J Arthropod-Borne Dis, June 2015, 9(1): 22–34 M Fathian et al.: Susceptibility of Culicidae … 22 Original Article Susceptibility of Culicidae Mosquitoes to Some Insecticides Recommended by WHO in a Malaria Endemic Area of Southeastern Iran Mousa Fathian 1, *Hassan Vatandoost 1, *Seyed Hassan Moosa-Kazemi 1, Ahmad Raeisi 1,2, Mohammad Reza Yaghoobi-Ershadi 1, Mohammad Ali Oshaghi 1, Mohammad Mehdi Sedaghat 1 1Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2Malaria Unit, CDC, Ministry of Health and Medical Education, Iran (Received 30 May 2012; accepted 14 May 2014) Abstract Background: According to the national strategy plan on monitoring of insecticides resistance, this study was carried out to determine the base line susceptibility of the Culicidae mosquitoes to the WHO-recommended insecticides in an endemic focus of malaria in southeastern Iran. Methods: Larval collection was carried out by dipping method and adult collection occurred by suction tube from January to December 2010. The susceptibility test was assessed to DDT 4 %, malathion 5 %, propoxur 0.1 %, deltamethrin 0.05 %, lambda-cyhalothrin 0.05 %, and cyfluthrin 0.15 % at different interval times (discriminative dose) followed by 24 h recovery period . The LT50 and LT90 values were calculated for plotting the regression line using Microsoft office Excel software ver. 2007. Results: Anopheles stephensi was quite resistant to DDT and showed susceptible or tolerant to other insecticides. The LT50 and LT90 values to DDT in this species were 29.07, and 98.26 minutes, respectively. Anopheles culicifacies and Anopheles dthali were found susceptible or tolerant to insecticides. Culex pipiens was found resistance to DDT, propoxur, lambda-cyhalothrin and cyfluthrin whereas observed susceptible to malathion and tolerant to deltamethrin. Ochlerotatus caspius sl. was resistant to DDT, whereas found susceptible to other insecticides. Culisita longiareolata was susceptible to deltamethrin, whereas tolerant to other insecticides. The LT50 and LT90 values of Cs. longiareolata to DDT were 17.82, and 51.26 minutes. Conclusion: We suggested the same study in different parts of the country for monitoring and evaluation of control measures. Keywords: Culicidae, Malaria, Susceptibility test, Southeastern Iran Introduction Arthropod borne diseases are very im- portant in the world. Malaria occurs often in poor, tropical and subtropical areas and still took an estimated 627 000 lives in 2012, mostly those of children under five years of age in Africa. In fact 1300 young lives lost to malaria every day (WHO 2013). The Culicidae mosquitoes are responsible for transmission of the worm parasites of heart dogs such as Dirofileria immitis, Dirofileria repense, Wuchereria bancrofti, some Arboviral diseases such as Japanese Encephalitis, Rift valley fever, Western equine encephalitis and Eastern equine encephalitis, Tahyna, Sagiyama, Trivitatus, Lymphocytic Choriomanangitis, West Nile virus, St. Louis encephalitis, Cal- ifornia encephalitis. About 70 Anopheles spe- cies in the world are able to transmit malaria and of these 40 species have been identified as the major vectors (Azari-Hamidian and Harbach 2009). By now, 64 species of Culicidae mos- quitoes due to 3 sub genera and 7 genera are reported from Iran (Azari-Hamidian and Harbach 2009). Anopheles stephensi is the main malaria vector in Iran. By now, seven *Corresponding author: Prof Hassan Vatandoost, E- mail: hvatandoost1@yahoo.com, Dr Seyed Hassan Moosa-Kazemi, E-mail: moosakazemi@tums.ac.ir http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 22–34 M Fathian et al.: Susceptibility of Culicidae … 23 species of Anopheles reported as the malaria vectors in the country including: An. fluviatilis s.l, An. culicifacies s.l, An. sacharovi, An. maculipennis s.l, An. superpictus, An. stephensi and An. dthali (Faghih 1969, Vatandoost et al. 2004c, Vatandoost and Borhani 2004a, Vatandoost and Moinvaziri 2004b, Vatandoost et al. 2005b, Sedaghat and Harback 2005, Hanafi-Bojad et al. 2006, Oshaghi et al. 2006, Vatandoost et al. 2006a, 2006b, 2007, 2008, 2009a, 2009b, Moosa-Kazemi et al. 2009, 2010, Omrani et al. 2010). In addition, Zaim et al. reported the An. pulcherrimus as sec- ondary vectors of malaria in the South East of Iran (Zaim et al. 1993). Oocyt of Plasmo- dium found at the first time in An. multicolor, while not found in salivary glands (Eshghy 1978). Avian malaria reported in Iran by Ghaffari (Ghaffari 1955). Spraying with residual insecticide (IRS) con- sidered an important mosquito control meas- ure. Twelve insecticides recommended by WHO for IRS currently, which belong to four chemical groups