J. Exp. Zool. India Vol. 17, No. 2, pp. 669-674, 2014 ISSN 0972-0030 IDENTIFICATION OF COLLECTED SAND FLIES FROM ROFAYYEH AREA, SOUTH WEST OF

Elham Jahanifard1,2, Shahrohk Navidpour3, Babak Vazirianzadeh*4,2, Seyed Abbas Moravvej5, Jasem Saki4,6, Mehdi Zarean6 1,2 Department of Medical Entomol. & Vector Control, School of Public Health, University of Medical Sci. Tehran, Iran 3Department of Venomous Animals and Toxins, Razi Vaccine and Serum Research Institute, , Iran 4 Health Research Institute, Infectious and Trop. Dis. Research Center, Jundishapur University of Medical Sci, , Iran 5Department of Plant Protection, College of Agriculture, Chamran University, Ahvaz, Iran 6Department of Medical Parasitology, School of Medicine, Ahvaz Jundishapour University of Medical Sciences, Ahvaz, Iran * e.mail: [email protected] (Accepted 22 May 2014)

ABSTRACT : An assay was conducted to determine the sand fly fauna in Rofayyeh city, where is situated in common border line with country, in 2009. Totally, 1420 Phlebotomous and 117 Sergentomyia were collected using sticky traps from outdoor and indoor resting places. In present study P. papatasi, P. alexandri, P. caucasicus, P. mongolensis and S. sintoni were identified. P. papatasi (69.68%) and P. alexandri (12.36%) were dominant species, respectively. The highest sex ratio was found in P. caucasicus which was collected in outdoor resting place. Further epidemiological research should be done to investigate vectors and reservoirs of Zoonotic Cutaneous Leishmaniasis (ZCL) in this area for prevention of disease outbreak. Key words : Sand fly fauna-Zoonotic Cutaneous Leishmaniasis-Khuzestan, Iran.

