J Arthropod-Borne Dis, December 2014, 8(2): 156–162 M Hesam-Mohammadi et al.: Efficacy of … 156 Original Article Efficacy of Different Sampling Methods of Sand Flies (Diptera: Psychodidae) in Endemic Focus of Cutaneous Leishmaniasis in Kashan District, Isfahan Province, Iran Marzieh Hesam-Mohammadi 1, *Yavar Rassi 1, *Mohammad Reza Abai 1, Amir Ahmad Akhavan 1, Fatemeh Karimi 1, Sina Rafizadeh 2, Alireza Sanei-Dehkordi 1, Maryam Sharafkhah 3 1Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2Ministry of Health and Medical Education, Tehran, Iran 3Department of Epidemiology and Biostatistics, Tehran University of Medical Sciences, Tehran, Iran (Received 4 Mar 2013; accepted 2 Oct 2013) Abstract Background: The aim of the study was to evaluate and compare the efficiency and practicality of seven trapping methods for adult phlebotominae sand flies. The results of this investigation provide information to determine the species composition and nocturnal activity pattern of different sand fly species. Methods: The study was carried out in both plain region (about 5km far from northeast) and mountainous region (about 40km far from southwest of Kashan City). Seven traps were selected as sampling methods and sand flies were collected during 5 interval times starting July to September 2011 and from 8:00PM to 6:00AM in outdoors habitats. The traps include: sticky traps (4 papers for 2 hours), Disney trap, Malaise, CDC and CO2 light traps, Shannon traps (black and white nets) and animal-baited trap. Results: A total of 1445 sand flies belonging to 15 species of Phlebotomus spp. and five of Sergentomyia spp. were collected. Females and males comprised 44.91% and 55.09% of catches, respectively. Of the collected specimens, Se. sintoni was found to be the most prevalent (37.86%) species, while Ph. papatasi, accounted for 31.76% of the sand flies. Conclusion: Disney trap and sticky traps exhibited the most productivity than other traps. In addition, in terms of the efficiency of sampling method, these two trapping methods appeared to be the most productive for both estimating the number of sand flies and the species composition in the study area. Keywords: Psychodidae, Sand flies, Nocturnal activity, Trapping, Sampling methods, Iran Introduction Phlebotomine sandflies (Diptera: Psy- chodidae) are widespread in the tropics and subtropics, and they transmit Leishmania, protozoan parasites which cause visceral leishmaniasis (VL) and various forms of cutaneous leishmaniasis (CL) infecting more than 350 million people in more than 80 countries. They have a wide distribution, though mainly in the tropics and subtropics (Lane 1993). Approximately 700 species of phlebotomine sand flies distributed among 6 genera but only two of them, Phlebotomus in the old world and Lutzomyia in the new world are introduced medically importance (Lewis 1982, Lane 1987, Lane 1993, Sharma and Singh 2008), and only 10% of them act as the disease vector. Further, about 30 spe- cies of these are important from public health standpoint (WHO 1990, Desjeux 2000, Sharma and Singh 2008). The blood-feeding females of phlebotomine sand flies (Diptera: Psy- chodidae, Phlebotomine) include natural vectors of protozoa of the genus Leishmania (Kinetoplastida: Trypanosomatidae), which are the parasitic causative agents of mam- malian leishmaniasis (Killick-Kendrick 1990). *Corresponding author: Prof Yavar Rassi, E-mail: rassiy@sina.tums.ac.ir, Mr Mohammad Reza Abai, E-mail: abaimr@sina.tums.ac.ir http://jad.tums.ac.ir Published Online: April 09, 2014 J Arthropod-Borne Dis, December 2014, 8(2): 156–162 M Hesam-Mohammadi et al.: Efficacy of … 157 Cutaneous leishmaniasis are reported from more than 50% of the 31 provinces in Iran (Yaghoobi-Ershadi et al. 1999, Rassi et al. 2004, Rassi et al. 2006, Rassi et al. 2007, Rassi et al 2007, Rassi et al. 2011). In the Middle East, phlebotomine sand fly populations occasionally occur in great numbers, resulting in biting pressure of 1,000 bites per person per night (Coleman et al. 2006). An extraordinarily high number com- pared with CDC light traps. Although the impact on adult sand fly biting activity was not determined (Blow et al. 2007), these pre- liminary results suggested that commercial mosquito traps might be good candidates for inclusion into integrated sand fly control or suppression programs in desert settings (Hoel et al. 2000). In this study, sampling of sand flies were carried with Disney trap, Malaise, Sticky traps, CDC light trap, CO2 Light trap, Shannon traps and Animal-baited