including one organochlorine, six pyrethroids, three organophosphates and two carbamates (Pluess et al. 2010). DDT re- sistance in the adult of An. stephensi was re- ported in Iran in 1957, subsequently to dieldrin in 1960, and then to malathion in 1976 (Eshghy 1978, Manouchehri et al. 1974). Propoxur was used after reports about to malathion toler- ance of An. stephensi in 1978 (Eshghy 1978). Release of larvivorous fish, using the Bacillus thuringiensis, and larviciding by chlorpyrifos-methyl are the main larval con- trol measures and pyrethroid as new insecti- cides are being used as IRS and LLINs in Iran (Vatandoost and Hanafi Bojd 2005a, Moosa-Kazemi et al. 2007). In spite of more than 50 years malaria- control programming, 42%–60%of the total malaria cases reported from Sistan and Ba- luchistan Province.Out of 112 malaria cases representing 79 males and 33 females have been reported in Chabahar County, south- eastern Iran in 2010. Out of 38 cases were from Bangladesh, Pakistan and Afghanistan and the rest were the local Aborigines (MOH and ME 2010). Chabahar is commercial and industrial free zone as well as important due to agricultural and husbandry in Sistan and Baluchistan Province in southeastern Iran and also the border line of Pakistan. There are scatter data about susceptibility level of malaria vectors in Iran, the suscepti- bility level of Culicinae mosquitoes in south- eastern Country had not been reported. There- fore, this study was carried out to determine susceptibility level of mosquitoes to moni- toring and evaluation of insecticides re- sistance. This information could provide an essential clue for judicious use of insecti- cides and will be very useful to health au- thorities for future planning of vector control in this endemic malarious area. Materials and Methods Study area The study was carried out in Chabahar City, Siatan and Baluchistan Province, south- eastern Iran (25˚17’GN, 60˚37’GE) (Fig. 1). The people engaged to agriculture, horticul- ture, livestock, fishing sailing, and hand Crafts including needlework, making carpet and musical instruments. The absolute maxi- mum and average of temperature was re- ported 36 ˚C and 16.5 ˚C, respectively (Me- teorological Organization of Iran, 2010). Av- erage annual rainfall and humidity was 90mm and 79 %, respectively. Kajoo and Bishmont rivers are the largest rivers located in this area. Chabahar County with 24, 729 square kilometers (19.3%) comprised 3 cities, 5 dis- tracts and 11 rural districts. The population of Chabahar reported 211,081 including 143,535 rural (68%) and 67,546 urban (32%). Naval Base near the 22 Bahman Squar (25° 18’GN,60°37’GE) in Chabahar city selected as fix station including, 30 households, and 114 population. Baluchi-Adam village (25˚ 47’GN, 61˚16’GE) with 71 households, and http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 22–34 M Fathian et al.: Susceptibility of Culicidae … 24 379 populations, and Bahoo-kalat village (25˚43’GN, 61˚25’GE), 198 households and 1064 population selected randomly as fixed stations. Sampling methods such as larval collection, Hand catch was carried out dur- ing January to December 2010 (WHO 1975, WHO 1975). These studies were conducted once every 15 days and collected mosquitoes were identified by specific systematic keys (Shahgudian 1960, Zaim and Cranston 1986). Larval collection and rearing In each fixed and variable station larvae was collected from January to December 2010. Larval mosquitoes picked up from the water using a dropper, pipette or fine net and inserted into the bulb. The related data such as water temperature, larval type, number and date sampling was recorded. Larvae and pupae in holding container filled with water were transferred to the laboratory for rear- ing. Mosquito larvae feed by dry fish food. Adult mosquitoes live quite well on bowl of sucrose 10% in bottom of the cage. The adults were kept in the temperature (22–24 ˚C), relative humidity (60–70%) and 12L- 12D photoperiod (WHO 1975a, 1975b). Hand collection Culicidae mosquitoes were collected from the villages that spraying was not performed before, between 06.30 and 09.30 am. Sam- pling was carried out in each human dwell- ing, cattle and goat sheds for 15 min using suction tube and torch (WHO 1975b). The mosquitoes were inserted in the cage as di- mensions of 40× 40 cm and sent to the labor- atory. Total of 200 to 250 mosquitoes were entered in each cage and covered with wet towel. The sucrose 5 % solution was placed inside the cage. The mosquitoes were kept in standard condition (2 5˚C, 75% H). Insecticide impregnated papers Impregnated papers with DDT 4 %, malathion 5 %, propoxur 0.1 %, deltamethrin 0.05 %, lambda-cyhalothrin 0.05 %, cyfluthrin 0.15 %, and control papers were supplied by World Health Organization. Adult susceptibility test The adult susceptibility test kit including holding and exposure tubes, slides, copper and silver rings and related covers were washed with detergent, and then washed three times with tap water to be free of de- tergents and insecticides. After drying of the pipe holders, standard paper impregnated with insecticides fixed inside the tubes marked with red dot and copper ring. For holding tube used paper control (no insecti- cide) tube with a green sign and silver pipe fixed to the wall (WHO 2006). Each time 4– 5 mosquito collected and insert to holding tube overall 20–25 mosquito were kept into holding tube. The susceptibility tests per- formed on their standard condition (22–26 ˚C, 60% H). The susceptibility of the wild strain of Culicidae mosquitoes was assessed to the in- secticides impregnated papers. The mosqui- toes were exposed to different insecticides by different interval times and 24 hours re- covery period. Smoking and use of pesti- cides during the test was strictly prohibited. WHO criteria for susceptibility test The following criteria have been used for interpretation and classification of results, based on WHO recommendations (African Network on Vector Resistance 2005). A. Susceptibility test carried out with less than 20 mosquitoes per test have not been considered B. At least 80 mosquitoes used per test, three resistance classes as defined. Resistant when the mortality was lower than 80 %, susceptible when mortality was 98 % or higher and possible resistant or tolerant when mortality was between 97 % and 80 %. C. Three resistance classes as defined when twenty to 79 mosquitoes used per test, sus- http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 22–34 M Fathian et al.: Susceptibility of Culicidae … 25 ceptible when mortality was 98 % or higher, resistance suspected, when mortality was be- tween 95 % and 97 %, and resistant when the mortality was lower than 95 %. Identification of mosquitoes using morpho- logical characteristics The mosquitoes after the test were mount- ed and identified by specific systematic keys. The samples were recorded in the special forms by and the appropriate time of deaths associated with history of collection, relative humidity and temperature (Shahgudian 1960, Zaim and Cranston 1986, Azari-Hamidian and Harbach 2009). Statistical analysis Results were considered reliable if the control mortality was less than 5 % and re- jected if more than 20 %. Results were cor- rected by Abbott's formula when mortality rates of control group were between 5 to 20 % (Abbott 1925, WHO 2006). Data were analyzed by probit analysis (Finney 1971, 1978). Regression lines of the species were measured through the χ2 test. The LT50 and LT90 were calculated for plot- ting the regression line using Microsoft Ex- cel software ver. 2007. Results Totally, nine species were collected includ- ing: An. stephensi, An. dthali, An. culicifacies, An. fluviatilis, Cx. pipiens, Cx. quinquefasciatus, Cx. theileri, Cs. longiareolata, Oc. caspius s.l (Fig. 2). In adult collection An. stephensi was dominant species 34.76 % allocated mosquitoes collected. Anopheles dthali and An. culicifacies were followed 15 %, 12.92 %, respectively. Culiseta longiareolata had the lowest density with 1.09 %. An. culicifacies, An. stephensi, Cx. pipiens, Cx. theileri were collected in all months. In larval collection, An. stephensi, with 1495 specimens (28.9%) was predominant followed by Cx. pipiens 753 (14.1%), An. culicifacies 12.8 %, Cx. quinquefasciatus 6.3 % in the same month. It should be noted that An. fluviatilis larva was collected in May and December. Mortality of Culicidae mosquitoes ex- posed to DDT and other insecticides has shown in tables 1 and 2. LT50 and LT90 val- ues of An. stephensi to DDT 4% were 29.70 and 98.26 minutes, respectively. This spe- cies was quite resistant to DDT while sus- ceptible to deltamethrin and tolerant to other insecticides (Fig. 3). It is concluded that An. culicifacies is tolerant to DDT, malathion, and propoxure whereas susceptible to delta- methrin, cyfluthrin and lambda-cyhalothrin (Table 2). The LT50 and LT90 values of this species to DDT 4 % were 18.12 and 46.42 minutes (Table 1, Fig. 3). Anopheles dthali was tolerant to DDT and propoxur whereas susceptible to deltamethrin, lamda-cyhalotrin, cyfluthrin and malathion (Table 2, Fig. 3). The LT50 and LT90 values to DDT 4 % were 17.86 and 48.42 minutes (Table 1). It is concluded that Cx. pipiens is quite re- sistance to DDT, propoxur, lambda-cyhalothrin, and