J Arthropod-Borne Dis, December 2020, 14(4): 408–415 Y Rassi et al.: Efficiency of Two … 408 http://jad.tums.ac.ir Published Online: December 31, 2020 Original Article Efficiency of Two Capture Methods Providing Live Sand Flies and Assessment the Susceptibility Status of Phlebotomus papatasi (Diptera: Psychodidae) in the Foci of Cutaneous Leishmaniasis, Lorestan Province, Western Iran Yavar Rassi1; Hamed Asadollahi1; *Mohammad Reza Abai1,2; Mohammad Hassan Kayedi3; *Hassan Vatandoost1,2 1Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2Department of Chemical Pollutants and Pesticides, Institute for Environmental Research, Tehran University of Medical Sciences, Tehran, Iran 3Faculty of Medicine, Department of Parasitology, Lorestan University of Medical Sciences, Khorramabad, Iran *Corresponding authors: Mr Mohammad Reza Abai, E-mail: abaimr@tums.ac.ir, Prof Hassan Vatandoost, E-mail: hvatandoost1@yahoo.com (Received 24 June 2020; accepted 22 Dec 2020) Abstract Background: The aims of this study were to evaluate the efficiency of two capture methods for providing live sandflies used for determining the susceptibility level of Phlebotomus papatasi, the main vector of zoonotic cutaneous leishmani- asis in Lorestan Province, west of Iran. Methods: The sand flies were collected from indoor and outdoor by hand-catch and baited traps during the peak of sea- sonal activity. The susceptibility level of sand flies was assessed using insecticide-impregnated papers against DDT 4%, bendiocarb 0.1%, permethrin 0.75%, deltamethrin 0.05%, and cyfluthrin 0.15%. Results: A total of 2486 live sandflies were caught from both indoor and outdoor places. Totally 849 sand flies were caught from outdoors with a sex ratio(SR) 0.1 versus 1637 sand flies collected from indoor using the hand-catch method with SR= 0.6. The dominant species of sand flies was Ph. papatasi in the study area. Mortality rates of outdoor-col- lected sand flies were exposed to DDT 4%, deltamethrin 0.05%, permethrin 0.75%, and bendiocarb 0.1%, and mortality rate ranged from 92.0–97.9% and for indoor-collected sand flies were 87.7–96.8%. Both outdoor and indoor collected sand flies were susceptible to cyfluthrin 0.15% that caused 100% mortality. Conclusion: Based on the findings, the most appropriate method for collecting the live female Ph. papatasi is the baited traps due to providing enough females is necessary for conducting the susceptibility tests. The finding indicated that Ph. papatasi was resistant to DDT, under ’verification required’ status to deltamethrin, permethrin, bendiocarb, and susceptible to cyfluthrin. Keywords: Phlebotomus papatasi; Insecticides; Resistance; Baited traps; Hand-catch Introduction Phlebotomus papatasi was the main vec- tor of zoonotic cutaneous leishmaniasis (ZCL) and papatasi fever in the past. This species is prevalent in many areas in thewestern Palae- arctic Region including Europe (France, Spain, Greece, Poland, Czech Republic, and Italy), Rus- sia, and Central Asia as well as all countries in the Eastern Mediterranean Region, the west- ern and northern Ethiopian Region (Saudi Ara- bia, Yemen, Ethiopia, and Sudan) and the Ori- ental Region (India and Bangladesh). Phleboto- mus papatasi is found almost in all provinces of Iran (1). It prefers the climatical conditions with hot summers, temperate winters, and mon- soon rains and the minimum temperature should not less than -6 ˚C. This species prefers the en- vironmental moisture and warmth and is fre- quently caught in areas where the water has a high level (2). Numerous biological observa- tions on Ph. papatasi was indicative of the high- er abundance in the plains compared to high mountainous regions. It has been recurrently 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:abaimr@tums.ac.ir mailto:hvatandoost1@yahoo.com https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, December 2020, 14(4): 408–415 Y Rassi et al.: Efficiency of Two … 409 http://jad.tums.ac.ir Published Online: December 31, 2020 found in rodent burrows, human and domestic animal shelters in the central plateau of Iran (3). Phlebotomus papatasi is more prevalent at indoor compared to other species of sand flies. The larval stage develops at animal shelters and rodent burrows (4). In terms of endophilic be- havior, Ph. papatasi generally rests in human and animal shelters