INTRODUCTION Sergentomyia. Javadian and Nadim have added two more Sand flies are biological vectors of Leishmania species of Sergentomia to the list of sand flies in this species in human and between human and animals in the province in their study (Javadian and Nadim 1975). In old world and new world. Different species of another study, natural leptomond infection was found in Leishmania, a Protozoa, cause leishmaniasis in man and P. papatasi, P. alexandri and S. sintoni which were animals. Three forms of leishmaniasis including: Zoonotic collected from rodent burrows in some parts of this region Cutaneous Lieshmaniasis (ZCL), Anthroponotic (Javadian and Mesghali 1974). In a research study, three Cutaneous Lieshmaniasis (ACL) and Visceral species of Phelebotomus were identified in two large Lieshmaniasis (VL) have been causing some health and marshlands of (Jahanifard et al. medical problems related to humans and animals in Iran 2009). In the latest study of sand flies in Khuzestan, and its adjoining countries: Iraq, Afghanistan , Pakistan Kavarizadeh et al. (2011) have recognized four species and Turkey. Cutaneous leishmaniasis (CL) due to of Phelebotumus sand flies from Maleh area, Shoush Leishmania major is endemic in Iran and causes a major County in the north of Khuzastan province.All sand fly public health problem in 17 of 31 including species in Kavarizadeh et al. (2011) and (Jahanifard et Khuzestan, a south west province (Rassi et al. 2004, al. 2009) studies have already been reported from the Shakila et al. 2006, Akhavan 2007, Simsek 2007, Azizi other parts of Iran. and Fekri 2010, Saki and Khademvatan 2011, Yaghoobi- Estimations are showing an increasing in the rate of Ershadi 2012). Vazirianzadeh et al (2013) as the latest ZCL cases in the region among the human population molecular Leishmaniasis study in Khuzestan have isolated including army staff in border line with Iraq during 2005- L.major from both reservoir and vector, Tatera indica 2008 (Jahanifard, 2010). Therefore, in respect to (Rodentia: Gerbillidae) and P.papatasi at the same time importance of ZCL, abundance of sand flies, high density from Roffaye area. of rodent (reservoir) and heavy infection (Nadim 1994) The status of sand flies in the different parts of in this area we arranged a new study on sand fly Khuzestan province has been published by Nadim et al. identification in Rofayyeh. In fact, there has been little (1974), Javadian and Nadim (1975), and Yaghoobi-Ershadi survey about species composition of Phlebotominae in (2012). In their research 20 species of sand flies have this border line of Rofayyeh area. Therefore, the purpose been reported of two genera Phelebotomus and 670 Elham Jahanifard et al of this research was to determine the composition of sand morphological characteristics in females and males flies in Rofayyeh area, west part of Khuzestan province, (Theodor and Mesghali 1964). First, sex of all specimens Iran. The special objectives of the present study regarding was determined. Then, they were identified by species to the sand flies were to determine the species diversity, level using the keys of Lewis (1982), Nadim and Javadian relative population density and sex ratio of sand flies in (1976) and Nadim et al. (1994). Finally, sex ratios of the Rofayyeh area. These factors provide basic epidemiologic predominant species were determined by formula of information to make vector control programs to reduce number of males / females × 100 (Kakarsulemankhel the incidence of ZCL in the region. 2010).Sex ratios are calculated based on the female MATERIALS AND METHODS population, therefore, the larger size of vector sand fly female population provides more probobility of leishmania Study area transmission in a region. It should be mentioned that Dasht-e-Azadegan County is located in North West because females two species of P. mongolensis and P. of Ahvaz near to Iran- Iraq border grounds. Latitude and caucasicus are not morphologically distinguishable from longitude of this county are 31Ú 33' N and 48Ú 10' E, each other, therefore when from one site only males of P. respectively. Altitude from sea level is 10 m. It involves mongolensis or P. caucasicus were collected, the three regions: central, and Boustan. Hoveyzeh collected females were indentified as same as male region has two cities: Rofayyeh and Hoveyzeh. Rofayyeh species. (31Ú 55' N, 47Ú 40') is situated in 25 km from west of RESULTS Hoveyzeh and 50 km from southwest of city, the center of Dasht-e-Azadegan, in border line between In this entomofaunistic study, totally 1537 sand flies Iran and Iraq (Fig1). There is not any synoptic meteorology (31.75% males and 68.25% females) were collected station in Roffayeh, therefore month average of during four months from Rofayyeh area. In the current environmental temperature and relative humidity of research study five species of sand flies were recognized, Boustan and Ahvaz and are considered as meteorology four belonging to Phlebotomus and one belonging to information rather than Roffayeh area, in the present Sergentomyia genera. The most frequent Phlebotomos study. The month average of temperature(ºC, June- species was P. papatasi Scopoli 1786, followed by P. September) in Boustan (1986-2005) and Ahvaz(1957- alexandri Sinton 1928, P. caucasicus Marzinowsky 1917, 2005) have been recorded : 33.30±1.26, 35.50± P. mongolensis Sinton; Theodor, 1958 and S. sintoni 1.37,35.10± 1.43 , 31.20 ±0.87 and 35.10±1.01,37.1 Pringle 1953 with 69.68%, 12.36%, 7.35%, 2.99% and ±1.11,36.5±1.28 and 32.8 ±1.01,repectiveley. The month 7.62%, respectively. Table 1 presents the sex ratio of the average of relative humidity (%, June-September) in specimens in outdoor and indoor resting places, Rofayyeh Boustan (1986-2005) and Ahvaz (1957-2005) have been area of Khuzestan province. The highest sex ratio was recorded : 28, 29, 30,31and 23, 24, 28 and 29, repectiveley observed in P. caucasicus which was collected in outdoor (I.R OF IRAN METEOROLOGICAL resting place. P. papatasi was recognized as dominant ORGANIZATION (IRIMO,2013). species of the region that formed 57.61% and 42.39% of indoor and outdoor traps, respectively. The results are Sand flies sampling and identifying presented in details in Figure 2. Sand flies were captured using 580 of sticky traps Table 1 shows the sex ratio of the sandfly specimens (papers 20 cm × 30 cm coated with castor-oil) from outdoor in outdoor and indoor resting places, Rofayyeh area of (rodent burrows) and indoors (stables and trench) once a Khuzestan province. Figure 2 illustrates the abundance month during June till September 2009. Trapping were of Phlebotomus species in outdoor and indoor resting conducted in two different rural areas of Rofayyeh places. including two indoor places per area (30 sticky traps per place) and 10 sticky traps for outdoors in each areas. DISCUSSION The sticky traps were set before sunset and gathered in Khuzestan is an endemic focus of Z C L (Saki and the next morning before sunrise. All traps were transferred Khademvatan 2011). Forty seven years ago, a survey to Razi Vaccine and Serum Research Institute in Ahvaz. was carried out for determining human infection rate in The specimens were removed by needle and washed in , Susangerd and Dasht-e-Mishan.These rate was acetone then they reserved in ethanol 70%. For calculated 25%,10%, and 5-10% for mentioned areas, preparation of permanent microscopy slides, the samples respectively.Furthermore, in Mussian area, north of Dasht- were mounted by the use of Puri’s medium. The sand e-Mishan, most of soldiers who were in military service flies were identified using the external and internal infected by Leishmania major (Nadim et al. 1974). Identification of sand flies 671

Fig. 1 : Map of Iran and place of the current study in Roffaye ,Khuzestan province.