cyfluthrin whereas susceptible to Malathion (Table 2, Fig. 3). In our study Oc. caspius found to be resistant to DDT whereas susceptible to other insecticides (Table 2, Fig. 3). The LT50 and LT90 values to DDT 4 % were 44.68, and 164.01 minutes (Table 1). Culiseta longiaerolata observed tolerant to DDT, propoxur, cyfuthrin, lambda-cyhalothrin whereas susceptible to deltamethrin (Table 2, Fig. 3). The LT50 and LT90 values to DDT 4 % were 17.82 and 51.26 minutes (Table 1). http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 22–34 M Fathian et al.: Susceptibility of Culicidae … 26 Table 1. Regression line parameters of various species exposed to DDT 4% in Chabahar County, Sistan and Balu- chistan Province, southeastern Iran, 2010 Y=A+ BXP valueX2 (df)****LT90 95%C.I. (minute) ***LT50 95%C.I. (minute) **B±SE*ASpecies Y= -8.0186+ 2.4666 X<0.0598.062(2)98.2629.702.4666±1.655-8.0186An. stephensi Y= -9.5283+ 3.1379 X<0.05265.565(2)46.4218.123.1379±3.222-9.5283An. culicifacies Y= -8.9653+ 2.9588 X<0.05397.042(2)48.4217.862.9588±3.713-8.9653An. dthali Y= -7.3266+ 2.2321 X<0.0559.087(2)119.7231.932.2321±1.042-7.3266Cx. pipiens Y= -8.2580+ 2.3891 X<0.05409.134(2)164.0144.682.3891±3.687-8.2580Oc. caspius Y= -8.4636+ 2.7939 X<0.0541.551(2)51.2617.822.7939±1.086-8.4636Cs. longeareolata *intercept. **B± SE= slope and its standard error. ***LT50, 95 % CI= lethal time causing 50 % mortality and its 95 % confidence interval. ****LT90, 95 % CI= lethal time causing 90 % mortality and its 95 % confidence interval. Fig. 1. Location of study area in southeastern Iran, 2010 Fig. 2. The species composition of adult Culicidae mosquitoes collected by different sampling methods in Chabahar County, Sistan and Baluchistan Province, southeastern Iran, 2010 http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 22–34 M Fathian et al.: Susceptibility of Culicidae … 27 Table 2. Susceptibility level of various Culicidae mosquitoes exposed to some insecticides in Chabahar County, Sistan and Baluchistan Province, Southeastern Iran, 2010 Species Insecticide *MR±ER **Resistance status An. culicifacies DDT 4% 96±2 T Malathion 5% 95±2 T Propoxur 0.1% 97±2 T Deltamethrin 0.05% 100 S Cyfluthrin 0.15 % 98±1 S Lambda-cyhalothrin 0.05% 99±1 S An. dthali DDT 4% 97±2 T Malathion 5% 98±1 S Propoxur 0.1% 97±2 T Deltamethrin 0.05% 100 S Cyfluthrin 0.15 % 98±1 S Lambda-cyhalothrin 0.05 % 99±1 S An. stephensi DDT 4% 67±5 R Malathion 5% 90±3 T Propoxur 0.1% 95±2 T Deltamethrin 0.05% 100 S Cyfluthrin 0.15 % 96±2 T Lambda-cyhalothrin 0.05 % 96±2 T Cx. pipiens DDT 4% 54±5 R Malathion 5% 100 S Propoxur 0.1% 20±4 R Deltamethrin 0.05% 93±2 T Cyfluthrin 0.15 % 72±4 R Lambda-cyhalothrin 0.05 % 72±5 R Cs. longiareolata DDT 4% 92±3 T Malathion 5% - - Propoxur 0.1% 92±3 T Deltamethrin 0.05% 100 S Cyfluthrin 0.15 % 88±3 T Lambda-cyhalothrin 0.05 % 96±2 T Oc. caspius DDT 4% 33±5 R Malathion 5% 100 S Propoxur 0.1% 100 S Deltamethrin 0.05% 100 S Cyfluthrin 0.15 % 100 S Lambda-cyhalothrin 0.05 % 100 S *Mortality rate ±Error Bar **R Resistance, S Susceptible, T Tolerance http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 22–34 M Fathian et al.: Susceptibility of Culicidae … 28 Fig. 3. Regression line of adult Culicidae mosquitoes exposed to DDT 4 % in Chabahar County, Sistan and Baluchi- stan Province 2010 Discussion In our study, 4 genera and 9 species of mosquito larvae and adults were identified based on morphological characters. Culicidae species were belongs to the genus of Anoph- eles, Culex, Culisita and Ochleratatus. The Species of Cx. quinquefasciatus and Cs. longiareolata found by larval collection. The most predominant species was An. stephensi with 34.76 % of adult and 29.36 % of larvae collection. Vatandoost et al. (2004c) report- ed three biological forms of this species in- cluding type, intermediate and mysorensis in southern Iran. Type and intermediate forms cited as vector in urban areas whereas, mysorensis form as vector in rural area (Vatandoost et al. 2006b). In Iran, indoor residual spraying (IRS) with DDT was carried out for malaria con- trol during 1950–1968. In this species, re- sistance to DDT was first recognized in 1958 (Mofidi et al. 1958, 1960, Manuchehri et al. 1976a, 1976b) and subsequently to dieldrin in 19609 (Mofidi and saminmi 1960) and matlathion in 1976 (Eshghy 1978). Following the emergence of resistance of Anopheles stephensi to DDT, other organophosphours, carbamate and pyrethroid insecticides were used. The susceptibility level of An. stephensi to DDT and Dieldrin was studied at various parts of Iran bordered in Persian Gulf and Oman Sea during 1985–88. The results re- vealed that this species was resistant to