Phlebotomine Sand Flies (Diptera: Psychodidae) Population of Particular Ecosystem in the Urban and Periurban Area in Morocco

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Phlebotomine Sand Flies (Diptera: Psychodidae) Population of Particular Ecosystem in the Urban and Periurban Area in Morocco

https://doi.org/10.21203/rs.3.rs-4613838/v1

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The Khettara is an abandoned irrigation system localized in Marrakech region, where no entomological investigations have been made. In the present study, sand flies (Diptera: Psychodidae) were collected with the aim to determine its abundance and species composition in this particular ecosystem.

A total of 477 specimens were collected belonging to six species; Phlebotomus (Phlebotomus) papatasi (28.72%) was the predominant species followed by Sergentomyia (Grassomyia) dreyfussi (23.06%), S. (Sergentomyia) fallax (18.87%), S. (S.) minuta (10.69%), P. (Larroussius) longicuspis (9.85%), and P. (Paraphlebotomus) sergenti (8.81%). When, S. (G.) dreyfussi was collected for the first time in the urban area of Marrakech.

The results showed important sand fly diversity in Khettara ecosystem compared to sand fly composition in urban area of Marrakech. In addition, of the six species of phlebotomine sand flies caught, three are proven vectors of leishmaniasis in Morocco. Thus, Marrakech region, and especially its Khettara ecosystem, should be regarded as a potential focus for leishmaniasis.

Parasitology

Phlebotomine sand flies

Khettara ecosystem

Irrigation system

Morocco

Marrakech is the most important former imperial city in Morocco's history. It’s the capital of the mid-southwestern economic region of Marrakech-Safi, near of the Atlas Mountains. The KHETTARA systems were established in the 10th century under the reign of Sultan Youssef Ibn Tachfine and Sultan Yaacoub El Mansour.

KHETTARA is a system of drainage galleries; a succession of wells aligned and regularly spaced. It’s about a drain that captures the surface horizon of the water sheet and allows it to flow upwards. From this point of intake to where the water naturally surfaces, there are three structures: a drainage tunnel, an open cut, and a network of “Seguias”. KHETTARA irrigation system is abandoned and localized in an urban area in Marrakech city.

As far as we know, no sand fly (Diptera: Psychodidae) investigations have been made on this particular ecosystem. Phlebotomine sand flies are a proven vector of leishmaniasis, parasitic diseases caused by Leishmania sp.

In Morocco, leishmaniasis is endemic. The epidemiological situation of leishmaniasis in 2014 was characterized by 2556 cases of cutaneous leishmaniasis and 86 cases of visceral leishmaniasis (Moroccan Ministry of Health, 2014). Additionally, the local distribution of different disease forms has changed. Increasing numbers of cases reported from the peri-urban and urban areas where the disease was once unknown or relatively rare. The KHETTARA contains, besides of various insect populations, Mammal and Reptile species that may be considered as hosts of leishmaniasis (Rioux et al., 1984).

Thus, the objective of the present study is analysing the sand fly biodiversity in urban and periurban KHATTARA systems where no entomological investigation has ever been conducted before.

1. Study area

Sand fly trapping was performed urban and in a per-urban Marrakech city KHETTARA ecosystem (Fig. 1).

In this area, the climate is arid, the mean maximum and minimum monthly temperature are 41.1°C and 4°C in July and December respectively.

The Khettara is a system of drainage galleries, a succession of wells aligned and regularly spaced (Fig. 2A). “Khettara” irrigation system is abandoned, and localized in an urban and periurban areas of Marrakech (Fig. 2B).

2. Sand fly collection:

In Marrakech area, the sand fly species differed in their activity periods. Two seasonal patterns were identified; a mono-modal one (peaking on May), and a bi-modal one (peaking on October-November and May) according to Boussaa et al. (2005). Thus, we have chosen to collect sand fly during October and May in order to maximize species diversity. Sticky paper (paper sheets A4 coated with castor oil) and CDC light traps were combined for specimen collection. All caught specimens were preserved in 70% ethanol, cleared in 20% potash and Marc–André solution, then slide mounted in Hoyer’s medium.

Sand fly species were then identified by the morphological examination of the male genitalia, the pharyngeal armature and female spermthecae, using the keys and descriptions published by Moroccan Ministry of Health (MMH, 2010). For Larroussius species, the females were identified by examining the dilatation of distal parts of spermathecal ducts and males by examining the shape of the copulatory valves and counting the number of coxite hairs (Pesson et al., 2004; Boussaa et al. 2008).

3. Soil analysis

Soil samples were taken in order to characterize the Khettara ecosystem. About 500g of soil was collected from the depth (Khettara main channel) and the wall (Qanat shafts) of the Khettara. PH, humidity, total organic carbon and granulometry measurements were noted (Table 4). For each parameter, three repetitions were performed to confirm the results.

a. Granulometry

The purpose of this study is to determine the different size classes of the mineral soil fraction. For this purpose, a sample preparation is necessary in order to remove the aggregates.

