J Arthropod-Borne Dis, September 2020, 14(3): 277–285 Y Rassi et al.: Insecticide Susceptibility Status of … 277 http://jad.tums.ac.ir Published Online: September 30, 2020 Original Article Insecticide Susceptibility Status of Wild Population of Phlebotomus kandelakii and Phlebotomus perfiliewi transcaucasicus Collected from Visceral Leishmaniasis Endemic Foci in Northwestern Iran Yavar Rassi1; *Eslam Moradi-Asl2,3; *Hassan Vatandoost1,4; Malek Abazari2, Abedin Saghafipour5 1Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2Department of Public Health, School of Public Health, Ardabil University of Medical Sciences, Ardabil, Iran 3Arthropod Borne Diseases Research Center, Ardabil University of Medical Sciences, Ardabil, Iran 4Department of Chemical Pollutants and Pesticides, Institute for Environmental Research, Tehran University of Medical Sciences, Tehran, Iran 5Department of Public Health, School of Public Health, Qom University of Medical Sciences, Qom, Iran *Corresponding authors: Dr Eslam Moradi-Asl, E-mail: moradiasl83@yahoo.com, Prof Hassan Vatandoost, E- mail: hvatandoost1@yahoo.com (Received 08 Apr 2020; accepted 28 Sep 2020) Abstract Background: Phlebotomus kandelakii and Phlebotomus perfiliewi transcaucasicus sand flies are the vectors of visceral leishmaniasis in Iran. The aim of this study was to evaluate the susceptibility of Ph. kandelakii and Ph. perfiliewi trans- caucasicus, collected from an endemic focus of visceral leishmaniasis (VL) in the northwest of Iran, to different select- ed insecticides. Methods: Sand flies were collected from the villages of Meshkinshahr and Germi Counties using light traps and aspira- tors from May to October 2019. The sand flies were identified as Ph. kandelakii and Ph. perfiliewi transcaucasicus using standard identification keys. Susceptibility test was carried out against DDT (4%), Malathion (5%), Propoxur (0.1%) and Lambda-cyhalothrin (0.05%) according to the WHO standard method. MedCalc statistical software was employed to calculate LT50 and LT90 and to compare the chances of sand flies surviving the exposure to the studied insecticides. Results: A total of 1,278 female specimens were used for the susceptibility tests. Out of which 1,063 samples were used for the test and 215 for the control group. The estimated LT50 values for DDT (4%), Malathion (5%), Propoxur (0.1%), and Lambda-cyhalothrin (0.05%) for Ph. kandelakii were 15.1, 13.4, 15.4 and 5.8 minutes respectively, and for Ph. per- filiewi transcaucasicus were 11.9, 15.6, 15.9 and 5.8 minutes respectively. Conclusion: This susceptibility studies revealed different LT50 values for different insecticides efficient against Ph. kandelakii and Ph. perfiliewi transcaucasicus. The regular monitoring for the resistance of Ph. kandelakii and Ph. per- filiewi transcaucasicus sand flies seems necessary in diseases control programs in this area. Keywords: Phlebotomus kandelakii; Phlebotomus perfiliewi transcaucasicus; Insecticide resistance; Insecticide sus- ceptibility; Lambda-cyhalothrin Introduction Leishmaniasis is transmitted to human in 98 countries and 3 territories on 5 continents in the world and approximately 0.2 to 0.4 cas- es of Visceral leishmaniasis (VL) and 0.7 to 1.2 million cases of cutaneous leishmaniasis (CL) occur each year. Leishmaniasis is a par- asitic disease found in tropical and subtropical areas around the world. It has been categorized as a neglected tropical disease (NTD). Leish- maniasis is triggered by infection with Leish- mania parasites, which spread through the bite of phlebotomine sand flies of Phlebotomus and Lutzomyia. There are several distinct types of leishmaniasis in humans. The most prevalent Copyright © 2020 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:moradiasl83@yahoo.com https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, September 2020, 14(3): 277–285 Y Rassi et al.: Insecticide Susceptibility Status of … 278 http://jad.tums.ac.ir Published Online: September 30, 2020 types are CL (causing skin sores) and VL (caus- ing impacts on several internal organs) (1). VL, also known as kala-azar, is a severe health problem in endemic areas. The disease is of great significance to the health care system around the globe and can be fatal without ad- equate therapy (2). The primary causative agent of VL is Leishmania infantum in Mediterra- nean areas such as Iran (3). In the Mediterra- nean region, there are two major forms of severe and fatal visceral leishmaniasis and cutaneous leishmaniasis. The worldwide mortality