DDT and it is quite susceptible to Dieldrin (Ma- nouchehri et al. 1976). In a similar study in south of Iran, the results showed that this species was resistant to DDT (Eshghy and Janbakhsh 1976). Prior of this study, Vatan- doost et al. (2004) reported three biological forms of An. stephensi was susceptible to Bendiocarb, Propoxur, Malathion, Fenitrothion, Deltamethrin, Permethrin, Cyfluthrin and Lambda-cyhalothrin, resistance to DDT and tolerant to Dieldrin in Hormozgan Province, south of Iran. The same study was carried out on An. stephensi of Bandar Abbas strain, showed a relatively high adult resistance of http://jad.tums.ac.ir Published Online: July 16, 2014 An. stephensi An. culicifacies An. dthali Cx. pipiens TIME Cs. longeareolata Oc. caspius P R O B IT J Arthropod-Borne Dis, June 2015, 9(1): 22–34 M Fathian et al.: Susceptibility of Culicidae … 29 4.66-fold (LT50= 108min) to 4 % DDT (Davari et al. 2007). In our study, the LT50 values of DDT 4.0 % against this species were 29.7 minutes, a manifestation of DDT resistance. Some investigations indicated An. stephensi, the main malaria vector, resistant to pyrethroid insecticides in southeastern Iran (Vatandoost and Hanafi-Bojd 2012). Anopheles culicifacies comprised of five sibling species of A, B, C, D and E in North and South of India reported as A and B, in north east found D associated sympatric with A and B whereas in central area reported all of sibling species (Goswami et al. 2006). Polytene chromosome examination has been reported only available method that able to differentiate four members of this complex in areas where species E is not prevalent (Goswami et al. 2006). Species A has been reported in Oman and Iran (Zaim and Javaherian 1991). This species is known as vector of malaria in East Afghanistan (Zahar 1990). In our study, An. culicifacies was tol- erant to DDT, malathion and propoxur while susceptible to other insecticides. In contrast, DDT resistance reported for the first time after residual spraying in Iran by Vatandoost et al. (2011). They reported susceptibility of An. culicifacies to pyrethroid and irritability to fenitrothion, cyfluthrin, and permethrin while high extremely resistance to DDT. There are several reports revealing DDT re- sistance of An. culicifacies in Afghanistan, and also resistance to dieldrin, malathion and DDT in India (Goswami et al. 2006). In our study, An. dthali found tolerant to DDT and propoxur while susceptible to Deltamethrin, Lambda-cyhalothrin, Cyfluthrin and Malathion. In contrast, there are also some reports emphasizing to susceptibility levels of this species in different countries (WHO 1992, Hanafi-bojd et al. 2007). In spite, the existence of many reports about to susceptibility of An. dthali to insecticides, re- sistance reported to malathion, chloropyriphos, bromophos, carbamate in Egypt and temephos in Jordan (WHO 1992). In our research, Cx. pipiens larvae with 1073 (51.14%) collection, was found one of the most predominant species. In adult col- lection, Cx. pipiens catches 12.14 % and Cx. quinquefasciatus found as 4.82 % of total collection Gjullin and Peters (1952) cited decrease of susceptibility level of Cx. pipiens com- plex to DDT and organophosphorus insecti- cide. First report related to resistance of the Cx. quinquefasciatus to organophosphorus insecticide was cited by Isaak in 1961 (Isaak 1961). Increasing in the resistance level of Cx. pipiens to organophosphorus insecticides reported by Toma et al. (2011). In our study, Cx. pipiens was quite resistance to DDT, propoxur, lambda-cyhalothrin, and cyfluthrin, whereas tolerant to deltamethrin and suscep- tible to malathion. In parallel, there are many reports in relation to some organophosphorus and pyrethroid insecticides resistance in Tu- nisia (Ben Cheikh et al. 1998), Cuba (Bisset et al. 1991), Burkina Faso (Chandre et al. 1998), Saudi Arabia (Amin and Hemingway 1998) and China (Jinfu 1999). Another study showed, pyrethroid insec- ticides had tolerance of Cx. pipiens pipiens var molestus in North America (McAbee et al. 2003). In contrast, DDT resistance of Cx. pipiens complex reported in southern Tehran (Nazari and Janbakhsh 2000). LT50 value for propoxur and malathion calculated as 51, and 31 minutes respectively (Nazari and Janbakhsh 2000). The species reported quit susceptible to dieldrin, propoxur and malathion (Nazari and Janbakhsh 2000). In our study, Cx. pipiens was highly resistance to DDT, propoxur, cyfluthrin and lambda-cyhalothrin as evidenced by the discriminating concen- trations of times. The results may be related to the present of the species complex, ap- plication of IRS and distribution of LLINs in