Article pp.105-113 du Vol.112 n°2 (2019)

(1)

MEDICAL ENTOMOLOGY /ENTOMOLOGIE MÉDICALE

Quantitative Inventory of Mosquito Larvae (Diptera: Culicidae)

and Physicochemical Analysis of Aquatic Habitats in the Region of Fez, Morocco*

Inventaire quantitatif des larves de moustiques (Diptera: Culicidae) et analyse physicochimique des habitats aquatiques dans la région de Fès, Maroc

T. Filali Mouatassem · C. Faraj · R. Guemmouh · N. Rais · A. EL Ouali Lalami

Received: 4 May 2019; Accepted: 15 July 2019

AbstractMosquitoes occupy a major health problem, as a potential vector for the transmission of many pathogens, the most important of which in Morocco are Plasmodium para- sites and West Nile fever virus. Acquiring knowledge about the biotypology of these mosquitoes is an essential step in any control strategy. Thus, the objective of this study is to draw up an inventory of the culicidian fauna in the Fez region (North-central part of Morocco) and to determine the physicochemical parameters influencing the production of mosquito larvae of aquatic habitats in this area. Mosquito larvae of the extant biotopes were sampled every two months between November 2015 and December 2016, by means of the“dipping”method. The physicochemical parameters of the various larval biotopes were collected six times during

the year of follow-up. Mosquito species were identified using morphological criteria. The results were analyzed using ecological indices and the generalized linear model (Poisson model) on R software. The identification of the 1,122 mosquito larvae collected from the various studied breeding areas revealed the presence of 11 species belonging to two subfamilies (Culicinae and Anophelinae) and five genera (Anopheles, Culex, Culiseta, Aedes, and Uranotae- nia). Anopheles were observed in small numbers in permanent, stagnant, and shallow habitats. The larvae of An. maculipenniss. l. (major vector of malaria in Morocco) were collected. Culex larvae, especially Cx. pipiens (the main vector of WNV in Morocco), were collected in the majority of the larval habitats and on the different types of environments. Two other species of Culex, which plays a significant role in the transmission of WNV, were also collected, particularly, Cx. perexiguus and Cx. theileri. The results found,viathis first study of its kind at the regional level, also made it possible to update the list of Culicidae species reported in the Fez region. Four new species have been recorded:Aedes flavescens,Uranotaenia unguiculata, Culiseta longiareolata, andCulex perexiguus. The results of this study will contribute to the provision of information for entomological surveillance and to better plan and guide vector control activities at local and national levels.

KeywordsInventory · Monitoring · Biotypology · Larvae · Mosquitoes Culicidae · Fez · Morocco · Maghreb · Northern Africa

RésuméLes moustiques constituent un problème majeur de santé, en tant que vecteur potentiel de la transmission de nombreux agents pathogènes dont les plus importants au Maroc sont le Plasmodium et le virus de la fièvre du West Nile. Acquérir des connaissances sur la biotypologie de ces moustiques est une étape essentielle dans toute stratégie de

T. Filali Mouatassem · R. Guemmouh · A. EL Ouali Lalami (*) Laboratory of Biotechnology and Preservation

of Natural Resources,

Sidi-Mohamed-ben-Abdellah University,

Faculty of Sciences Dhar-El-Mahraz, 30000 Fez, Morocco e-mail : eloualilalami@yahoo.fr

C. Faraj

Laboratory of Medical Entomology,

National Institute of Hygiene, Rabat, Morocco N. Rais

Laboratory of Computer Science, Modelling and Systems, Sidi-Mohamed-ben-Abdellah University,

Faculty of Sciences Dhar Mahraz, Fez, Morocco A. EL Ouali Lalami

Higher Institute of Nursing Professions and Health Techniques of Fez,

Regional Health Directorate, El-Ghassani Hospital, Fez 30000, Morocco

* This work was published with the support of National Institute for Hygiene (INH) [Morocco].

DOI 10.3166/bspe-2019-0084

(2)

