Evaluation of Susceptibility Status of Phlebotomus papatasi, the Main Vector of Zoonotic Cutaneous Leishmaniasis, to Different WHO Recommended Insecticides in an Endemic Focus, Central Iran http://jad.tums.ac.ir Published Online: Dec 31, 2021 Original Article Evaluation of Susceptibility Status of Phlebotomus papatasi, the Main Vector of Zoonotic Cutaneous Leishmaniasis, to Different WHO Recommended Insecticides in an Endemic Focus, Central Iran Zahra Saeidi1, Hassan Vatandoost1,2, Morteza Zaim1, Reza Ahmadkhaniha3, Yavar Rassi1, Alireza Zahraei-Ramazani1, Mohammad Hossein Arandian4, Reza Jafari4, Yamdeu Joseph Hubert Galani5,6, Alireza Sanei-Dehkordi7, *Mohammad Reza Yaghoobi-Ershadi1,* Amir Ahmad Akhavan1,2 1Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2Department of Environmental Chemical Pollutants and Pesticides, Institute for Environmental Research, Tehran University of Medical Sciences, Tehran, Iran 3Department of Human Ecology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 4Esfahan Research Station, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 5School of Food Science and Nutrition, University of Leeds, Leeds, LS2 9JT, UK 6Section of Natural and Applied Sciences, School of Psychology and Life Sciences, Canterbury Christ Church University, Canterbury, CT1 1QU, UK 7Infectious and Tropical Diseases Research Center, Hormozgan Health Institute, Hormozgan University of Medical Sciences, Bandar Abbas, Iran *Corresponding author: Dr Mohammad Reza Yaghoobi-Ershadi, E-mail: yaghoobi.reza@gmail. com, Dr Amir Ahmad Akhavan, E-mail: aaakhavan@tums.ac.ir (Received 29 June 2021; accepted 1 November 2021) Copyright © 2021 The Authors. Published by Tehran University of Medical Sciences.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/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.by- nc/4.0/). Non-commercial uses of the work are permitted, provided the original work is properly cited. Abstract Background: Among neglected zoonotic diseases, leishmaniases caused by Leishmania parasite through infected female sand fly bite, are a group of diseases found in 98 countries and territories representing a critical burden of disease worldwide. Vector management plays a crucial role in reducing the burden of vector-borne diseases by WHO’s global plan. The objective of the current study was to assess the susceptibility status of wild phlebotomine sand flies from Esfahan Prov- ince, central Iran, to the recommended insecticides by WHO. Methods: Sand flies were collected by mouth aspirator in Matin Abad desert Eco-resort and were tested using WHO adult mosquito test kit against Dichlorodiphenyltrichloroethane (DDT) 4%, Deltamethrin 0.05%, Malathion 5% and Propoxur 0.1%. The number of knockdown sand flies were recorded during exposure time in ten minutes interval for DDT and Deltamethrin and they were allowed to recover for 24 hours. Knockdown Time50 (KD50) and KD90 were generated for them using Probit software. They were mounted and identified by valid keys. Results: Among the tested insecticides against female Phlebotomus papatasi, DDT, Deltame- thrin, and Malathion recorded the highest mortality rate of 100%, followed by Propoxur with 92.2% mortality for a one-hour exposure. For DDT, KD50 and KD90 were calculated 21.87 and 42.93 and for Deltamethrin, they were 23.74 and 56.50 minutes respectively. Total sand flies ex- posed with DDT and Deltamethrin shed their leg(s). Conclusion: It is concluded that Ph. papatasi from central Iran is susceptible to DDT, Deltame- thrin, Malathion, and Propoxur. Keywords: Phlebotomus papatasi; Insecticide; Susceptibility; Iran J Arthropod-Borne Dis, Dec 2021, 15(4): 366–379 Z Saeidi et al.: Evaluation of Susceptibility Status... 367 http://jad.tums.ac.ir Published Online: Dec 31, 2021 J Arthropod-Borne Dis, Des 2021, 15(4): 366–379 Z Saeidi et al.: Evaluation of Susceptibility Status... Introduction Leishmaniases caused by parasite (Protozoan) are a group of neglected zoonotic diseases (NZDs) that draw more attention among all the neglected tropical diseases (NTDs). There are over 20 Leishmania species that are transmitted by the female phlebotomine sand flies through infected bite; a total of 98 sand fly species are identified as medically important vectors (1– 3). The most common form of the disease is cutaneous leishmaniasis (CL) that causes on the exposed parts of the body skin lesion/s mostly ulcer/s and long-life scares (2). Although CL is a self-healing form of the disease, it creates permanent scars and serious disability (4). Approximately 95 % of CL cases occur