J Arthropod-Borne Dis, June 2021, 15(2): 171–178 A Sofizadeh et al.: Resistance Status of … 171 http://jad.tums.ac.ir Published Online: June 30, 2021 Original Article Resistance Status of Anopheles maculipennis and Anopheles superpictus to the Conventional Insecticides in Northeastern Caspian Littoral, Iran Aioub Sofizadeh1; *Mohammad Reza Abai2,3; Hassan Vatandoost2,3; Ahmad Raeisi4; Mo- hammad Sistanizadeh-Aghdam2 1Infectious Diseases Research Center, Golestan University of Medical Sciences, Gorgan, 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 4Malaria Control Unit, Center for Communicable Diseases Control, Ministry of Health and Medical Educa- tion, Tehran, Iran *Corresponding author: Mr Mohammad Reza Abai, E-mail: abaimr@tums.ac.ir (Received 22 Mar 2020; accepted 23 May 2021) Abstract Background: Malaria resurgence has occurred in the northern half parts of Iran. The resurgence of malaria in the prone area could arise from various factors, e.g. wide use of pesticides in the agriculture sector and factors such as habitual patterns of movement of local people from problematic southeastern foci in Iran toward the Caspian Littoral. There are no new data on the resistance status of main malaria vectors in the Caspian Littoral, and this study was aimed at renewal data on conventional insecticides. Methods: The field strain of adult Anopheles superpictus and Anopheles maculipennis were collected using the hand catch method and transferred to the laboratory. The susceptibility tests were carried out against DDT 4%, Malathion 5%, Permethrin 0.75%, Deltamethrin 0.05%, and Lambda-cyhalothrin 0.05%, followed by the WHO’s procedure. Results: The primary malaria vector in Caspian Littoral is An. maculipennis, revealed to be still resistant to DDT and mortality rate, LT50 and LT90 of female mosquitoes were 75.0%, 54.2, minutes and 111.3 minutes. The under ’verifica- tion required’ status of An. maculipennis was also revealed to Lambda-cyhalothrin based on recent WHO’s criteria. The malaria vector An. superpictus is also considered the second malaria vectors in the west parts of the studied area, which showed to be susceptible to all insecticides tested. Conclusion: DDT resistance is persisted in An. maculipennis despite stopping residual spraying with DDT since 1978 in the Caspian Littoral, but the occurrence of pyrethroid under ’verification required’ status is a progressive threat to the possible development of cross-resistance in the future. Keywords: Anopheles maculipennis; Anopheles superpictus; Insecticide resistance; Malaria; Caspian Littoral Introduction Malaria is one of the most important vec- tor-borne diseases globally, especially in de- veloping countries, and Iran is located in the Eastern Mediterranean Region with lower ma- laria endemicity. The country's southeastern parts, including the provinces of Sistan- Balu- chistan, Hormozgan, and southern Kerman are characterized by "refractory malaria". Later on up to the year 1944, malaria epidemiology was studied by some Iranian and overseas inves- tigators, and it was found the hypo-endemic situation at some littoral parts of the Caspian Sea in North of Iran (1). In the past years, stud- ies were carried out in the Golestan Province from 1949 to 1957 and, the spleen index was measured in 21 villages. The classical malar- iometric measure causing splenic enlargement rate was estimated at 52.1% in the Bandar-e- Copyright © 2021 The Authors. Published by Tehran University of Medical Sciences. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International license (https://creativecommons.org/licenses/by- nc/4.0/). Non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:abaimr@tums.