trap. The purpose of the current study was to assess which of these traps could catch a larger number or show the most diversity of sand flies in the interval times. Some of them are currently used in rou- tine sand fly surveillance programs (Alexander 2000). Previous studies aiming at determining the species composition and distribution pat- terns of sand flies in Kashan revealed eleven Phlebotomus spp. and six Sergentomyia spp. Some ideas for precise sampling in order to determine species composition, nocturnal ac- tivity and biodiversity of different sand fly was species in CL focus at different topographic condition e.g. mountain/plain. Furthermore the effects of some environmental factors such as temperature, relative humidity and height above sea level were studied in relation to fluctuation considered in pattern of nocturnal activity as well as sand fly biodiversity. Materials and Methods Study area The study was carried out in both plain region about 5km far from northeast and moun- tainous region about 40 km far from south- west of Kashan District, Isfahan Province, in central Iran (Fig. 1). The geographical co- ordinates is 51º 29' 54.6" E and 33º 58' 52.9" N for plain region and 51º 13' 54.6" E and 33º 58' 52.9" N mountainous region with at an altitude of 951m and 1823m respec- tively. The climate is hot and dry in summer and cold during the winter. The maximum and minimum temperatures were 38 °C and 19 °C in plain region and were 45 °C and 15 °C in mountainous region during the study period. Sand fly sampling Seven traps were employed for sampling of sand flies. The sampling was repeated three times during the peak activity of sand flies in both plain and mountainous regions. Turning of sand flies sampling were 5 times per night starting 8:00PM and ending 6:00 AM at outdoors using sticky traps (4 papers for 2 hours, totally 20 papers per day), Dis- ney trap, Malaise, CDC light trap, CO2 Light trap (0.5kg dry ice), Shannon traps (both black and white nets) and animal-baited trap (Fig. 2). The study was extended from July to September 2011. The traps were set up at least 15m from each other. Sticky traps were changed every two hours and as the traps were changed, the new ones were replaced in the same location. Other collection meth- ods were also performed over the same 10 hours period using mouth aspirator to col- lect the sand flies. All sand flies at these traps were captured during 10–15min in eve- ry two hours. Collected sand flies for sticky traps (20×30cm papers coated with castor oil) and Disney trap were removed from sticky papers using entomological needles or fine brushes, preserved in 70% ethanol, and kept in microtubes before identifica- tion. At other traps, sand flies were also col- lected at interval of 2 hours by aspirator start- ing 8:00 PM ending 6:00 AM. The sand http://jad.tums.ac.ir Published Online: April 09, 2014 J Arthropod-Borne Dis, December 2014, 8(2): 156–162 M Hesam-Mohammadi et al.: Efficacy of … 158 flies were anesthetized with chloroform and transferred to microtube containing 70% eth- anol. Then specimens transported to labor- atory research center for mounting and iden- tification. Mounting and Morphological identification After recording the sampling data and locations, some sand fly specimens which collected with sticky traps were washed in acetone and rest of them transferred to 70% ethanol. At appropriate time, the sand flies were mounted in Puri’s medium. All the identifications were carried out based on their genus and then species. The identifi- cation keys of Theodor and Mesghali 1964, Lewis 1982, Rassi et al. 2006 and Absavaran et al. 2009 were used. The permanent mount- ed, labelled and identified microscope slides were deposited in Medical Entomology and Zoology Museums, School of Public Health, TUMS under code No. GC22ST5-92. Recording climatic conditions To determine the relationship between temperature and relative humidity (RH) and the abundance or activity of sand flies in both study regions (plain and mountain) as- sociated with traps some meteorological data were recorded. The temperature and relative humidity were recorded every two hours dur- ing the night. Statistical Analysis Data were analyzed using SPSS 20. In order to ensure normality and homogeneity of variances before subjecting to statistical analysis, data on the number of flies collect- ed were square-root transformed. The sig- nificance difference of effect of the traps and time was analyzed in relation to sand flies species caught in two regions