in the Middle East Mediterranean basin in the old world, and central Asia and the Americas in the new world, and 70% of worldwide cases are related to the Eastern Mediterranean region (2). In 2018 it was reported that 85 % of cases occurred in 10 countries including Iran (2). According to the 2018 WHO report, 98 countries and territories are endemic for leishmaniasis (5). More than 200,000 new cases reported in 2018 and the disability adjusted life years (DALYs) were about 260,000 in 2017(6). Also CL is one of the skin NTDs affecting subcutaneous tissue and skin resulting in disfigurement, disability, stigmatization, and other socio-economic problems (7). In Iran, leishmaniasis is endemic in many rural areas of 18 provinces out of 31 (CDC, Ministry of Health and Medical Education, Iran, Unpublished data) in the way that several research groups have worked on different aspects of the disease. In addition, some international courses about the disease and its control were conducted which attracted lots of interest among different countries (8– 21). Phlebotomus papatasi is the first line incriminated vector of zoonotic cutaneous leishmaniasis (ZCL) in Iran. Studies have shown there are 48 species of sand flies, among which 30 species belong to the genus Phlebotomus, and 18 species of the genus Sergentomyia. Four species of the family Cricetidae of rodents are considered as the main reservoir host including Rhombomys opimus, Meriones libycus, Tatera indica, and Meriones hurrianae in different parts of Iran (22). Diseases transmitted by vectors cause a critical burden in the world, especially in tropical and neotropical areas. Several important vector-borne diseases as parts of NTDs or Skin NTDs in public health continue to need to intensify vector control interventions aimed at monitoring and reducing transmission. The WHO has several global plans to combat NTDs for decades by the multi-intervention packages including integrated vector management (IVM) (23–28). Vector control has a vital role to play in reducing the burden of vector- borne diseases. However, vector control also has proven well-known weaknesses, including the development of insecticide resistance in vectors that played a critical role in the breakdown of the eradication, elimination, and even controlling. Today, there is a need to learn how to monitor and manage vector resistance in a better way (23). Control methods include insecticide spray, use of insecticide-treated nets, environmental management, and personal protection (2). Residual spraying for endophilc, exophilic, and peridomestic sand flies is recommended by World Health Organization Pesticides Evaluation Scheme (WHOPES). Various insecticide classes can be used for indoor residual spraying (IRS), such as organochlorines (for example DDT), synthetic Pyrethroids (for instance Deltamethrin and Lambda-cyhalothrin), organophosphates (for example Malathion), and carbamates (for example Propoxur) (29). Although major scientific breakthroughs have been made worldwide during recent decades in the different aspects of leishmaniases diagnosis, prevention, treatment and control, morbidity and mortality of that disease still show a worrying raising trend (29). Vector control with insecticides remains one of the most efficient approach to tackle the disease, and targeting 368 http://jad.tums.ac.ir Published Online: Dec 31, 2021 J Arthropod-Borne Dis, Des 2021, 15(4): 366–379 Z Saeidi et al.: Evaluation of Susceptibility Status... adult insects with insecticide compounds has shown good results on the spread of the disease. However, non-managed application of insecticide as harmful poisons in any way can result in long or short term toxicity. It is therefore critical to assess the susceptibility or resistance of vectors against the current or foreseen used insecticides. Toxicity cannot be defined as a single molecular event, it is a cascade of events that start with “Exposure”. It proceeds through “distribution and metabolism” and ends with “interaction with cellular macromolecules” and expresses with” toxic endpoint” (30). Insecticides susceptibility testing has a long history worldwide. Wood (1962) tested Aedes aegypti against DDT 1% and 2% and Dieldrin 0.1% and found females more tolerant than males, Pener and Wiliamovsky (1987) tested Ph. papatasi, a colony originating from the Jordan Valley, against DDT, Permethrin, and Methoxychlor. They found sand flies susceptible to DDT and Permethrin but tolerant to Methoxychlor, El-Sayed, et al (1989) worked on baseline susceptibility of Ph. papatasi and mechanism