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, June 2021, 15(2): 171–178 A Sofizadeh et al.: Resistance Status of … 172 http://jad.tums.ac.ir Published Online: June 30, 2021 Gaz and 32.5% in the Gorgan area during 1949–1959. However, the annual parasite in- dex was reported as 5.7% in the Gorgan area, northern Iran. The prevalence of malaria was stated as 100 per 10,000 populations in 1949 to 8 per 10000 populations in 1959, and the mor- tality decreased from 40% to 2%. Malaria cases were recorded 164 in the Gorgan, 50 in the Bandar-e-Shah, 57 in the Komish-Tapeh, 52 in the Gonbad-e-Kavous, 44 in the Haji- Lar, and 103 in the Gaz among the age group 2–12 years old in 1935. The percentage of malaria parasites was 50.0% Plasmodium ma- lariae, 46.1% Plasmodium vivax, and 3.9% Plasmodium falciparum (2). Seven Anophe- line mosquitoes, including Anopheles stephensi, Anopheles culicifacies, Anopheles fluviatilis, An. superpictus, Anopheles sacharovi, An. maculi- pennis complex and Anopheles dthali are in- volved in the transmission of malaria in Iran (3-5). A total of three species of malaria vec- tors was reported in North of Iran, and An. maculipennis was introduced as the primary vector (3) and An. superpictus as a secondary vector (6-7) and Anopheles sacharovi are also considered a malaria vector in the northwest- ern parts of Iran (8). Malaria was considered a significant health problem from 1941 to 1948, so that no other disease has caused such irrepa- rable financial and human losses in the coun- try. The disease has become more common in the populated areas of the Caspian territory, due to the presence of a favorite climate for the development of Anopheles mosquitoes. Con- trol strategies were using the residual spraying of indoor places with DDT, larval control with oil derivation at different habitats, and treat- ing the patients with quinine. Malaria eradica- tion program (MEP) was started in 1957 in Iran and from 1957–1971 caused interruption of transmission in the North of Iran (9). Due to prone condition of study area, and favorite climate for malaria vectors, routine movement of local people from the southeastern parts to northeastern of the Caspian area as well as the wide application of pesticides in the agricul- tural sector, this study aimed to determine the susceptibility level of two Anopheles species to the conventional insecticides in the Kalaleh district, northeastern of the Caspian territory. Materials and Methods Study area The study was conducted in the Kalaleh district (37° 22' N, 55° 29' E), Golestan Prov- ince, from April to October 2016. This prov- ince was split off from Mazandaran Province in 1998. The province is bounded by the Cas- pian Sea and the Mazandaran Province in the west, the Semnan Province in the south, the North Khorasan Province in the East, and a borderline with Turkmenistan in the North (Fig. 1). This study was carried out in three fixed villages and five randomly selected one in Kalaleh District. Most parts of the Golestan Province are plain, and more than 2/3 of the plains have arid and semiarid climates, and 1/3 of the others have a temperate climate. The dis- trict area is 1985km2 with 117660 population located in the northeast parts of the Golestan Province. The main agricultural products are alfalfa, rice, watermelon, and cotton. Maximum and minimum temperatures were recorded as 40.8 and -0.2 °C, respectively, and the mean annual relative humidity was recorded as 74.0 %. The total annual rainfall was 772mm, the minimum precipitation in August and maxi- mum in February. The sampling of mosquitoes carried out in 3 villages of Kalaleh District, in- cluding Aziz-Abad (37°32'45''N, 61°41'52''E), Gharanki-Jangal (37°34'31''N, 61°46'43''E) and Gorgandoz (37°31'24''N, 61°43'10''E) with the mean elevation of 65 meters above sea level. Mosquito collection The sampling plan for collecting of adult mosquitoes was carried out from April to Feb- ruary 2016. The fresh-fed mosquitoes were dom- inant compared to unfed and gravid physiologic conditions, so only the fresh-fed mosquitoes were used in order to the homogeneity of test http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 171–178 A Sofizadeh et al.: Resistance Status of … 173 http://jad.tums.ac.ir Published Online: June 30, 2021 data. The indoor-resting mosquitoes were col- lected by mouth aspirator before sunrise, trans- ferred into the wooden cages, and transported in a cool condition to the laboratory in the Health Center of Kalaleh District, Golestan Province, Northeast of Iran. Susceptibility Test Susceptibility levels of field-collected mos- quitoes to insecticides were determined by ex- posing freshly fed females to the diagnostic doses of insecticide-impregnated papers sup- plied by WHO, i.e. DDT 4%, Malathion 5%, Permethrin 0.75%, Deltamethrin 