Sand fly (Diptera: Psychodidae) distribution in the endemic and non-endemic foci of visceral leishmaniasis in northwestern Iran
Université de Reims Champagne-Ardenne, Transmission vectorielle et épidémiosurveillance de maladies parasitaires (VECPAR), Université de Reims Champagne-Ardenne, 51096 Reims Cedex, France, firstname.lastname@example.org
Pasteur Institute of Iran, Parasitology Department, Tehran, Iran
Université de Reims Champagne-Ardenne, Transmission vectorielle et épidémiosurveillance de maladies parasitaires (VECPAR), Université de Reims Champagne-Ardenne, 51096 Reims Cedex, France, email@example.com
An entomological study was conducted from June to September, 2010 in rural regions of Azarbayjan-e-sharqi, Azarbayjan-e-qarbi, and Ardabil provinces in northwestern Iran to determine sand fly fauna, diversity, and distribution in different habitats and altitudes using both sticky papers and light traps. Geographical distribution of sand flies and the similarity of populations in different locations were analyzed ecologically based on the Shannon-Wiener Index and Jacard Coefficient, respectively. A total of 3,982 specimens was collected and sixteen species recorded. They belonged to the genera Phlebotomus [subgenus Phlebotomus (P. papatasi), Paraphlebotomus (P. sergenti, P. mongolensis, P. caucasicus, P. jacusieli), Larroussius (P. major s.l., P. tobbi, P. perfiliewi transcaucasicus, P. kandelakii) and Adlerius (P. halepensis, P. brevis, P. longiductus, P. balcanicus)], and Sergentomyia [subgenus Sergentomyia (S. sintoni, S. dentata and S. theodori)]. P. papatasi was the predominant species in all the locations except Bileh Savar, Macu, and Meshkin Shahr, followed by P. perfiliewi transcaucasicus and P. kandelakii. The latter species were caught from different habitats and altitudes with higher frequency than other species of the subgenus Larroussius. The lowest abundance belonged to P. jacusieli. The predominant species of subgenus Adlerius was P. halepensis. Data analysis showed that Meshkin Shahr and Bileh Savar had high and low diversities of sand fly distribution, respectively. Meshkin Shahr and Sarab districts had the highest similarity. Both are located in the foothills of Sabalan Mountain, with high diversity and richness.
Phlebotomine sand flies (Diptera:Psychodidae) are vectors of Leishmania that are responsible for mammalian leishmaniasis (Killick-Kendrick 1990). Several studies in some restricted regions of Azarbayjan-e-sharqi and Ardabil provinces, two endemic foci of visceral leishmaniasis, have been carried out in order to identify the sand fly fauna and probable Leishmania infection. Hazratian et al. (2011) studied the phenology and population dynamics of sand flies in Azar shahr district. To our knowledge, no investigation has been carried out to study and quantify the possible effects of altitude and physical barriers on sand fly populations in these regions. The composition of sand fly populations was also unknown in Mianeh, Maragheh, Poldasht, Chaldoran, and Macu in Azarbayjan-e-sharqi and Azarbayjan-e-qarbi provinces.
Visceral leishmaniasis (VL) is reported sporadically throughout Iran. However, four foci are endemic: two of these are located in the northwest (Ardabil and Azarbayjan-e-sharqi) and two others in southern Iran (Fars and Bushehr) (Nadim et al. 1978). Two species of the subgenus Larroussius, P. kandelakii and P. perfiliewi transcaucasicus, were previously found to transmit VL in northwest Iran, whereas P. keshishiani and P. major were considered to be vectors of VL in the south of the country (Sahabi et al. 1992, Azizi et al. 2008).
