Albeit the short sampling period, the present entomological survey has shed some light on the diversity of sand fly fauna and their preferred habitats in CL endemic highland localities of Tigray, an area not previously surveyed. Ten sand fly species, from two genera, were identified from outdoor habitats during our exploration: four Phlebotomus (P. longipes and P. gibiensis, P. arabicus and P.sergenti) and six Sergentomyia species (S. bedfordi, S. multidens, S. clydei, S. africana, S. wansoni and S.schewetzi). All of the ten species identified here have been observed previously in Ethiopia (9, 19–22). The highest mean sand fly density, maximum species richness and species diversity was encountered in caves and crevices, followed by among rock falls, indicating that these are some of the most preferred sand fly habitats in the region.
Among the four Phlebotomus species identified in this study, three of them (P. longipes, P. sergenti and P. arabicus) are proven vectors of CL in several countries of the old World. While P. sergenti and P. arabicus are the vectors of L. tropica in North Africa and the Mediterranean Basin(23, 24), P longipes is the most important vector of CL caused by L. aethiopica in the highlands of Ethiopia (4). P. longipes has been reported only from the mountainous regions of northeast Africa, including Ethiopia, Kenya and Sudan (25).
In the present study, P. longipes was the predominant species (62.3%) among the Phlebotomus species and the second most abundant one (27.6%) of the entire sand fly collection after S. bedfordi (30.3%). It was collected from all the three sampling sites and represented in all the four outdoor habitats searched. However, it was most prevalent in caves and crevices (68.7%), followed by rock falls (15.4%) in proximity to human residence where rock hyraxes prevailed. This is consistent with several study reports from Ethiopia (3, 4) and Kenya (26–28) (where caves and rocky outcrops in highland cliffs were implicated as the main breeding sites of P. longipes. Rocks and crevices provide shelter for hyraxes, the main reservoirs of CL in Ethiopia. A close association between hyraxes and sand flies in both cliff and tree habitats are well known in Ethiopia. Female sand flies of P. longipes are known to feed on hyraxes and share their habitat (4) where the decaying hyrax manure serves as a source of food for larval development. This intimate ecological association between hyraxes and the two sand fly vectors (P. longipes and P. pedifer) is characteristic of Ethiopian CL and gives a clue on the existence of the disease in any locality, especially in the highlands of Ethiopia(29).
In this study, P. longipes was not encountered inside the sleeping quarters of humans. However, it was collected from animal sheds in the compounds of residents indicating that CL is transmitted in domestic and peri-domestic habitats in the study sites. In other studies, P. longipes is also known to occasionally occur in houses close to the cliffs (9, 30). In peri-domestic settings, sand flies rest in cool, dark and humid corners of animal shelters or human dwellings. Peri-domestic areas also provide ready access to blood-meals and the presence of livestock manure further creates favourable breeding grounds for the sand fly vectors in peri-domestic environments (31, 32).
The other two medically important Phlebotomus species identified in this study were P. sergenti and P. arabicus. P. sergenti is widely distributed in north Africa, the Mediterranean Basin eastwards through Israel, Iraq, Iran, Afghanistan, Pakistan, India and southwards to Saudi Arabia, Yemen and Ethiopian highlands; whereas P. arabicus has been reported from Egypt, Ethiopia, Yemen, Saudi Arabia (18) and Israel (33). In the present study, P. sergenti and P. arabicus, were exclusively collected from caves and crevices from one sampling site only (Kumasubuha subdistrict) where the prevalence of CL was previously reported to be high (37.1% − 2.7% for active lesions & 34.4% for healed lesions) (34). The affinity of P. sergenti and P. arabicus for rocky habitats is well known (17, 19, 20) and this landscape is strongly associated with the presence of rock hyrax populations.
P. sergenti is the confirmed vector of L. tropica throughout North Africa, Middle East and Central Asia (23, 35, 36). In Ethiopia, P. sergenti has been reported from several regions of the country, ranging in altitude from 1230 to 1590 meters above sea level(19). However, it was implicated in CL transmission over three decades ago when promatigotes of L. tropica and L. aethiopica (with new zymodemes) were isolated from specimens collected from eastern Ethiopia (Upper and Middle Awash) (20). Although L. tropica is predominantly considered to be anthroponotic (37), human-to-human transmission is very unlikely in this area, since no cases of CL had been reported from the area until then. It was after many years, that one case of CL due to L. tropica has been reported from the Awash valley (38). Therefore, it was suggested that some wild animals, notably the hyraxes prevalent in the area, might serve as a reservoir of the parasite (20). In Kenya, southern Africa (37) and Israel(33), L. tropica has also been isolated from hyraxes suggesting that the same zoonotic cycle involving the hyraxes might exist in the Awash Valley.
In this study, P. arabicus was the second most abundant (24%) species among the Phlebotomus after P. longipes. P. arabicus has been previously reported from the Langano and Sodere area in the Rift Valley (19) and from the Upper Awash Valley, in northeastern Ethiopia (20). Along with P. sergenti, two infected specimens of P. arabicus were also encountered in the Awash Valley of eastern Ethiopia, despite the failure to isolate in cultures and characterize the parasites. However, since the infected flies come from the same habitat as those of the infected P. sergenti, it was very likely that the parasites could have been either L. tropica or L. aethiopica or both (20). P. arabicus is a proven vector of CL caused by L. tropica in Israel (33) with hyraxes as reservoirs (24). P. arabicus was also found naturally infected with Leishmania-like flagellates in Al Baha, Saudi Arabia(39). Besides, laboratory experiments demonstrated that P. arabicus is a permissive vector, meaning it is susceptible to development of more than one species of Leishmania, including L. major and L. infantum (40). However, the role of P. arabicus and P. sergenti species in the transmission of CL in the present study area or in Tigray region needs further investigation into vector competence.
In this study the genus Sergentomyia dominated both in species diversity and numbers accounting for 56% of the total sand fly collection (n = 393). Sergentomyia species are known to be rich in diversity and numbers in Africa since they tolerate various biotopes and environments (41–43). Sand flies of the genus Sergentomyia are proven vectors of reptilian Leishmania species, non-pathogenic to humans (44). Although generally not considered to be medically important, to date, some Sergentomyia species have been implicated in the transmission of visceral leishmaniasis (VL). For example, S. garnhami in Kenya was suggested as a vector of VL because of its high infection rate with promastigotes in the anterior mid gut (16.4%), and its susceptibility to infection when fed on VL patients(45). Besides, the detection of natural infections of L. major in S. darlingi by molecular methods in Mali suggested the potential involvement of this species in the transmission of zoonotic cutaneous leishmaniasis (ZCL) in the country (46). These findings suggest that attention should also be given to these groups of sand flies, particularly, in some VL and ZCL foci where Sergentomyia species are abundantly present and exhibit anthropophilic tendencies for their potential roles in the transmission of mammalian leishmaniasis. S. multidens and S. clydei recorded in the present study are known to be occasional human biters in Ethiopia as well as elsewhere in Africa (5, 47)