Ecological Survey of the Peridomestic Sand Flies of an Endemic Focus of Zoonotic Cutaneous Leishmaniasis in the South-East of Morocco

Leishmaniasis is a parasitosis caused by parasites of the genus Leishmania and is transmitted by Phlebotominae sand flies. An entomological survey was carried out in different localities of Zagora Province. Our work allowed us to establish an inventory of sand flies to study potential vectors of leishmaniasis and to compare the composition and the specific abundance of different endemic stations. The sand flies were collected using CDC miniature light traps during the month of July 2019 in the ten studied villages. The results indicate the presence of thirteen species, belonging to the genera Phlebotomus and Sergentomyia. Phlebotomus papatasi was the predominant species (46.65%) followed by Ph. alexandri (17%), Ph. longicuspis (11.55%), Ph. bergeroti (1.53%) and Ph. sergenti (1.27%). Phlebotomus kazeruni (0.03%) was rare, and only one female was captured in Ifred. Sergentomyia schwetzi (8.69%) was the most prevalent species in the Sergentomyia genus followed closely by Se. fallax (6.84%). Sergentomyia africana was present with a proportion of (3.86%) followed by Se. clydei (1.96%). Sergentomyia dreifussi (0.46%), Se. antennata (0.08%), and Se. minuta (0.08%) were very limited. Phlebotomus papatasi, Ph. alexandri, Ph. bergeroti, Ph. longicuspis, Ph. sergenti, Se. schwetzi, Se. clydei, and Se. fallax are constant species, being present at least in 50% of the stations (occurrence> 50%). Common species (25%–49%) were Se. minuta and Se. africana and rare species were Ph. kazeruni and Se. antennata with a very limited distribution (occurrence <12%). The greatest species richness was found in Ksar Mougni and Ifred with the occurrence of 11 species, but overall, it was high (>9 species) in most of the villages. The Shannon–Wiener index was high (H′ > 1) in eight localities (Ksar Mougni, Tassaouante, Bleida, ZaouiteLeftah, Ifred, Timarighine, Ait Oulahyane, and Ait Ali Ouhassou). The high value of this index is in favor of the ZaouiteLeftah locality (Shannon–Wiener index = 1.679) which is explained by the presence of a stand dominated by Ph. papatasi. In order to avoid exposure to infections, a good epidemiological surveillance and vector with rodent control measures must be well maintained. Awareness campaigns are also required and must be conducted for better knowledge of the disease.

Leishmaniasis is a parasitosis caused by parasites of the genus Leishmania and is transmitted by Phlebotominae sand fies. An entomological survey was carried out in diferent localities of Zagora Province. Our work allowed us to establish an inventory of sand fies to study potential vectors of leishmaniasis and to compare the composition and the specifc abundance of diferent endemic stations. Te sand fies were collected using CDC miniature light traps during the month of July 2019 in the ten studied villages. Te results indicate the presence of thirteen species, belonging to the genera Phlebotomus and Sergentomyia. Phlebotomus papatasi was the predominant species (46.65%) followed by Ph. alexandri (17%), Ph. longicuspis (11.55%), Ph. bergeroti (1.53%) and Ph. sergenti (1.27%). Phlebotomus kazeruni (0.03%) was rare, and only one female was captured in Ifred. Sergentomyia schwetzi (8.69%) was the most prevalent species in the Sergentomyia genus followed closely by Se. fallax (6.84%). Sergentomyia africana was present with a proportion of (3.86%) followed by Se. clydei (1.96%). Sergentomyia dreifussi (0.46%), Se. antennata (0.08%), and Se. minuta (0.08%) were very limited. Phlebotomus papatasi, Ph. alexandri, Ph. bergeroti, Ph. longicuspis, Ph. sergenti, Se. schwetzi, Se. clydei, and Se. fallax are constant species, being present at least in 50% of the stations (occurrence> 50%). Common species (25%-49%) were Se. minuta and Se. africana and rare species were Ph. kazeruni and Se. antennata with a very limited distribution (occurrence <12%). Te greatest species richness was found in Ksar Mougni and Ifred with the occurrence of 11 species, but overall, it was high (>9 species) in most of the villages. Te Shannon-Wiener index was high (H′ > 1) in eight localities (Ksar Mougni, Tassaouante, Bleida, ZaouiteLeftah, Ifred, Timarighine, Ait Oulahyane, and Ait Ali Ouhassou). Te high value of this index is in favor of the ZaouiteLeftah locality (Shannon-Wiener index � 1.679) which is explained by the presence of a stand dominated by Ph. papatasi. In order to avoid exposure to infections, a good epidemiological surveillance and vector with rodent control measures must be well maintained. Awareness campaigns are also required and must be conducted for better knowledge of the disease.