0.05%, and Lambda-cyhalothrin 0.05%. The exposure time for all the insecticides tested was 60min, fol- lowed by a 24h recovery period. To calculate the LT50 for DDT, the logarithmic exposure times ranged 15, 30, 60, and 120 minutes, fol- lowed by 24h holding period were carried on. Each logarithmic exposure time was replicated four times using 25 female field-caught mosqui- toes. The susceptibility exposure tubes were held in the vertical position during testings with py- rethroids, organochlorine and organophosphate insecticides (12). The recovery period of ex- posed mosquitoes was kept in a room with a temperature of 25±2 °C. Simultaneously, the control group also was exposed 60 minutes to untreated papers. After exposure, the mosqui- toes spent the recovery period at 25±2 °C and 70–80% relative humidity with access to soaked cotton pads in 10% sucrose solution for 24h until scoring the mortality. If control mortality was within 5–20%, test mortality was correct- ed by Abbott's formula. The mortality rate was ranked as the susceptible, under ’verification re- quired’ status, and resistant, based on WHO’s criteria e.g., 98–100%, 90–97%, and below 90%, respectively (10-11). Results The dominant species were, An. superpic- tus and An. maculipennis that tested for re- sistance/susceptibility level to DDT, malathion, deltamethrin, permethrin, and lambda-cyhalo- thrin (Table 1). The response of 100 mosqui- toes of An. maculipennis to DDT 4.0% for 1h, followed by a 24h recovery period resulted in the survival of 25 mosquitoes, and the mortal- ity was 75.0%. The regression parameters of DDT time-response, including intercept (a), slope ± standard error (b±SE), heterogeneity of mor- tality data with the degree of freedom (χ2(df)), LT50 ±95% confidence interval (CI), and LT90 ± 95% CI were calculated (Table 2). The LT50 and LT90 values for An. maculipennis were 54.2 and 111.3 minutes, respectively. The re- gression line and the equation was shown in Fig. 2. The susceptibility level of An. superpic- tus to the tested insecticides is summarized in Table 1, which showed complete susceptible to all tested insecticides. Table 1. Susceptibility levels of dominant species of Anopheles at the diagnostic doses to different insecticides using WHO-recommended method (10), northeastern parts of the Caspian Littoral, Iran Insecticide Anopheles superpictus Anopheles maculipennis Total mosquito tested No. dead Mortality rate (%) Resistance status* Total mos- quito test- ed No. dead Mortality rate (%) Resistance status* DDT 4% 100 99 99.0 S 100 75 75.0 R Malathion 5% 100 100 100.0 S 100 100 100.0 S Deltamethrin 0.05% 100 98 98.0 S 100 98 98.0 S Permethrin 0.75% 100 100 100.0 S 100 100 100.0 S Lambda-cyhalothrin 0.05% 100 100 100.0 S 100 96 95.5 V Control 100 0 0.0 - 100 0 0.0 - *S=Susceptible; V=under ’verification required’ status; R=Resistant http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 171–178 A Sofizadeh et al.: Resistance Status of … 174 http://jad.tums.ac.ir Published Online: June 30, 2021 Table 2. Regression analysis of bioassay data of Anopheles maculipennis exposed to DDT 4.0% using WHO- recommended method, northeastern parts of the Caspian Littoral, Iran a b±SE LT50 (min)±95%CL LT90 (min)±95%CL χ2 (heterogeneity) χ2 Table (df) p -7.1132 4.1014±0.337 49.4392 54.2415 59.6185 97.2503 111.3806 132.7855 24.685 * 5.991 (2) 0.0 5 Fig. 1. Map of study area showing Kalaleh District, Golestan Province, northeast of Iran where two main species of Anopheline were collected Fig. 2. Regression parameters estimating the lethal time of Anopheles maculipennis exposed to DDT 4.0%, northeast- ern parts of the Caspian Littoral, Iran http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 171–178 A Sofizadeh et al.: Resistance Status of … 175 http://jad.tums.ac.ir Published Online: June 30, 2021 Discussion Currently, malaria is regarded as an infec- tious disease causes financial losses and work- force health. It is still concerned with health au- thorizes at the Sistan and Baluchistan, Hor- mozgan, and southern Kerman provinces (12). With attention to development achieved dur- ing five decades of vector control programs and the reduction of prevalence, malaria elim- ination is in the joint approach of the Iranian