lutte. Ainsi, l’objectif de ce travail est de dresser l’inventaire de la faune culicidienne dans la région de Fès (Centre Nord du Maroc) et de déterminer les paramètres physicochimiques influençant la production en larves de moustiques des habitats aquatiques dans cette zone. L’échantillonnage des larves a été réalisé tous les deux mois dans les différents biotopes existants, entre novembre 2015 et décembre 2016, à l’aide de la méthode dedipping. Les paramètres physicochimiques des différents biotopes larvaires ont été relevés six fois dur- ant l’année du suivi. L’identification des caractères morpho- logiques des larves a été déterminée. Les résultats ont été analysés moyennant des indices écologiques et le modèle linéaire généralisé (GLM), suite à un modèle de Poisson sur le logiciel R. L’identification des 1 122 larves de culi- cidés récoltées au niveau des différents gîtes prospectés a révélé la présence de 11 espèces appartenant à deux sous- familles (Culicinae et Anophelinae) et cinq genres (Anophe- les, Culex, Culiseta, Aedes et Uranotaenia). Les anophèles ont été observés en faible nombre dans des habitats perma- nents, à eaux stagnantes, et peu profondes. Les larves d’ An. maculipennis s. l. (vecteur majeur du paludisme au Maroc) ont été récoltées. Les larves de Culex, en particulier

de Cx. pipiens (principal vecteur du WNV au Maroc), ont été collectées dans la majorité des gîtes prospectés et dans les différents types de milieux. Deux autres espèces de Culex, dont le rôle dans la transmission du WNV est reconnu, ont été également collectées au cours de cette étude ; il s’agit de Cx. perexiguus et Cx. theileri. Les résultats trouvésviacette étude, première du genre au niveau de la région, ont permis également de mettre à jour la liste des espèces culicidiennes signalées au niveau de la région de Fès par l’ajout de quatre espèces : Aedes flavescens,Uranotaenia unguiculata Culi- seta longiareolataetCulex perexiguus. Les résultats de cette étude contribueront à l’apport d’informations pour la surveillance entomologique et pour mieux planifier et orienter les activités de la lutte antivectorielle au niveau local et national.

Mots clésInventaire · Surveillance · Biotypologie · Larves · Moustiques Culicidae · Fès · Maroc · Maghreb · Afrique du Nord

Introduction

Mosquitoes are a group of insects that have a significant role in the spread of diseases. Their geographical extension throughout the world is considerable. They occur in tropical and temperate zones, even above the Arctic Circle but are absent in the Antarctic [21]. Nowadays, more than 3,500 species have been described [1]. In Morocco, the culicidian fauna has been the subject of a great deal of research since the beginning of the twentieth century [27].

The continuous evolution of the geographical distribution of certain species, the appearance of new species or the dis- appearance of others under the impact of local modifications (environmental management, increase of hydraulic projects, extension of urbanization…), or general changes (global warming) may be at the origin of the re-emergence or emergence of vector-borne diseases.

Then, it is interesting to continually monitor mosquito populations in vulnerable areas such as the northern center of Morocco [10] in order to prevent the possible transmission of vector-borne diseases.

The present study aims to conduct an inventory of the mosquito species present in the region of Fez and to determine the physicochemical parameters affecting their prolif- eration in the aquatic habitats in this area. The present study also planned to better understand the diversity of Culicidae in this region and generate data of great interest that can be used by the vector control program to deliver targeted and cost-effective interventions.

Abbreviations

Ae: Aedes An: Anopheles

BPRN: Laboratory of Biotechnology and Preservation of Natu- ral Resources

C: Distribution

CondE: Electrical conductivity Cu: Culiseta

Cx.: Culex

F: Frequency of occurrence GLM: Generalized Linear Model

n: Number of sites where the species was found N: Total number of sites surveyed

OrigineN: Origine Natural OxyD: Dissolved Oxygen P: Plasmodium

pH: Potential of Hydrogen pi: Relative abundance Prof: Water depth Sal: Salinity T: Temporary TC: Water temperature TDS: Total Dissolved Solids Turb: Turbidity

U: Uranotaenia WNV: West Nile Virus

(3)

Material and Methods Study Region

The city of Fez is located in the North-central part of Morocco in the Saiss plain. It is crossed by several rivers such as Oued El Himmer, Oued El Maleh, Oued Aïn Chkef, Oued Mahrez, Oued Boufekrane, and Oued Fez which lead to Oued Sebou, the lead recipient of wastewater from the Fez agglomeration. These wadis contribute, during the dry seasons of the year, to the formation of numerous sites favorable to the development of different species of mosquitoes. The region also includes natural sources and artificial water stagnations that provide a breeding ground for mosquitoes.

Site Prospecting

The study was carried out from different biotopes on 27 larval habitats (Table 1): 12 watercourses of which 40% receive

sewage, 7 springs, 2 fountains, 3 swamps, 1 lake, 1 dam reservoir, and 1 water reservoir. Figure 1 shows the geographical location of mosquito breeding sites in the studied sites.

