J Arthropod-Borne Dis, June 2013, 7(1): 1–7 AR Zahraei-Ramazani et al.: Sand Flies of the… http://jad.tums.ac.ir Published Online: April 10, 2013 Original Article Sand Flies of the Subgenus Adlerius (Diptera: Psychodidae) in an Endemic Focus of Visceral Leishmaniasis and Introduction of Phlebotomus (Adlerius) comatus as a New Record for Iran Ali Reza Zahraei-Ramazani 1, Dinesh Kumar 1, *Mohammad Reza Yaghoobi-Ershadi 2, Abdollah Naghian 2, Reza Jafari 3, Mohammad Reza Shirzadi 4, Hamid Abdoli 3, Hassan Soleimani 5, Niloofar Shareghi 3, Maryam Ghanei 3, Mohammad Hossein Arandian 3, Ahmad Ali Hanafi-Bojd 2 1Department of Zoology, Centre of Advanced Study, Banaras Hindu University, India 2Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 3Esfahan Health Research Station, National Institute of Health Research, Esfahan, Iran 4Communicable Disease Management Center, Ministry of Health and Medical Education, Tehran, Iran 5Yazd Health Research Station, National Institute of Health Research, Yazd, Iran (Received 12 Dec 2012; accepted 22 Jan 2013) Abstract Background: Sand flies of subgenus Adlerius has a wide geographical distribution in Iran and are mostly found in wild form in mountainous areas. They are always considered as probable vectors of visceral leishmaniasis. The ob- jective of this study was to determine the Adlerius species and its composition in an endemic focus of zoonotic vis- ceral leishmaniasis in northwest of the country. Methods: Sand flies were collected from 6 different areas of Azarbaijan-e-Sharqi Province using sticky paper traps from August to September which is active season for sand flies in this area, in 2009. The flies were mounted and identified. The length of third antennal segments, ascoid, labrum, coxite, surstyle, style, aedeagus, genital filament, genital pump, width of style, and the end of aedeagus were measured and the number of costal hairs group was also counted as the morphological characters. Results: A total of 30 adult sand flies, (26 males and 4 females) including Phlebotomus halepensis (46.8%), P. longiductus (13.3%), P. balcanicus (23.3%), P. comatus (3.3%), and Adlerius spp. (13.3%) belong to subgenus Adlerius were identified respectively in 6 counties. One P. comatus male was captured in front of a cave located in the hillside of a mountain covered with the vegetation in Varzeqan area. Conclusion: The presence of at least 5 species of the subgenus Adlerius in Azarbaijan-e-Sharqi Province, an en- demic focus of zoonotic visceral leishmaniasis in Iran, shows that the risk of parasite transmission among man and reservoir animals is high during the active season of sand flies. P. comatus is a new record for Iran and needs to be added to the list of Iranian phlebotomines of subgenus Adlerius. Keywords: Phlebotomus (Adlerius) comatus, Phlebotomine sand flies, Visceral leishmaniasis, New species, Iran Introduction Visceral Leishmaniasis (VL) is a fatal proto- zoan disease caused by Leishmania donovani complex (Kinetoplastidae: Trypanosomatidae) transmitted by the bites of phlebotomine sand flies (Diptera: Psychodidae) of the genus Phlebotomus in The Old World and Lutzomyia in The New World. It is a serious illness that gives rise to epidemics and causes high mor- tality if left untreated. Visceral leishmaniasis appears as an opportunistic disease associated with the HIV infection and in other immune suppressed patients (Desjeux and Alvar 2003). *Corresponding author: Dr Mohammad Reza Yaghoobi-Ershadi, Email: yaghoobia@tums.ac.ir 1 J Arthropod-Borne Dis, June 2013, 7(1): 1–7 AR Zahraei-Ramazani et al.: Sand Flies of the… http://jad.tums.ac.ir Published Online: April 10, 2013 Leishmania infantum Nicolle, 1908 can also cause cutaneous lesions (WHO 2010). There are at least 7 endemic foci of Zoonotic Viscer- al Leishmaniasis (ZVL) in Iran as follows: In Iran, some parts of Fars Province in the south and Ardabil Province in the northwest are indicated as the highly endemic