Leishmaniasis is a group of protozoan disease caused by the members of genus Leishmania, and transmitted by Phlebotomine (Diptera: Psychodidae) sand flies. Vector arthropods such as sand flies have great importance due to their transmission ability of pathological agents from/to humans and animals. In Turkey, L. infantum Nicolle, 1908 (Kinetoplastida: Trypanosomatida) is the main causative agent for visceral (VL), while cutaneous (CL) caused by L. tropica, L. infantum, and L. major Yakimoff and Schokhor, 1914 are the main causative agents for CL. Recently, L. donovani Ross, 1903 was also reported as a causative agent of CL in Turkey (Özbilgin et al. 2017). Previous studies related to sand fly fauna in Turkey were conducted in more than 45 provinces and 28 sand fly species belonging to Phlebotomus and Sergentomyia genera were identified (Kasap et al. 2019). At least 18 of these species are proven or probable vectors of human/canine leishmaniasis in the Old World (Volf et al. 2002, Sadlova et al. 2003, Yaman and Özbel 2004, Ertabaklar et al. 2005, Toprak and Özer 2007, Svobodova et al. 2009, WHO 2010). Phlebotomus tobbi is the proven vector of CL caused by L. infantum in East Mediterranean parts of Turkey. Phlebotomus sergenti and P. similis Perfil'ev, 1963 are the probable vectors of L. tropica in south-eastern and western parts of Turkey, respectively (Alptekin et al. 1999, Depaquit et al. 2002, Svobodova et al. 2009, WHO 2010).

Phlebotomine sand flies are biological vectors harboring different developmental stages of Leishmania parasites from amastigotes to infective metacyclic promastigotes. The detection of these stages by microscopical examination of the midguts is an important evidence for the vectorial status of a particular species in an endemic area (Killick-Kendrick 1990). The biggest advantage of this technique is isolation of the parasite strain by culture; however, the midgut dissection takes relatively long time and needs skilled personnel, and limited in sensitivity. PCR methods targeting different gene regions are also in use for detecting the Leishmania DNA in sand flies. As these techniques are not able to determine the developmental stages of the parasite in sand fly, the use of these methods is not sufficient to determine vectorial role of the sand fly. However, positive result in PCR indicates the parasite circulation and natural infection in wild caught sand flies in a particular study area.

Cutaneous leishmaniasis has posed a public health problem in Mersin province. In total, 420 (66.9%) out of 627 cases were recorded in three towns of Mersin between 1994 and 2001; Anamur (36.5%), Mut (26.1%), and Silifke (4.3%) (Baz et al. 2002, Kokturk et al. 2002, Ministry of Health [MoH] 2014). Additionally, 1,578 CL cases were also recorded between 1990 and 2010 (Gürel et al. 2012). Therefore, we aimed to carry out an entomological survey in endemic areas for CL in Mersin province for the determination of sand fly fauna and possible vector species.

Materials and Methods

Study Area

The present study was carried out in eight villages belonging to three towns in Mersin province where CL cases had been reported over the past 10 yr; Anamur (Çarıklar, Malaklar), Mut (Ortaköy, Kelce, Balabanlı, Topluca), and Silifke (Keben, Ortaören) (Table 1). Mersin province is 15.853 km² in area, and it is a major port city located in the southern part of Mediterranean coast (latitude 36°48′0″N, longitude 34°38′0″E; (Fig. 1). The province has 321 km coastline and flatland along the coast, but the mountainous area covers 87% of the land. The altitude is changing between sea level and >3,500 m. The coastlines of the province usually have Mediterranean vegetation while there are forests in higher areas. The climate is typical of the Mediterranean; very hot and very humid in summer, warm and wet in winter. In total, 65 small mammal or rodent species including 6 Meriones species that are well-known reservoirs especially for L. major were reported in Turkey. Beside Meriones species, endemic species Acomys cilicicus and Apodemus mystacinus were recorded in Mersin province (Çetintaş et al. 2017).

Table 1.