Sampling Technique

Larvae were sampled every two months in the different biotopes selected. At the same time, the physical and chemical parameters of larval habitats as well as the current weather conditions were recorded in situ (pH, temperature, dissolved oxygen, total dissolved solids, salinity, electrical conductivity, depth, turbidity, and the flow of water) using a portable multiparameter analyzer (Consort Multiparameter analyser C561, Lovibond^®turbidimeter Turbidirect, and water qual- ity instrument YSI scientific).

Larval sampling was done using the standard dipping method in shallow water [24]. In deep waters, they were collected, using a bucket of 30 cm in diameter attached to a cord [9]. The recorded larvae were collected by a pear pipette and stored in glass containers in a 70% ethanol solution.

Table 1 Coordinates of the studied collection sites /Coordonnées des gîtes étudiés

Place of collection Origin Nature Latitude Longitude Altitude

Wislane Oued Natural Permanent 34°02.289ʹN 004°58.132ʹW 339

Aîn Knssara source Natural Permanent 34°08.732ʹN 004°49.909ʹW 367

Sbou Oued Natural Permanent 34°06.051ʹN 004°53.943ʹW 181

Sidi Harazem Natural Permanent 34°02.459ʹN 004°52.866ʹW 216

Jnan Sbil lake Artificial Permanent 34°03.461ʹN 004°59.214ʹW 382

Jnan sbil Oued Natural Permanent 34°03.507ʹN 004°59.264ʹW 383

Jnan Sbil Fountain Artificial Permanent 34°03.532ʹN 004°59.275ʹW 385

Grand Canal Natural Temporary 34°02.091ʹN 005°05.697ʹW 386

Fez Oued Natural Permanent 34°02.419ʹN 005°03.676ʹW 381

Marjat Lwazani puddle Natural Temporary 34°00.516ʹN 005°02.310ʹW 411 Marjat Lwazani source Natural Permanent 34°00.516ʹN 005°02.287ʹW 409

Lmzah Oued Natural Temporary 33°59.672ʹN 005°02.629ʹW 427

Diamant Vert Natural Permanent 33°59.638ʹN 005°02.657ʹW 442

Douwar Lhandiya Artificial Permanent 34°02.049ʹN 004°58.512ʹW 410 Montfleurie II Oued Natural Permanent 34°00.067ʹN 004°58.897ʹW 445 Montfleurie II Sheepfold Artificial Temporary 34°00.085ʹN 004°58.952ʹW 450

Awinat Elhajaj Natural Permanent 34°01.226ʹN 004°57.105ʹW 382

Lgaâda dam Artificial Permanent 34°01.155ʹN 004°57.213ʹW 400

Al mamounia Artificial Temporary 34°01.170ʹN 004°59.229ʹW 469

Aîn Amier source Natural Temporary 34°00.416ʹN 005°00.131ʹW 437

Lmsefer Natural Temporary 34°00.134ʹN 005°00.028ʹW 425

Aîn Chkef Natural Permanent 33°58.436ʹN 005°01.210ʹW 447

EL Heimmer Oued Natural Temporary 33°58.458ʹN 005°59.171ʹW 450

Sais Airport Natural Permanent 33°56.035ʹN 004°59.085ʹW 566

Jnan EL Alami Natural Permanent 34°04.421ʹN 004°57.710ʹW 242

Jnan Edrder Source 1 Natural Permanent 34°04.181ʹN 004°58.183ʹW 270 Jnan Edrder Source 2 Natural Permanent 34°04.066ʹN 004°58.169ʹW 266

(4)

In the Laboratory of Biotechnology and Preservation of Natural Resources (BPRN), faculty Dhar Elmehraz, the specimens were identified using an optical microscope brand OPTIKA using the identification software for The Mosqui- toes of Mediterranean Africa [5] and the identification key of the Moroccan Culicidae [17].

Data Analysis

Analysis of Data by Ecological Indices

The data were processed by ecological indices of composi- tion (relative abundance and frequency of occurrence) [4].

These indices show the distribution of the larval population and account for species diversity. They were calculated as follows:

•

the relative abundance (pi) is the relative number of individuals of each species from all collected number of individuals;

•

frequency of occurrence (F): This is the ratio expressed as a percentage of the number of samples where this species is recorded to the total number of samples collected [7];

•

constant species (F≥50%), accessory species (25% < F <

50%), and accidental species (F≤25%).

The distribution of larval species was determined according to the formula:

Distribution: C =n/N × 100, where

n: number of sites where the species was found, N: total number of sites surveyed.

Distribution classes; C1 Sporadic appearance (con- stancy 0–20%), C2 infrequent (20.1–40%), C3 moderate (40.1–60%), C4 frequent (60.1–80%), and C5 constant (80.1–100%) [28].