The subgenera Larroussius and Adlerius (Diptera, Psychodidae, Phlebotomus) include vectors of both cutaneous and visceral leishmaniasis. They have been defined by Nitzulescu (1931) based on their specific morphological characters. Their type species are Phlebotomus major Annandale and P. chinensis Nitzulescu, respectively. The identification of male Larroussius is based on the morphology of the aedeagus and is usually not very difficult. The females are distinguished by a careful examination of the base of their spermathecal ducts (Leger et al. 1983, Killick-Kendrick et al. 1991). Some closely related species are difficult to identify, like the P. perniciosus complex (Pesson et al. 2004), P. perfiliewi transcaucasicus (Lewis 1982, Artemiev and Neronov 1984), or P. major s.l. (including P. major s. st., P. neglectus, P. syriacus, P. wenyoni, and P. notus). The latter group needs a revision suggested by Léger and Pesson (1987). The subgenus Adlerius includes twenty described species, located mainly in the Middle East. Its origin may be from this area, where its biodiversity is the most abundant (Léger and Pesson 1987). The identification of males is based on the morphology of the genitalia, whereas the females are indistinguishable (Artemiev and Neronov 1984).
In this study, we report the species composition and distribution of sand flies in endemic and non-endemic foci of visceral leishmaniasis in Ardabil, Azarbayjan-e-sharqi, and Azarbayjan-e-qarbi provinces in northwest Iran. Large scale sampling was performed using both sticky papers and light traps from different locations, habitats, and altitudes. The data were analyzed ecologically using the Shannon-Wiener Index, with the aim of investigating and quantifying the possible effects of altitude and physical barriers on the sand fly populations.
MATERIALS AND METHODS
A sampling was carried out from June to September, 2010 in ten counties in an area of approximately 100,503 km2 belonging to three provinces of Ardabil (Meshkin Shahr: Aghbolagh, Dooshanloo, Mizan, Ghassabeh, Uor kandi, Niaz, and Ghurt tapeh), (Bileh Savar: Damirchi, Gog Tapeh, Odlou, Fouladlou, Nazar Blagh, and Goon Papagh), and (Germi: Tazeh Kand, Ghasem Kandi, and Qabaleh Kandi), Azarbayjan-e-sharqi (Sarab: Sanziq, Razliq, Ardahan, Mehraban, and Asbforoushan), (Kaleybar: Abdorazagh, Bashkalan, Gharavanlou, Joaghaj, Kalantar, and Shekhamlou), (Maragheh: Kahaq, Dash Atan, Alavian, Gol Tapeh, and Yengejeh) and (Mianeh: Sheykhdarabad, and Bashmaq) and Azarbayjan-e-qarbi (Macu: Ghal'e Joogh, and Golali), (Chaldoran: Ravaz, and Shadloo) and (Poldasht: Bohlool Kandi, and Poldasht) in northwest Iran (Figure 1). These provinces share international borders with Armenia, Azerbaijan, and Turkey. All districts except Bileh Savar, Poldasht, Chaldoran, Mianeh, and Maragheh are located in the mountainous sites of Sahand, Sabalan, and Ararat mountains. We focused our sampling on rural regions, houses, animal shelters, rodent burrows, plains, mountainous regions, forests, and caves.
Sampling was conducted in 40 villages distributed throughout the three provinces with an altitude of up to 1,830 m above sea level (Figures 1, 2). The climate varies from one locality to another but it is generally hot (about 35–40º C) in summer and cold (−10º C to −27º C) during the winter. The warm season is short in these provinces and normally lasts from mid-May to the end of September. The mean annual rainfall varies from 300–480 mm. Three provinces of Azarbayjan-e-sharqi, Azarbayjan-e-qarbi, and Ardabil are important mountainous areas of the country. However, their topography varied with plains, mountains, pastures, and forests. They constitute a wet part of Iran, often under the influence of the moist air from a Mediterranean climate. Severe chill and snowfall occur in autumn and winter in this coldest part of Iran (Akhani et al. 2010, Fallahchai 2011). The vegetation of these provinces is scattered in the highlands and is mainly in the foothills of mountainous regions. Considering the climate, geographic location, and relatively abundant rainfall, various vegetation types can be seen in these provinces (Akhani et al. 2010, Fallahchai 2011).
