDT, cyfluthrin and Lambda- cyhalothrin, However, susceptible to perme- thrin and Etophenprox. The indication of resistant to deltamethrin at the early stages of evolution has also be documented. Our findings reveal that An. stephensi is resistant to DDT which is in line with previous re- searches results that have been performed in our study area (Vatandoost and Hanafi-Bojd 2012, Fathian et al. 2015). Majority of sus- ceptibility tests which performed during the past decade in different malarious area re- vealed resistance to DDT in southern part of Iran (Borhani 2004, Vatandoost et al. 2005, Vatandoost et al. 2006) as well as in the most distribution area of An. stephensi in the world (Rathor et al. 1980, Thavaselvam et al. 1993, Tikar et al. 2011, Chang et al. 2014, Singh et al. 2014). Furthermore, there are many resistance reports to DDT in other species of Anopheles mosquitoes from dif- ferent part of the world (Hemingway and Ranson 2000, Hemingway et al. 2002, Za- hirnia et al. 2002, Lak et al. 2002, Balkew et al. 2006 , Raghavendra et al. 2010, Tikar et al. 2011, Vatandoost et al. 2011, Nardini et al. 2013, Wang et al. 2013). In the present study resistance to cyfluthrin and lambda- cyhalothrin were indicated and these find- ings are in line with previous research re- sults that have been conducted in the same area (Vatandoost and Hanafi-Bojd 2012). On the other hand our finding about cyfluthrin susceptibility status is not in con- cordance with another research that has been performed previously in the same area by Fathian et al. (2015) that showed this species is susceptible to cyfluthrin. (Fathian et al. 2015). It may be due to different sampling localities. Resistance of An. stephensi to py- rethroid compounds were reported from its different distribution regions, for instance in the study was performed by Rathor et al. (2013) in Punjab Province of Pakistan, re- sistance to three commonly used pyrethroids, permethrin, lambda-cyhalothrin, and del- tamethrin were indicated from the majority of test localities (Rathor et al. 2013). In the present study An. stephensi was susceptible to Etophenprox and Permethrin that these findings are in parallel with other previous conducted researches results (Vatandoost et al. 2005). In the current study deltametthrin was indicated as resistant candidate so that more investigation is needed. Molecular and biochemical assays for this species as a main malaria vector must be conducted for accu- rate evaluating of resistance status of pyre- throid insecticides specially those commonly used in malaria control program. Conclusion In the present study An. stephensi was found resistant to DDT and some pyrethroid insecticides. This enhanced resistance status may be due to previous chemical control programs against malaria vectors, such as IRS/ITNs or insecticide application in agri- culture. However, more investigation for determination of resistance mechanisms is necessary. Furthermore regular monitoring of resistance status by standard bioassay tests and other complementary methods es- pecially in active foci of malaria transmis- sion is suggested. J Arthropod-Borne Dis, December 2016, 10(4): 493–500 MA Gorouhi et al.: Current Susceptibility … 498 http://jad.tums.ac.ir Published Online: October 04, 2016 Acknowledgements This article is a part of the first author’s dissertation for fulfillment of a PhD degree in Medical Entomology and Vector Control from Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Teh- ran, Iran. This study was financially sup- ported by the Deputy for Research, Tehran University of Medical Sciences. Authors are grateful for Dr Amini head of Chabahar health center and all his staff for their kind collaboration. The authors declare that there is no conflict of interest. References Abbott WS (1965) A method of comparing the effectiveness of an insecticide. J Econ Entomol. 18: 265–267. 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