of resistance by comparing the process with DDT-resistant and susceptible strains of Culex quinquefasciatus and Anopheles gambiae (31– 33). The susceptibility investigation on sand flies is less explored due to lack of specific protocol, and researchers who studied it had to follow the test procedure of mosquitoes. In this study, the susceptibility of sand flies as the main vector of ZCL in Iran was tested against DDT, Deltamethrin, Malathion, and Propoxur, following the commonly used mosquitos’ protocol, to pave the way towards further studies on sand fly susceptibility with a specific protocol. Materials and Methods Study area This study was conducted in Matinabad Desert Eco-Resort, Matinabad Village, Badroud Rural District, Natanz County (33.753584 N, 51.990596 E), located 60 Km southeastern of Kashan City, Esfahan Province, central Iran (Fig. 1). This area is one of the most important endemic focus of ZCL and one of the most popular touristic desert Eco-resort in Iran which received the peace and environment award of 2015 (34). The average annual rainfall was 15.44 mm, the average temperature was between 11.3 to 21.3 °C, and relative humidity reported Fig. 1. Map of study area, Esfahan Province, Iran Fig. 1. Map of study area, Esfahan Province, Iran 369 http://jad.tums.ac.ir Published Online: Dec 31, 2021 J Arthropod-Borne Dis, Des 2021, 15(4): 366–379 Z Saeidi et al.: Evaluation of Susceptibility Status... between 30.3–50.7% (Meteorological Organization - Esfahan Province). Sand fly collection Sand flies were collected using a filtered mouth aspirator, most of them on a car trap inside Matinabad Desert Eco-Camp before sunset till early in the next morning around Haloxylon bushes and rodent burrows, from July to September 2019. Sand flies were kept in the cage with a wet towel and were transported to the sand fly insectary in Esfahan Health Station. Susceptibility tests were conducted the day after in the laboratory. Sand flies were fed with cotton soaked in 10% sucrose solution, and the insects were kept at 25–28 ºC temperature, 70–90% relative humidity, and 14:10 L:D photoperiod. Insecticides All WHO test- kit tubes and impregnated papers were procured by CDC, Ministry of Health and Medical Education, Iran by the WHO collaborating center in University Sains Malaysia, Penang, Malaysia. The choice of insecticides was based on highly recommended WHO insecticides at least one from each class such as Organochlorine: Dichlorodiphenyltrichloroethane (DDT) 4% (BATCH No: DD 265), Organophosphate: Malathion 5% (BATCH No: MA 234), Carbamate: Propoxur 0.1% (BATCH No: PR 123) and Pyrethroid: Deltamethrin 0.05% (BATCH No: DE 527). Bioassay (susceptibility) tests Since there is no integrated standard protocol for susceptibility testing of sand flies, they were tested using adult susceptibility test procedures of adult mosquitoes based on WHO the latest protocol of 2018. (28) The WHO susceptibility tube test is a kind of “direct response-to-exposure” test. It measures mosquito mortality to a known standard concentration of a given insecticide, either with a discriminating concentration or with intensity concentrations.(28) Control papers were prepared using ‘acetone and silicone oil’-impregnated paper (0.66 ml oil + 1.34 ml acetone) as a control for DDT and Pyrethroid group and ‘acetone and olive oil’- impregnated paper (0.71 ml oil + 1.29 ml acetone) as a control for Organophosphate and Carbamates according to the standard method of World Health Organization Pesticides Evaluation Scheme (WHOPES) Institute of Research for Development (IRD), Montpellier, France. Standard procedure Sand flies were offered a 10 % sucrose solution for water and energy sources and kept in insectary condition, then transferred to the tubes about one hour prior to starting the test. Insecticide impregnated papers inside test tubes kept refrigerated in a plastic bag were put at room temperature about 1 hour prior to the test. All sand flies were exposed to insecticides for one-hour paralleling with control tubes for each replication. At the end of exposure time, all tubes were kept in insectary condition (T: 25–28 °C- RH: 70– 90%) for 24 hours to recover after exposure, with a cotton pad containing 10% sucrose on the top net. Then the mortality of sand flies in both test and control tubes was read and recorded the day after (28). All sand flies that had the ability to fly were considered alive, regardless of leg losing. The number of knocked down sand flies was recorded every 10 minutes for sand flies exposed to DDT and Deltamethrin. If observed