rate for leishmaniasis is between 26,000 and 65,000 cases per year (4). VL is revealed to be en- demic in 7 areas in Iran and the most significant endemic foci are Ardabil Province in north- western Iran (5). Untreated VL has a 100% mor- tality rate (6). Of 44 confirmed species of sand flies in Iran, three of them have been identi- fied as the main or probable vectors of VL in Ardabil Province (7-10). To date, 22 Phleboto- mus species have been recognized in Ardabil Province, and two major vector species, Ph. kandelakii and Ph. perfiliewi transcaucasicus, have been recorded from various regions with a large population (11). Leishmania infection of the two listed species with L. infantum and Leishmania donovani from the north and cen- ter of the province has been reported using par- asitological and molecular techniques (12). At present, the chemical control of sand flies, in- cluding insecticide residual spraying (IRS) or the use of insecticide-impregnated bed nets (ITNs), are effective means to control visceral leishmaniasis (13). In order to use insecticides against leishmaniasis, the WHO recommends the use of a safety insecticide previously test- ed for vectors’ susceptibility (14-20). Accord- ingly, this is the first study to test the sensi- tivity of two major vectors of visceral leish- maniasis in an endemic foci in northwestern Iran to four types of insecticides based on the WHO guidelines. Materials and Methods Study sites This research was performed in Ardabil Province located in northwestern Iran from May to October 2019. The geographic coor- dinates of the province are 38.4853 °N, 47.8911 °E. The province is 17,953km2 in area (about 1.09 percent of the country's total re- gion), 1,338 meters above sea level and has a population of 1,249,000 people. Sandflies collection A total of 1,278 sand flies were collected for testing and control purposes. Samples were gathered from the villages of Meshkinshahr and Germi Districts using light traps and as- pirators from human homes, dog nests and livestock stables. Sand flies were immediately transferred to the Arthropod-Borne Research Center in Ardabil University of Medical Sci- ences. The specimens were kept in a labora- tory in a maintenance cage under 25±2 °C and 60±10% humidity. The specimens were fed with sugar-soaked cotton during storage. They were recognized by using valid keys (21-23). Procurement of insecticide impregnated pa- pers and their concentration Impregnated papers with DDT (4%) (Batch No. DD186, Exp. date: July 2016); Malathion (5%) (Batch No. MA 177, Exp. date: April 2018); Propoxur (0.1%) (Batch No. PR 104, Exp. date: April 2018), and Lambda-cyhalo- thrin (0.05%) (Batch No. LA 262, Exp. date: July 2018), as well as papers for control were supplied by the collaborating center of the World Health Organization in Penang, Malaysia. Susceptibility tests Female sandflies were subjected to suscep- tibility tests according to the guidelines of WHO. Sandflies were exposed to impregnated papers (DDT (4%) (Organochlorine), Malathi- on (5%) (Organophosphate), Propoxur (0.1%) (Carbamate) and Lambda-cyhalothrin (0.05%) (Pyrethroid) at different logarithmic times. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2020, 14(3): 277–285 Y Rassi et al.: Insecticide Susceptibility Status of … 279 http://jad.tums.ac.ir Published Online: September 30, 2020 The sample exposure period was 3.75, 7.5, 15, 30 and 60 minutes. Samples were recov- ered at a suitable temperature for 24 hours under laboratory circumstances and supplied with sugar water. The mortality rate was rec- orded after 24 hours. Dead and live samples were gathered and stored in 70% alcohol and then mounted in a drop of Puri's medium and recognized using valid morphological keys. Four replicates were performed for each ex- periment. Data analysis Data collected in Microsoft Excel 2016 were used to plot slopes and graphs. The MedCalc statistical software was used to calculate LT50 and LT90 and to compare the chances of sand flies surviving the exposure to insecticides in the study areas. Results During the study period, 1,278 sand fly specimens were used for sensitivity test on two main proven vectors species of VL in the Ardabil Province of which 1,063 were for the test and 215 for the control group. Seven hun- dred and forty-nine specimens of Ph. kan- delakii and 529 specimens of Ph. perfiliewi transcaucasicus were evaluated respectively. All the samples of the above-mentioned spe- cies were found fully susceptible to Lambda- cyhalothrin (0.05%) insecticides