using univariate analysis of variance (ANOVA) and Chi-square tests. Total sand fly species caught consti- tuted the dependent variable, while sex and traps and time were chosen as the fixed fac- tor. Basic correlation matrices were used to determine the existence of a correlation be- tween the abundance of the sand flies, aver- age hourly temperature and relative humid- ity in the study area. Some of graphs were prepared using Microsoft Excel 2010. Results A total of 1445 sand flies belonging to 15 species of the genus Phlebotomus and 5 of the genus Sergentomyia were collected. Females and males comprised 44.91% and 55.09% of catches during the study period respectively. Of the collected total, S. sintoni was found to be the most prevalent (37.86 %) species while Ph. papatasi the proven vector of L. major, L. turanica and L. gerbili in Isfahan accounted for 31.76% of the sand flies that were identified. Other species were S.(Ser.) dentata (0.3%), S.(Ser.) antennata (0.2%), S.(Ran.) pawlowskyi (0.3%), S.(Par.) palestinensis (0.1%), Ph.(Syn.) ansarii (0.1%), Ph.(Para.) kazeruni (1.7%), Ph.(Par.) cau- casicus group (0.4%), Ph.(Par.) alexandri (1.1%), Ph.(Lar.) wenyoni (0.6%), Ph.(Lar.) tobbi (1%), Ph.(Lar.) major (3.5%), Ph.(Lar.) keshishiani (0.2%), Ph.(Lar.) kandelaki (0.1%), Ph.(Adl.) longiductus (0.1%), Ph.(Adl.) hale- pensis (0.1%), Ph.(Adl.) chinensis group (0.1%) and Ph.(Adl.) brevis (0.3%). During the study period, the population of sand flies was found to be the lowest in CDC light trap and CO2 Light trap. Population size was raised with two highest peaks in Disney trap and sticky trap at 22:00–24:00 PM. The average monthly temperature and rel- ative humidity values ranged between 21.83– 28.33 °C and 24.83–36.33% respectively. Dur- ing the night, when the maximum number of sand flies (29.25%) was collected at 22:00– 24:00 PM, the average temperature and RH were found to be 26.92 °C and 26.5%, respec- tively, while the minimum number of sand flies was sampled at 02:00–04:00 AM (11.62 %) with an average temperature of 23.08 °C and 33.5% relative humidity (Fig. 3). http://jad.tums.ac.ir Published Online: April 09, 2014 J Arthropod-Borne Dis, December 2014, 8(2): 156–162 M Hesam-Mohammadi et al.: Efficacy of … 159 The number of sand flies collected using different types of traps during the study pe- riod is presented in Table 1. Almost all sand fly species were commonly caught with Disney trap and sticky traps. Approximately 37.3% of the total collection was made up using Disney trap. 24.2% and 10.5% of sand flies were collected using sticky traps and Shannon trap (black net), respectively. Collections using animal baited trap and Shannon trap (white net) constituted 8.5% and 7.3% of the total collection. Neither CO2 Light trap (5.0%) nor CDC light trap (3.3%) and Malaise trap (0.1%) showed a high efficiency during the study. A signifi- cant interaction was revealed between the collection methods and the number of in- dividuals collected using of univariate anal- ysis of variance (ANOVA) (P< 0.05), and there was significant interaction between the trapping methods and the proportion of males and females collected (P< 0.05). When significant effects of traps were established (P< 0.05), differences among traps were ex- posed that CO2 and standard CDC light traps, Shannon trap (black and white net) and animal baited trap displayed some simi- lar efficiency, whereas sticky papers and Dis- ney trap differed from the others (P< 0.05). Results with respect to the nocturnal ac- tivity indicated that even though the number sand flies declined rapidly between 02:00 and 04:00h. There was no significant differ- ence between hourly pattern either in the species prevalence or in the activity of the species in different traps (Fig. 4). Fig. 1. Kashan District, Isfahan Province, central Iran Fig. 2. Different methods used for sand fly sam- pling Fig. 3. Correlation of two environmental conditions with nocturnal activity of sand flies Fig. 4. Correlation between collection methods and density of abundance sand flies species in the study area in Kashan City http://jad.tums.ac.ir Published Online: April 09, 2014 J Arthropod-Borne Dis, December 2014, 8(2): 156–162 M Hesam-Mohammadi et al.: Efficacy of … 160 Table 1. Prevalent sand flies collected by different traps, Kashan, Isfahan Province, 2011 Species Traps Total Baited trap CDC CO2 Light trap Disney trap Black Shannon trap White Shannon trap Sticky trap Ph.(Lar.) major 19 3 0 12 2 4 9 49 Ph.(Phl.) papatasi 14 18 56 208 38 22 86 442 Ph.