Distribution and Bionomic of Sand Flies in Five Ecologically Different Cutaneous Leishmaniasis Foci inMorocco

Cutaneous and visceral leishmaniases are endemic inMorocco.eir geographical distribution is well known. It has been related to bioclimatic factors. However, in 2008, two new cutaneous leishmaniasis foci appeared out of the known risk areas. Hence, we have undertaken this entomological survey in �ve foci situated in different bioclimatic areas to better understand the distribution and the epidemiology of this disease. Results of this study, undertaken during 2011, showed the predominance of Phlebotomus sergenti (55.8%) followed by Ph. perniciosus (12.9%), Ph. longicuspis (12.3%), and Ph. papatasi (12.0%). Ph. sergenti was active during 6 months in subhumid area and 8 months in arid, semiarid, and Saharan areas. Compared to previous studies, our results show a rise inPh. sergenti abundance and an extension of its activity period.ese results could explain the increase in anthroponotic cutaneous leishmaniasis incidence in Morocco and its extension to new nonendemic regions. Ph. perniciosus and Ph. longicuspis have also displayed a large distribution and a long activity period indicating a high potential risk of Leishmania infantum transmission in the studied areas.


Background
Leishmaniases are endemic in Morocco with two described forms; visceral (VL) and cutaneous (CL).Two types of CL are observed: anthroponotic (ACL) and zoonotic (ZCL) leishmaniases.Mucocutaneous leishmaniasis was described in Morocco but remains rare [1].
e geographical distribution of different leishmaniasis forms in Morocco has been clearly de�ned [2,3].It was related to bioclimatic areas described in Morocco.According to Rioux et al. [4], bioclimate affects the vector's distribution and density and hence disease prevalence.VL due to Leishmania infantum is sporadic but extending on large parts of Morocco in all bioclimatic areas because of the large distribution of its sand�ies vectors; Phlebotomus longicuspis, Ph.Perniciosus, and Ph.ariasi.e ZCL caused by L. major is endemic especially in the southern slopes of the Atlas Mountains where its unique vector, Ph. papatasi and its reservoir host, Meriones shawi, are prevalent.While the ACL due to L. tropica is endemic in arid and semiarid regions in the center and mainly in northwestern slopes of the Atlas Mountains where its proven vector Ph.sergenti is dominant.
Over the last years, leishmaniases have emerged or spread in many countries of Europe especially in the Mediterranean region.Most of declared outbreaks have been linked to global and local changes resulting of climatic changes or growth in human populations and their activities [5].In Morocco, leishmaniasis distribution has also gradually spread since 2001.In 2008, two new ACL foci appeared out of the known risk areas, previously de�ned by Public Health Services.e �rst one in the northeast of the country on the eastern hillside of the Middle Atlas mountains in a subhumid region (province of Boulmane) and the second in the southeast of the country on the east slopes of the High Atlas mountains in a Saharan region (province of Tinghir).Knowledge of vectors' population's distribution and dynamics is important in predicting the spatial and temporal variations in the risk of disease.However, information on dynamics of Leishmania vectors is limited in Morocco.Few localized and short-term studies are published [6][7][8].Hence, to better understand epidemiology and distribution of leishmaniasis in Morocco, we have undertaken this study in �ve different stations that represent the main biotopes in the country.Results of this study will help to establish effective and appropriate vector control measures.is study aims also to update entomological data by providing information on richness, abundance, seasonal trend, and spatial distribution of sand �ies species.(5) the Sahara extending on the south part of the country.e large latitudinal extension of Morocco, the importance of its maritime facades and its relief involve a large geographical variation in its climate.So, the annual means of precipitation vary from less than 25 mm in Sahara to about 2000 mm in the Rif.e analysis of the climatic map [9] showed �ve ecological zones, the Saharan bioclimate in the south and east gradually turning to arid, semiarid, subhumid, and humid in high altitudes of Atlas and Rif mountains.is study was carried out in �ve leishmaniasis areas located at different altitudes and in different ecological niches (Figure 1).

Sand Flies Collection.
Sand �ies were collected bimonthly in animal shelters during their optimum development period in Morocco, between April and November 2011.Five collecting sites were randomly chosen in each village.In each trapping night, an average of 40 sticky traps were set for maximum 24 h in each sampling localities.All sand �ies collected were identi�ed morphologically to species level using morphological key [12].Morphological differentiation between males of Ph. perniciosus and Ph.longicuspis was made according to description of Pesson et al. [13].

Data Analysis.
In order to characterize the sand �y populations in different sites, three parameters were calculated: (i) density of sand �ies collected: number of specimens/m 2 of sticky traps; (ii) relative frequency: number of specimens of species ()/total number of specimens × 100; (iii) species richness: number of species in a given area.
e greatest species richness was found in Ait Chribou, a semiarid area with fresh winter located at 1200 m altitude, with the occurrence of nine species against eight species occurring in Azrou, a Saharan biotope at 1500 m altitude.