mortality in control groups after 24 h recovery time ranged between 5 to 20%, mortality in the test tubes of that group should be corrected using Abbott’s formula (35). If observed mortalities in control groups exceeded 20%, the entire tubes of that group were discarded. For mortality percentage calculation and correction of mortality the following formulas, adopted from WHO (2016) were used (28). Total number of dead sand flies Observed mortality *100 Total sample size = ( ) ( ) % observed mortality % control mortality Corrected mortality *100 100 % control mortality − = − Based on the 2018 WHO test procedure 370 http://jad.tums.ac.ir Published Online: Dec 31, 2021 J Arthropod-Borne Dis, Des 2021, 15(4): 366–379 Z Saeidi et al.: Evaluation of Susceptibility Status... if the mortality recorded equal or more than 98%, the tested group will be categorized as susceptible; if the mortality ranged between 90 to 97% it shows the resistance possibility. When it happens, the test must be repeated. If the second test mortality is less than 98% the resistance is confirmed. If the mortality recorded less than 90%, we are facing a confirmed resistance. Then researchers can determine the intensity of resistance or mechanism of resistance by applying following the protocol (28). Sand flies testing Total number of 1316 unfed female Ph. papatasi sand flies have been tested. Since they were wild, all fed, gravid, semi-gravid females, all males, and other species were excluded at the time of transferring to the test tubes, checking mortality, mounting and also during identification. Susceptibility tests were carried out on six to fifteen replications in several rounds to obtain enough sand flies tested (at least 100 for each insecticide) with relevant enough control tube/s in each group in parallel. Sand fly mounting and identification All sand flies tested after recording the mortality results, transferred to ethanol 70% for mounting and identification. They were mounted in Pouri’s media and mounted sand flies’ species were identified based on valid identification keys (36, 37). Knockdown effect and leg loss The number of knocked down sand flies was counted in the DDT and Deltamethrin test tubes and recorded every ten minutes during the exposure time. Sand flies leg loss was investigated and recorded after 24h recovery in males and females. Data analysis/ Knockdown curve The knock down time regression line was created for DDT and Deltamethrin using Probit software and data analysis was made with 95% confidence interval and the KD50 and KD90 were calculated (Table 1, Fig 3,4) (38) Number of sand flies tested shows in Table 2. Results Knock down Time50 (KD50) and KD90 The number and percent of knock down sand flies are shown in Fig. 2 and 3. The Probit parameters and the KD50 and KD90 with 95% confidence interval were calculated (Table 1). Table 1. The Parameters of Probit regression line of knockdown times for wild-caught sand flies Matinabad desert Eco- resort, Esfahan Province, 2019 Insecticide Name A B ± SE KD50, (LCL-UCL) 95% C.I. KD90, (LCL-UCL) 95% C.I. X2 (df) P value Deltamethrin 0.05% -4.68 3.4 ± 0.326 19.9 46.75 12.93 (4) <0.05 23.74 56.5 27.44 75.43 DDT 4% -5.86 4.38 ± 0.495 17.74 35.81 23.78 (4) <0.05 21.87 42.93 25.69 56.84 A = y-intercept B = the slope of the line; SE = Standard error; KD50, 95 % CI = Time causing 50 % Knockdown and its 95 % confidence interval KDT90, 95 % CI = Time causing 90 % Knockdown and its 95 % confidence interval LCL: Lower Confidence Limit UCL: Upper Confidence Limit X2 = Heterogeneity about the regression line df = degree of freedom P value = Represent heterogeneity in the population of tested Table 1. The Parameters of Probit regression line of knockdown times for wild-caught sand flies Matinabad desert Eco-resort, Esfahan Province, 2019 371 http://jad.tums.ac.ir Published Online: Dec 31, 2021 J Arthropod-Borne Dis, Des 2021, 15(4): 366–379 Z Saeidi et al.: Evaluation of Susceptibility Status... Total sand flies exposed with DDT and Deltamethrin shed their leg(s). Additionally, sand flies exposed to these two insecticides experienced the “knockdown effect” evidently by muscle spasm, involuntary movement/move less (convulsion or erratic movement or paralysis) during the exposure time (39). It was observed that sand flies exposed with DDT had more involuntary movements and then the ones exposed with Deltamethrin who were more moveless. Susceptibility status The susceptibility status of female sand flies is shown in Table 2. The mortality rate of sand flies exposed to Propoxur has shown a