Study of the biotypology of culicidian fauna in the region of Fez by the generalized linear model:

•

statistical analyses were performed with R, version 3.4.3 (2017) statistic software package;

•

the study of the biological typology of the culicidian species was carried out by a generalized linear model (GLM);

•

the Poisson model was adopted to evaluate the effect of different factors on the abundance of each species;

•

then, an analysis of variance was carried out on each model to test the effects of the significant parameters.

The choice of this analysis is justified by the interest of knowing how a count variable depends on one or more explanatory variables that can be quantitative or qualitative.

The data from this study were adjusted on 128 outings.

The eleven species are the variables studied according to nine physicochemical parameters, which are the explanatory Fig. 1 Geographical location of collection site on a topographic background (extracted from the maps of Fez Est and of Fez West) [source: Direction of Geology. Ministry of Energy, Mines, Water and Environment. Rabat] /Localisation géographique des gîtes suivis sur un fond topographique (extrait de la carte de Fès Est et celle de Fès Ouest) [source : Direction de la géologie. Ministère de l’Énergie, des Mines, de l’Eau et de l’Environnement. Rabat]

(5)

variables (water temperature (°C), pH, salinity (‰), dissolved oxygen (PPM), electrical conductivity (μs/cm), flow rate m³/s, total dissolved solids (g/l), turbidity (MTU), water depth (cm)), based on four factors (sampling date, name of the mosquito breeding site area, origin, and nature of the mosquito breeding areas). The sampling date and name of mosquito breeding site area were treated separately in order to group their levels together.

Results

Of the 27 aquatic habitats that were sampled, 20 (74%) are natural habitats, of which 14 are permanent and six temporary, and seven (26%) are artificial habitats, of which five are permanent and two temporary. Sixteen (59%) were found positive and 11 (41%) were always found negative.

A total of 1,122 larvae from 11 species were collected;

four species of the genus Culex, three of the genus Anophe- les, two of the genus Uranotaenia, one of the genus Culiseta, and one of the genus Aedes (Table 2).

This study has shown that Culex species were the most abundant larvae (89.92%) (especially Cx. pipiens andCx.

perexiguus) and were collected in the various habitats from the region.Cx. pipiensis by far the most abundant species (pi: 70.68%), and it was a predominant species (F: 19.44%) and the most present (C: 48.15%) among the Culicidae group in Fez. An. maculipennis s. l. and An. sergentii occurred in low numbers with relative abundance of 0.53%

and 1.25%, respectively.

The use of the generalized linear analysis test showed that the distribution of the four species,Cx.pipiens,Cx.perexiguus,Cx.theileri, and Cs. longiareolata; was found to be significantly influenced by physicochemical parameters and habitat types (Table 3). The other species,An. cinereus,

An. maculipennis s. l., An. sergentii, Ae. flavescens, Cx. hortensis,Ur. unguiculata, andUr. balfouri, which are less abundant in the present study, have shown no statisti- cally significant relationship between their distribution and the physical and chemical parameters and the habitats sampled.

The results of the statistical analysis of the relationship between the “number of species”and the factor“breeding site”showed that space has a significant influence on the distribution of species (with a risk of error of 9% for Cx.

perexiguus, 8% for Cx.pipiens, 4 × 10⁻⁵for Cx.theileri, 3% for Cs. longiareolata, and 5.4% for Ur. unguiculata).

This means that the breeding sites of these species are different, while the results of the distribution, of larvae of An. cinereus, An. maculipennis s. l, An. sergentii, Ae. flavescens, Cx. hortensis, and Ur. balfouri, according to breeding sites, are nonsignificant results. Note that the sampling of larvae does not allow to draw the least conclusion of their distribution because it is too small. However, this observation should not be taken into account given the weakness of the number of sampling. This is the main limi- tation of this work.

The statistical results of the variable“date”according to species explain the temporal distribution of Culicidae species. The factor“sampling date”is not involved in the occurrence of Cx. pipiens, Cx. theileri, and An. sergentii. It is not significant even for An. cinereus, Cx. hortensis, and Ur. balfourirepresented by a single record larva. However, it is significant for Ae. flavescens, An. maculipennis s. l, Cx. perexiguus,Cs. longiareolata, andUr. unguiculata.