with 100% mortality. The estimated LT50 values for DDT (4%), Malathion (5%), Propoxur (0.1%), and Lambda-cyhalothrin (0.05%) for Ph. kan- delakii were 15.1, 13.4, 15.4 and 5.8 minutes (Table 1), respectively, and for Ph. perfiliewi transcaucasicus were 11.9, 15.6, 15.9 and 5.8 minutes respectively (Table 2). The mortality rates for Ph. kandelakii after 60 minutes of exposure to DDT (4%), Mala- thion (5%), Propoxur (0.1%) and Lambda- cyhalothrin (0.05%) were 91.17%, 93.18%, 97.61% and 100%, respectively. The mortality rates for Ph. perfiliewi transcaucasicus were 95.83%, 90.90%, 91.30% and 100%, respective- ly (Table 3). The slope diagrams and the trend of the mortality rates are shown in Fig. 2. Fig. 1. The study area and sand fly collection sites in the northwest of Iran http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2020, 14(3): 277–285 Y Rassi et al.: Insecticide Susceptibility Status of … 280 http://jad.tums.ac.ir Published Online: September 30, 2020 Table 1. Lethal Time, LT50 and LT90 values for Phlebotomus kandelakii to DDT (4%), Malathion (5%), Lambda- cyhalothrin (0.05%) and Propoxur (0.1%), in Ardabil Province, Iran, 2019 Insecticide a b±SE LT50±95% CI (minutes) LT90±95% CI (minutes) Wald statistic p-value DDT (4%) -3.063 2.596±0.367 11.884 34.783 49.855 <0.0001 15.123 47.113 18.943 76.134 Malathion (5%) -3.144 2.789±0.338 10.911 29.906 68.032 <0.0001 13.397 38.582 16.235 56.021 Lambda-cyhalothrin (0.05%) -2.501 3.278±0551 4.084 10.012 35.296 <0.0001 5.794 14.253 8.221 20.292 Propoxur (0.1%) -3.060 2.579±0326 12.375 36.518 62.416 <0.0001 15.351 48.183 18.811 72.833 Fig. 2. Comparison of regression lines, equations and LT50 of Phlebotomus kandelakii and Ph. perfiliewi transcau- casicus as exposed to DDT (4%), Malathion (5%), Propoxur (0.1%), and Lambda-cyhalothrin (0.05%) http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2020, 14(3): 277–285 Y Rassi et al.: Insecticide Susceptibility Status of … 281 http://jad.tums.ac.ir Published Online: September 30, 2020 Table 2. Lethal Time , LT50 and LT90 values for Phlebotomus perfiliewi transcaucasicus to DDT (4%), Malathion (5%), Lambda-cyhalothrin (0.05%) and Propoxur (0.1%), in Ardabil Province, Iran, 2019 Insecticide a b±SE LT50±95% CI (minutes) LT90±95% CI (minutes) Wald statistic p-value DDT (4%) -2.621 2.436±0.402 8.703 27.897 36.682 <0.0001 11.901 39.949 15.725 73.761 Malathion (5%) -3.207 2.690±0.431 11.757 32.870 38.864 <0.0001 15.565 46.619 20.495 84.098 Lambda-cyhalothrin (0.05%) -3.430 4.506±0.908 4.467 8.821 24.606 <0.0001 5.772 11.111 7.059 17.158 Propoxur (0.1%) -3.089 2.568±0.404 12.221 35.565 40.336 <0.0001 15.944 50.291 20.679 90.236 Table 3. Susceptibility tests using the WHO bioassays for Phlebotomus kandelakii and Ph. perfiliewi transcaucasicus in endemic foci of visceral leishmaniasis in the northwest of Iran, 2019 Insecticide Exposer time (min) Ph. kandelakii Ph. perfiliewi transcaucasicus No. of sandflies exposed No. of sand- flies dead Mortality rate (%) No. of sand- flies exposed No. of sand- flies dead Mortality rate (%) DDT (4%) 3.75 20 1 5 18 3 16.67 7.5 26 5 19.23 21 5 23.80 15 31 16 51.61 19 11 57.89 30 29 24 82.75 22 19 86.36 60 34 31 91.17 24 23 95.83 Malathion (5%) 3.75 29 2 6.89 21 1 4.76 7.5 32 4 12.5 17 3 17.64 15 38 26 68.42 19 9 47.36 30 35 30 85.71 20 17 85 60 44 41 93.18 22 20 90.90 Lambda- cyhalothrin (0.05%) 3.75 24 3 12.5 18 2 11.11 7.5 26 21 80.76 21 18 85.71 15 31 30 96.77 25 23 92 30 28 26 92.85 19 19 100 60 38 38 100 26 26 100 Propoxur (0.1%) 3.75 26 2 7.69 18 1 5.56 7.5 34 8 23.52 24 4 16.67 15 32 14 43.75 21 11 52.38 30 40 29 72.50 26 20 76.92 60 42 41 97.61 23 21 91.30 Control - 110 2 1.81 105 3 2.85 Discussion Since Ardabil Province, along with East Azerbaijan, is known as the primary focal point of VL in Iran (24) and 100–300 new cases of the disease are recorded annually (5), most of which are from the northwest (25), care must be taken against any sudden epidemics. One http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2020, 14(3): 277–285 Y Rassi et al.: Insecticide Susceptibility Status of … 282 http://jad.tums.ac.ir Published Online: September 30, 2020 way to control the epidemic is to use suitable and safe insecticides to kill sand flies (22). Sev- eral trials have been performed in separate ar- eas of Iran on vectors of cutaneous leishman- iasis, including Ph. papatasi and Ph. sergenti, and it has been verified that Ph. papatasi is susceptible to the assessed insecticides (26- 29), except tolerance to DDT in Isfahan Prov- ince, central