and becomes as a domes- tic species in many regions, so that no sand fly could ever be so accustomed to adapting to the residential places (5). With development the construction of human dwelling in the plains, abundance of Ph. papatasi rapidly increases and when the settlements are near to ZCL fo- ci, the transmission condition is more likely to become epidemic (6). It seems that the sand- flies, which were caught from residential places, moved from adjacent infected rodent burrows, where Ph. papatasi is considered as the pre- vailing species (7). In terms of biting and blood-feeding behaviors, Ph. papatasi can re- peatedly feed on its hosts both indoor and outdoor. The presence of multiple lesions on affected persons indicating evidence for re- current bites of humans by sand flies (8). Con- sidering the blood-feeding preference of Ph. papatasi, it can be argued that it mainly feeds on any host available nearby with more ten- dency to humans and rodents. The seasonal activity of this species starts from the begin- ning of May to the end of October in the northern and central regions of the country and from mid-April to January in southern regions with peaks in July and September (10). The lepomonad infection of Ph. papa- tasi has been reported from Isfahan, Lotfabad, Esferayen, Torkaman Sahra, Ahvaz, Dezful, Shush, Abardej, Balochistan, Sarakhs, Sem- nan, Damghan and Shahrud in Iran, all of which are known as the foci of ZCL (11-13). Transmission of ZCL by Ph. papatasi occurs outside the residential places, especially on the rooftops of houses or outdoor in front of the house in the regions with high endemicity for ZCL, whilst in regions with low endemicity, the transmission is more common in agricul- tural fields and in localities near the rodent bur- rows (10). Therefore, the transmission chain of ZCL seems to be interrupted by the use of py- rethroids impregnated bed nets, curtains, or uniforms and personal protection using repel- lents and occasionally application of residual spraying at indoors (14). The preliminary tri- als for determining the susceptibility levels of Ph. papatasi were reported in the Jordan Val- ley in 1967 (15) and the subsequent report re- vealed the resistance in northern Bihar, India (16) and followed in Turkey (17). Although numerous susceptibility tests have been carried out in the foci of ZCL in Iran, no evidence has yet been presented for the resistance of Ph. papatasi to insecticides (18-22). This study was the first comparative trial to determine the susceptibility of Ph. papatasi collected from both indoor and outdoor in ZCL foci in Lorestan Province, west of Iran. Materials and Methods Study area This study was conducted in Pol-e Dokh- tar, Rumeshgan, and Kuhdasht districts where the highest prevalence of ZCL was reported. The geographical coordinates of the research sites were 47º27´–48º22´E, 32º37´–33º20´ N, the mean elevation 662 meters above sea level (AMSL) for Pol-e Dokhtar, 47º20´–48º65´E, 33º16´–33º35´N, 1089m AMSL for Rumesh- gan, and 46º51´–47º50´E, 33º09´–33º56´N, and 1191m AMSL for Kuhdasht. Sand fly collections Sand flies were caught from indoor using hand catch method with mouth aspirators in the early morning as well as from outdoor us- ing baited traps in the evening till midnight. Each baited double net trap had equipped with mini gas lamps as a light attraction as well as a rooster in a cage as an attractant host for blood-feeding of sand flies. The baited traps set up in the evening using four wooden ba- ses, so that the lower edges of the nets lie 10 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 408–415 Y Rassi et al.: Efficiency of Two … 410 http://jad.tums.ac.ir Published Online: December 31, 2020 cm above the ground. The location of traps was close to the rodent burrows and fox nests adjacent to the agricultural lands in the stud- ied villages. The attracted sand flies were col- lected with the mouth aspirators also released into paper cups with net cover and transferred in a cool-box to the laboratory. A cotton pad soaked in 10% sucrose solution was put on the top of cups for feeding of sand flies. Susceptibility tests The insecticide-impregnated papers were purchased from the WHO’s Collaborating Cen- ter, Malaysia. Susceptibility tests were carried out on both indoor and outdoor collected sand flies and exposed to DDT 