The plant cover in northwest Iran is affected by altitude and plains, foothills, and high mountains. Each level has its own vegetation. Alnus glutinosa, Parrotia persica, Shumard Oak, Racosperma mangium and Abies balsamea are frequent plants in the lowlands and on the plains, ranging from sea level to 1,100 m a.s.l. From 1,100 to 2,400 m a.s.l., the tree vegetation includes Acer tataricum, Crataegus ambigua, Crataegus crus-galli, Fraxinus pennsylvanica, and Populus alba. At altitudes above 2,400 m a.s.l., the tree vegetation usually decreases and plant cover consisted of low-growing shrubs and small Downy Birch (Betula pubescens czerepanovii) (Akhani et al. 2010, Fallahchai 2011). Some parts of the regions we studied are forested, particularly in Kaleybar, Germi, Meshkin shahr, and Poldasht counties.
Sand fly collection and identification
During this survey, we selected two trapping methods: sticky papers and CDC miniature light traps. In order to maximize the opportunity to catch all the species from a specific location, the two trapping methods were used concomitantly in different habitats and various biotopes in mountainous regions and plains. They included indoor places (houses, stores) and outdoor places (scuppers, wall cracks, burrows, vegetation, forests, animal shelters, and caves). Sticky papers consisted of 21×30 cm white sheets coated with castor oil. The traps were placed on the ground 1 h before sunset, collected 1 h after sunrise, and placed in the interstices of stone or clay walls, or placed vertically in cracks, crevices, and large boulders. CDC miniature light traps were placed to catch photophilic species alive and well preserved for morphological analysis. All traps were installed before sunset and remained functional until the next morning. A total of 50 sticky papers and three CDC light traps were used equally for sand fly collection at each sampling site.
Sand flies were stored in 96% ethanol. Head and abdominal terminalia of each sand fly were mounted dorsally in a drop of Berlese fluid (Nadim and Javadian 1976, Lewis 1982). Male genitalia were mounted laterally and female spermathecae were dissected before mounting. Our identification followed the key of Artemiev and Neronov (1984) and Killick-Kendrick et al. (1991). The identification of specimens of the subgenus Larroussius showing a long aedeagus rounded at the tip for males and spermathecae having a basal common duct has been considered as P. major s.l., due to difficulties of identification described by Leger and Pesson (1987). The specimens belonging to P. perfiliewi s.l. have been identified as P. perfiliewi transcaucasicus, according to previous records in Iran and the Caucasus in the literature (Lewis 1982, Artemiev and Neronov 1984). No molecular identification was carried out for sample identification.
The data and ecological comparisons among sub-regions in the study areas and different altitudes were analyzed using the computer program SAS JMP Statistical Discovery v8.0.2. The Shannon-Weiner (H), evenness (E), and richness (S) diversity indices of the collected sand flies within different localities were estimated as follows:
Shannon-Wiener: H =Σ Pi log Pi where “s” is the number of species, Evenness: SHEI= H/ln (S) where “H” is the value of Shannon-Wiener Index and “s” is the number of species in sample (simple species diversity). “Pi” is the proportion of total samples belonging to “i-th” species.
Similarity among different habitats was estimated using the Jaccard Coefficient (Sij) formula: Sij = a/(a + b + c), where a is the number of species present in both replicates, b is the number present in replicate i only and c is the number of species present in replicate j.
A total of 3,982 sand flies was caught using sticky papers (2,701 specimens) and CDC light traps (1,281 specimens) (Table 1). In total, 2,217 were males and 1,765 were females, of which 1,401 specimens (761 males and 640 females) belonged to the subgenus Larroussius and 454 (333 males and 121 females) to the subgenus Adlerius, and sampled from different locations and various habitats. Of 418 specimens caught indoors, 253 specimens were found in stores and 91 specimens at the entrance of houses. P. papatasi was the most frequent species among sand flies caught indoors (Figure 3).