(Par.) sergenti 21 4 2 114 73 22 36 272 S.(Ser.) sintoni 45 17 14 179 31 46 207 539 Total 99 42 72 513 144 94 338 1302 Discussion This study is the first detailed research in terms of species composition, density and nocturnal activity of sand flies using differ- ent methods of capturing in an endemic fo- cus of cutaneous leishmaniasis in Kashan dis- trict, Isfahan Province, Iran. Trapping over the summer season during the peak of sand flies activity revealed that S. sintoni (37.9 %), P. papatasi (31.8%) and P. sergenti (20.1%) are the most abundant and prevalent species in these regions respectively (Doroudgar et al. 1999). Both the previous and recent find- ings showed that P. papatasi the main vec- tor of L. major, L. turanica and L. gerbili in Isfahan Province, P. sergenti the proven vector of L. tropica and L. gerbili in this re- gion, also S. sintoni were represented with high populations in the study areas (Seyedi Rashti and Nadim 1992, Doroudgar et al. 1999, Yaghoobi-Ershadi 2012). Although sand fly abundance was not strongly and significantly influenced by the var- iations in average monthly temperature and rel- ative humidity during the six months of survey in Cukurova Plain, south Anatolia, Turkey, the maximum number of sand flies was rec- orded in the hottest and driest seasons between June and September when the average tem- perature was comparatively high, the relative humidity was low and the rainy days ranged between 1.5 to 3.5 days. (Kasap et al. 2009). The role of climatic factors on the seasonal distribution of sand flies in the arid areas of India exhibited that the majority of species preferred comparatively higher temperatures and low RH%, a prerequisite for survival in arid and semi-arid conditions (Singh 1990). Our study results are concordance with the other survey in relation to effects of temper- ature and relative humidity on abundance of sand flies. It seems that special range of both high temperature and low relative humidity caused the high abundance of sand fly spe- cies in our study regions. The results showed that before midnight (between 22:00–24:00 PM) sand flies were very active. Although CDC and CO2 light traps are used extensively in the field studies of sand flies (Alexander 2000), in Cukurova Plain in Turkey sticky traps have no known attrac- tiveness and have generally been used for determining species composition of an area as they randomly sample the species where they are set (Kasap et al. 2009), whereas our find- ings showed that Disney trap and sticky traps have been attractiveness more than other traps. Our findings in terms of the ef- ficiency of the sampling methods, showed these two trapping methods appeared to be the most productive for both estimating the number of sand flies and the species compo- sition in the study area, in agreement with some previous studies. It seems that the sticky traps provide more realistic results than CDC light traps that could attract additional pho- totropic sand flies. The effective range of http://jad.tums.ac.ir Published Online: April 09, 2014 J Arthropod-Borne Dis, December 2014, 8(2): 156–162 M Hesam-Mohammadi et al.: Efficacy of … 161 CDC light traps was less than 5m (Wheeler et al. 1996). Study in northern Italy showed that when compared to sticky traps, CO2 traps were more effective in collecting sand flies and addition of a light source improved the catches (Veronesi et al. 2007). The nocturnal activity patterns of sand flies have been reported for Old World spe- cies (Roberts 1994, Guernaoui et al. 2006, Coleman et al. 2007). According to our re- sults, sand flies activity was not significantly different over time even though total counts decreased between 04.00–06.00h during late summer. No significant difference was found in the species prevalence throughout the nights. It seems to be the most important factor affecting sand fly nocturnal activity in similar studies was low humidity, fol- lowed by low wind velocity and high tem- perature (Roberts 1994, Guernaoui et al. 2006, Kasap et al. 2009). Conclusion Disney trap and sticky traps exhibited the most productivity than other traps. Therefore, more detailed studies with respect to the sea- sonal variations and the effects of abiotic con- ditions, other than temperature and relative hu- midity, such as cloud cover, wind velocity and lunar cycle on the nocturnal activity of sand flies may improve knowledge of the behav- ior of sand flies for important epidemiologi- cally surveys. Acknowledgements This study was funded and supported