J Arthropod-Borne Dis, September 2016, 10(3): 330–336 T Hazratian et al.: Diversity of Sand Flies … 330 http://jad.tums.ac.ir Published Online: January 06, 2016 Original Article Diversity of Sand Flies (Diptera: Psychodidae) in Endemic Focus of Visceral Leishmaniasis in Azar Shahr District, East Azarbaijan Province, North West of Iran Teimour Hazratian 1, Hasan Vatandoost 2, Mohammad Ali Oshaghi 2, Mohammad Reza Yaghoobi-Ershadi 2, Esmael Fallah 1, Sayena Rafizadeh 3, Mohammad Reza Shirzadi 3, Mansoreh Shayeghi 2, Kameran Akbarzadeh 2, *Yavar Rassi 2 1Departmemt of Parasitology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran 2Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 3Ministry of Health and Medical Education, Tehran, Iran (Received 3 Aug 2014; accepted 17 Aug 2014) Abstract Background: There are nearly 1000 species of Phlebotomine sand flies in 6 genera, of which only two, Phlebotomus in the old world and Lutzomyia in the new world are medically important. Globally, leishmaniasis prevalent in 98 countries and affects estimated 12 million people with almost two million new cases per year. Some rural areas of Azarshahr District in East Azarbaijan Province have been reported to be endemic for visceral leishmaniasis. This study is the first attempt to determine the species diversity and density in a new focus of visceral leishmaniasis in Azarshahr District, East Azarbaijan Province, Iran. Methods: Sand flies were collected form indoor and outdoor biweekly using sticky traps. Diversity index of the collected sand flies within different villages were estimated by the Shannon-Weaver. Results: The activity of the sand flies extended from April to October with one peak in August. Diversity of sand flies within study area were estimated as 0.917, 1.867, 1.339, 1.673, and 1.562 in Almalodash, Jaragil, Segaiesh, Amirdizaj, and Germezgol Vvillages, respectively. Conclusion: Identifying the diversity and seasonal abundance of the collected species is of importance for prediction of the period of maximum risk for leishmaniasis transmission and for the successful implementation of a control program. Species diversity is one of the most important factors in ecological studies. Keywords: Diversity, Density, Sand flies, Visceral Leishmaniasis, Azar Shahr, Iran Introduction Phlebotomine sand flies belong to the family Psychodidae (Young and Duncan 1994). Psychodidae family is characterized by numerous parallel veins running to wing margin and the presence of dense hairs on the wings and thorax. From 5 Psychodidae subfamilies, Phlebotominae is known to Phlebotomine sand flies by biting mouth parts that are longer than the head, five- segmented palps, nearly cylindrical anten- nae, a five-brached radial vein on the wing, and the absence of an eye-bridge (Lane and Crosskey 1993). Generally, sand flies are distinguished from other small flies by their size (2.5–3.5mm in length), characteristic hop- ping flight, and the "V" position in which they hold their wings while resting (Triplehom and Jhonson 2005). In the old world, about 40 species of Phlebotomus have been proven or suspected vectors of Leishmania (Rassi 2004). In Iran, 44 species of the sand flies are found and the *Corresponding author: Prof Yavar Rassi, E- mail: rassiy@tums.ac.ir J Arthropod-Borne Dis, September 2016, 10(3): 330–336 T Hazratian et al.: Diversity of Sand Flies … 331 http://jad.tums.ac.ir Published Online: January 06, 2016 presence of other 10 species is doubtful. In the endemic center of northwest of Iran, two species of Ph. kandelakii and Ph. Perfiliewi and in the south of Iran, three species of, Ph. keshishiani, Ph. alexandri and Ph. major are the vectors of the disease (Rassi 2007, Azizi 2009). Leishmaniasis is prevalent in 98 count-ries. This parasitic disease is known one of ten major diseases by WHO (Maia et al. 2011). It has widely spread not only