Ministry of Health and the World Health Or- ganization (12). Due to the risk of malaria re- emergence in northeastern parts of the Caspi- an Littoral, which is caused by numerous cli- matic, environmental, and social factors, de- termining of susceptibility level of Anopheles vectors was noticed. The Maculipennis complex comprised 12 Palearctic members that dis- tributed in different provinces including West Azarbaijan, East Azarbaijan, Ardabil, Guilan, Mazandaran, Golestan, Isfahan, Fars, Koh- giluyeh and Boyer-Ahmad, Kermanshah, Kur- distan, Zanjan, Tehran and Khuzistan but the exact distribution of each member of Maculi- pennis complex as well as its bioecology is not clear (3). The Resistance ratio (RR) which calculated by dividing the LC50 of the resistant population by the LC50 of the susceptible strain had been calculated for An. maculipennis in different localities of Iran during 1970–1977, e.g., central parts (Isfahan Province, RR= 64.2 min), the northwestern parts adjusting to bor- derlines of Republic Azarbaijan and Armenia (Ardabil Province, RR= 57.5min, the East Azarbaijan Province, RR= 58.4min), Caspian littoral (Guilan Province, RR= 77.1min; Ma- zandaran Province, RR= 58.1min; Golestan Province, RR= 63.7min) and northeastern parts (Razavi Khorasan, RR= 74.6min) (13-14). Dur- ing the malaria resurgence at the Caspian lit- toral in 2008, it was shown that An. maculi- pennis (strain Astara, Guilan Province, Caspi- an littoral) exhibited low resistance (84.0%) to DDT, whereas susceptible to Malathion, Lamb- da-cyhalothrin, and Deltamethrin during 1998– 1999 (15). A similar study conducted at differ- ent villages of the Mazandaran Province, in the Caspian plateau during 1988–1989 and the sus- ceptibility level of An. maculipennis was de- termined against DDT 4% after 60min of ex- posure time using WHO’s method. The results showed the resistance of An. maculipennis to DDT ranged 72.5–94.4%, which followed 93.9% mortality after 120min exposure. The latter spe- cies was susceptible to Dieldrin 4% and Mala- thion 5.0% but surprisingly showed under ’ver- ification required’ status to Deltamethrin 0.025 % with a mean of mortality of 96.5% (16). Al- so, the susceptibility level of An. maculipennis to DDT 4.0% was also determined in the Gui- lan Province, in the west of the Caspian littoral during 1987 with the mortality rate of 87.5– 91.7%, 90.5–94.3%, and 96.1–97.1% after 120, 150, and 180min exposure time which indicated a high resistance level of An. maculipennis to DDT 4% in Guilan Province (16). Another study conducted on the susceptibility level of Anoph- eles messeae against DDT 4% using the WHO’s method at 60min in Sari, Amol, and Toneka- bon districts, Mazandaran Province, Caspian littoral during 1989–1990. The results also re- vealed a high resistance level of An. messeae to DDT ranged 8.9–61.2%, with a mean of 40.1%. The latter species was reported as sus- ceptible to Dieldrin 4% and Malathion 5.0% (17). During a recent trial in the northwestern part of Iran, it was indicated that An. maculi- pennis (strain West Azarbaijan, the borderline of Turkey) displayed high resistance (50.0%) against Malathion and under ’verification re- quired’ status to Permethrin and Deltamethrin (18). In neighboring countries of Iran, suscep- tibility tests on An. maculipennis were carried out since 1974 in Turkey, revealing resistance to organophosphate insecticides (19). The re- sistance of Anopheles artemievi, one member of the Maculipennis complex, was established to DDT (26.7%) at different parts of Uzbeki- stan. The variations in susceptibility level of http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 171–178 A Sofizadeh et al.: Resistance Status of … 176 http://jad.tums.ac.ir Published Online: June 30, 2021 An. maculipennis were shown related to sea- sonal change and mosquito collection months (20-21). The resistance of five strains of An. maculipennis was confirmed to DDT, Mala- thion, Permethrin, and Deltamethrin in Turkey (20, 22). During this study, it was revealed that An. superpictus still remained susceptible to all tested insecticides from different groups. A similar situation of the susceptibility of An. superpictus was shown in different parts of Iran during 1971–74 (23) and