Table 1. Species composition and relative proportions of adult sand flies caught by CDC light traps and sticky papers in northwestern Iran between June and September, 2010.
Percentage of total sand fly population
Proportions (numbers) in the two type of traps
P. perfiliewi transcaucasicus
P. major s.l.
P. (Adlerius) sp.
Out of 3,564 specimens caught outdoors, 1,628 and 593 specimens were captured in animal shelters (close to the houses) and rodent burrows, respectively. Also 876 specimens were sampled in mountainous regions including the caves. P. papatasi was the most frequent species among species captured outdoors (Figure 3). Sampled specimens belonged to 13 species of Phlebotomus and three of Sergentomyia. Of the entire collection, 90.8% belonged to the genus Phlebotomus, with only 9.2% of the genus Sergentomyia. P. papatasi was the predominant species in all of the locations except in Bileh Savar (25.4%), Macu (22.6%), and Meshkin Shahr (17.2%), followed by P. perfiliewi transcaucasicus and P. kandelakii. The lowest abundance was observed for P. jacusieli (2.5%). P. perfiliewi transcaucasicus (22.5%) was the predominant species captured by CDC light traps. The majority of species caught by CDC light traps were female and nulliparous (Phlebotomus: 61.8% and Sergentomyia: 53.4%) (Table 1).
The specimens captured by sticky papers show that P. papatasi is the most abundant species in sampled areas (27.2%), followed by P. perfiliewi transcaucasicus (13.1%). The majority caught by sticky papers were males (Phlebotomus: 71.7% and Sergentomyia: 63.2%). The sampling ratio (SP/CDC) showed more of the genus Sergentomyia (4.2) was captured than Phlebotomus (1.98). Sex ratios indicated that males were more frequent than females: 1.3 for the Phlebotomus and 1.1 for the Sergentomyia. The highest number of Larroussius was caught in Macu (51.1%) and Chaldoran (46.9%), and the lowest in Maragheh (12%), whereas many Adlerius were caught in Kaleybar (17.5%) and Sarab (16.3%) and only a few caught in Poldasht (4.2%) (Figure 2). Among Larroussius species, P. perfiliewi transcaucasicus (45.9%) and P. tobbi (5.7%) had the highest and lowest abundances in ‘the sampled sites respectively, whereas P. halepensis (36.3%) and P. balcanicus (5.7%) were the highest and lowest abundant species among Adlerius species. Except for P. jacusieli, all the species have been caught both indoors and outdoors (Figure 3). P. perfiliewi transcaucasicus and P. kandelakii are the Larroussius mostly caught from outdoor places. Concerning Adlerius, the highest and lowest frequent species were P. halepensis and P. balcanicus, respectively.
There were differences in species diversity, as indicated by the values of Shannon-Wiener index (H), evenness (E), and richness of the sand fly fauna among the locations (Table 2). The richness, diversity, and evenness were maximal in Meshkin shahr (H’=2.3; E=0.87; S=14) and Sarab localities (H’=2.25; E=0.85; S=14), whereas the estimated diversity (1.68), evenness (0.73), and richness (10) were lowest in Bileh savar (Table 2). The sand fly community similarity as measured by Jacard's coefficient (IJacard) indicated that the similarity of the sand fly communities was highest (100% of the sand fly communities or IJacard = 1) between Sarab (1,620 m) and Meshkin Shahr (1,830 m) and also between Chaldoran (1,680 m) and Macu (1,750 m). The lowest (40%=0.4) was between Bileh Savar (85 m) and Germi (1,050 m) (Table 3).
Table 2. The Shannon-Weiner diversity index (H), evenness (E) and richness (S) for the sand fly species in different study areas.
Table 3. Similarity of population composition of sand flies captured from different localities of northwestern Iran.