areas while some parts of Azarbaijan-e-Sharqi Province in the northwest, Saveh and Qom in the cen- tral, Khorramabad area of Lorestan Province in the west, some parts of Khuzestan Province in the southwest and Khorassan-e-Shomali area in the northeast are known as the low en- demic areas. In all foci, kala-azar is caused by L. infantum. Dogs, foxes and Jackals are main reservoir hosts (Yaghoobi-Ershadi 2012). Kala-azar is endemic in four counties of Azarbaijan-e-Sharqi province including Ahar, Sarab, Kaleybar and Azarshahr. Five species of sand flies including P. kandelakii, P. perfiliewi, P. keshishiani, P. neglectus and P. alexandri are considered as probable vector species of ZVL in Iran (Yaghoobi-Ershadi 2012). The subgenus Adlerius Nitzulescu includes about 20 species which some are suspected or proven vectors of ZVL in the old world, as follows: Phlebotomus balcanicus in Armenia and Georgia, P. longiductus in China, Kazakhestan, Kyrgyzstan, Ukrine and Uzbekistan, P. halepensis in Syrian Arabic Republic and Georgia (WHO 2010). The main key to species identification of subgenus Adlerius of Iran was prepared by Seyedi-Rashti and Nadim (1992). They be- lieved that females of the subgenus are uni- dentifiable. So the key is based on male mor- phology as follows: 1- Coxite with less than 29 hairs. Subterminal tubercle of aedeagus 19–28 μm from tip. Aedeagus short and thick. P. brevis - Coxite with more than 30 hairs. Aedeagus long. 2 2- Aedeagus with rectangular subterminal notch, coxite with 50 hairs or less (39– 40). P. halepensis - Subterminal tubercle of aedeagus not rectangular. Coxite with more than 50 hairs. 3 3- Coxite with 50–80 hairs. Genital fila- ments very long 8–15 times length of pump. P. longiductus - Coxite with 69–105 hairs. Genital filament 8 times length of pump. P. balcanicus In the last 3 decades ZVL has become an endemic disease in Azarbaijan-e-Sharqi Prov- ince, northwest of the country. The objective of this study was to determine the Adlerius species and their population size in an endemic focus of ZVL in northwest of the country. Materials and Methods Study area The Azarbaijan-e-Sharqi Province is lo- cated in the northwest of Iran (38.0766 o N, 46.2800 o E) bordering Armenia and the Re- public of Azerbaijan countries and the prov- inces of Ardabil, Western Azarbaijan, and Zanjan (Fig. 1). It covers an area of ap- proximately 47830 km2, with altitude range of 1310 m in plain locale to 3722 m in highland locale. It has a cool and dry climate being in the main a mountainous region. Temperatures run between -17 °C in the winter and up to 40.6 °C in the summer. The annual precipitation is about 255 mm. It has a population of 3691270 people (SCI, 2006). Sand flies collection Sand flies were collected monthly from 6 dif- ferent areas including Tabriz, Ahar, Sharabian- Esmaeilabad, Varzeqan, Kaleibar and Marand in Azarbaijan-e-Sharqi Province using 50 sticky paper traps (castor oil coated white papers 15×21 cm) from the beginning (August) to the end (September) of the active season in 2009. Traps were installed before sunset and collected before sunrise. Collected sand flies 2 J Arthropod-Borne Dis, June 2013, 7(1): 1–7 AR Zahraei-Ramazani et al.: Sand Flies of the… http://jad.tums.ac.ir Published Online: April 10, 2013 were removed from sticky papers using nee- dle dipped and stored in 75% ethanol. Females of sand flies belong to the subgenus Adlerius are more or less morphologically identical and there is no key for identification so only male specimens were selected for morphological study (Seyedi-Rashti and Nadim 1992). Mounting The head and genitalia of individual male sand flies were cut off within a fresh drop of sterile saline on a clean slide then they were transferred to a drop of puri’s media between a slide and cover slip (Smart et al. 1965). Identification of specimens was carried out after 24 h using the keys of Theodor and Mesghali 1964, Artemiev 1973, Lewis 