then in Ilam Prov- ince, west of Iran during 2000 (24), whereas a record of resistance (56.0%) of An. superpic- tus was recently recorded in the Sistan and Baluchistan Province, southeastern Iran (25). In the piedmont and mountainous districts of Uzbekistan, An. superpictus was also highly sensitive to the insecticides, while the diapaus- ing female An. superpictus mosquitoes in the population were found to be resistant to DDT (82.8%) and highly resistant to Malathion (43.8%) (26). More than a half-century has passed since the newer investigations revealed the An. superpictus still remained susceptible to DDT, Malathion, and pyrethroids (27). In Tajikistan, An. superpictus was proved to be exophile and completely susceptible to the Mal- athion, but with a low DDT resistance (28). A different pattern of susceptibility was shown a low resistance (85.0%) to the Deltamethrin, but susceptible to the DDT, Malathion, and Per- methrin between the field population of An. superpictus collected from the Badakhshan Province, Afghanistan (29). The adult An. su- perpictus that collected from the Jordan in the Middle East showed a transit susceptibility (96.0%) to the Deltamethrin, whereas complete- ly susceptible to the Lambda-cyhalothrin (30). The study's results and comparison of the past and present data in different countries indicated a serious alert status for pesticide management both in health and agriculture arthropod control. Conclusion Susceptibility level of An. maculipennis to DDT remained with the least change in the eastern part of Caspian littoral despite with- drawal of indoor spraying with DDT since 1971, but under ’verification required’ status to pyrethroids could be considered a threat to the possible development of resistance in the fu- ture. The results of the tests on Malathion, Deltamethrin, Permethrin revealed suscepti- bility to both An. maculipennis and An. super- pictus to these insecticides. Acknowledgments This study was supported by the Research Deputy of Golestan University of Medical Sci- ences (GUMS) under research code 35/678. The authors would like to appreciate the kind col- laboration of GUMS Deputy and the staff of the Health Center of Kalaleh District. The au- thors declare that there is no conflict of interest. References 1. Edrissian GH (2006) Malaria in Iran: Past and present situation. Iranian J Parasitol. 1: 1–14. 2. Jalali M (1956) History of malaria and it's control in Iran to 1955. Institute Malari- ology and Parasitology, Tehran Univer- sity and Ministry of Health. Iran. 225: 60–74. 3. Djadid N, Gholizadeh S, Tafsiri E, Romi R, Gordeev M, Zakeri S (2007) Molecu- lar identification of Palearctic members of Anopheles maculipennis in northern Iran. Malar J. 17: 1–10. 4. Manouchehri AV, Zaim M, Emadi AM (1992) A review of malaria in Iran, 1957–1990. J Am Mosq Control Assoc. 8: 381–385. 5. Salari-Lak SH, Vatandoost H, Entezarmah- di MR, Ashraf H, Abai MR and Nazari M (2002) Monitoring of insecticide re- sistance in Anopheles sacharovi (Favre, 1903) in borderline of Iran, Armenia, Naxcivan and Turkey. Iran J Public Health. 31: 96–99. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 171–178 A Sofizadeh et al.: Resistance Status of … 177 http://jad.tums.ac.ir Published Online: June 30, 2021 6. Vatandoost H, Hanafi-Bojd AA, Raeisi A, Abai MR, Nikpour F (2018) Bioecology of the dominant malaria vector, Anophe- les superpictus s.l. Grassi (Diptera: Cu- licidae) in Iran. J Arthropod Borne Dis. 12(3): 196–118. 7. Oshaghi MA, Shemshad K, Yaghobi-Ershadi MR, Pedram M, Vatandoost H, Abai MR, Akbarzadeh K, Mohtarami F (2007) Ge- netic structure of the malaria vector Anopheles superpictus in Iran using mi- tochondrial cytochrome oxidase (COI and COII) and morphologic markers: a new species complex. Acta Trop. 101: 241–248. 8. Oshaghi MA, Vatandoost H, Gorouhi A, Abai MR, Madjidpour A, Arshi S, Sadeghi H, Nazari M, Mehravaran A (2011) Anophe- line species composition in borderline of Iran-Azerbaijan. Acta Trop. 119(1): 44– 49. 9. Fekri S, Vatandoost H, Daryanavard A, Shahi M, Safari R, Raeisi A, Omar AS, Sharif M, Azizi A, Ali AA, Nasser A, Hasaballah I, Hanafi-Bojd AA (2013) Malaria situation in an endemic area, southeastern Iran. J Arthropod Borne Dis. 8: 82–90. 