•

The species Culex pipiens was present throughout the period of study; its distribution is diversified and adapted to almost all aquatic larval habitats. Its density was high in temporary, deep, and turbid habitats loaded with ions and

Table 2 List of Culicidae identified in the region of Fez (Morocco) /Liste des Culicidae identifiés dans la région de Fès (Maroc)

Subfamily Genus Species Number Relative

abundance (%)

Frequency of occurrence (%)

Distribution of larval populations (%)

Anophelinae Anopheles Anopheles cinereus 1 0.09 0.69 3.70

Anopeles maculipenniss. l. 6 0.53 2,08 7.41

Anopheles sergentii 14 1.25 2,08 7.41

Culicinae Aedes Aedes flavescens 2 0.18 1.39 7.41

Culex Culex perexiguus 155 13.81 9.03 3.7

Culex pipiens 793 70.68 19.44 48.15

Culex hortensis 1 0.09 0.69 3.70

Culex theileri 60 5.35 11.11 33.33

Culiseta Culiseta longiareolata 83 7.40 5.56 22.22

Uranotaenia Uranotaenia unguiculata 6 0.53 1.39 3.70

Uranotaenia balfouri 1 0.09 0.69 3.70

(6)

dissolved solids and characterized by a high temperature and a pH basic, while it was low in more oxygenated environments, saline, and natural sites;

•

Culex perexiguus was more frequent in summer and autumn (August–October), especially in permanent site.

The number of larvae captured is inversely proportional to the values of the parameters: pH, TDS, dissolved oxygen, and depth, while it varies proportionally to the conductivity values;

•

Culex theilerihas been found throughout the year in almost all environments. It has been more common with increas- ing salinity and turbidity, and decreasing dissolved oxygen.

Note, however, that salinity has a p-value of 0.0614, so very close to the default threshold of 0.05, which suggest that its influence could be near that of turbidity. So this variable should be considered more carefully;

•

Culiseta longiareolatawas present in all types of breeding site and was more common in autumn and summer, espe-

cially in artificial water deposits. The increase in the number of larvae is closely related to the increase in pH and the depth of the water, and the decrease in turbidity;

•

Uranotaenia unguiculatawas found only in the Douwar Lhandiya source, known by an artificial and permanent aquatic biotope, during the months of September and October;

•

the genus Aedes is represented only byAedes flavescens;

it is weakly found in two natural breeding area, one of which is permanent and the other temporary, notably in December 2015 and October 2016;

•

the larvae ofAn. maculipenniss. l. were encountered in two permanent deposits (Lgaâda dam and Awinat El Hajaj puddle) characterized by waters with pH basic (ranging between 7.96 and 9.93) during the months of March, June, and July;

•

Anopheles cinereusandUranotaenia balfouri were collected only once in the rainy season from natural habitat (permanent and temporary, respectively);

Table 3 Significant results of the distribution of species according to the physicochemical parameters of the water and the habitat types /Résultats significatifs de la répartition des espèces en fonction des paramètres physicochimiques de l’eau et du type de gîtes

Species Parameter Estimate Std. Error zvalue Pr(>z)

Culex perexiguus Intercept 6.26e + 00 4.18e–01 14.98 <2e–16

OrigineN −3.31e + 00 2.07e–01 −15.97 <2e–16

pH −6.43e–02 4.82e–03 −13.33 <2e–16

OxyD −4.55e–01 7.18e–02 −6.34 2.4e–10

CondE 5.69e–04 8.19e–05 6.95 3.7e–12

TDS −1.80e + 00 2.87e–01 −6.28 3.5e–10

Prof −6.84e–03 3.23e–03 −2.11 0.034

Culex theileri Intercept −0.393506 0.281661 −1.40 0.1624

OxyD −0.251778 0.084465 −2.98 0.0029

Turb 0.002982 0.000613 4.86 1.1e–06

Culex pipiens Intercept −2.36e–01 2.04e–01 −1.16 0.24697

OrigineN −3.32e–01 1.23e–01 −2.70 0.00697

NatureT 5.85e–01 1.70e–01 3.45 0.00056

TC 2.97e–02 8.38e–03 3.55 0.00039

pH 4.47e–03 2.22e–03 2.01 0.04403

Sal −5.47e–01 1.06e–01 −5.17 2.4e–07

OxyD −1.13e–01 2.31e–02 −4.90 9.5e–07

CondE 1.63e–04 4.51e–05 3.62 0.00029

TDS 7.57e–01 1.14e–01 6.63 3.3e–11

Turb 2.69e–03 2.14e–04 12.58 < 2e–16

Prof 1.85e–02 1.20e–03 15.51 < 2e–16

OrigineN: NatureT 7.91e–01 2.01e–01 3.93 8.5e–05

Culiseta longiareolata Intercept −2.87217 0.50529 −5.68 1.3e–08

NatureT −1.44319 0.34129 −4.23 2.4e–05

pH 0.07592 0.00808 9.39 < 2e–16

Turb −0.11265 0.01766 −6.38 1.8e–10

Prof 0.00885 0.00287 3.08 0.0021

CondE: Electrical conductivity; OxyD: Dissolved Oxygen; OrigineN: Origine Natural; pH: Potential of hydrogen; Prof: Water depth;

Sal: Salinity; T: Temporary; TC: Water temperature; TDS: Total dissolved solids; Turb: Turbidity

(7)

•

Anopheles sergentiiwas present in October and Septem- ber in two different permanent breeding sites. It was more common in the artificial source (Douwar Lhandiya) than in the Awinat El Hajaj swamp.

