Diversity and Dynamics of Sand Flies (Diptera: Psychodidae) of Two Cutaneous Leishmaniasis Foci in the Fes-Boulemane Region of Northern Morocco

1Faculty of Sciences Dhar El Mahraz, Laboratory Biotechnology and Preservation of Natural Resources, Sidi Mohamed Ben Abdellah University, 30000 Fez, Morocco 2Regional Diagnostic Laboratory of Epidemiological and Environmental Health, Regional Health Directorate, El Ghassani Hospital, 30000 Fez, Morocco 3Laboratory of Medical Entomology, National Institute of Hygiene, 27 Avenue Ibn Battuta, Agdal, 11400 Rabat, Morocco 4Laboratory of Environment and Renewable Energies, Faculty of Science, University of Ibn Tofail, 14000 Kenitra, Morocco 5Institute of Nursing Professions and Health Techniques, El Ghassani Hospital, 30000 Fez, Morocco


Introduction
Cutaneous leishmaniases (CL) are parasitic diseases that remain today as a serious public health problem throughout the world.The parasitosis is caused by a "Protist Euglenobionte" that belongs to the Trypanosomatidae family, genus Leishmania.Three forms are observed: anthroponotic leishmaniasis caused by Leishmania tropica and zoonotic leishmaniasis due to L. major and L. infantum [1,2].L. tropica is the most widespread and most frequently encountered.It is transmitted by Phlebotomus sergenti [3].
In Morocco, there is an annual emergence of new foci of leishmaniasis.Indeed, in 2011 and 2012, the Ministry of Health recorded, respectively, 4426 and 2990 cases with 4.92% and 7.19% in the region of Fes-Boulemane [4,5].The first LC focus recorded from the Fes-Boulemane region was in 2001 with 1,600 cases only in the province of Moulay Yacoub [6,7].
The objectives of our study were to identify and to make an inventory of the principle sand-flies and also to compare the specific composition and population dynamics of two biotopes located on the western and eastern slopes of the International Journal of Zoology Middle Atlas, correspondingly at Aichoun and Bouasseme (Figure 1).All these steps are necessary for the surveillance and treatment of existing leishmaniases in these two foci of Fes-Boulemane region and thus contribute to its control.From an epidemiological standpoint, 1242 cases of LC in the province of Sefrou were reported between 1997 and 2011 [8].The majority of cases are due to L. tropica registered in the municipalities of El Menzel, Sefrou, Aghbalou, Tazouta Sidi Lahcen, and Ain Cheggag [9].

Materials and Methods
Bouasseme (33 ∘ 31  N, 04 ∘ 33  W) is located in the Northeast of the Middle Atlas.It is characterized by a subhumid climate receiving about 700 to 800 mm of rainfall from October to May.This town is in the province of Boulemane where the first endemic cases of leishmaniasis in L. major were reported [10] (Figure 1).

Collection of Sand
Flies.Sand flies were captured once every two weeks from January to December using sticky paper traps [11].Five stations were randomly chosen.In each station, ten traps were deposited in different biotopes (stables, barns, chicken and sheep pens, etc.) and recovered the next day.The captured sand flies are kept in airtight plastic tubes containing 70% ethyl alcohol.
The females were identified based on the examination of spermathecae, cibarium, and posterior pharynx.Male identification was mainly based on examination of the genital armature.The identification was done by using morphological identification keys [8,12].The morphological differentiation between the two sympatric species Ph. longicuspis and Ph.perniciosus was made according to Berchi et al. (2007) [13].The identification of all sand flies species was confirmed at the Laboratory of Medical Entomology in National Institute of Hygiene (the national reference laboratory in Medical Entomology).
Results are analyzed by the following ecological indices [14]: (i) Specific richness is the number of species that make up the stand.
(ii) The relative abundance is the relative number of individuals of each species from all collected sand flies.
(iii) Shannon-Weiner index (  ) is follows: where "  " can be calculated by dividing   , the number of species, by , the total number of collected individuals.