The villages and towns selected as study area in Mersin province

Results

Sand Fly Fauna and Midgut Dissection

Throughout the field works, a total of 912 sand fly specimens were captured using 52 CDC light traps in three towns of Mersin province where 637 CL leishmaniasis cases were reported between 2005 and 2011. The 90% of trap locations were open and closed animal shelters. In total, 9 Phlebotomus species, P. sergenti 30.15%, P. alexandri 18.20%, P. neglectus/syriacus 12.06%, P. tobbi 11.73%, P. papatasi 10.20%, P. simici Nitzulescu, 1931 3.18%, P. halepensis Theodor, 1958 0.55%, P. mascittii Grassi, 1908 0.88%, P. brevis, Theodor & Mesghali 1964, 0.11%, and three Sergentomyia species, S. minuta 11.29%, S. dentata Sinton 1933, 1.21%, S. fallax Parrot, 1921 0.44%, were identified in the study area (Table 2 and Fig. 2). Phlebotomus sergenti was found as dominant species in the province level. Phlebotomus sergenti, P. alexandri, and S. minuta were identified as dominant species in Mut, Silifke, and Anamur, respectively. The male/female ratio was 1.19 in the total capture.

Fig. 1.

The location of Mersin province in Turkey and geographical towns of the province (the study area was shown in red color, for color figure refer online version).

Table 2.

The sand fly species identified in the study area

The midguts of 81 female sand fly specimens were checked microscopically for natural Leishmania infection, and no promastigotes were detected.

In Mut town, P. sergenti was found as dominant species with 45.25%, while P. tobbi (12.5%), P. alexandri (12.32%), and P. papatasi (11.79%) were other dominant species. All nine species detected in this study were only present in Mut town. In Silifke town, the ratio of P. sergenti was only 8.27% and P. alexandri was the dominant species with 55.17%. In Anamur town, S. minuta was the dominant species with 42.78% while P. neglectus/syriacus (21.39%) and P. tobbi (16.04%) were second and third dominant species (Table 2).

Discussion

Sand fly fauna studies have been mainly performed in both cutaneous and visceral leishmaniasis endemic areas in Turkey. Mersin province has divided into 13 administrative towns and 627 CL cases were reported from 10 towns between 1994 and 2001. In total, 420 (66.9%) out of 627 cases were recorded in three towns; Anamur (n = 229; 36.5%), Mut (n = 164; 26.1%), and Silifke (n = 27; 4.3%) (Baz et al. 2002, Köktürk et al. 2002). Additionally, 1,578 CL cases were also recorded between 1990 and 2010 (Gürel et al. 2012). For this reason, to detect sand fly fauna and better understand the possible vector species, these three towns were selected as the study area and sand fly collections were carried out in three different time intervals between 2010 and 2011.

Fig. 2.

The sand fly species collected in three towns.

Fig. 3.

Melting curves of (a) L. tropica positive control, (b) pool 2 and (c) pool 6. (Arrows indicate the pick of sample).

Fauna studies, in Turkey, revealed 23 and 5 species belonging to Phlebotomus and Sergentomyia genera, respectively. In the present study, 9 Phlebotomus and 3 Sergentomyia species were identified and Mut town had the richest species diversity among three areas. In Turkey, sand fly species belonging to Larroussius (Diptera: Psychodidae) subgenus such as P. tobbi have usually seen in the regions that having Mediterranean climate and species belonging to Adlerius Nitzulescu (Diptera: Psychodidae) subgenus such as P. halepensis, usually seen in higher altitudes. Mut district resembles the middle of a bowl between the rising mountains. Although the Mediterranean climate is observed in Mut district, the climate is terrestrial due to the mountainous and hilly terrain structure as it goes toward the inner parts. Two villages selected within Mut town are in almost 500 m altitudes while the other two are around 200 m altitude. The proven vector species of L. tropica is P. sergenti and was found to be dominant with 45.2% in Mut town followed by, P. tobbi (12.5%), which is the proven vector species of L. infantum (Svobodova et al. 2009) and P. halepensis (12.3%). Besides P. sergenti, P. halepensis is also probable vector species for L. tropica (Sadlova et al. 2003, Kavur et al. 2018). The presence of the members of both subgenera can be increased the possibility of transmission of the parasite among people. The high number of the CL cases reported between 1994 and 2001 (Köktürk et al. 2002) is also supporting this phenomenon. Since the causative Leishmania species for CL in the study area is not known, we can only speculate on the basis of Leishmania species seen in neighboring provinces such as Adana and Antalya, where the agents are found as L. tropica and L. infantum in human (Svobodova et al. 2009) as well as in dogs (Balcıoğlu İC et al. 2009, Karakuş et al. 2015).

Table 3.