Seasonal
Occurrence of Sand Flies Species.e seasonal trends shown by each vector are illustrated in Figure 2.
Except in subhumid area where seasonal activity of sand �ies extended from May to October (6 months) (Figure 2(c)), the activity period of the species starts from April and lasts to November (8 months) in the others biotopes (Figures 2(a), 2(b), 2(d), and 2(e)).At Aichoune, Ait Bousdouk, Ait Chribou, and Bouassem, the majority of sand �ies, dominated by Ph. sergenti, were collected between April and June.At Azrou locality, where Ph. papatasi is predominating, the majority was collected between August and September.
Ph. sergenti displayed a biphasic trend with a �rst and more important peak, situated between end of spring and beginning of summer, depending on sites.A second peak is observed in the beginning of autumn.Ph. longicuspis was more abundant in May and in August, respectively at Ait Bousdouk (Figure 2(b)) and Azrou (Figure 2(e)).Ph. perniciosus was more prevalent in September and October, respectively at Bouassem (Figure 2(c)) and Ait Chribou (Figure 2(a)).In saharan areas, activity of Ph. papatasi begins at the end of April, lasting to November with two peaks, a �rst one in June and a more important one in August-September (Figure 2(e)).

Discussion
Among 23 species described in Morocco, eleven sand �y species (48%) were identi�ed in this study.e identi�ed species belong to two genera: Phlebotomus, and Sergentomyia.Species in Phlebotomus genus belong to three different subgenera: Phlebotomus, Paraphlebotomus, and Larroussius.
In Larroussius subgenus, we reported three species: Ph. longicuspis, Ph.Perniciosus, and Ph.ariasi.ese three species are proven vectors of L. infantum [14].In Paraphlebotomus subgenus, we also identi�ed three species: Ph. sergenti, Ph.Alexandri, and Ph.chabaudi.e �rst one is the proven vector of L. tropica in Morocco [11].Ph. papatasi was the sole species belonging to Phlebotomus subgenus in our collection.is species is the proven vector of L. major in Morocco [15].e four species belonging to Sergentomyia genus (S. minuta, S.     fallax, S. antennata, and S. dreyfusi) were weakly represented in sand �y fauna caught in this study.ese species are known to be less abundant in human settings [16,17].
e species richness was the highest in Ait Chribou locality (semiarid area at 1200 m asl).Nine out of eleven species collected from the study area are present in this locality.However, 77.3% of specimens collected were Ph.sergenti and Ph.Perniciosus, and only 22.7% are belonging to the seven remaining species.Azrou locality (saharan area at 1500 m asl) also displayed a rich sand �y fauna with eight species.Previous studies in the High-Atlas Mountains have linked biodiversity to altitude [18,19].e high species diversity has been found at about 800 m altitude.Boussaa et al. [19] highlighted, moreover, the importance of aspect in sand �y diversity.ey noted that the east-facing sites were generally richer in sand �y species than the west-facing sites in Morocco.Similar data were found in southern Anatolia, Turkey [20] where the highest biodiversity was found at about 900 m asl.Our results do not meet completely with these �ndings, we have recorded a high diversity at 1500 m in Saharan area.Rioux et al. [4], by structuring species on bioclimate basis, have also noticed the high diversity in Saharan areas.
Of the �ve vectors we described, Ph. sergenti, Ph. perniciosus, Ph. longicuspis, and Ph.papatasi have been the most common species found in this study.ey were collected at all biotopes.Other studies also indicated that these species are the most common sand �ies in Morocco [2,18,19].ese species are known to be dominant in anthropic environments and are easily collected inside and in the vicinity of human dwellings [16,17].
Ph. sergenti was the most fre�uently collected sand �y representing 55.8% of the specimens caught from various habitats.It was the most abundant species in arid (51.9%), semiarid (64.9%), and subhumid (60.4%) areas.Its highest density (72%) was registered at Aichoune locality, a semiarid area at 750 m asl.In Saharan areas, it represented the second most abundant species, aer Ph. papatasi.Adults of Ph. sergenti were active throughout 8 months (April-November) in arid, semiarid, and Saharan areas and during 6 months (May-October) in subhumid areas showing a bimodal distribution.Previous studies showed that this species is largely widespread in Morocco without being the most abundant.It was proved to be more associated with arid and semiarid bioclimates [4,21,22].Its activity period was investigated in Taza, a semi arid area in northern Morocco, where it was collected from June to November showing two density peaks [6].e same activity period has been reported in Chichaoua province, a mountainous semi arid area in the southwest of High Atlas Mountains, but with only one density peak [4].While, in Al Haouz plain, an arid area in southwestern Morocco, this species was active only during the period April-June and absent in the rest of the year [8].Compared to these studies, our �ndings show an increase in Ph. sergenti abundance, even in biotopes where the species was less prevalent (subhumid and Saharan areas) and an extension of its activity period.ese results could explain the increase in ACL incidence in Morocco and its extension to new nonendemic regions (Boulmane and Tinghir).In fact, 15 years ago, Rioux et al. [22] have predicted, such expansion in subhumid areas using a model in which temperature parameters were varied to simulate global warming.