The distribution models of P. papatasi, subgenera Paraphlebotomus, Larroussius, and Adlerius regarding the physical factor of altitude were different. P. papatasi and P. sergenti had a gradually decreasing gradient with altitude in our ten studied locations, whereas P. kandelakii and P. perfiliewi transcaucasicus had an increasing gradient with altitude (Figure 2). P. papatasi, P. kandelakii, P. perfiliewi transcaucasicus, P. halepensis, P. (Adlerius) sp., S. dentate, and S. sintoni were caught in all sites, while P. caucasicus, P. sergenti, P. mongolensis, P. jacusieli, P. major s.l., P. tobbi, P. brevis, P. longiductus, P. balcanicus, and S. theodori were only found in some locations.
Phlebotomine sand flies are abundant and widespread in the three provinces of Ardabil, Azarbayjan-e-sharqi, and Azarbayjan-e-qarbi in northwest Iran (Figure 1). More importantly, all proven or suspected vectors of visceral leishmaniasis that belong to the subgenera Larroussius and Adlerius are also present in these provinces as sympatric species. Several investigations have been carried out in limited regions of Azarbayjan-e-sharqi and Ardabil provinces which are the endemic foci of visceral leishmaniasis in Iran. P. perfileiwi and P. kandelakii were reported as the proven vectors of VL in these districts (Rassi et al. 2009, Oshaghi et al. 2009, Sanei Dehkordi et al. 2011).
Species composition differs from one locality to another and may reflect changes in the environment within the large-scale area. Overall, while P. papatasi, P. perfiliewi transcaucasicus, P. kandelakii, P. halepensis, P. (Adlerius) sp., S. sintoni, and S. dentata were collected from all counties and altitudes, other species were restricted to some sampled localities. Doha and Samy (2010) studied the bionomics of Phlebotomine sand flies in Saudi Arabia. They reported high species richness and diversity at altitudes between 800–1,200 m. They also reported P. papatasi, P. bergeroti, and P. arabicus as the most abundant species both indoors and outdoors and that P. papatasi was found at all altitudes with maximum abundance between 800–1,200 m a.s.l. In the present study, we found P. papatasi in all studied localities in greater numbers than other species both indoors and outdoors (Figure 3). P. papatasi is the main vector of zoonotic cutaneous leishmanisis, often abundant in areas of steppe and semi-arid zones where temperatures are high but humidity is not extremely low (Belazzoug 1991). This species also showed widespread distribution with decreasing altitude gradient throughout our study areas (Figure 2). We observed fewer P. papatasi in Bileh Savar district (85 m) that there would seem to be, based on the high abundance of P. perfiliewi transcaucasicus in this district that causes low evenness (0.73) and richness (10) (Table 2). The high occurrence of P. papatasi in the lowlands and its low frequency in the mountains could be explained by its preference for semi-arid areas (Simsek et al. 2007).
Concerning the subgenus Paraphlebotomus, P. sergenti is the main vector of L. tropica in several countries around the Mediterranean (Jacobson et al. 2003), including Iran, despite a doubtful taxonomic status (Moin-Vaziri et al. 2007). This species showed widespread distribution throughout the study area with lower populations except in Germi County (Figure 2). Seyedi Rashti and Nadim (1992) described P. sergenti as a “mountainous” species. Simsek et al. (2007) reported the presence of P. sergenti at an altitude ranging from 1,132 to 1,385 m a.s.l. in Konya province in Turkey, whereas in this investigation it was more abundant in Maragheh (19%) with an altitude of about 850 m. It had a nearly negative correlation with altitude and was minimal in Meshkin Shahr (1,830 m) (Figure 2).
The distribution model of P. caucasicus has shown the nearly flat-shaped graph between Kaleybar (1,000 m) and Meshkin Shahr (1,830 m). However, it seems that this species is more abundant than other Paraphlebotomus in mountainous regions and it is recorded in all sampled locations except in Bileh Savar (85 m) (Figure 2). Specimens caught in stores and animal shelters constituted a large number of specimens indoors and outdoors, respectively. P. papatasi was the most frequent species both indoors and outdoors. Outdoors, the Larroussius species were more abundant than the Adlerius species in all sampled sites with similar habitats and biotopes (Figure 3).