Discussion

This study documented the presence of 11 culicine and anopheline mosquito species among the 43 species already reported in Morocco [25] and constitutes the first report of Ae.flavescens, the presence of which was considered dubi- ous, as it has been identified twice by the same authors, only on the basis of larval specimens [26,27]. Two larvae belonging to this species were collected in two different habitats (Diamant vert and Lmsefer) in two different seasons (Octo- ber and December months). Unfortunately, confirmation of identification on adult specimens was not made because the rearing of these larvae did not succeed; however, molecular confirmation on larval specimens is possible. Given the lack of tools of molecular identification in our laboratory, this is a research area that we are considering in perspective of this work. Subsequent surveys in these biotopes are to be considered in order to argue the addition of this species to the list of Moroccan culicidian fauna.

The present study also made it possible to update the list of culicidian species reported at the level of the Fez region, since it allowed the addition of four species;Ae. flavescens, Ur. unguiculata,Cs. longiareolata, and Cx. perexiguus, to the last inventory made in 2010 [12].

Anopheles is represented by three species, two of which are considered major potential vectors of malaria (An. maculipenniss. l. andAn.sergentii) [16].

An. maculipennis s. l., the main vector of malaria in Morocco [15], was relatively rare (0.53%) in the study area. It was recorded only three times during the months of March, June, and July. These results are consistent with what is reported in the literature indicating that this mosquito is widespread in the country but predominates in the coastal plains where it constitutes the majority of the anopheline population [16,18,25].

Anopheles sergentiiis the most abundant anopheline species in the Fez region, accounting for 66.7% of the total Anopheles captures. This species, whose geographical distribution extends throughout Morocco with the exception of the Atlantic coastal areas in the west of the country, was considered to be the main vector of malaria in southern Morocco [16]. In addition to its involvement in malaria transmission due toPlasmodium vivax,An. sergentiiis considered an important vector ofP. falciparummalaria in several countries such as Egypt, West and Southeast Saudi Ara- bia, Yemen, Jordan, and Libya [6,16,29]. The coexistence of

this species in the Fez region with potential reservoirs of the P. falciparum parasite is a risk that needs to be assessed.

Indeed, in addition to the continuous increase in the number of African immigrants settling in the Fez region and who constitute potential reservoirs of tropical malaria, the region reports about ten imported malaria cases each year, mainly due toP. falciparumfrom tropical countries [10]. The prob- ability of these reservoirs coming into contact with the vector in areas of potential transmission and during the favorable season for transmission should be assessed.

Culex was represented in this study by four species but Cx.pipiensis by far the most abundant (70.7%) and the most frequent species (19.44%). Indeed, this mosquito was collected throughout the year, in different biotopes: in natural and artificial, temporary and permanent breeding sites, with and without vegetation, and in clean and polluted waters, though it exhibited a very marked preference for polluted deposits. The findings expressed herein are those known about this mosquito in the literature at world level [3,23]

and are consistent with the observations made in the center of Morocco [12], in the region of Tetouan [9] and in the northern Morocco [20]. This mosquito is known for its great nuisance to the populations living near its breeding sites [14]. It is also considered to be the main vector respon- sible for the West Nile virus outbreaks that have affected Morocco [13]. As a result, its abundance in the Fez region must be a subject of attention to overcome the health risks it can engender.

The analysis of the relationship between Culicidian species and the physical and chemical parameters of the breeding sites made it possible to characterize those that are favorable to the development of species that present a health risk for the region.

Anopheles, vectors of malaria, develops particularly in stagnant, shallow, and sufficiently sunny water (Douar Lhan- diya source, Awinat Elhajaj swamp, and Lgaâda dam). Larvae ofAn. maculipenniss. l. were collected only from clear waters with submerged vegetation, those ofAn.sergentiifrom waters containing decomposing plant substances. These results are in line with those of other studies carried out in other regions of Morocco [18,19].