Specific Composition.
The sand flies' activity period runs from April to October-November at the two sites.Catches by adhesive traps allowed harvesting 1120 sand flies of six species in Aichoun and 255 specimens belonging to seven species in Bouasseme.Five species belonged to the Phlebotomus genus while the other two were from the Sergentomyia genus.These species are Ph.sergenti which is widely distributed in the two foci, Ph. perniciosus, Ph. longicuspis, Ph. papatasi, Sergentomyia minuta, and Sergentomyia fallax.These species are common in both localities except for Ph.ariasi which was collected only at Bouasseme (Figures 2 and 3).These species are considered the most abundant among the 23 species recorded in Morocco [8].
At Bouasseme, 255 sand flies were captured with species of Phlebotomus genus predominating both in the number of specimens captured and in their diversity (3 subgenera, 5 species).Only two species of the genus Sergentomyia were captured (Sergentomyia minuta and Sergentomyia fallax).
There was a difference in the diversity of the sand fly fauna between Aichoun and Bouasseme, as indicated by the values of Shannon-Weiner index (  ).Both localities have a value of   less than 1, with a difference of 0.1.We compared the Shannon diversity index of all the species in each study area.The values of   at Aichoun range from 0.02 to 0.13.The most significant value was reported in Ph. sergenti and Ph.longicuspis with 0.13.But the value of this index in Bouasseme for all species varies only from 0.01 to 0.09.This difference explains why the population is more diversified at Aichoun with the predominance of Ph. sergenti and Ph.longicuspis in comparison to Bouasseme.

Population Dynamics of Sand Flies at Aichoun and
Bouasseme.The study of the activity of sand flies in these two areas was followed from January to December 2011.According to the capture sessions conducted in Aichoun and Bouasseme, we found a monthly evolution that varies from one species to another, biphasic in some species and monophasic in others.Throughout the study period, the activity of sand flies was marked by the dominance of Ph. sergenti with two peaks in June and September in both localities.In Aichoun, the activity of sand flies starts in April, coming after a gradual development observed for both Ph.sergenti and Ph.longicuspis.After June, the sand flies' activity experienced  a fall followed by a subsequent peak for Ph.papatasi and then fell during the following months giving rise to an emergence of Ph. perniciosus with a high percentage of 4.28% in September (Figure 4).The activity of sand flies at the Aichoun locality is spread over a period of eight months stopping in November.
At Bouasseme, sand flies appeared from May to October.Compared to Aichoun, this focus has known another representation of the seasonal fluctuation of collected species.Indeed, during the period of collections, the three species Ph. sergenti, Ph. perniciosus, and Ph.longicuspis experienced a consistent, biphasic course with peaks, June and September, as follows: Ph sergenti (26.66%, 5.88%), Ph. longicuspis, and Ph.perniciosus.
For Ph. ariasi, S. minuta, and S. fallax, the period of their activity was spread from June to September but with only small percentage variation (Figure 5).