in tropical countries but also in some regions with very different topography (Faiman 2000, WHO 2010). Clinically, leishmaniasis is observed in four types: Visceral leishmaniasis (VL), cu- taneous leishmaniasis (CL), mucocutaneus leishmaniasis (ML) and difuse cutaneous leishmaniasis (DCL) (WHO 1994). From 4 mentioned types, only two types of visceral and cutaneous leishmaniasis are common in Iran. Visceral leishmaniasis is considered as the main problems in Iran (Rassi 2011). This disease is observed in all areas of Iran spo- radically but the endemic foci of the disease have been reported from Ardabil, Fars, Boshehr and East Azerbaijan Provinces (Rassi et al. 2005, Oshaghi et al. 2009, Rassi et al. 2010). Some rural areas of Azarshar District in East Azarbaijan Province have been reported to be endemic for visceral leishmaniasis with more than 34 new cases. Performing the control plans without sufficient information about the fauna and ecological parameters is not possible. Therefore, this study was car- ried out during late April to late October 2010 in rural areas of Azarshahr District, East Azarbaijan Province, Northwestern Iran, to identify diversity, species composition and other ecological parameters. Material and Methods Study area Azar Shahr is located 45 km far from Tab- riz. The geographical coordinates is 45 30΄E and 37 ̊30΄N. The height of this county from the sea level is 1368 meters. It is limited to Lake of Urmia from the west, to Ajabshir City from the south, to Osko from the north and to Sahand Mountains from the east. In this county, the average rainfall and the rel- ative humidity were 303 and 49 millimeters, respectively. The mean of the annual tem- perature is 15.25 and the mean of monthly maxi-mum and minimum temperatures were 27.7 and 3.7 °C respectively. The population of this county has been 103952 people in 2006. Selection of Study villages According to reported cases of the disease, a number of villages including: Segaiesh, Amirdizaj, Almalodash, Jaragil and Germezgol were selected from the county of Azarshahr in east Azarbaijan Province. Segaiesh village: It is located 14 km from Azar Shahr with 183 families and 633 people in 2010. Amirdizaj Village: it is located 16 km from Azar Shahr with 250 families and 1046 people. Almalodash Village: It is located 20 km from Azar Shahr with 182 family and 860 people. Jaragil Village: It is located 15 km from Azar Shahr with 110 families and 422 people. Germezgol Village: It is 12 km from Azar Shahr with 70 families and 300 people in 2010. Sand fly sampling Sand flies were collected using sticky traps (castor oil coated white papers 20 cm× 30 cm) biweekly from selected villages indoors (bedrooms, guest room, toilet and stable) and outdoors (rodent burrow). Traps (60 papers per village) were installed in sunset and sand flies were collected during sunrise. Sample collection began in late J Arthropod-Borne Dis, September 2016, 10(3): 330–336 T Hazratian et al.: Diversity of Sand Flies … 332 http://jad.tums.ac.ir Published Online: January 06, 2016 April and continued until late October 2010, when sand fly activities were reduced sharply. Collected sand flies were stored in 96% ethanol. In the laboratory for species identification, head and three last abdomen segments of females, head with whole body of males specimens were mounted in Puri’s medium. Species identification were carried out after one week using keys of sand flies (Rassi and Hanafi bojd 2006). Determination of Density The density of sand flies collected by sticky traps was calculated using the formula number of specimens / of sticky traps and numbers of specimens/ number of traps. Logistic regression used to investigate the existence of a correlation between the abun- dance of the sand flies, and average monthly temperature in the study area using SAS software (Kasap et al. 2009, Tarallo 2009). The