Organic matter destruction has been made by H₂O₂. The classification of the particles is done by using the Stokes relation (1851) which makes it possible to determine the sedimentation rate (V) as a function of the radius (r) V = 2gr2 (ds ± de) / 9n or else V = k r2 (k constant = 2g (ds-de) / 9n).

Where, V: sedimentation rate (cm /s), G: gravity (cm /s²), Ds: density of the soil, De: density of water, N: viscosity of the soil and R: spherical ray of the particle (cm)

b. Soil moisture

The gravimetric method is used to determine humidity soil. The soil sample was weighed in the humid state (ph) and dry state (ps) after having passed at 105°C. Humidity is calculated according to the following ratio: H = (ph - ps) 100 /ps

c. Organic matter and pH

The Anne method (Aubert, 1978) is used to estimate the rate of organic matter (o.m) in our samples. Thus, from the determination of total organic carbon (c.o), we calculated the percentage of organic matter according to the following ratio: o.m% = c.o% x 1.724. in addition, we proceeded to calculate the PH for soil samples from both urban and peri-urban Khettaras.

4. Data Analysis:

Various ecological parameters were calculated to characterize the sand fly populations in the different sites and habitats:

Relative frequency = (Number of specimens of one species / Total of specimens) × 100%
Shannon Index = H’ = - sum pi log2 pi. Where pi is the proportional abundance of species i
Biodiversity index = (S-1)/LnN; where N is the number of individuals, S refers to the number of species in the sample.

A total of 477 specimens were collected. The number of specimens collected and the relative abundance of species identified are shown in (Table 1).

Table 1

The number of sand fly species per site and sex
	Site (code)	Marrakech Khettara (S1)	Tameslohte Khettara (S2)	Total
	Altitude (m)	420	560	Total
	Co-ordinates	31°40’03.9’’N	31°29’34.8’’N	M/F	All	%
	Co-ordinates	8°01’21.6’’ W	8°05’07.7’’W	M/F	All	%
Phlebotomus papatasi	M	41	37	78	137	28,72
Phlebotomus papatasi	F	39	20	59	137	28,72
P. sergenti	M	10	13	23	42	8,81
P. sergenti	F	8	11	19	42	8,81
P. longicuspis	M	14	10	24	47	9,85
P. longicuspis	F	20	3	23	47	9,85
Sergentomyia fallax	M	22	26	48	90	18,87
Sergentomyia fallax	F	26	16	42	90	18,87
S. minuta	M	20	18	38	51	10,69
S. minuta	F	3	10	13	51	10,69
S. dreyfussi	M	32	9	41	110	23,06
S. dreyfussi	F	63	6	69	110	23,06
Total		298	179	477

Morphological identification showed three species of the genus Phlebotomus, and three species of the genus Sergentomyia: P. papatasi, P. longicuspis, P. sergenti, S. dreyfussi, S. fallax and S. minuta. In addition, 4 males and 1 female showing P. perniciosus morphology characters were collected; this observation must be verified with a supplementary sampling and confirmed via molecular and or enzymatic typing. S. dreyfussi was identified for the first time in urban area of Marrakech, and it is the most abundant species in the Khettara system in urban area, while P. papatasi is the most abundant species in periurban Khattara (Table 2).

Table 2

Biodiversity Indices
Sites	S (Species richness)	Individuals	Biodiversity index	Dominant species	Shannon Index
S1	6	298	0,88	S. dreyfussi	2,35
S2	6	179	0,96	P. papatasi	2,32

Table 3 shows the substrate characterization of Khattara system in urban and periurban area. Both Khattara systems have almost even granulometry composition as well as Humidity rate and Organic matter proportion, while slight change in pH was noted between urban and periurban Khattaras. In contrast, significant variation was found between wall and depth in both Khattara systems, especially regarding Humidity and Organic matter.

Table 3

Substrate characterization of urban and peri-urban Khettara.
	Granulometry (%)
	Coarse sands	Fine sands	Clays	Coarse silts	Fine silts
Urban Khettara (wall)	10,89	6,53	1,73	1,75	1,8	24,22	11,55	8,84
Urban Khettara (depth)	7,61	8,78	1,65	1,74	1,78	6,38	9,05	8,8
Periurban Khettara (wall)	10,81	7,06	1,74	1,78	1,76	29,7	11,2	7,98
Periurban Khettara (depth)	6,67	5,6	1,65	1,65	1,66	7,53	9,48	7,7

In this study, we report the results of an entomological investigation in the Khettara system located in the urban and periurban area of Marrakech with the aim to study the current species composition and to find out, if exists, any risk for Leishmania transmission in this particular area.