e emergence of ACL in Tinghir (Saharan area) could be linked to local changes.Indeed, this region has undergone a high level of environmental degradation over the last years.e human population has increased, especially in areas where housing conditions are unfavorable.e establishment of animal settlements in the vicinity of households and the population's movement from neighboring endemic areas has favored leishmaniasis transmission in this province.
Aer Ph. sergenti, Ph. perniciosus was the second most collected species in this study.Its highest density was noted at Ait Chribou at 1200 m. is result is in line with previous reports in High Atlas mountains [18,19].However, contrary to the results of Guernaoui et al. [18] who have suggested the absence of this species below 600 m asl, we have found it in the lowland at 250 m.As noted by the last authors, the male of Ph. perniciosus was mainly found in atypical form in the �ve study sites.Its density was underestimated in earlier studies because, in this form, it was confused with its sympatric species Ph. longicuspis.Ph. longicuspis was strongly indicated as the most abundant Larroussius species, while Ph.perniciosus was considered to be much less prevalent in Morocco [2,21].Ph. perniciosus was more abundant in our collections from early summer and reached its maximum density in September-October.Guernaoui et al. [7] found similar results in Chichaoua province.
Ph. longicuspis shows also a wide distribution including all bioclimatic zones and all altitudes.is species was most abundant in arid (Ait Bousdouk, 250 m) and saharan (Azrou, 1500 m) bioclimate which is consistent with previous distribution reports [4,21].ese results are in contradiction with those of Guernaoui et al. [18] who showed, in the same region of High Atlas Mountains, that Ph. longicuspis was more abundant between 600 and 799 m altitude, while its density became much lower outside this range.e altitude alone does not seem to be a selective factor for sand �y distribution in Morocco, characterized by a large climatic and ecologic diversity.Bioclimate, taking in account, many factors, mainly climatic factors, seems to better explain sand �y distribution in Morocco.Adults of Ph. longicuspis displayed a bimodal annual abundance pattern with peaks in May and September in arid areas.In saharan areas, it showed only one peak in August.According to Guernaoui et al. [7], this species showed a monophasic cycle in Chichaoua province, southwestern of Morocco, with one density peak in August-September.e large distribution and the occurrence with considerable density and long activity period of members of the subgenus Larroussius are a cause for concern and indicate the high potential risk of L. infantum transmission in the studied areas.
Ph. papatasi, was found in all biotopes and at all altitudes but at relatively low number except in Azrou (saharan area, 1500 m) where it was the most collected sand �y, representing 45.7% of the specimens.Ph. papatasi has been described as a sub-Saharan vector.It is the most commonly collected species in arid and per arid areas [4,8,21].In a study conducted in 2002 in southwestern Morocco, Guernaoui et al. [18] reported negative association between altitude and Ph.papatasi abundance.ey indicated that the species was predominant in plain (400-599 m), rare at the other altitudes and absent from 1200 m asl.Boussaa et al. [19] indicated, in the same region, that Ph. papatasi was most abundant at sites lying at 400-800 m asl.In southern Anatolia, Turkey, Simsek et al., [20] indicated that the species was more abundant at the range of 0-600 m altitude (76.8%).Our results are clearly in con�ict with these �ndings.As for the others species, Ph. papatasi distribution seems to be determined mainly by bioclimate.In Azrou, where it is abundant, Ph. papatasi populations were prevalent from April to November with two peaks.ese data are in accordance with earlier results from Marrakech area in southwestern Morocco [8].
e complexity of leishmaniasis in Morocco make their management challenging.Data obtained in this study which represented spatial and seasonal trends of the most important sand �y vectors con�rm a serious concern.ey might be helpful in prediction of areas and period of maximum risk for leishmaniasis transmission.Prevention and control should be an integrated approach including case treatment and application of appropriate reservoir and vectors control.

Con�ict of �nterests
e authors declare that they have no con�ict of interests.

Authors' Contribution
C. Faraj conceived and designed the study and draed the paper.E. Adlaoui carried out the data analysis and participated in the review of the paper.S. Ouahabi carried out sand �ies identi�cation and participated in the review of the paper, M. ElKhohli, L. Lakraa, M. ElRhazi carried out the �eld work and participated in the sand �ies identi�cation.B. Ameur has given �nancial support and participated in the review of the paper.All authors read and approved the �nal version of the paper.

F 1 :
Map showing the �ve study site locations. 0

T 1 :
Species di�ersity� abundance� and relati�e frequency of sand �ies caught in the ��e studied areas.and F: female.
Azrou Locality.(31 ∘ 30 � N, 05 ∘ 31 � W): situated in the south of the high Atlas Mountains in the province of Tinghir within the Saharan zone.e area has a Saharan climate with an altitude of about 1500 m. e mean annual precipitation is less than 200 mm.Tinghir is an emerging focus where the �rst human cases were declared in 2008.