possible resistance in the first round of test and according to the most recent test protocol, the test was repeated in 2 more Fig. 2. The number of knockdown sand flies in ten-minute intervals during exposure time with DDT and Deltamethrin. Matinabad desert Eco-resort, Esfahan Province, 2019 0 50 100 150 200 250 0 10 20 30 40 50 60 70 N U M B E R O F K N O C K D O W N D TIME (MIN) Number of knockdown sand flies in ten minute interval DDT 4% Deltamethrin 0.05% Fig. 2. The number of knockdown sand flies in ten-minute intervals during exposure time with DDT and Deltamethrin. Matinabad desert Eco-resort, Esfahan Province, 2019 Fig. 3. Knockdown percent of sand flies exposed to DDT and Deltamethrin for one hour in ten- minute intervals. Matinabad desert Eco-resort, Esfahan Province, 2019 0 10 20 30 40 50 60 70 80 90 100 10 20 30 40 50 60 Pe rc en t o f K no ck do w n (± SD ) Time (Min) Percent of knockdown sand flies in ten minute interval DDT 4% Deltamethrin 0.05% Fig. 3. Knockdown percent of sand flies exposed to DDT and Deltamethrin for one hour in ten-minute intervals. Matinabad desert Eco-resort, Esfahan Province, 2019 372 http://jad.tums.ac.ir Published Online: Dec 31, 2021 J Arthropod-Borne Dis, Des 2021, 15(4): 366–379 Z Saeidi et al.: Evaluation of Susceptibility Status... rounds and replications obtaining enough number sand flies. In the current study, 1248 female Ph. papatasi were exposed to different standard discriminative concentrations of insecticides. One hundred and thirty-four unfed adult Ph. papatasi were exposed to DDT 4% which resulted in 100% mortality, showing that this species is susceptible to DDT insecticide. For Deltamethrin 0.05% and Malathion 5%, 138 and 223 females were tested respectively, and both of them resulted in 100% mortality that was determined as a totally susceptible population. One hundred and five sand flies were tested against Propoxur 0.1% and resulted in 90.4% mortality which was shown as resistance at the first glance. According to the 2018 WHO guideline, the second round of tests was done using 133 females and resulted in 99.2% mortality that was evaluated as susceptible species. While control groups were tested in parallel for each batch accordingly by recorded mortality of 1.1 and 0.9% for acetone/ silicone oil and 1.3% and 1.2% for acetone/ olive oil respectively. Survival curve Regression analysis was performed for Ph. papatasi to estimate KD50 and KD90 Fig. 4. The regression line for DDT and Deltamethrin for sand flies exposed for one-hour. Matinabad desert Eco-resort, Esfahan Province, 2019   3 3.5 4 4.5 5 5.5 6 6.5 7 10 100 PR O B IT TIME (Min) DDT 4% Deltamethrin 0.05% Fig. 4. The regression line for DDT and Deltamethrin for sand flies exposed for one-hour. Matinabad desert Eco- resort, Esfahan Province, 2019 Table 2. Susceptibility status of female Phlebotomus papatasi to different insecticides- Matinabad desert Eco-resort, Esfahan Province, 2019 Insecticide/ Concentration Test Control Result Total No. of unfed females No. of dead Mortality rate (%) Total No. of unfed females No. of dead Mortality rate (%) Susceptibility status DDT 4% 134 134 100 174 2 1.1 Susceptible Deltamethrin 0.05% 138 138 100 109 1 0.9 Susceptible Malathion 5% 223 223 100 75 1 1.3 Susceptible Propoxur 0.1% 1st round 105 95 90.47 75 1 1.3 Propoxur 0.1% 2nd round 133 132 99.24 82 1 1.2 Susceptible Table 2. Susceptibility status of female Phlebotomus papatasi to different insecticides- Matinabad desert Eco- resort, Esfahan Province, 2019 373 http://jad.tums.ac.ir Published Online: Dec 31, 2021 J Arthropod-Borne Dis, Des 2021, 15(4): 366–379 Z Saeidi et al.: Evaluation of Susceptibility Status... for DDT and Deltamethrin with a 95% confidence interval. A knockdown time regression line was created for them showed in Fig. 4. Discussion The objective of the current research was to investigate the susceptibility/resistance of Ph. papatasi to certain insecticides from various chemical classes using the WHO test kit. World Health Organization bioassay susceptibility test kit is a direct response- to-exposure test that is vital in insecticide resistance management worldwide (24). The discovery of DDT in 1939 was one of the most meaningful developments in the history of pest control. Deltamethrin also was the most active insecticide ever known at the time of its discovery. Continuous use of many insecticides is a potential threat in the field of emerging resistance in insects (40). Wild-caught Ph. papatasi in the current study was found to be susceptible to Organochlorine (DDT 4%), Organophosphate (Malathion 