Six species of phlebotomine sand flies were caught: they belonging to the Phlebotomus and Sergentomyia genera: P. papatasi, P. sergenti, P. longicuspis, S. dreyfussi, S. fallax, and S. minuta.

These Phlebotomus species are proven vectors of leishmaniasis forms in Morocco. Cutaneous leishmaniasis (CL), caused by Leishmania major Yakimoff and Schokhor (Kinetoplastida: Trypanosomatidae), is prevalent in arid and semiarid zones of northern Africa, the Middle East, and central Asia (Ashford, 1996). In Morocco, CL is widespread, investigations in the zoonotic cutaneous leishmaniasis foci showed that Phlebotomus papatasi Scopoli is the proven vector of L. major (Rioux et al., 1986), and Meriones shawi Duvernoy is the main reservoir host (Rioux et al., 1982).

P. papatasi was recorded in Morocco in 1932 (Ristorcelli, 1939), and it is considered as ubiquitous species (Bailly-Choumara et al., 1971). It is well adapted to the conditions of arid climate (Rioux et al., 1997), and its density increases with the aridity (Croset et al., 1974). P. papatasi is the dominant species in the arid plain, but it exists at low frequencies in the mountains (Kahime et al., 2015). Although active throughout the year in the urban area of Marrakech city, two density peaks have been identified; summer (June) and autumn (November) (Boussaa et al., 2005). In Marrakech city, it has been shown that P. papatasi is distributed equally among all types of human habitats, and its establishment is favored by urbanization (Boussaa et al., 2007). The greatest number of specimens was collected at the urban Khettara. However, despite of the high density of P. papatasi, the omnipresence of M. shawi and the movement of large number of patients from zoonotic cutaneous leishmaniasis foci around, there is at present, no documented active transmission in the study area.

P. sergenti is the proven vector of L. tropica in North Africa, Middle East and Central Asia (Al-Zahrani et al., 1988; Killick-Kendrick, 1990; Boubidi et al., 2011; Tabbabi et al., 2011) the vector was abundant in these Moroccan households. Evidence of its vector role in Morocco was first in the Tanant outbreak (Guilvard et al., 1991). Actually, P. sergenti is the proven vector of the anthroponotic CL by L. tropica, the most widespread CL form in Morocco.

The sub-genus Larroussius remains an individualized group within the genus Phlebotomus and this is due to their morphology and epidemiology status of which three species are vectors of L. infantum, namely P. perniciosus, P. ariasi and P. longicuspis in Morocco (Rioux et al., 1984; Killick-Kendrich, 1990). In the present study, P. longicuspis presence was confirmed, in addition, specimens showing P. perniciosus morphology characters were collected in the Khattara system. Knowing the P. perniciosus complex status in Morocco, these observations must be verified with a supplementary sampling.

Among Sergentomyia species S. minuta is widely distributed around the Mediterranean Sea. In Morocco, S. minuta is a ubiquitous species (Bailly-Choumara et al. 1971) and it was collected up to 2000 m (Kahime et al., 2015). S. fallax has a large distribution in southern Morocco (Bailly-Choumara et al., 1971) and it was collected up to 2000 m in Moroccan High-Atlas Mountains (Kahime et al., 2015). S. dreyfussi is the only Grassomyia species present in Marrakech area. It was collected only in rural sites. S. dreyfussi seems to be not affected by altitude, but it is probably more sensitive to urbanization effect (Boussaa et al., 2009).

S. dreyfussi is collected in the rural area of Marrakech, and never been mentioned in the urban area of the city. Boussaa et al., (2009), showed that S. dreyfussi is collected starting from the altitudinal range of 300 meters in arid area. In this study, we add information about S. dreyfussi occurrence in urban area with altitude of 436 m. The Khettara, the abandoned irrigation system, is still considered as a microecosystem regulated by its own ecological micro-condition, its temperature and its physiognomy. Thus, we consider that the Khettara ecosystem -even if it is located in the urban area of Marrakesh-has protected S. dreyfussi population from the urbanization effect.

Khettara ecosystem is very specific and present a considerable sand fly diversity. This preliminary study offers interesting results about sand fly species composition, local environmental conditions and consequently, the leishmanian risk in the study area. More investigations are need to study more precisely and deeply this ecosystem by using molecular techniques to characterize this specific sand fly populations.

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The authors declare no competing interests.

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Phlebotomine Sand Flies (Diptera: Psychodidae) Population of Particular Ecosystem in the Urban and Periurban Area in Morocco

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Abstract

Figures

BACKGROUND

METHODS

1. Study area

2. Sand fly collection:

3. Soil analysis

a. Granulometry

b. Soil moisture

c. Organic matter and pH

4. Data Analysis:

RESULTS

DISCUSSION

CONCLUSIONS

References

Additional Declarations

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