density of species was corrected according to the formula (Guernaoui 2009, Rajider 2010) Density = Determination of the Species Diversity To calculate the species diversity in a population, there are different methods but in most resources, Shannon-Weaver index has been used. Shannon-Weaver index is shown as    S 1i piLogePiH' . In this formula, S is the Richness of the species and pi is the proportion of species Ith in the population and log e is common logarithm. To convert it to common logarithm, it should be multiply in 2.303. In the present study, it is used Shannon-Weaver formula ( H'). Species diversity composed of two fac- tors, species richness and species evenness. There are many kind of species diversity indexes, a common one is the Shannon- Weaver index: (H'=   s i 1 Pilogepi) H΄= The diversity index Pi= The proportion of the Ith species Loge= Natural log S= Number of species in the community Results According to formula, monthly average density, number of specimens/ m2 of sticky traps were 37.60, 41.10, 40.23, 30.38 and 30.67 in Almalodash, Jaragil, Segaieesh, Amirdizaj and Germezgol, respectively. Corrected monthly average density, number of specimens/ number of traps, in mentioned villages were 1.47, 1.51, 1.39 and 1.40. Table 1. Density of sand flies in studied villages in Azarshahr District, Eastern Azarbaijan, Iran, 2010 Mean density= trapsofNumber SpecimensofNumber 1 Mean density= trapsofM SpecimensofNumber 2 Mean density= trapsofNumber SpecimensofNumber Village 1.4737.601.18Almalodash 1.3930.380.95Amirdizaj 1.5141.101.29Jaragil 1.5140.231.26Segaiesh 1.430.670.96Germezgol To calculate the species diversity, we used the formula Shannon-Weaver because it has been used in most resources to calculate diversity. The results are shown in tables 2, 3, 4, 5 and 6. J Arthropod-Borne Dis, September 2016, 10(3): 330–336 T Hazratian et al.: Diversity of Sand Flies … 333 http://jad.tums.ac.ir Published Online: January 06, 2016 Table 2. Diversity of sand flies in Almalodash Village in Azarshahr District, Eastern Azarbaijan, Iran, 2010 Community A (Almalodash) Piloge piLoge piProportion (Pi)NumberSpecies -.333-.6660.54554Ph. papatasi -.202-2.5260.0877Ph.sergenti -.169-2.5140.0658Ph.kandelakii -.046-4.6060.019Ph.(Adlerius) spp -.316-1.6610.19203Ph.C.group -.186-2.6500.0778Ph.halepensis -.078-3.9130.0219Ph.longiductus -.078-3.9130.0219Ph.major -.046-4.6060.019Ph.mongolensis    S i piLogePiH 1 454/1'1026Total Table 3. Diversity of sand flies in Amirdizaj Village in Azarshahr District, Eastern Azarbaijan, Iran , 2010 Community B (Amirdizaj) Piloge piLoge piProportion (Pi)NumberSpecies -.328-1.5560.211214Ph. papatasi -.337-1.4740.229232Ph.sergenti -.442-1.7440.0099Ph.mesghali -.303-1.7540.173175Ph.c.group -.278-1.9450.143145Ph.mongolensis -.278-1.9450.143145Ph.tobbi -.042-0.4110.0099Ph.kandelakii -.072-4.0810.01819Ph.halepensis -.124-3.2710.03839Ph.andrejevi -.072-4.0180.01819Ph.caucasicus -.042-4.7110.0099Ph.pawlowskyi    S i piLogePiH 1 918/1'1015Total Table 4. Diversity of sand flies in Jaragil Village in Azarshahr District, Eastern Azarbaijan, Iran, 2010 Community C (Jaragil) Piloge piLoge piProportion (Pi)NumberSpecies -.159-2.9010.00567Ph. papatasi -.363-1.1260.313378Ph.sergenti -.087-3.7330.02328Ph.tobbi -.006-4.1360.01619Ph.kandelakii -.035-4.9630.0079Ph.major -.362-1.1870.305369Ph.c.group -.110-3.3430.03239Ph.hodsoni -.286-1.8840.152184Ph.mongolensis -.202-2.2520.08097Ph.halepensis -.035-4.9630.0079Ph.longiductus -.035-4.9630.0079Ph.andrejevi    S i piLogePiH 1 74/1'1208Total J Arthropod-Borne Dis, September 2016, 10(3): 330–336 T Hazratian et al.: Diversity of Sand Flies … 334 http://jad.tums.ac.ir Published Online: January 06, 2016 Table 5. Diversity of sand flies in Segaiesh Village in Azarshahr District, Eastern Azarbaijan, Iran, 2010 Community D (Segaiesh) Piloge piLoge piProportion (Pi)NumberSpecies -.333-0.6160.540438Ph. papatasi -.049-4.5140.0119Ph.tobbi -.144-3.0580.04738Ph.sergenti -.342-1.4320.239194Ph.c.group -.049-4.5110.0119Ph.hodsoni -.144-3.050.04738Ph.kandelakii -.167-2.8310.05948Ph.mongolensis -.167-3.7330.02319Ph.halepensis -.049-4.5110.0119Ph.perfiliewi -.049-4.5110.0119Ph.major    S i piLogePiH 1 413/1'811Total Table 6. Diversity of sand flies in Germezgol Village in Azarshahr District, Eastern Azarbaijan, Iran, 2010 Community E (Germezigol) Piloge piLoge piProportion (Pi)NumberSpecies -.353-1.3130.269156Ph. sergenti -.066-4.1360 .0169Ph.ansari -.066-4.1360٠0169Ph.kandelakii -.066-4.1360٠0169Ph.c.group -.207-2.2480٠08348Ph.halepensis -.368-.9550.385223Ph.mongolensis -.113-3.4120.03319Ph.tobbi -.251-2.1460.11768Ph.andrejevi -.149-2.9960.05029Ph.caucasicus -.066-4.1360٠0169Ph.perfiliewi    S i piLogePiH 1 413/1'811Total Due to the tables of species diversity, it is obvious that the species diversity in Amirdizaj Village is in highest level than other villages and the diversity of the sand flies in this village (Population B) has more diversity and better results. Discussion This study is the report of the results of entomology investigations on sand flies in Azarshahr District. The geographical loca- tion of this district is appropriate for the population being potentially at risk for visceral leishmaniasis. According to Table 1, the corrected den- sity of sand flies in Jaragil and Segaiesh vill- ages had highest level (1.51) and we found the lowest level (1.39) in Amirdizaj village. The monthly activities of sand flies started from first half of June and have been continued to second half of October. The most level of density was related to August. Whereas, there was a peak of temperature in this month. In another words, J Arthropod-Borne Dis, September 2016, 10(3): 330–336 T Hazratian et al.: Diversity of Sand Flies … 335 http://jad.tums.ac.ir Published Online: January 06, 2016 the population of the sand flies dynamically was direct correlation to the temperature of the area but to the humidity. Given the history of the emergence of the sand flies adults in the region (20 May, 2010) and the density peak of in this region, it can be determined the control programs. Especially, if there is chemical control program, we can determine exact date of residual spraying reasonably and avoid unreasonable residual spraying, further costs and extreme residual spraying. Among 16 species collected and iden- tified, Ph. ansarii and Ph. mesghali had lowest density (0.18%). According to the studies of Rassi et al. 2000 on the fauna of the sand flies in west north of Iran and Kaverizadeh et al. 2000 on the fauna of sand flies in Ahar, all 16 species have been reported first time in Azarshahr. Further- more, two species of S. hodgsoni and P. mesghali have been observed in East Azerbaijan. Two main factors of species diversity are: Richness of species and Evenness of species. Richness of species is the species diversity in the population and the Evenness of species is the species diversity of the people in the species. The results are shown in tables 2, 3, 4, 5 and 6. In Al-Baha Province in the Saudi Arabia, a similar study has been performed (DohaandSomy 2010) with bionomics rela- tion of Phlebotomine sand flies population with temperature and species diversity in different places that its result is compatible to our results of this study. Conclusion According to the results, most species diversity was observed in Amirdizaj, there- fore, this village can be the best choice for determine of species composition of sand flies in the study area. It should be men- tioned, identifying the diversity and seasonal abundance of the collected species is very important for prediction of the period of maximum risk for leishmaniasis transmis- sion and for the successful implementation of a control program. 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