4%, deltamethrin 0.05%, permethrin 0.75%, bendiocarb 0.1%, and cyfluthrin 0.15% impregnated papers using WHO test kits. At each replicate, 20–25 sand flies were exposed for 60min. If the mortality criterion was in the resistance border, the tests were continued using an increasing trend of logarithmic times until observing 98–100% mor- tality. Both living and dead sand flies treated with each insecticide were preserved in etha- nol 70% till mounted in Puri’s medium for species identification. Data Analysis Data of susceptibility tests including the number of live, dead, and total sand flies were grouped based on physiological conditions for each of the treated and control groups. The sex ratio is calculated by dividing the number of males by females. Mortality was calculated by counting the dead and live mosquito spe- cies after 24h exposure to insecticide paper. The mortality was then corrected by applying Abbott’s formula when control mortality was observed between 5% and 20%, whereas tests with more than 20% control mortality were discarded and repeated. Median and mean of mortality used for plotting box and whisker plot used for showing the efficiency of two capture methods providing live sand flies and the independent-samples t-test used for show ing a significant difference between two cap- ture methods. WHO criteria were used for in- terpretation, with 98–100% mortality indicat- ing susceptibility, 90–97% indicating needs further confirmation or verification required, and mortality < 90% indicating resistance. The Probit analysis was used in analyzing the time- mortality response to estimate LC50 for a fixed concentration of insecticides to kill a defined proportion of sand flies, known as lethal time (LT). Result Efficacy of two collecting methods A total of 2486 live sandflies were caught using hand-catch and baited trap methods both from indoor and outdoor places with spending 12 and 4 working rounds. From indoor, 1637 sand flies (65.4%) were collected with sex ra- tio (SR) 0.6vs from outdoors 859 sand flies (34.6%) with SR= 0.1 (Table 1). A significant difference was shown between the SR values of sand flies collected in the hand-catch and baited trap methods (t= 7.245, df= 3, p= 0.005) (Fig. 1). Susceptibility levels of Phlebotomus papa- tasi collected indoor and outdoor The susceptibility of Ph. papatasi collected from both indoor and outdoor studied areas were assessd at discriminative doses and 60 min exposure time. The comparative percent mortality were 87.7±1.1 and 92.0±0.4 for DDT, 96.8±0.3 and 97.8±1.4 for deltamethrin, 92.4±0.9 and 97.9±0.0 for permethrin, 93.6± 0.9 and 94.0±0.7 for bendiocarb, and 100±0.0 and 100±0.0 for cyfluthrin while the values in the control group were 1.1±0.1% and 1.3±0.1 respectively (Fig. 2). http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 408–415 Y Rassi et al.: Efficiency of Two … 411 http://jad.tums.ac.ir Published Online: December 31, 2020 Table 1. Efficacy of two capture methods used for collectiong live sand flies for susceptibility tests both indoors and outdoors, Lorestan Province, western Iran C a p tu r e m e th o d T y p e o f c o lle c tio n p la c e R e p e titio n o f tr ia l T o ta l liv e c o lle c te d P e r c e n ta g e M a le F e m a le S e x r a tio M e a n o f T e m p e r a tu r e (°C ) R e la tiv e h u m id ity (% ) Baited trap Outdoor 4 849 34.6 83 766 0.1 30 47 Hand-catch Indoor 12 1637 65.4 1042 595 0.6 32 38 Fig. 1. Box plot showing sex ratio of sand flies abundance collected using hand-catch and baited traps methods, Lorestan Province, Iran Fig. 2. Comparison of susceptibility level of Phlebotomus papatasi collected from indoor and outdoor with hand-catch and baited trap methods following the WHO's Criteria (32), Lorestan Province, Iran http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 408–415 Y Rassi et al.: Efficiency of Two … 412 http://jad.tums.ac.ir Published Online: December 31, 2020 Discussion Due to the widespread outbreaks of ZCL in Iran and the world, the practical approaches are interrupting the transmission chain using different individual protection methods such as insecticide-impregnated bed nets and cur- tains, application of repellent when working outdoors, and in limited scale application of indoor residual spraying (14). Prior to any control measures during outbreaks of ZCL, cer- tain