5%), Pyrethroid (Deltamethrin 0.05%), and Carbamate (Propoxur 0.1%). There are studies conducted worldwide on baseline susceptibility of various species of sand flies in different countries. Old world In India Phlebotomus argentipes reported resistant to DDT in 1992 and in different parts of Bihar they found developing resistance to DDT 4% in 2001. (41, 42) In the latter study they do their research on a different species from our study. In North Africa and the Middle East researchers worked on Bendiocarb, Cyfluthrin, DDT, Malathion, Permethrin, and Resmethrin on Phlebotomus bergeroti, Phlebotomus langeroni, Ph. papatasi and Phlebotomus sergenti in 2001. They worked on four different species from ours and various insecticide and reported 3 least toxic insecticides in order of toxicity Permethrin, Malathion and DDT, with DDT being the least toxic. It has been stated that the response to three other insecticides: Bendiocarb, Cyfluthrin and Resmethrin has not been as uniform among species (43). In Italy (2002) Phlebotomus perniciosus and Ph. papatasi were susceptible to DDT 2%, Lambda-cyhalothrin 0.06% and Permethrin 0.2% (44). It agree with our study while the percentage of DDT is less than current experiment. In some parts of India and Nepal border Ph. argentipes in 2010–2012 reported resistant to DDT 4% and susceptible to Deltamethrin 0.05% and Malathion 5%. They conducted the study on different species and their result about DDT was not same as ours. It is explained that the use of DDT in IRS measures for VL control could effect on sand flies susceptibility (45, 46). Also, in 2012 Ph. papatasi and Ph. sergenti were susceptible to DDT and Lambda-cyhalothrin in Morroco, in parallel with our report (47). In some area of Sudan populations of Ph. papatasi was susceptible to DDT, Permethrin, Malathion, and Propoxur that is agree to our research and in some area sensitive to DDT and Permethrin but highly resistant to Malathion and Propoxur in 2012 which is probably due to anti-malaria activities during last 50 years in the area and it is on the contrary to our experiments (48). In 2015 Ph. argentipes in West Bengal, India reported developing resistance to DDT (49). In Kerala Ph. argentipes, Ph. sintoni, Sergentomyia bagdhadis, Se. zeylonica and Se. babu were susceptible to DDT and Deltamethrin (50). Phlebotomus argentipes in 2016 reported resistance in Kala-azar endemic region and susceptible to DDT in the non-endemic region in India (51). All of these last-mentioned studies have been done by different sand flies species from ours. In two different Western provinces of Turkey with and without a background of insecticides use, populations of sand flies found resistant and susceptible to Deltamethrin 0.05% and Permethrin 0.75% respectively (2017) as a result of long term application of both insecticides in the region (52). They did not mention the species of sand flies tested. In 13 villages of Bihar Ph. argentipes as a different species from our region was highly susceptible to Deltamethrin, 374 http://jad.tums.ac.ir Published Online: Dec 31, 2021 J Arthropod-Borne Dis, Des 2021, 15(4): 366–379 Z Saeidi et al.: Evaluation of Susceptibility Status... Lambda-cyhalothrin, Alpha-cypermethrin (2016) (53). In Nepal and Bangladesh Ph. argentipes was highly susceptible to Alpha- cypermethrin 0.05%, Deltamethrin 0.05%, Lambda-cyhalothrin 0.05%, Permethrin 0.75%, Malathion 5% and Bendiocarb 0.1% in 60 min of exposure (2017) (54). In the last two studies they tested Ph. argentipes that this species is a vector on that area but not in Iran. Also in Iran, there are studies on the susceptibility status of sand flies. During 1985–88 Seyedi Rashti et al experimented on various areas of Iran with the treatment background with DDT which discontinued from 1969. They expressed that sand flies from Esfahan showed more tolerance against DDT in comparison to other areas. (8) But our experiments show different condition in this area now. Yaghoobi Ershadi and Javadian found Ph. papatasi tolerant to DDT 4% in Borkhar County in Esfahan Province due to DDT or related compound application in public health or in agricultural pest control which is in contrary to our results, but susceptible to Dieldrin 4% and in Varzane they were susceptible to DDT 4% similar to our research results (9, 10). It is reported that Ph. papatasi and Ph. sergenti was susceptible to DDT 4% in Kerman province. (11). It agree with our result about Ph. papatasi. In 1998 a study showed that Ph. kandelakii and Ph. perfiliewi as a probable vector of zoonotic visceral leishmaniasis (ZVL) were susceptible to DDT 4% during 1994 in Ardabil province, Northwest of Iran (12). These species are in different area where it is a ZVL foci with different vectors from ZCL. In Arsanjan County of Fars Province, Ph. papatasi recorded sensitive to DDT 4% in 1999 same as current report (13). It is showed that Ph. sergenti was susceptible to DDT 4% in Esfahan city in 2005 it is a study on a different species in same province (14). In 2004 and 2005 in Bam City, Kerman Province Ph. papatasi and Ph. sergenti were susceptible to DDT 4% and Deltamethrin 0.05% similar to this reports (15). Wild-caught Ph. papatasi in Badrood, Esfahan Province and their progeny were found susceptible to Permethrin 0.75%, Deltamethrin 0.1%, Cyfluthrin 0.15% and Lambda-cyhalothrin 0.05% and to DDT 4% During summer 2010 (16, 17). Our research also confirm the susceptibility of them in this area to DDT and Deltamethrin. Another study in the same place during summer 2015 showed that there is susceptibility to Cyfluthrin 0.15%, Lambda-cyhalothrin 0.05%, Permethrin 0.75%, and Deltamethrin 0.05% same as our report and tolerant to DDT 4% unlike to our study (18). A study in North Khorasan showed the development of resistance against DDT (4%) in the wild strain of Ph. sergenti but susceptible to Bendiocarb 0.1% and Permethrin 0.75% (19). This report is about another species with various insecticide differ from our experiment. During 2016 and 17 Laboratory reared of Ph. papatasi were found susceptible to Permethrin 0.75%, Deltamethrin 0.05%, Cyfluthrin 0.15%, and Lambda-cyhalothrin 0.05% but resistant candidate to DDT 4% (20). This study reported likely result about Deltamethrin and unlike result from current research about DDT. New world In 1997 a comprehensive study carried out on field population of Lutzomyia longipalpis of Venezuela against DDT 2%, Propoxur 0.01 %, Malathion 2%, Fenitrothion l%, Pirimiphos methyl l%, Deltamethrin 0.06%, Lambda-cyhalothrin 0.06%, and permethrin 0.2% insecticides and compered with laboratory population of reference strain and reported highly susceptible (55). The species used in this experiment is different from ours because in new world Lu. longipalpis has medical importance as a vector but there is no in the old world and the concentration of Deltamethrin, Malathion and DDT used in their study are not same as concentration used in current study. In 2009 researchers reported two wild populations of Lu. longipalpis with different exposure backgrounds susceptible to Malathion, Fenitrothion, Lambda- cyhalothrin, Permethrin, and Deltamethrin in Brazil (56). In 2015 another study in Brazil reported Lu. longipalpis highly 375 http://jad.tums.ac.ir Published Online: Dec 31, 2021 J Arthropod-Borne Dis, Des 2021, 15(4): 366–379 Z Saeidi et al.: Evaluation of Susceptibility Status... susceptible to Alpha-cypermethrin (57). Also Brazil located in new world and the vector is Lu. Longipalpis and the only common insecticide was Deltamethrin. In the United State, some tests performed on laboratory populations of Ph. papatasi and Lu. longipalpis using CDC bottle bioassay against different concentrations of Cypermethrin, Deltamethrin, Lambda- cyhalothrin and Permethrin, Chlorpyrifos, Fenitrothion, and Malathion, Bendiocarb, Propoxur and DDT and they documented as susceptible population (39). Same species and same insecticide tested by different methods of CDC bottle bioassay but reported the same result. In Colombia in a study with the same method on Lu. longipalpis, Lambda- cyhalothrin showed the highest degree of toxicity followed by Alpha-cypermethrin and Deltamethrin (58). There is another study in Brazil using a modified method of WHO comparing laboratory population of Lu. longipalpis with some population in the field with different exposure background and reported that Lab-reared sand flies were more tolerant to field-collected ones against Lambda-cyhalothrin (0.05%), Deltamethrin (0.5%) and control was (Silicone oil) (59). The sand fly species is different and also the concentration of Deltamethrin is not the same. It can be observed that the only resistant Phlebotomus registered in The Arthropod Pesticide Resistance Database is Ph. argentipes in 23 locations of Bihar state in India (60– 62). It is reported as resistance to DDT in VL endemic area of Bihar and also developed resistance/ tolerant to Malathion in a larger area but susceptible to Deltamethrin and the wild-caught and their seven offspring’s is reported resistant to DDT (60, 61). They also experimented another species in different location and the