As forCx. pipiens, its high abundance has been particularly noted in water heavily polluted by domestics (Oued Lmzah, Montfleurie II Sheepfold, Lmsefer, and the Grand Canal) or industrials sewages (Oued Wislane, Oued Sebou, and Oued Jnan EL Alami). These results corroborate those found by other authors in the regions of Tetouan, Fez, Tang- ier, and Kenitra [8,12,20,22].

The effect of physicochemical characteristics of larval breeding site on the development of different culicidian species has already been noted by several authors [2,11], pH, for example, seems to play a role in controlling larval development andCx.pipienslarvae develop in water rich in organic

(8)

matter at basic pH [3], while Anopheles larvae prefer well- oxygenated water at an acidic [22] or neutral pH [11,18].

Conclusion

The results of this study made it possible to update the list of mosquito species recorded in the Fez region. Eleven species belonging to five different genera were found: Anopheles, Culex, Aedes, Culiseta, and Uranotaenia. Among these species, three are known for their major role in the transmission of important diseases, namely, An. maculipennis s. l andAn. sergentii in malaria transmission and Cx. pipiens in West Nile virus fever transmission. The biotypology of the breeding sites made it possible to characterize the sites specific to each of the three vector species. The results thus generated will be an essential prerequisite and will contribute to the development of any entomological surveillance and vector control strategy in the region.

AcknowledgementsWe warmly thank the persons who con- tributed to the execution of this work; I mean Mohammed El Kohli, El Houssein Lakraa, Fouad El-Akhal, and Fatima zahra Faqihi for their advice, help in the field, and their collaboration.

Conflict of Interests:The authors do not have any conflict of interest to declare.

References

1. Aichetou Mint ML, Ould Lemrabott MA, Hasni Ebou M, et al (2017) Mosquitoes (Diptera: Culicidae) in Mauritania: a review of their biodiversity, distribution and medical importance. Parasi Vectors 10:35 doi 10.1186/s13071-017-1978-y

2. Benhissen S, Habbachi W, Rebbas K, et al (2018) Études entomologique et typologique des gîtes larvaires des moustiques (Diptera: Culicidae) dans la région de Bousaâda (Algérie).

Bull Soc Roy Sci Liege 87:112–20

3. Berchi S, Aouati A, Louadi K (2012) Typologie des gîtes pro- pices au développement larvaire de Culex pipiens L. 1758 (Diptera-Culicidae), source de nuisance à Constantine (Algérie).

Ecologia Mediterranea 38:2

4. Bouabida H, Djebbar F, Soltani N (2012) Étude systématique et écologique des Moustiques (Diptera: Culicidae) dans la région de Tébessa (Algérie). Entomologie Faunistique–Faunistic Entomol- ogy 65:99–103

5. Brunhes J, Rhaim A, Geoffroy B, et al (1999) Les moustiques de l’Afrique méditerranéenne. Logiciel d’identification et d’en- seignement. IRD & IPT, CD-Rom collection didactique, Éditions IRD. Montpellier, France

6. Cope SE, Gad AM, Presley SM (1995) New record of the malaria vector Anopheles sergentii in the southern Nile Valley of Egypt. J Am Mosq Control Assoc 11:145–6

7. Damerdji A (2008) Contribution à l’étude écologique de la mala- cofaune de la zone Sud de la région de Tlemcen (Algérie). Afr Sci 4(1):138–53, 16 p

8. El Joubari M, Hajji K, Himmi O, et al (2015) Étude des macroin- vertébrés (Gastéropodes, Diptères et Odonates) des marais de Smir- Restinga (Nord-Ouest du Maroc). Entomologie Faunistique–Fau- nistic Entomology 68:17–31

9. El Joubari M, Louah A, Himmi O (2014) Les moustiques (Dip- tera, Culicidae) des marais de Smir (nord-ouest du Maroc):

inventaire et biotypologie. Bull Soc Pathol Exot 107:48–59. doi:

10.1007/s13149-014-0327-4. Epub 2014 Jan 8 [http://www.

pathexo.fr/documents/articles-bull/131490048.pdf]

10. El Ouali Lalami A, Cherigui M, Ibnsouda Koraichi S, et al (2009) Le paludisme importé au Centre Nord du Maroc entre 1997 à 2007. Sante 19:43–7

11. El Ouali Lalami A, El Hilali O, Benlamlih M, et al (2010) Étude entomologique, physicochimique et bactériologique des gîtes larvaires de localités à risque potentiel pour le paludisme dans la ville de Fès. Bulletin de l’Institut Scientifique, Rabat, Section Sciences de la Vie 32(2):119–27

12. El Ouali Lalami A, Hindi T, Azzouzi A, et al (2010) Inventaire et répartition saisonnière des Culicidae dans le centre du Maroc.