1982, Seyedi-Rashti and Nadim 1992. Morphometric measurements Morphometric measurements were done by Olympus microscope (CH-2) and photographs were taken by camera of Leica microscope (Model: DM 2000). The specimen characters on the length of third antennal segments, ascoid, labrum, coxite, surstyle, style, aedeagus, genital fil- ament, genital pump, and also the width of style were measured with different magnifi- cations. In addition, for the P. comatus male, the end of aedeagus was measured. Number of costal hairs group, a very important mor- phological character, for all Adlerius speci- mens were counted. The entire given meas- urements are in micrometer (μ.m). Results Adult sand flies belong to genus Phlebotomus were collected and identified. Males were sig- nificantly larger numbers than females from the area sampled (30:4). Thirty four Phlebo- tomus sand flies belong to the subgenus Ad- lerius (30 males and 4 females) were identified. Phlebotomus halepensis (46.8%), P. longiduc- tus (13.3%), P. balcanicus (23.3%), P. comatus (3.3%), and females of Adlerius spp. (13.3%) have been captured during this study in 6 areas of the Azarbaijan-e-Sharqi Province (Table 1). Phlebotomus halepensis was present in the all areas. The relative abundance of this spe- cies was 46.8%, higher than of the other spe- cies in comparison. The most captured Adlerius specimens, including P. halepensis, P. longi- ductus and P. balcanicus, were related to Varzeqan area. The altitudinal distribution and abundance of all Adlerius species collected from the study areas are shown in Table 1. Phlebotomus halepensis was found at an altitude range of 1200–1670 m, P. longiductus was captured from two different altitudes at 1200 and 1670 m, while P. balcanicus and P. comatus were only trapped at 1670 m. As the table shows different species of subgenus Adlerius have been collected at altitude of 1670 m in Varzeqan area. It seems that the highest al- titudes are preferred for breeding places of these phlebotomine sand flies. We found only one Phlebotomus comatus male from Varzeqan area, which was a new record for Iran. It was collected in front of a cave located in the hillside of a mountain covered with the vegetation in Varzeqan area. The humidity was measured as normal (60%). Table 2 shows the measurements of the spe- cies. In this specimen, the coxite is wide with dense group of about 176 hairs. The hairy spots are located almost the middle of the coxite. The paramere is pigmented at the end. Aedeagus with subterminal tooth at 23 μ.m from the end. The length of genital filament is 931 μ.m and F/P=6.5. Figure 2 shows pho- tos of some important characters on the head eg, antennal segment 3, clypeus, epipharynx and palp and also the genitalian parts at the end of the abdomen eg, genital filament, genital pump, length and width of aedeagus, dense group of 176 hairs on coxite and pigmented paramere. 3 J Arthropod-Borne Dis, June 2013, 7(1): 1–7 AR Zahraei-Ramazani et al.: Sand Flies of the… http://jad.tums.ac.ir Published Online: April 10, 2013 Table 1. Sand flies of subgenus Adlerius and their relative abundance (%) in 6 areas of Azarbaijan-e-Sharqi, an endemic province of visceral leishmaniasis in northwest of Iran Row Area Altitude* Species of subgenus Adlerius (male) P. halepensis P. longiductus P. balcanicus P. comatus Adlerius sp. (Female) Total No. % No. % No. % No. % No. % No. 1 2 3 4 5 6 Tabriz Ahar Mianeh Varzeqan Kaleibar Marand 1200 m 1367 m 1300 m 1670 m 1240 m 1334 m 1 3 1 6 2 1 25 100 25 40 100 50 3 0 0 1 0 0 75 0 0 6.7 0 0 0 0 0 6 0 1 0 0 0 40 0 50 0 0 0 1 0 0 0 0 0 6.7 0 0 0 0 3 1 0 0 0 0 75 6.6 0 0 4 3 4 15 2 2 Total 14 46.8 4 13.3 7 23.3 1 3.3 4 13.3 30 *(Statistical Center of Iran, 2006) Fig. 1. Map of Azarbaijan-e-Sharqi province, showing sand fly sampling areas 4 J Arthropod-Borne Dis, June 2013, 7(1): 1–7 AR Zahraei-Ramazani et al.: Sand Flies of the… http://jad.tums.ac.ir Published Online: April 10, 2013 Table 2. The