result also is unlike to current research. In the current study, it was found that sand flies from Esfahan Province, were highly susceptible to Deltamethrin and DDT and it was also noted that during the exposure time and counting the knockdown numbers of sand flies, those who exposed with DDT had more involuntary movement in their place but the vast majority of those who exposed to Deltamethrin was moveless. Pyrethroids as a major class of neurotoxic insecticide and DDT, fairly slow-acting on the protein of voltage-gated sodium channels in the cell membrane of the insect nerves. Exposing insects to DDT and Deltamethrin disrupts the normal process leading to paralysis and finally death. Peripheral nervous system influenced by DDT causing tremors in appendages or entire body called “DDT Jitters” then leads to excitatory paralysis and eventually death. Deltamethrin affects both the central and peripheral nervous systems by producing repetitive discharge and cause paralysis the same as DDT but more obvious. After exposure with Deltamethrin, the channels remain open and leads to abnormal hyperexcitability but “Knockdown” is its sub-lethal effect (40). Sand flies in response to exposure to DDT and Deltamethrin manifested evident leg shedding in the current study. The same observation was made by Denlinger and Alexander (39, 56). Sand flies with shedding legs, as a significant sub-lethal effect, will not be able to transmit the parasite as a consequence of disabling for blood-feeding (56). On the other hand, the authors reported that sand flies after shedding legs could still be capable of blood feed (39). We did not check the ability to have blood meals for leg shedded sand flies because the mortality rate was high, they were wild-caught, and we needed to identify them after keeping in alcohol and mounting. Nevertheless, this will be considered in further studies. Conclusion This study revealed that Ph. papatasi from central Iran is susceptible to DDT, Deltamethrin, Malathion, and Propoxur. Knowing about the susceptibility/ resistance of sand flies in this endemic area can play a vital role in the field of vector control and pesticide management. Excessive use of insecticide with unsuitable concentration can cause resistance in vector sand flies and complicate 376 http://jad.tums.ac.ir Published Online: Dec 31, 2021 J Arthropod-Borne Dis, Des 2021, 15(4): 366–379 Z Saeidi et al.: Evaluation of Susceptibility Status... disease control. This result brings additional data to the worldwide need to assess the insecticide susceptibility status of sand flies, in order to strengthen vector surveillance and integrated vector management. We strongly recommend performing susceptibility tests on sand flies in various parts of the world as systematic monitoring and evaluating the status of leishmaniasis vectors against various insecticides, as regular or periodic susceptibility tests can ring a timely alert regarding early resistance. Also doing some further tests on the resistant ones is recommended to determine the resistance intensity and mechanism according to standard protocols of WHO. Acknowledgements The authors would like to appreciate Dr Nikpoor at TUMS for procurements of the susceptibility test kit, and standard impregnated filter papers. We also appreciate Professor Corbel and his team at IRD, Montpellier, France for their kind support and for generous sharing and training the Impregnation methods and principles. Many thanks to the personnel and manager of Matinabad Desert Eco-resort particularly Mr SH Vaghefi for their precious cooperation and kind support. 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Rama A, Kumar V, Kesari S, Singh V, Das P (2015) Monitoring Susceptibility Status of Phlebotomus argentipes (Diptera: Psychodidae) at Bihar (India) for the Procurement of Homozygous DDT Resistant Colony. J Trop Dis. 3: 4. https://www.pesticideresistance.org/display.php?page=species&arId=676 https://www.pesticideresistance.org/display.php?page=species&arId=676 Evaluation of Susceptibility Status of Phlebotomus papatasi, the Main Vector of Zoonotic Cutaneous L Abstract Keywords Introduction Materials and Methods Study area Sand fly collection Insecticides Bioassay (susceptibility) tests Standard procedure Sand flies testing Sand fly mounting and identification Knockdown effect and leg loss Data analysis/ Knockdown curve Results Knock down Time50 (KD50) and KD90 Susceptibility status Survival curve Discussion Old world New world Conclusion Acknowledgements Ethical considerations Conflict of interest statement References