by Tehran University of Medical Sciences (TUMS), Grant No. 13967. The authors de- clare that they have no conflicts of interest. References Alexander B (2000) Sampling methods for phlebotomine sandflies. Med Vet Entomol 14: 109–122. Absavaran A, Rassi Y, Parvizi P, Oshaghi MA, Abaie MR, Rafizadeh S, Mohebali M, Zarea Z, Javadian E (2009) Identifi- cation of sand flies of the subgenus Larroussius based on molecular and mor- phological characters in north western Iran. Iran J Arthropod-Borne Dis. 3(2): 22–35. Coleman RE, Burkett DA, Sherwood V, Caci J, Spradling S, Jennings BT, Rowton E, Gilmore W, Blount K, White CE, Putnam JL (2007) Impact of phlebotomine sand- flies on US military operations at Tallil Air Base, Iraq: 2. Temporal and geo- graphic distributions of sandfies. J Med Entomol. 44: 29–41. Desjeux P (2000) Leishmania/HIV coinfection, south-western Europe, 1990–1998 Ge- neva, Switzerland: WHO, pp.1–12. Doroudgar A, Seyedi-Rashti MA, Rassi Y (1999) Study on sandflies fauna in Kashan District during 1990–1997. Per- sian. J Kashan Uni Med Sci. 9: 79–85. Guernaoui S, Boussaa S, Pesson B, Boumez- zough A (2006) Nocturnal activity of phlebotomine sandflies (Diptera: Psy- chodidae) in a cutaneous leishmaniasis focus in Chichaoua, Morocco. Parasitol Res. 98: 184–188. Hoel DF, Kline DL, Hogsette JA, Watany N, Obenauer PJ, Bernier UR, Fawaz EY, El-Hossary SS, Hanafi HA, Furman BD, Szumlas DE (2010) Efficacy of com- mercial mosquito traps in capturing phelbotomine Sand Flies (Diptera: Psy- chodidae) in Egypt. J Med Entomol. 47 (6): 1178–1184. Kasap OE, Belen A, Kaynas S, Simsek FM, Biler L, Ata N, Alten B (2009) Activity patterns of sand Fly (Diptera: Psycho- didae) species and comparative perfor- mance of different traps in an endemic cutaneous leishmaniasis focus in Cu- kurova Plain,southern Anatolia, Turkey. Acta Vet Bron. 78: 327–335. http://jad.tums.ac.ir Published Online: April 09, 2014 J Arthropod-Borne Dis, December 2014, 8(2): 156–162 M Hesam-Mohammadi et al.: Efficacy of … 162 Killick-Kendrick R, Tang, Killick-Kendrick M, Sang DK, Sirdar MK, KE L, Ash- ford RW, Schorscher J, Johnson RH (1991) The identifcation of female sand- flies of the subgenus Larroussious by the morphology of the spermatechal ducts. Parasitologia. 33(l): 335–347. Lewis DJ (1982) A taxonomic review of the genus Phlebotomus (Diptera: Psycho- didae). Bull Br Mus Nat Hist Entomol. 45: 121–129. Theodor O, Mesghali A (1964) On the Phle- botominae of Iran. J Med Entomol. 1(3): 285–300. Rassi Y, Kaverizadeh F, Javadian E, Mohebali M (2004) First report on natural pro- mastigote infection of Phlebotomus caucasicus in a new focus of visceral leishmaniasis in northwest of Iran. Ira- nian J Publ Health. 33(4): 70−72. Rassi Y, Hanafi-Bojd AA (2006) Sand flies (Leishmaniasis vectors) with pictorial indentification key. Noavaran-e-elm Press, Tehran, Iran, pp. 156–169. Rassi Y, Ghasemi MM, Javadian E, Rafizadeh S, Motazedian H, Vatandoost H (2007) Vector and reservoir of cutaneous leish- maniasis in Marvdasht District, south- ern Islamic Republic of Iran. Eastern Meditter Health J.13: 686–693. Roberts DM (1994) Arabian sandfies (Diptera: Psychodidae) prefer the hottest nights? Med Vet Entomol 8: 194–198. Seyedi Rashti MA, Nadim A (1992) The genus Phlebotomus (Diptera: Psychodidae: Phlebotominae) of the countries of the eastern Mediterranean region. Iranian J Publ Health 21: 11–50. Sharma U, Singh S (2008) Insect vectors of Leishmania: distribution, physiology and their control. J Vector Borne Dis. 45: 255–272. Singh KV (1990) Studies on the role of cli- matological factors in the distribution of phlebotomine sandflies (Diptera: Psy- chodidae) in semi-arid areas of Raja- sthan, India. J Arid Environ. 42: 43–48. Veronesi E, Pilani R, Carrieri, Bellini R (2007) Trapping sandflies (Diptera: Psychodidae) in the Emilia-Romagna region of north- ern Italy. J Vector Ecol. 32: 313–318. Wheeler AS, Feliciangeli MD, Ward RD, Maingon RDC (1996) Comparison of sticky-traps and CDC light-traps for sampling phlebotomine sandflies en- tering houses in Venezuela. Med Vet Entomol. 10: 295–229. WHO (2010) Control of the leishmaniasis: re- port of a meeting of the WHO Expert Committee on the Control of Leishma- niases Control. Technical Report Series, No. 949. World Health Organization, Geneva, Switzerland, pp. 32–138. Yaghoobi-Ershadi MR (2012) Phlebotomine sand flies (Diptera: Psychodidae) in Iran and their role on Leishmania transmis- sion. J Arthropod-Borne Dis. 6(1): 1–17. http://jad.tums.ac.ir Published Online: April 09, 2014