Among the Larroussius and Adlerius species, P. kandelakii and P. perfiliewi transcaucasicus and also P. halepensis were abundant outdoor species. These first two species are proven vectors of visceral leishmaniasis in northwestern Iran and it seems that these species have more predomestic and anthropophilic preferences than other Larroussius species (Rassi et al. 2005, 2009, Oshaghi et al. 2009). The subgenus Larroussius, P. perfiliewi transcaucasicus, and P. kandelakii follow the same distribution model and were positively correlated with altitude. Their abundances decreased at altitudes over 1,750 m (Figure 2). Distribution of P. (Larroussius) major s.l. is restricted to locations at altitudes above 850 m, with the model of flat form from Germi (1,050 m) to Meshkin Shahr (1,830 m) (Figure 2).
Seyedi-Rashti and Nadim (1992) recorded few P. tobbi in mountainous areas. In agreement, the greatest abundance of P. tobbi was in the Kaleybar district at an altitude of about 1,000 m. It was absent in Germi, Maragheh, Mianeh, Macu, and Chaldoran districts. The distribution of Adlerius species includes both mountains and plains with higher abundances in mountains. The numbers of Adlerius were high in Kaleybar, Germi, Sarab, and Meshkin Shahr with mountainous locations. We also found Adlerius species with high frequencies in the plains of Bileh Savar County. Interestingly, Adlerius species had lower abundances in the plain regions of Maragheh, Poldasht, Mianeh, and Chaldoran (Figure 2).
P. halepensis and P. brevis were reported as the suspected vectors of L. infantum in the south of the former USSR and Kazakhstan, respectively, and we found them with high frequencies indoors in Kaleybar and Bileh Savar (Lewis and Ward 1987, Lesnikova and Sabitov 1995). P. halepensis and P. brevis share a similar distribution and they had higher frequencies in Kaleybar, Germi, Sarab, and Meshkin Shahr in the foothills of Sabalan Mountain (Figure 2). Low numbers of Sergentomyia species were also caught in different locations and various sites. They had less diversity and were restricted to S. dentata, S. sintoni, and S. theodori. Most Sergentomyia species were catured from rodent burrows (44%).
The main advantage of the use of sticky traps is that they are cheap, numerous, and are not attractive. They indicate the relative abundance of the different species. Unfortunately, captured flies are dead and traps are sometimes difficult to use inside houses. The CDC miniature light traps have the main advantage of catching live sand flies; they are easy to use and allow many species attracted by light to be caught. Unfortunately, the latter point is also a bias in sampling. The captures do not reflect the relative abundance of the different species due to the attractivity of the trap.
Sand fly distributions are highly disjunctive within their range, depending on local environmental factors such as precipitation and temperature, physical factors like geographical barriers and habitat availability, and biotic factors such as the distribution and abundance of vertebrate hosts (Cross et al. 1996, Ghosh et al. 1999). This study shows that altitude influences the spatial distribution of sand flies. Although it seems that altitude above sea level is not a selective factor, biotic and abiotic properties of the environment are highly correlated with altitudinal gradients, the most obvious of which is climate (Karan et al. 2000, Simsek et al. 2007). Moreover, the geographical and ecological richness of the region provides numerous adult resting and larval sites for flies. It seems the altitude is one of several important factors that is necessary to predict the sand fly fauna in a region, but it is not a sufficient factor.
The authors are grateful to Sylvette Gobert for proofreading this manuscript. We also thank the staff of health centers in the counties of Bileh Savar, Meshkin shahr, Germi, Sarab, Myianeh, Maragheh, Poldasht, Macu, and Chaldoran in Azarbayjan-e-sharqi, Azarbayjan-e-qarbi, and Ardabil provinces for their help in sampling.