Discussion
This research is a comparative study of two leishmaniasis foci in the Fes-Boulemane region of Northern Morocco in terms of abundance, species diversity, and seasonality of sand flies.During this year long entomological survey in 2011, we collected 1,120 sand flies in Aichoun and 255 in Bouasseme, two localities of the Fes-Boulemane region.The identified species belong to two genera: Phlebotomus and Sergentomyia.Species in Phlebotomus genus belong to the following three subgenera: Phlebotomus, Paraphlebotomus, and Larroussius.
In the subgenus Larroussius, we reported three species: Ph. longicuspis, Ph. perniciosus, and Ph.ariasi.In subgenus Paraphlebotomus, we identified one species: Ph. sergenti.Ph.  papatasi was the sole species belonging to subgenus Phlebotomus in our collection.The two species from Sergentomyia (S. minuta, S. fallax) were poorly represented in sand fly fauna caught in this study.
Indeed, there is a dependency between the development of leishmaniasis and density of sand flies [15].
All proven and suspected vectors of leishmaniasis in Morocco are present in the study area: Ph. sergenti is confirmed as a vector of L. tropica anthroponotic leishmaniasis in Northern Africa, the Middle East, and Central Asia [16,17], as well as in Morocco [18].This species, which was also suspected to be responsible for CL in Chichaoua [19], is dominant over the other species, widely dispersed in both localities and present from May to November, with peaks of activity in June and September.This vector prefers a semiarid bioclimate, even though it was captured in all bioclimatic zones and was widespread throughout Morocco [20].
These results are in agreement with those obtained by Guessouss-Idrissi et al. [21] in the province of Taza, and El Miri et al. [22] in the province of Sidi Kacem in northern Morocco.Ph. perniciosus is one of the most competent vectors of L. infantum in Mediterranean foci [23], responsible for visceral leishmaniasis in the Northeast slope of the Rif mountains where dogs are the main reservoir of the parasite [24].
In Bouasseme, seven species were trapped.They come from two genera: Phlebotomus and Sergentomyia with Ph. sergenti being dominant.Ph. ariasi and Ph.perniciosus, the usual two vectors of L. infantum in the North of the Mediterranean [25], are also represented here.The taxonomic inventory and biodiversity sand flies obtained in Aichoun during the study period in 2011 were also obtained in 2012 [26].This confirms continuity in the sand fly development cycle.
Similar work was carried out in the Fes-Boulemane region in a focus for cutaneous leishmaniasis at Ouled Aid in the province of Moulay Yakoub in 2011.This study showed the same period of sand fly activity between April and November with the presence of the same species, but with different abundance [27].
From an epidemiological point of view, considering the vector-parasite relationship, we can consider Ph. sergenti as a potentially causative agent of L. tropica transmission in both Aichoun and Bouasseme bearing in mind that it is the only potential vector species of L. tropica in the study area.Given the low seasonality of Ph. ariasi and the proven role of Ph. perniciosus and Ph.longicuspis in L. infantum [25,28], these two species are likely the vectors of L. infantum in the study area.Ph. papatasi, the proved vector of L. major in Morocco [29], has a low density at both sites; this could be explained by its preference for an arid climate [30].The high abundance of the sand flies species in Aichoun and Bouasseme can be explained by environmental conditions, especially temperature and humidity which are conducive to a rapid biological development.

Conclusion and Perspectives
The wide distribution of sand flies and the long activity period of Ph. sergenti, Ph. longicuspis, and Ph.perniciosus in both foci Aichoun and Bouasseme indicate that the risk of transmission of leishmaniasis is potentially higher in this region.These results will serve as a database for further work in these two localities.They suggest the necessity for continuous entomological surveillance to lessen the risk of transmission of cutaneous leishmaniasis.
In perspective, we propose that further studies of sand fly diversity be conducted through molecular analyses, in order to positively identify sand fly species.
The determination of climatic factors favorable for sand flies development and the study of host-blood feeding preferences are also useful for developing a control program, directing operations, and evaluating their effectiveness.

Figure 1 :
Figure 1: Map showing the two study site locations.

Figure 3 :
Figure 3: (a) Male genitalia and the extremity of penile valves in Phlebotomus, subgenus Larroussius; (b) male genitalia and the extremity of penile valves in Phlebotomus perniciosus; (c) male genitalia and the extremity of penile valves in Phlebotomus papatasi (personal photography; G ×40).

Figure 4 :
Figure 4: Seasonal distribution of sand flies captured at Aichoun by sticky paper traps from January to December 2011.

Figure 5 :
Figure 5: Seasonal distribution of sand flies captured at Bouasseme by adhesive traps in the period from January to December 2011.

Table 1 :
Percentage and the Shannon-Weiner diversity index () of the sand flies collected during the study period at both localities of the Fes-Boulemane region.