Entomologie Faunistique–Faunistic Entomology 62(4):131–8 13. Failloux A, Bouattour A, Faraj C, et al (2017) Surveillance of

arthropod-borne viruses and their vectors in the Mediterranean and Black Sea regions within the MediLabSecure Network.

Curr Trop Med Rep 4:27–39. doi: 10.1007/s40475-017-0101-y.

Epub 2017 Mar 17

14. Faraj C, Elkohli M, Lyagoubi M (2006) Cycle gonotrophique de Culex pipiens (Diptera: Culicidae), vecteur potentiel du virus West Nile, au Maroc : estimation de la durée en laboratoire.

Bull Soc Pathol Exot 99:119–21 [http://www.pathexo.fr/documents/articles-bull/T99-2-2846-3p.pdf]

15. Faraj C, Ouahabi S, Adlaoui E, et al (2008) Risque de réémer- gence du paludisme au Maroc : Étude de la capacité vectorielle d’Anopheles labranchiaedans une zone rizicole au nord du pays.

Parasite 15:605–10

16. Faraj C, Ouahabi S, Adlaoui E, et al (2010) État actuel des connaissances sur les anophèles du Maroc (Diptera: Culicidae): sys- tématique, distribution géographique et compétence vectorielle.

Rev Epidemiol Sante Publique 58:349–57. doi: 10.1016/j.respe.

2010.05.002

17. Himmi O, Dakki M, Trari B, et al (1995) Les Culicidae du Maroc : clés d’identification, avec données biologiques et écolo- giques. Travaux de l’Institut Scientifique, Série Zool Rabat 44:51 18. Laboudi M, Faraj C, Sadak A, et al (2012) Some environmental factors associated withAnopheles Labranchiaelarval distribution during summer 2009, in Larache Province, Morocco. Afr Ento- mol 20:229–38

19. Laboudi M, Sadak A, Ouahabi S, et al (2014) Molecular charac- terization of Anopheles maculipennis complex (Diptera: Culici- dae) in Northern Morocco. Entomologie Faunistique–Faunistic Entomol 67:37–42

20. Louah A, Ramadani M, Saoud Y, et al (1995) Biotypologie de la faune culicidienne de la péninsule tingitane. Bull Inst Sci Rabat 19:93–102

21. Manguin S, Boete C (2011) Global impact of mosquito biodiversity, human vector-borne diseases and environmental change. In:

Pujol JL (ed). The importance of biological interactions in the study of biodiversity. InTech Winchester, UK. biodiversity human-vector-borne-diseases-and-environmental-change, pp 27–50 22. Marc I, Chibani A, Alemad A, et al (2016) Étude écologique et entomologique des culicides larvaires des gîtes de la province de Kenitra (Maroc). Eur Sci J 12:398–409. doi: 10.19044/esj.

2016.v12n32p398

23. Martínez-de la Puente J, Ferraguti M, Ruiz S, et al (2016) Culex pipiens forms and urbanization: effects on blood feeding sources and transmission of avian Plasmodium. Malar J 15:589.

doi 10.1186/s12936-016-1643-5

(9)

24. Service SW (1976) Mosquito ecology: fields sampling methods.

Applied science publishers, London, 583 p

25. Trari B, Dakki M, Harbach RE (2017) An updated checklist of the Culicidae (Diptera) of Morocco, with notes on species of his- torical and current medical importance. J Vector Ecol 42:94–104.

doi: 10.1111/jvec.12243.

26. Trari B, Dakki M, Himmi O, et al (2003) Les moustiques (Dip- tera Culicidae) du Maroc. Revue bibliographique (1916-2001) et inventaire des espèces. Bull Soc Pathol Exot 96:329–34 [http://www.pathexo.fr/documents/articles-bull/T96-4-2405- 2552.pdf]

27. Trari B (2017) Les moustiques (Insectes, Diptères) du Maroc : Atlas de répartition et études épidémiologiques. Thèse de doctorat d’état, faculté des sciences de Rabat, université Mohammed-V, Maroc, 356 p

28. Vanlalruia K, Senthilkumar N, Gurusubramanian G (2014) Diversity and abundance of mosquito species in relation to their larval habitats in Mizoram, North Eastern Himalayan region. Acta Trop 137:1–18.

doi: 10.1016/j.actatropica.2014.04.026. Epub 2014 Apr 30 29. WHO (1991) Vector bionomics in the epidemiology and control

of malaria. Part II. The WHO European region and the WHO eastern Mediterranean region. WHO, Geneva, 352 p