southern Iran. http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 22–34 M Fathian et al.: Susceptibility of Culicidae … 30 In the present study, Oc. caspius found to be quite resistance to DDT whereas suscep- tible to other insecticide. The LT50 and LT90 values to DDT 4 % were 47.68 and 164.01 minutes. In addition, our study showed Cs. longiaerolata quite resistance to DDT and susceptible to other insecticides The LT50 and LT90 values to DDT 4 % were 17.82 and 50.26 minutes. Based on the literature, no reports were available on the susceptibility levels of Oc. caspius and Cs. longiaerolata to WHO recommended insectidides in the world. Conclusion Iran is in the malaria elimination stage (WHO 2011). By now, IRS in human dwell- ings and animal shelters, space-spraying, per- sonal protection through distribution of LLINs and curtains (ICNs), repellents measures used to control of malaria vectors in Iran. In addition, some biological and chemical agents against larval and adult stages of Culicidae mosquitoes had been evaluated in the labor- atory. Results obtained from susceptibility tests of the malaria vectors on DDT revealed that only An. stephensi was highly resistance to DDT in Chabahar area, precautionary measures should be taken in future vector control operations. Moreover, the status of re- sistance in other locations in malaria en- demic area should be investigated. Since the country relies on deltamethrin for IRS oper- ation, tolerant populations of Cx. pipiens im- plies careful consideration and regular mon- itoring of susceptibility level of mosquitoes in the future. Acknowledgement The authors are grateful to Dr. Nazari head of Chabahar health center for coop-oration during the study. The authors thank to Dr Hanafi-Bojd, School of Public Health, Tehran University of Medical Sciences, for reviewing the manuscript. We express thanks to Mr Gorgij, Mr Jadgal, Mr Amiri, Mr Lalo field technician of Chabahar health center. This study was financially supported by grant of Tehran University of Medical Sciences (ID: No: 10513-27-01-89). References Abbott WS (1925) A method of computing the effectiveness of an insecticide. J Econ Entomol. 18: 265–267. African Network on Vector Resistance (2005) World Health Organization African Region. Atlas of insecticide resistance in malaria vectors of the WHO African region. Harare, Zimbabwe Amin AM, Hemingway J (1998) Preliminary investigation of the mechanisms of DDT and pyrethroid resistance in Culex quinquefasciatus Say (Diptera: Culicidae) from Saudi Arabia. Bull Entomol Res. 79: 361–366. Azari-Hamidian S, Harbach RE (2009) Keys to the adult females and fourth-instar larvae of the mosquitoes of Iran (Dip- tera: Culicidae) Zootaxa. 2078: 1–33. Barr AR, Kabtman L (1951) Biometrical notes on the hybridization of Culex pipiens L. and C. quinquejasciatus Say. J Parasit. 37: 419–420. Ben Cheikh H, Ben Ali-Haouas Z, Marquine M, Pasteur N (1998) Resistance to or- ganophosphates and pyrethroid insec- ticides Culex pipiens (Diptera: Culicidae) from Tunisia. J Med Entomol. 35: 251– 260. Bisset JA, Rodriguez MM, Hemingway J, Diaz C, Small GJ, Ortiz E (1991) Malathion and pyrethroid resistance in Culex quinquefasciatus from Cuba: efficacy of pirimiphos-methyl in the presence of at least three resistance mechanisms. Med Vet Entomol. 5: 223–228. http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 22–34 M Fathian et al.: Susceptibility of Culicidae … 31 Chandre F, Darriet F, Darder M, Cuany A, Doannio JMC, Pasteur N, Guillet P (1998) Pyrethroid resistance in Culex quinquefasciatus from west Africa. Med Vet Entomol. 12: 359–366. Davari B, Vatandoost H, Oshaghi MA, Ladonni H, Enayati AA, Shaeghi M, Basseri HR, Rassi Y, Hanafi-Bojd AA (2007) Selection of Anopheles stephensi with DDT and dieldrin and cross-re- sistance spectrum to pyrethroids and fipronil. Pest Biochem Physiol. 89: 97– 103. Dehghan H, Sadraei J, Moosa-Kazemi SH (2010) The morphological variations of Culex pipiens larvae (Diptera: Culicidae) in Yazd Province, Central Iran. Iran J Arthropod-Borne Dis. 4(2): 42–49. Eshghy N (1978) Tolerance of Anopheles stephensi to Malathion in the province of Fars, southern Iran. Mosq News. 38(4): 580–583. Eshghy N, Janbakhsh B (1976) Insecticide resistance of Anopheles stephensi my- sorensis in the province of Fars, south- ern Iran. Mosq News. 36(3): 336– 339. Faghih MA (1969) Malaria and malaria erad- ication. Tehran University Press, p. 726. Finney DJ (1971) Probit analysis (3rd ed): Cambridge University Press, Cam- bridge, UK. Finney DJ (1978) Statistical method in bio- logical assay (3rd ed): Hodder Arnold press, London, UK. Ghaffari AN (1955) The classification