Nadim et al. (1974) published a paper about sand flies in human, whilst P. alexandri, P. andrejevi, P. caucasicus, in which they showed the majority of the P. mongolensis and P. ansari are vectors among rodents captured sand flies were P. papatasi. It is interesting in rural areas (Nadim and Faghih 1968, Javadian et al. that similar result was found in the current study regarding 1977, Nadim et al. 1994, Yaghoobi-Ershadi et al. 1994, P. papatasi density. Jahanifard et al. (2009) have pointed 1995). to three species (P. papatasi, P. alexandri and P. It is important that P. papatasi is the main vector of mongolensis) in their study which were collected using Cutaneous Leishmanasis among countries in neighboring insect collecting net from Hovizeh and or near to Iran such as Afghanistan (Nadim et al. 1979), Marshlands of Khuzestan province. (Killick-Kendrik et al. 1985), (Lane Our finding determined that the density of P. and Al-Taql 1983) and Iraq (Coleman et al. 2007). In alexandri, P. mongolensis, P. caucasicus and S. sintoni addition, it has known as the main vector of ZCL in the were high in rodent burrows. This result was accordance old world (Gramiccia and Gradoni 2005).The highest to this fact that P. papatasi is the main vector of ZCL to collected specimens in this essay were P. papatasi 672 Elham Jahanifard et al species after P. papatasi in Roffayae. Studies of Jahanifard et al. (2009), Kavarizadeh et al. (2011) and Kavarizadeh et al (2013) support the related result of our study in Roffaye. It is assumed that the presence P. alexandri in the present study is accordance to the second explanations of Rassi and Hanafibojd (2006) which indicated this species was fitted to live in the regions with high percentage of relative humidity and warmer niches, because Roffaye meets both mentioned environmental conditions. Sergentomyia sintoni has been reported from different parts of Iran(Azizi et al, Fig. 2 : Comparison of species in outdoor and indoor resting places of study 2010).However, they were with many other area, Rofayyeh city, Khuzestan province. species of Sergentomyia, but in the current study only this species has been reported. It is Table 1 : The sex ratio of the sandfly specimens in outdoor and assumed that this finding has come from small sampling indoor resting places, Rofayyeh area of Khuzestan of the current study and restriction of seasonal sampling. province. Moreover, P. papatasi was dominant sand flies which Sex ratio Species No. were collected in indoor resting places during the present Outdoor Indoor study. It shows that P. papatasi is a predomestic, P. papatasi 1071 45.98 28.27 endophilic and endophagic species. Therefore, elimination P. caucasicus 113 196.55 not calculated of P. papatasi population in this area is a recommended P. mongolensis 46 not calculated 0 health protocol as Khuzestan is recognized as an endemic focus of ZCL.However, control important of this species P. alexandri 190 26.47 50 is not only due to transmission of zcl , but also reffered to S. sintoni 117 40.96 not calculated transmission of sandfly fever as an arbovirus disease in middle east (Colacicco-Mayhugh et al, 2010).In this term (69.68%) and then P. alexandri (12.36%). Findings of control population of ph.alexandri is also important the current study is supported by that of Jahanifard et al. because this sandfly is recognized as vector of VL (2009), Kavarizadeh et al. (2011) and Kavarizadeh et al (Colacicco-Mayhugh et al, 2010). (2013) who found these two species as the most frequent samples in two large marshlands of Khuzestan province, Finally, it is requested with respect to two forms of Maleh area of Shoush (Khuzestan province) and Musian cutaneous and visceral leishmaniasis in Iraq, existence of district of (South west of Iran), respectively. P. papatasi in this country, having border line and similar weather to the south west of Iran and reporting natural P. papatasi which normally prefers to live in plains leptomonad infection in P. papatasi and P. alexandri area rather than in mountains (Belen et al., 2004, Rassi (Javadian and Mesghali 1974), more epidemiological and Hanafi-bojd, 2006), has been collected from all parts investigation is needed to determine vectors and reservoirs of Iran including Rofayyeh (10 m above level of sea) of disease in this area, using molecular method. from both indoor and outdoor places. In the current study this species was collected from Rofayyeh area where ACKNOWLEDGMENTS the maximum and minimum temperature were recorded We offer our sincerest gratitude to Infectious and 44.3°C in July and 31.7°C in September, respectively(I.R Tropical Diseases Research Center of Ahvaz Jundishapur OF IRAN METEOROLOGICAL ORGANIZATION University of Medical Sciences and Razi Vaccine and (IRIMO,2013).From both ecologic points including sea Serum Research Institute in Ahvaz for financial level and temperature of environment,it is concluded that supports.We also would like to express the deepest this species has a wide ecological valance. appreciation to Mr. Habibzadeh and Mr. Bahrani who However, Rassi and Hanafi-bojd ( 2006) have helped us throughout this survey. appointed P. alexandri as a rare species in different REFERENCES areas of Iran including plain and highlands , our findings Akhavan A A, Yaghoobi-Ershadi M R, Hasibi F, Jafari R, Abdoli H, of the current study showed that it was the most frequent Arandian M H, Soleimani H, Zahraei-Ramazani A R, Mohebali Identification of sand flies 673 M and Hajjaran H (2007)Emergence of cutaneous leishmaniasis Nadim A and Faghih M A (1968) The epidemiology of CL in due to Leishmania major in a new focus of southern Iran. 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