comparison of morphometric measurements (μ.m) on males of Phlebotomus (Adlerius) comatus from Iran and Afghanistan Country Iran Present study Afghanistan (Seyedi- Rashti and Nadim) * Number of Coxal setae 176 - Ascoid 3 Length 49 - Labrum Length 270 270–300 Clypeous Length 180 - Cibarium teeth Unarmed Unarmed Palp1 Length 60 - Palp2 Length 175 - Palp3 Length 190 - Coxite Length 450 420 Surstyle Length 450 - Style Length 220 200 Style width 50 - Aedeagus Length 170 - Aedeagus End Subterminal tooth - A3 Length 399 310–370 Genital Filament 931 - Genital Pump 144 - *(Seyedi-Rashti and Nadim 1992) Fig. 2. Phlebotomus (Adlerius) comatus Artemiev male: 1A, Antennal 3(×400), 1B, Pharynx and pharyngeal armature (×400), 1C, Epipharynx (labellum, maxilla) and palp (×400), 1D, Genital filament, genital pump, Surstyle (×100), 1E, dense group of 176 hairs on coxite (×1000), 1F, Length and width of aedeagus, paramere (×630), 1G, Style and the seta(×400), 1H, Pigmented paramere (×400), 1I, Clypeus(×400) 5 J Arthropod-Borne Dis, June 2013, 7(1): 1–7 AR Zahraei-Ramazani et al.: Sand Flies of the… http://jad.tums.ac.ir Published Online: April 10, 2013 Discussion Most species of the subgenus Adlerius were considered as subspecies of Phlebotomus chinensis (Lewis 1978). Theodor and Mesghali (1964) supposed that some of the subspecies may have to be made into species. After the examination of numerous specimens from Af- ghanistan, central Asia and Caucasus it was concluded that the supposition of these sci- entists was right (Artemiev 1973). Males of all described Adlerius species from Iran can be easily identified according to an- tennal formula, position of the coxal tuft and its number of setae and shape of aedeagus, but females are much more uniform. Prior to this study six species including P. halepensis, P. brevis, P. longiductus, P. balcanicus, P. turanicus and P. salangensis were recorded from Iran (Seyedi-Rashti and Javadian 2008, Akhundi et al. 2012). Whether P. kabulensis has also been recorded in the past (Kasiri et al. 2000) but it has not been confirmed by senior entomologists in the country yet. Some morphomethric measurements have been pro- cessed to characterize another new species for the list of phlebotomine sand flies of Iran (Table 3). Artemiev revised the Adlerius of Afghanistan in 1978. He caught and iden- tified 44 males of P. comatus from Northern and Central Afghanistan including Kala-i- Naw (Badghis), Khulm, Bamyan and Gorband valley. He mentioned that it is a species of rocky mountain (1000–2600 m) and it seems to be moderately thermophilic and hydro- philic. He described that male of P. comatus has antennal formula 2/3–5, 1/6–15, coxite very wide, with dense group of 165(126- 203) hairs, distal border of the hairy spot at 0.47 (0.41–0.51) of coxite, paramere with wide pigmented end, aedeagus rather short, with subterminal tooth a 20 (16–24) μ.m from the end, Genital filaments 1040 (800–1280) μ.m long, F/P=8.2 (6.9-9.4). Seyedi-Rashti and Nadim (1992) mentioned that in Afghan specimens of P. comatus, the measurements are as follows: the length of antennal segment 3, 310–370 μ.m, labrum 270–300 μ.m, coxite 420 μ.m and style 200 μ.m (Table 3). The Iranian male specimen of P. comatus has 176 numbers of coxal hairs. It is in the range record numbers of the Afghan speci- men by Artemiev in 1978. Antenna 3 ob- served to be longer than flies recorded by Seyedi-Rashti and Nadim (1992) but labrum is in the range record of the latter authors. The length of coxite is larger and style length is also bigger than which was de- scribed by Seyedi-Rashti and Nadim in 1992. According to the literature P. halepensis was recorded by Theodor and Mesghali from Tabriz county, Azarbaijan-e-Sharqi Province in 1964 and from Sarab county by Mesghali in 1961. During an entomological study of visceral leishmaniasis P. halepensis, P. brevis, P. longiductus and P. balcanicus were rec- orded in Kaleybar county at the same