Introduction
Sand fies are vectors of various pathogens responsible for human and/or animal diseases. Among these diseases, leishmaniasis is a public health problem in Morocco [1]. Tey are endemic in several regions of the country with two major clinical forms: cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL). Cutaneous leishmaniasis caused by Leishmania (L.) tropica, endemic in the center and north of the country, and CL, caused by L. major and is endemic especially in the south and east of the country, are the most widespread diseases [2]. Visceral leishmaniasis caused by L. infantum occurs sporadically throughout the country but is particularly common in the north [3,4]. Te sand fy species responsible for the transmission of each of these forms are diferent; Phlebotomus papatasi is the vector of L. major; Ph. sergenti is responsible for the transmission of L. tropica, while L. infantumis is transmitted by three species Ph. perniciosus, Ph. longicuspis and Ph. ariasi [5]. Until now, 24 sand fy species have been identifed in Morocco, of which 14 species belong to the Phlebotomus genus and 10 to the Sergentomyia genus [6]. Te diversity of sand fies, their abundance, and distribution are infuenced by several geographic and bioclimatic factors. Tus, entomological feld investigations are of great importance for identifying these factors and their impact on each species and especially on the vector species. Tis study is considered as the frst research on the diversity of sand fies in diferent biotopes of Zagora Province which is the most infected focus of zoonotic cutaneous leishmaniasis (ZCL) in Morocco [7]. It aims to compare the composition of sand fies species in ten localities and to identify the main high-risk biotopes due to the presence of vector species.

Study Region.
Te study was conducted in the province of Zagora (30°19′50″ N, 05°50′17'' W) in the south-east of Morocco. Te weather conditions are typical of the Saharan climate, i.e., hot in summer and quite cold in winter. Te mean annual precipitation is 61 mm, and the mean annual temperature is 23°C. A variety of domestic animals that are potential sand fy hosts (sheep, goats, donkeys, chickens, rabbits, cats, and dogs) are present in this area. Ten villages were monitored for the sand fy presence (Figure 1).

Sand Flies Collections.
Te sand fies were collected using CDC miniature light traps (LT) during July 2019 in the ten study villages. July was chosen to conduct this survey since it corresponds to the period when sand fies reach their optimal abundance in Zagora Province [8,9]. Five houses were randomly chosen in each village where two CDC traps were set for three consecutive nights from sunset to sunrise, i.e., one inside (bedrooms) and the other outside (courtyard). In the ten prospected localities, a total of 300 LT were set up during 30 nights of trapping. Captured specimens were transferred into tubes with 90% ethanol and were labeled. Prior to species identifcation, sand fies were cleared in the Marc André solution (chloral hydrate/acetic acid) [10]. Specimen identifcation was individually verifed based on the morphology of the pharynges and/or the male genitalia or female spermathecae, as described by Faraj and Himmi [6]. In a second step, they were counted and separated by sex.
ni is the number of individuals of the given species, and N is the total number of individuals. (vi) Degree of presence: C � n/N × 100, where n is the number of localities where the species was found, and N is the total number of study localities. According to this index, sand fy species were classifed as constant species that were found in 50% or more of the study localities; common species were captured in 25%-49% of the study localities, accidental species were found in 12.5%-24% of the study localities, and very accidental species were captured in less than 12% of the study localities [11]. (vii) Te sex ratio (male/female) was also calculated for each species collected during this study.
We used descriptive statistics with graphics and tables to interpret results. We studied the distribution of sand fies species using Qgis 2.18 software by integrating data on leishmaniasis in the geographic information system.