Iran (30). Furthermore for the first time, resistance against DDT was observed in wild strain of P. sergenti population in North Khorasan Prov- ince, Iran (14, 27). According to the results of current study, Lambda-cyhalothrin 0.05% with 100% lethality had the greatest impact on Ph. kandelakii and Ph. perfiliewi transcaucasicus in Ardabil region. This revealed that these vec- tors are extremely susceptible to this insecti- cide and can be used to regulate vectors at the period of the epidemic. The results of the Lambda-cyhalothrin (0.05%) insecticide in Bra- zil against lutzomyia longipalpis, the main vec- tor of VL in the Americas, showed that this vector was also highly sensitive to insecti- cides (31). The Ph. kandelakii and Ph. perfiliewi trans- caucasicus vectors’ resistance to insecticides has not been recorded to date in Iran. In 1994, susceptibility tests were conducted on Ph. kan- delakii and P. perfiliewi transcaucasicus, in Ar- dabil Province (northwest), Iran. The results revealed that the both species were suscepti- ble to the insecticides and mortality rate with 60min exposure to 4% DDT was 100%. In In- dia, in numerous areas, Ph. argentipes showed resistance to DDT (32-34), but was suscepti- ble to malathion and deltamethrin with 98% and 100% mortality, respectively (20). The re- sistance of the main vectors of VL against DDT (4%) and deltamethrin (0.05%) was re- ported in borderline of Nepal (17). The reports on insecticide susceptibility status of Ph. kandelakii and Ph. perfiliewi trans- caucasicus are limited and there are no records of insecticide resistance till now. According to results of current study and the WHO guide- lines for malaria vectors at the diagnostic dose Ph. kandelakii and Ph. perfiliewi transcaucasi- cus, proven vectors of visceral leishmaniasis in northwestern Iran were possible resistance to Propoxur (0.1%), Malathion (5%) and DDT (4%) insecticides. So the results of earlier stud- ies were contradictory to this study. Phleboto- mus kandelakii distributed in some important VL foci of Iran and leishmania infection re- ported previously so further studies should be conducted in other regions (8). The strength of this study was that we con- ducted insecticide susceptibility status of two main vectors of VL in an endemic area of Iran with four different kinds of insecticides and in five different logarithmic times but because of limitation, the enough amount of sand flies col- lection was not possible. Also there are spe- cific guidelines approved by WHO for diag- nostic dose of mosquitoes, and there are no such guidelines for phlebotominae sandflies; however, it is of great value to establish a base- line data and to assess the insecticide suscep- tibility to different insecticides in sand fly vec- tors. There are several novel investigations of main vector borne diseases in the country (35- 38). Evaluation of diseases monitoring is a vi- tal responsibility of corresponding ministries. Conclusion The findings of this study showed that ac- cording to the criteria for resistant level of Ph. kandelakii and Ph. perfiliewi transcaucasicus, the proven vectors of VL in northwestern Iran were susceptible to Lambda-cyhalothrin (0.05 %), however these species are possible re- sistance to Propoxur (0.1%), Malathion (5%) and DDT (4%) insecticides. Regular monitor- ing for the resistance of Ph. kandelakii and Ph. perfiliewi transcaucasicus sand fly popu- lations are necessary for VL control programs in this area. Furthermore, these insecticides should also be assessed for Ph. papatasi and Ph. sergenti, as the main vectors of CL in en- demic foci in Iran. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2020, 14(3): 277–285 Y Rassi et al.: Insecticide Susceptibility Status of … 283 http://jad.tums.ac.ir Published Online: September 30, 2020 Acknowledgements This study was financially supported by the Ardabil University of Medical Sciences (Pro- ject No. 41147) and the Department of En- vironmental Chemical Pollutants and Pesti- cides, National Institute for Environmental Re- search, School of Public Health, and Tehran University of Medical Sciences. The study was approved by the Ethical Committee of Ardabil University of Medical Sciences, Iran (Code of ethics: IR. ARUMS. REC.1397.268). The au- thors declare that there is no conflict of interest. References 1. Torres-Guerrero E, Quintanilla-Cedillo MR, Ruiz-Esmenjaud J, Arenas R (2017) Leish- maniasis: a review. F1000Res. 6: 750. 2. 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