of Culicidae (Diptera: Nematocera): the study of Culex linneanues in Iran. Teh- ran University, School of Medicine, Iran, p. 189. Gjullin CM, Peters RF (1952) Abstract of recent studies of mosquito resistance to insecticides in California. Proceed- ings and Papers of the Twenty Ann Conf Calif Mosq Control Assoc, Vol. 1. Visalia press, Cleveland, Ohio, USA, pp. 44–45. Goswami G, Singh OP, Nanda N, Raghavendra K, Gakhar SK, Subbarao SK (2006) Identification of all members of the Anopheles culicifacies complex using allele-specific polymerase chain reac- tion assays. Am J Trop Med Hyg. 75 (3): 454–60. Hanafi-Bojd AA, Azari-Hamidian S, Vatan- doost H (2011) Spatio-temporal distri- bution of malaria vectors (Diptera: Culicidae) across different climatic zones of Iran. Asian Pacific J Trop Med 4(6): 498-504. Isaak LW (1961) Review of insecticide resistance in Kern Mosquito Abate- ment District. Proc and Papers of the Twenty-nine Ann Conf Calif Mosq Control Assoc. Vol. 1. Visalia press, Cleveland, Ohio, USA, pp. 105–106. Jinfu W (1999) Resistance to deltamethrin in Culex pipiens pallens (Diptera: Culi- cidae) from Zhejiang, China. J Med Entomol. 36: 389–393. Manouchehri N, Eshghi F, Rouhani F (1974) Malathion susceptibility test of Anoph- eles stephensi mysorensis in southern Iran. Mosq News. 34: 440–442. Manouchehri AV, Janbakhsh B, Rouhani F (1976b) Studies on the resistance of Anopheles stephensi to Malathion in Bandar Abbas, Iran. Mosq News. 36 (3): 320–322. McAbee RD, Kang KD, Stanich MA, Christiansen JA, Wheelock CE, Inman AD, Hammock BD, Cornel AJ (2003) Pyrethroid tolerance in Culex pipiens pipiens var molestus from Marin Coun- ty, California. Pest Manag Sci. 60: 359– 368. Ministry of Health (MOH) Medical Educa- tion (ME) of Iran (2010) Annual report of malaria control department. Tehran: CDC, Iran, pp. 50–65. Mofidi Ch, Samimi B, Eshghy N, Ghiasedin M (1958) Further study of Anophelinae susceptibility to insecticides in Iran. Re- http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 22–34 M Fathian et al.: Susceptibility of Culicidae … 32 sult of Busvine and Nash method, Iran. Publ 585. Institute of Parasitology and Malariology, Tehran, pp. 1–7. Mofidi Ch, Samimi B (1960) Resistance of Anopheles stephensi to dieldrin in Iran. Publ 650. Institute of Parasitology and Malariology, Tehran, pp. 3–4. Moosa Kazemi SH, Karimiana F, Davari B (2010) Culicinae mosquitoes in Sa- nandaj County, Kurdistan Province, western Iran. J Vector Borne Dis: 103– 107. Moosa-Kazemi SH, Vatandoost H, Nikookar H, Fathian M (2009) Culicinae (Dip- tera: Culicidae) Mosquitoes in Cha- bahar County, Sistan and Baluchistan Province, Southeastern Iran. Iran J Arthropod-Borne Dis. 3: 29–35. Moosa-Kazemi SH, Vatandoost H, Raeisi A, Akbarzadeh K (2007) Deltamethrin impregnated bed nets in a malaria con- trol program in Chabahar, Southeast Baluchistan, Iran. Iran J Arthropod- Borne Dis. 1: 43–51. Nazari M, Janbakhsh B (2000) A survey of the susceptibility level of Culex theileri and Cx. pipiens to DDT, Dieldrin, Propoxur and Malathion in the south- ern area of Tehran. J Uromia Univ Med Sci. 11(1): 13–19. Nikookar SH, Moosa-Kazemi SH, Oshaghi MA, Yaghoobi-Ershadi MR, Vatandoost H, Kianinasab A (2010) Species com- position and diversity of mosquitoes in Neka County, Mazandaran Province, Northern Iran. Iran J Arthropod-Borne Dis. 4(2): 26–34. Omrani SM, Vatandoost H, Oshaghi MA, Shokri F, Guerin PM, Yaghoobi-Ershadi MR, Rassi Y, Tirgari S (2010) Fabri- cation of an olfactometer for mosquito behavioural studies. J Vector Borne Dis. 47: 17–25. Oshaghi MA, Chavshin AR, Vatandoost H (2006) Analysis of mosquito bloodmeals using RFLP markers. Exp Parasitol. 114: 259–264. Pluess B, Tanser FC, Lengeler C and Sharp BL (2010) Indoor residual spraying for preventing malaria. Cochrane Database Syst Rev. 4: 4. Sedaghat MM, Harbach RE (2005) An annotated checklist of the Anopheles mosquitoes (Diptera: Culicidae) in Iran. J Vector Ecol 2005. 30: 272–276. Shahgudian ER (1960) A key to the Ano- phelines of Iran. Acta Med Iran. 3: 38– 48. Toma L, Menegon M, Romi R, De Matthaeis E, Montanari M, Severini C (2011) Status of insecticide resistance in Culex pipiens field populations from north- eastern areas of Italy before the with- drawal of OP compounds. Pest Manag Sci. 67(1): 100–106. Vatandoost H, Borhani N (2004a) Suscep- tibility and irritability levels of main malaria vectors to synthetic pyrethroids in the endemic areas of Iran. Acta Med Iranica. 