Analysis of Sand Flies by Ecological
Indices. Te greatest species richness was found in MO and IF with the occurrence of 11 species, but overall, it was high in most of the villages. However, there were diferences in species diversity, as indicated by the values of the Shannon-Wiener index (H′), evenness, and Simpson index among the villages (Table 1).
In order to characterize the population of sand fies captured in the diferent surveyed localities, fundamental ecological parameters were used: total richness (S), Shannon index (H′), and equitability index (E). A low value refects a stand dominated by one species or a stand with a small number of species with a high representativeness. Te Shannon-Wiener index is high (H′ > 1) at eight localities (MO, TAN, BL, FA, IF, TIM, YAN, and OUH). Te high value of this index is in favor of the FA locality (Shannon-Wiener index � 1.679) which is explained by the presence of a stand dominated by Ph. papatasi.
By analyzing the distribution and frequency of species present in the surveyed sites, three groups of species can be distinguished: constant species, that are defned as being present in at least 50% of the stations (occurrence> 50%) were Ph. papatasi, Ph. alexandri, Ph. bergeroti, Ph. longicuspis, Ph. sergenti, Se. schwetzi, Se. clydei, and Se. fallax. Common species, present in 25%-49% of stations were Se. minuta and Se. africana, and rare species(occurrence <12%) were Ph. kazeruni and Se. antennata (Table 3).