10. World Health Organization (2016) Test pro- cedures for insecticide resistance moni- toring in malaria vector mosquitoes–2nd ed. WHO Document Production Services, Geneva, Switzerland. 11. World Health Organization (2014) Malaria entomology and vector control, partici- pants’ guide. WHO, Geneva. Available at: http://apps.who.int/iris/handle/10665/85 890. 12. Raeisi A, Gouya MM, Nadim A, Ranjbar M, Hasanzehi A, Fallahnezhad M, Sakeni M, Safari R, Saffari M, Mashyekhi M, Ahmadi Kahnali A, Mirkhani V, Al- masian E, Faraji L, Paktinat Jalali B, Nikpour F (2013) Determination of ma- laria epidemiological status in Iran's ma- larious areas as baseline information for implementation of malaria elimination program in Iran. Iran J Public Health. 42: 26–36. 13. Eshghi N, Zaini A, Yazdanpanah H (1980) Susceptibility of Anopheles maculipennis to insecticides in Iran. Mosq News. 40: 510–513. 14. Manouchehri AV, Zaini A, Mottaghi M (1976) Susceptibility of Anopheles mac- ulipennis to insecticides in northern Iran. Mosq News. 36: 51–55. 15. Vatandoost H, Zahirnia AH (2010) Respon- siveness of Anopheles maculipennis to dif- ferent imagicides during resurgent ma- laria. Asian Pac J Trop Biomed. 360–363. 16. Dinparast Djadid N (1989) Ecology of Anopheles maculipennis at north parts of Iran. Unpublished MSc thesis, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran, p. 243. 17. Momeni S (1990) Bioecology and suscep- tibility level of Anopheles maculipennis complex to the insecticides at Mazanda- ran Province. Unpublished MSc thesis, School of Public Health, Tehran Univer- sity of Medical Sciences, Tehran, Iran, p. 197. 18. Chavshin AR, Dabiri F, Vatandoost H, Mohammadi Bavani M (2015) Suscep- tibility of Anopheles maculipennis to dif- ferent insecticides classes in West Azarbai- jan Province, Northwestern Iran. Asian Pac J Trop Biomed. 5: 403–406. 19. Ramsdale CD, Herath PR, Davidson G (1980) Recent developments of insecti- cide resistance in some Turkish anophe- lines. J Trop Med Hyg. 83: 11–19. 20. Akiner MM, Caglar SS, Simsek FM (2013) Yearly changes of insecticide suscepti- bility and possible insecticide resistance mechanisms of Anopheles maculipennis Meigen (Diptera: Culicidae) in Turkey. Acta Trop. 126: 280–285. 21. Zulueta J, Joliver P (1957) Seasonal varia- tion in susceptibility to DDT of An. maculipennis in Iran. Bull World Health Organ. 16: 475–479. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 171–178 A Sofizadeh et al.: Resistance Status of … 178 http://jad.tums.ac.ir Published Online: June 30, 2021 22. Akıner MM (2014) Malathion and pro- poxur resistance in Turkish populations of the Anopheles maculipennis Meigen (Diptera: Culicidae) and relation to the insensitive acetylcholinesterase. Turkiye Parazitol Derg. 38: 111–115. 23. Eshghi N, Janbakhsh B, Mottaghi M (1977) Susceptibility of Anopheles superpictus to insecticides in Iran. Mosq News. 27: 490–493. 24. Jalilian M, Mussavi Ivanaki A, Aiwazi A, Jalali A (2001) Susceptibility level of Anopheles superpictus to DDT, Malathion and Lambda-cyhalothrin insecticides in Ilam Province. lIam J Med Sci. 9: 25–29. 25. Nejati J, Vatandoost H, Oshaghi MA, Salehi M, Mozafari E, Moosa-Kazemi SH (2013) Some ecological attributes of malarial vector Anopheles superpictus Grassi in endemic foci in southeastern Iran. Asian Pac J Trop Biomed. 12: 1003–1008. 26. Zhakhongirov SHM, Abdullaev IT, Pono- marev IM, Muminov MS (2004) Moni- toring of the insecticidal resistance of main malaria vectors in Uzbekistan. Med Parazitol (Mosk). 7: 29–33. 27. Zhakhongirov SHM, Saifiev SHT, Abidov ZI (2016) Insecticide resistance in major malaria vectors in Uzbekistan. Med Parazitol (Mosk). 12: 31–34. 28. Sorokin NN, Mingaleva GN (1992) A com- parison of the level of resistance and ir- ritability in Anopheles hyrcanus and An. superpictus to insecticides. Med Parazitol (Mosk). 1: 15–17. 29. Ahmad M, Buhler C, Pignatelli P, Ranson H, Nahzat SM, Naseem M, Sabawoon MF, Siddiqi AM, Vink M (2016) Sta- tus of insecticide resistance in high-risk malaria provinces in Afghanistan. Malar J. 5: 98. 30. Khalil A, Kanani KA, Katbeh-Bader A, Al-Abdallat M, Shadfan B (2015) Sus- ceptibility tests on insecticides used to control mosquitoes in Jordan. Jordan J Biol Sci. 8: 180–183. http://jad.tums.ac.ir/