prov- ince in 2000 (Rassi et al. 2000). Our captures and identifications showed that P. halepensis, P. longiductus and P. balcanicus are com- mon species in this province. Phlebotomus brevis which had recorded from the area previously was not found in this study. Phlebotomus halepensis and P. brevis have wide distribution in Iran at alti- tudes between 800 and 1700 m above sea level mostly in mountainous regions. Phle- botomus longiductus and P. balcanicus have limited distribution in comparison of P. halepensis and P. brevis in the country. These four species are present in sympatry in the northwest of Iran (Ardehali et al. 1995). We caught the Iranian specimen of P. comatus from Varzeqan area in elevation of 1670 m. Its coxite is also wide, paramere with wide pigmented ends. In comparison with other species aedeagus is short with sub- terminal tooth at 23 μ.m from the end and it is in the range record numbers of Artemiv’s 6 J Arthropod-Borne Dis, June 2013, 7(1): 1–7 AR Zahraei-Ramazani et al.: Sand Flies of the… http://jad.tums.ac.ir Published Online: April 10, 2013 specimens. The ratio of F/P in Iranian spec- imen is also in the range of his record from Afghanistan. It should be mentioned that Varzaqan is the mining center for base metal mining as well as gold and farming is the predominant oc- cupation in the rural areas. Recently Spruce trees have also been planted as the start of a forestry industry at the same place. More investigations are needed to reveal the distribution of P. comatus in Iran and its role in the transmission of L. infantum in the study area remains to be specified. Acknowledgments Authors thank the leishmaniasis staff of Es- fahan, Meshkinshahr and Yazd health re- search stations, National Institute of Health Research for their contribution in sand fly collections. The study was funded by Deputy of Research of Tehran University of Medical Sciences through project number: 5146-27- 01-86. The authors declare that there is no conflict of interest. References Akhundi M, Parvizi P, Baghaei A, Depaquit J (2012) The subgenus Adlerius Nitzulescu (Diptera: Psychodidae, Phlebotomus) in Iran. Acta Trop. 122: 7–15. Ardehali SV, Rezaei HR, Nadim A (1995) Leishmania Parasite and Leishmaniasis. Academic Press Center, Tehran, Iran, pp. 208 (in Persian). Artemiev V MM (1973) Sand flies (Diptera, Psychodidae, Phlebotomine) of Afghan- istan, Malaria and Leishmania Insti- tute, Afghanistan. 174 pp. Desjeux P, Alvar J (2003) Leishmania/HIV co-infections: epidemiology in Europe. Ann Trop Med Parasitol. 97(1): S3–S15. Kasiri H, Javadian E, Seyedi-Rashti MA (2000) Liste des phlebotomes (Diptera: Psychodidae) d’ Iran. Bull Soc Pathol Exot. 93(2): 129–130. Lewis DJ (1978) The Phlebotomine sand flies (Diptera: Psychodidae) of the ori- ental region. Bull Br Mus Nat Hist (Entomol). 37(6): 434 pp. Lewis DJ (1982) A taxonomic review of the genus Phlebotomus (Diptera: Psycho- didae). Bull Br Mus Hist (Ent). 45(2): 121–209. Rassi Y, Javadian E, Abai MR, Naghian A, Fallah A (2000) Fauna of sand flies in northwest of Iran. Hakim Res J. 3(1): 11–18. Seyedi-Rashti MA, Nadim A (1992) The Genus Phlebotomus (Diptera: Psycho- didae: Phlebotominae) of the countries of the Eastern Mediterranean Region. Iranian J Publ Health. 21(1–4): 11–50. Seyedi-Rashti MA, Javadian E (2008) Vectors of human leishmaniasis in Iran, sand flies of genus Phlebotomus. In: Nadim A, Javadian E, Mohebali M and Zamen- Momeni A (Eds) Leishmania Parasite and Leishmaniasis. Academic Press Cen- ter, Tehran, Iran. pp: 162–176 (in Per- sian). Smart J, Jordan K, Whittick RJ (1965) Insects of Medical Importance. 4th ed. British Museum, National History, Oxford, Adlen Press. 286–288. Theodor O, Mesghali A (1964) On the phle- botominae of Iran. J Med Entomol. 1: 285–300. World Health Organization (2010) Control of Leishmaniasis. Technical Report Se- ries 949 of WHO Export Committee. Geneva. 186 pp. 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. 7