Discussion
We conducted this entomological survey in the Province of Zagora, which is a pre-Saharan region in south-eastern Morocco and is considered as an endemic focus of ZCL, where the last outbreak was between 2008 and 2010 which included 4,437 cases [12]. In 2010, Zagora experienced an epidemic Zoonotic Cutaneous Leishmaniasis (ZCL) caused by L. major with 1134 cases. Even during the year 2017, a last epidemiological study reported the emergence of a new outbreak with a high incidence in the region [13]. Tis survey revealed the presence of 13 species of sand fies among the 23 species listed in Morocco (Faraj et Himmi, 2019). Te two genus present in the country, Phlebotomus and Sergentomyia, are represented by, respectively, six and seven species.
Phlebotomus papatasi was the predominant species, and it was present in all the studied localities in large proportions. Phlebotomus papatasi and Ph. bergeroti have been recognized as sympatric in Morocco, Algeria, Egypt, Sudan, and Ethiopia with possibilities of crosses [14].Ph. papatasi species that was frst described in Morocco in 2015 [15] is present throughout the country and is among the most common species [16]. Its density has been shown to increase with aridity [17], and it is present in all peridomestic biotopes, including houses, animal shelters, and caves [18], preferably between 400 and 800 m of altitude [19]. Phlebotomus papatasi is a confrmed vector of L. major in Morocco [20][21][22] and has always been found in the vicinity of ZCL foci in the arid region of the northern edge of the Sahara Desert [24]. In Morocco, the province of Zagora is considered as an endemic focus for L. major. Te type of climate in this province characterized by periods of drought, intermittently interspersed with violent storms deeply mark the biological cycles. In this study area, several factors which have been assumed to be important in favor of phlebotomine proliferation were present: vegetation cover, soil type, abiotic factors, etc. as well as the biological development of the reservoirs (rodents) [21,25,26]. In addition to this, other risk factors could participate in ZCL epidemic, for example, dumping pits, open sewers, and cattle manure near houses. Te rodent reservoir host incriminated in the transmission of ZCL due to L. major is Meriones shawi [27].
A study was conducted at Zagora Province, the study of seasonal dynamics of sand fies, especially at Mougni locality, noted a seasonal abundance of adult Ph. papatasi with a frst peak in June, and then sand fy numbers decreased steadily in July-September as the climate became hotter, and then increased slightly to mark a second peak clearly less important than the frst peak durinh late September-October [8]. In another locality Touna in the same province, Ph. papatasi showed a similar trend, but with only one peak in early July [8].  In Morocco, the period of activity of Ph. pappatasi was determined in Marrakech in the southwest of the country [28], in Tinghir adjacent to Zagora province [29] and in Zagora [8].
In the three studies, Ph. papatasi evolves in two generations with two peaks of abundance, the frst in late spring and the second in early autumn.
Phlebotomus alexandri is considered a proven vector of kala-azar in China, where it was found infested at a rate of 2% in the Xiujiang-Uygan region [30,31]. Te anthropophilic behavior of Ph. alexandri implicates it as a potential vector of L. tropica [32][33][34]. In Iraq, Ph. alexandri is a proven vector of VL caused by L. infantum [35]. In Iran, 108 individual and pooled samples of Ph. papatasi and Ph. alexandri from Khuzestan Province showed infection with L. major and L. infantum [36]. Phlebotomus alexandri is the   third most common species caught in Iran (accounting for more than 17%), and it is clearly anthropophilic and can be infected by L. infantum. Although other sand fy species have been found to be naturally infected with promastigotes, Ph. alexandri is the frst to be proven to be infected with L. infantum [37]. Phlebotomus alexandri is a proven vector of L. donovani in China [37] and of the parasites, causing cutaneous leishmaniasis in Iran, Iraq, Algeria, Djibouti, Greece, Morocco, Tunisia, and Yemen [38]. No study has confrmed the vector role of this species in Zagora Province. In our study, from an entomological point of view, the locality Timarighine had the highest number of specimens (35.82%). In Iran, en se basant sur alpha biodiversité, the analysis of this parameter showed that agricultural ecosystem of Khorramshahr County had the highest diversity on Ph. alexandri and Ph. papatasidue to maximal richness and diversity and also relatively high evenness [39]. Te factors for having high diversity of sand fies in the plain area studied may be due to higher annual precipitation, the related land use, and land cover. Te changes on the composition of sand fies are perhaps due to human intervention in their natural habitats. Phlebotomus longicuspis is native to the western part of the Mediterranean basin [5]. Tis species is considered to be widespread, preferentially in the subhumid to semiarid climatic stage. Phlebotomus longicuspis was the suspected vector of VL in Morocco [40]. In Zagora Province, this species represents only 11.55% of the total catch. Mougni locality has the highest abundance of Ph. longicuspis with 68%.
Phlebotomus sergenti is the main vector of L. tropica. Its role as a vector has been proven frst in Saudi Arabia [41] and then in Morocco [42]. Tis role was also confrmed in Israel [43] and Ethiopia [44]. In Morocco, Ph. sergenti is known as a widespread species throughout the country and predominant in the arid and Saharan areas [45][46][47]. Te vector is abundant in rural areas as well as in the habitats of the urban population [48]. In the present study, Ph. sergenti was present in seven localities, contributing to only 1.27% of the total sand fy catches. Tassaouante was the locality with the highest abundance of this species.
Phlebotomus kazeruni covers the sub-Saharan area. It is known in studies from Afghanistan [49], Iran [50], and Saudi Arabia [51]. Rioux [52]. Although some species feed on humans, they do not cause any epidemiological risk to humans until proven otherwise [53].
According to the distribution of species in the diferent localities, we can distinguish three groups of species. Ph. papatasi, Ph. alexandri, Ph. longicuspis, Ph. bergeroti, Ph. sergenti, Se. schwetzi, Se. fallax, Se. clydei, and Se. dreifussi are qualifed constant because they were found in more than 50% of the localities surveyed. While Se. africana and Se. minuta are considered constant because they represent a frequency of occurrence between 25% and 49%. Ten, the accidental species which are Ph. kazeruni and Se. antennata have a frequency of occurrence between 10% and 25%. In general, the diversity index is an ecological index that refects the way individuals are distributed among species at a given time. In general, a high index refects a balanced and diverse community, in which species are numerous, with no one species being truly dominant [54]. In our study, Zaouite Leftah (FA) is the locality with the highest index (H′ � 1.679), where eight species were found. Te equitability in this environment is 70%, and it appears to be the best organized and structured station.
In order to verify risk factors involved in the proliferation of sand fies and favoring the biological development of the parasite vector Ph. papatasi, additional ecological and environmental studies of the region should be developed.

Conclusion
Tis entomological study in the Province of Zagora, Morocco, revealed a specifc richness of phlebotomines of the order of 13 species including 6 of the Phlebotomus genus. Te predominance of Ph. papatasi constitutes the basis for this province to be an L. major endemic area. In ZCL endemic foci, good surveillance and vector and rodent control measures must be well maintained. In addition, awareness campaigns are required and must be conducted for better knowledge of the disease in order to avoid exposure to infections.

Data Availability
All the data analyzed during the study are included with links in the paper.

Conflicts of Interest
Te authors declare that they have no conficts of interest.