42: 240–247. Vatandoost H, Moinvaziri VM (2004b) Larvicidal activity of neem tree extract (Neemarin) against mosquito larvae in the Islamic Republic of Iran. East Mediterr Health J. 10: 573–578. Vatandoost H, Shahi H, Abai MR, Hanafi- Bojd AA, Oshaghi MA, Zamani G (2004c) Larval habitats of main malaria vectors in Hormozgan Province and their susceptibility to different larvicides. Southeast Asian J Trop Med Pub Hlth. 35: 22–25. Vatandoost H, Hanafi-Bojd AA (2005a) Current resistant status of Anopheles stephensi Liston to different larvicides in Hormozgan Province, southeastern Iran. Pakistan J Bio Sci. 8: 1568–1570. Vatandoost H, Mashayekhi M, Abai MR, Aflatoonian MR, Hanafi-Bojd AA, Sharifi I (2005b) Monitoring of insecti- cides resistance in main malaria vectors http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 22–34 M Fathian et al.: Susceptibility of Culicidae … 33 in a malarious area of Kahnooj district, Kerman Province, southeastern Iran. J Vec Borne Dis. 42: 100–108. Vatandoost H, Gholizadeh MR, Abai MR, Djavadian E (2006a) Laboratory effi- cacy of protection rate of torn nets treated with pyrethroids, cufluthrin, deltamethrin and permethrin against Anopheles stephensi (Diptera: Cu- licidae). J Biol Sci. 6: 331–336. Vatandoost H, Oshaghi M, Abai MR, Shahi M, Yaaghoobi F, Baghaii M, Hanafi- Bojd AA, Zamania G, Townsonc H (2006b) Bionomics of Anopheles stephensi Liston in the malarious area of Hormozgan Province, southern Iran. Acta Trop. 97: 196–205. Vatandoost H, Shahi M, Hanafi-Bojd AA, Abai MR, Oshaghi MA, Rafii F (2007) Ecology of Anopheles dthali Patton in Bandar Abbas. Iran J Arthropod-Borne Dis. 1: 21–27. Vatandoost H, Khazani A, KebriainZadeh A, Vatandoost H, Khazani A, Kebriain Zadeh A, Rafinejad J, Khoobdel M, Abai MR, Hanafi-Bojd AA, Akhavan AA, Abtahi. SM, Rafi F (2008) Com- parative efficacy of Neem and dime- thyl phthalate (DMP) against malaria vector, Anopheles stephensi (Diptera: Culicidae). Asian Pac J Trop Med. 1: 1–6. Vatandoost H, Ramin E, Rassi Y, Abai MR (2009a) Stability and wash resistance of local made mosquito bednets and detergents treated with pyrethroids against Anopheles stephensi. Iran J Arthropod-Borne Dis. 3: 19–28. Vatandoost H, Abai MR, Abbasi M, Shaeghi M, Abtahi M, Rafie F (2009b) Design- ing of a laboratory model for evalua- tion of the residual effects of delta- methrin (K-othrine WP 5%) on differ- ent surfaces against malaria vector, Anopheles stephensi (Diptera: Culicidae). J Vector Borne Dis. 46: 261–267. Vatandoost H, Emami SN, Oshaghi MA, Abai MR, Raeisi A, Piazzak N, Mahmoodi M, Akbarzadeh K, Sartipi M (2011) Ecology of malaria vector Anopheles culicifacies in a malarious area of Sistan va Baluchestan Province, south- east Islamic Republic of Iran. East Mediterr Health J. 17(5): 439–445. Vatandoost H, Hanafi-Bojd AA (2012) In- dication of pyrethroid resistance in the main malaria vector, Anopheles stephensi from Iran. Asian Pac J Trop Med. 5(9): 722–726. WHO (1975a) Manual of practical entomol- ogy in malaria. Part 1. Vector bionom- ics and organisation of antimalarial activities. WHO Press, Avenue Appia, Geneva, Switzerland WHO (1975b) Manual on Practical Ento- mology in Malaria. Part II. Methods and Techniques. WHO Press, Geneva, Switzerland. WHO (1992) Vector resistance to pesticides. Fifteenth report of the WHO expert committee on vector control. WHO Tech Rep Ser. No. 818. WHO Press, Avenue Appia, Geneva , Switzerland. WHO (2002) Entomology and vector con- trol. Training module on malaria con- trol. Guide for participants. World Health Organization: Global Malaria Programe, WHO Press, Avenue Appia, Geneva, Switzerland WHO (2006) Pesticides and their application. For the control of vectors and pests of public health importance. Volume WHO/ CDS/NTD/WHOPES/GCDPP/2006.1. WHO Press, Avenue Appia, Geneva, Switzerland , WHO (2011) World malaria report. WHO Press, Avenue Appia, Geneva, Swit- zerland. WHO (2013) Malaria report. WHO Press, World Health Organization, WHO Press, Avenue Appia, Geneva, Switzerland. http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 22–34 M Fathian et al.: Susceptibility of Culicidae … 34 Zahar AR (1990) Applied field studies Section 3, Vector bionomics, Malaria epidemiology and control by geo- graphical areas (B) Asia west of India. WHO VBC/60.3- Mal/90.3. Zaim M, Cranston PS (1986) Checklist and keys to the Culicinae of Iran (Diptera: Culicidae). Mosq Syst. 18: 233–245. Zaim M, Javaherian Z (1991) Occurrence of Anopheles culicifacies species A in Iran. J Am Mosq Control Assoc. 7(2): 324–326. Zaim M, Zahirnia AH, Manouchehri AV, Zaim M, Zahirnia AH, Manouchehri AV (1993) Survival rates of Anopheles culicifacies s.l and Anopheles pul- cherrimus in sprayed and unsprayed villages' in Ghassreghand district, Ba- luchistan, Iran. J Am Mosq Cont Assoc. 9: 421–425. http://jad.tums.ac.ir Published Online: July 16, 2014