The details about 29 sand fly pools and real-time ITS1 PCR results

In Silifke town (around 200 m a.s.l), P. alexandri that is reported as vector species of L. infantum and L. donovani in Iran (Azizi et al. 2006) and Xinjiang Uygur Autonomous Region (XUAR) of China (Li-Ren et al. 1986), respectively, was found to be the dominant species with the ratio of 55.17% (Table 2). It can be emphasized that P. alexandri seems to be a permissive vector like other vectors of VL such as P. argentipes (Annandale and Brunetti 1908) (Pimenta et al. 1994). In Turkey, P. alexandri was detected in many CL and VL endemic areas, but it was not reported as dominant species before (Simsek et al. 2007). However, P. sergenti (8.27%) is present in the town and causative agent is not known yet. Although P. alexandri could be probable vector species and causative agent(s) may be L. infantum and/or L. donovani (Özbilgin et al. 2017), new studies on the identification of Leishmania strains and additional entomological surveys are needed to clarify the vectorial status of the species.

Even though similar trapping locations were selected in Anamur town, interestingly S. minuta was detected as a dominant species with 42.7% (Table 2). The second and third dominant species were belonging to Larroussius subgenus, P. neglectus/syriacus (21.3%) and P. tobbi (16.0%), in this area. Anamur town is located in the Mediterranean coast in the westernmost part of Mersin province and sand fly fauna of this area was found to be similar to the neighboring town Gazipaşa where is an endemic area for CL, especially caused by L. infantum (Ser and Çetin 2013). Because of 229 CL cases were reported between 1994 and 2001, we can speculate that P. tobbi can be a vector species and the primer causative agent is L. infantum in this particular area.

The entomological study including midgut dissection was performed in the Cukurova region and resulted in 13 successful isolation of L. infantum from P. tobbi specimens (Svobodova et al. 2009). Then, L. infantum was detected in P. tobbi specimens by microscopic examination and its DNA was determined by real-time ITS1 PCR assay in the survey carried out in a VL and canine leishmaniasis focus in western Turkey (Özbel et al. 2016). In the present study, no promastigotes were found in the midguts of 81 dissected female sand fly specimens. On the other hand, two pools containing P. sergenti specimens in Mut town were found to be positive for L. tropica DNA (Fig. 3). One of the positive pools (no. 2) was containing gravid (n = 3) plus blood-fed (n = 13) plus blood fed/gravid (n = 4) P. sergenti specimens while the other one (no. 6) was containing 10 unfed P. sergenti specimens (Table 3). Although positivity with any kind of PCR is not sufficient for the incrimination as vector species, P. sergenti is a very well-known and proven vector species in many CL endemic areas of the Old World countries (WHO 2010) as well as in Turkey (Demir and Karakuş 2015).

It is hypothesized that L. infantum has two possible cycles in the neighboring province in the east Adana, zoonotic and/or anthroponotic, which needs to be clarified by further studies on other possible vector species (Karakuş et al. 2015). Leishmania tropica transmitted by P. sergenti is a dominant causative agent for CL, and additionally CL cases caused by L. major were also reported from this province recently (Koltaş et al. 2014). For this reason, several Leishmania parasites can also be present in Mersin province because it has a similar climate and vegetation to Adana province.

In the present study, the results showed that P. sergenti is probable vector species for CL transmission in Mersin province, especially in highly endemic Mut town, as well as P. tobbi and P. halepensis can also be involved in the parasite transmission, especially for L. tropica, in the province. For better understanding, further studies related to the causative agent(s) is necessary. The detection of certain or probable vector species can be helpful for applying control measures in particular endemic areas.

In conclusion, this study provides new data on the sand fly fauna of the southern Mediterranean coast of Turkey and the possible vectorial role of P. sergenti in the area. The variation observed in the Phlebotomine fauna composition among the towns are probably depending on geographical differences in the province. Our results showed that the dominant species P. sergenti is a probable vector for CL transmission in Mersin, especially in highly endemic Mut town. Phlebotomus tobbi, P. neglectus/syriacus, and P. alexandri may also be involved in the parasite transmission in the province. The presence of these possible vector species and high frequency of human movements coming from south-eastern region where different Leishmania parasites were identified suggests that more than one Leishmania species may be involved in the transmission of CL in Mersin. In order to apply effective control measures in the study area. Further epidemiological studies related to the identification of causative Leishmania species and vectorial role of P. tobbi, P. neglectus/syriacus, and P. alexandri is needed for better understanding of the transmission dynamics of Leishmania and the detection of possible vector species.