information is required about the bionomy of the vectors in order to take appropriate pre- ventive measures during natural disasters and epidemics. Insecticides play a crucial role in controlling the vectors of ZCL, and the type of insecticide should be determined using stand- ard tests before use (23). The larval habitats of sand flies are located in rodent burrows and other small mammals nests as well as in do- mestic animal shelters (24). Due to the exten- sive use of pesticides in animal husbandry and agricultural lands, the larval and adult sand flies are exposed to different chemicals which may lead to tolerant against various insecti- cides in the long term (25). Given that no com- parative study has been conducted as a concur- rent for determination on susceptibility level between two separate populations living indoors and outdoors using two live capture methods; this study was the first research attempt in this subject. According to the results of this study, the mortality of indoor-collected Ph. papatasi was 87%±1.1 when exposed 60min to DDT, and the for deltamethrin, permethrin, and ben- diocarb were ranged from 92.4 to 96.8%. With regard to the sufficiency of test replication in this study, it became evident that Ph. papatasi was resistant to DDT, under‘verification re- quired’ to bendiocarb, permethrin, and deltame- thrin, and susceptible to cyfluthrin. The main cause for the occurrence of resistance to DDT is believed to be the frequent use of this insec- ticide for controlling malaria vectors during past decades, and the gene responsible for organo- chlorine resistance was transferred to the next generations (26). It has been reported that the increased use of pyrethroid insecticide the health and agriculture sectors has resulted in cross-resistance to both organochlorine and py- rethroid insecticides (27). The sand flies col- lected from outdoor using baited traps were tol- erant to DDT 4%, deltamethrin 0.05%, perme- thrin 0.75%, and bendiocarb 0.1% and the mor- tality rates ranged from 92.0 to 97.8% com- parison of susceptibility of Ph. papatasi col- lected from both indoor and outdoor using hand-catch baited traps methods showed the under ’verification required’/susceptibility of sand flies caught from outdoor compared to indoor against DDT, deltamethrin, permethrin, and bendiocarb. Investigations on the suscep- tibility of sand flies to insecticides were initi- ated by Seyedi Rashti in Mashhad, northeast- ern Iran in 1970. He found that Ph. papatasi was susceptible to DDT and dieldrin (18). A study in Isfahan showed no resistance in Ph. papatasi (12). According to research in Mash- had and Isfahan (2006–2007), the LT50 of Ph. papatasi was increased up to 2.3–3% (22). The first occurrence of under ’verification required’ status to DDT was reported from Isfahan in 1992 (29), while another study in the Varzaneh County (Isfahan Province) showed complete susceptibility to DDT in the same period. In the latter study, it was concluded that the dis- continuation application of the organochlorine insecticides could result in returning the under ’verification required’ status (12). Some other studies conducted in Fars, Kerman, and Isfa- han during 1993–2001 reported the suscepti- bility of Ph. papatasi to DDT (18-21). Simi- larly, the susceptibility of Ph. papatasi to DDT, propoxur, and deltamethrin were confirmed in the studies conducted in Sabzevar, Isfahan, Baft, and Bam during 2002–2013 (20-21). This study was the first report on occurrence of re- sistance and under ’verification required’ sta- tus of Ph. papatasi to DDT, deltamethrin, per- methrin, and bendiocarb as well as susceptibil- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 408–415 Y Rassi et al.: Efficiency of Two … 413 http://jad.tums.ac.ir Published Online: December 31, 2020 ity to cyfluthrin in ZCL foci, west Iran. There are several novel investigations of the main vector-borne diseases in the country (28-31) and monitoring the susceptibility/resistance of related vectors is a vital responsibility of cor- responding ministries (28-31). Conclusion The findings of the present study indicated Ph. papatasi resistance to DDT, occurrence of under ’verification required’ status to bendio- carb, permethrin, and deltamethrin, and com- plete susceptibility to cyfluthrin. 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