Culicoides (Diptera: Ceratopogonidae) Fauna in Central Tunisia

For a better understanding of the Culicoides spp biodiversity of the Center of Tunisia, an entomological survey was carried out between 2009 and 2012 in four districts. A total of 9275 biting midges were collected from different sites in the Center of Tunisia using CDC and OVI light traps as well as emergence in the laboratory from mud sampling. 
Twenty two species were identified of which two were newly recorded for the Tunisian fauna. The most abundant Culicoides spp occurring on the Center of Tunisia were Culicoides imicola (6.9%) captured by light traps and Culicoides circumscriptus (70.93%) from mud. Other less abundant species were also identified including Culicoides paolae (15.04%); Culicoides sahariensis (12.45%); Culicoides jumineri (9.72%); Culicoides cataneii (66.07%); Culicoides puncticollis (5.34%); Culicoides newsteadi (3%); Culicoides kingi (1.36%); C. circumscriptus (0.71%); Culicoides spp near kibunensis (0.58%); Culicoides. pseudojumineri (0.31%); Culicoides heteroclitus (0.10%); Culicoides pseudopallidus (0.03%); Culicoides saevus (0.15%); Culicoides submaritimus (0.05%); Culicoides langeroni (0.05%); Culicoides punctatus (0.05%); Culicoides pseudolangeroni and C. jumineri var (0.03%). Culicoides kurensis and C. puncticollis (0.01%). 
During this study two species of Culicoides were reported for the first time: Culicoides sergenti (3.1%) (Region of Kairouan) and Culicoides semimaculatus (0.04%) (Region of Sidi Bouzid). An updated checklist is provided for the 35 species of Culicoides now known to occur on Tunisia. 
This study has the potential to significantly improve our understanding of the epidemiology of Bluetongue (BT) in Tunisia in recent years.


Introduction
Culicoides biting midges (Diptera:Ceratopogonidae) are small, blood-sucking insects that feed on a wide range of hosts, and act as vectors for pathogens responsible for animal and human diseases worldwide. Of more than 1316 biting midges species which have been described to date, approximatively 50 arboviruses have been isolated from species of Culicoides [1,2]. Most recently, Culicoides have been acknowledged as a vector of the newly identified Schmallenberg bunyavirus (genus Orthobunyavirus; Bunyaviridae) (SBV) in Europe [3,4].
An outbreak of BT (serotype 2) occurred in Tunisia between 1999 and 2002 [5]. The first such outbreak appeared during autumn 1999 in the eastern part of the country along the coast. The overall morbidity and mortality rates were 8.35% and 5.5% respectively [5]. In 2000, 72 outbreaks were reported between June and October affecting 6.120 sheep in the eastern and central parts of the country.
Entomological investigations have reported an increasing number of species during the last decades. Indeed, in the early eighties' 19 species were identified [6] including 10 new ones in Tunisia. In 2005, the number of the recorded species was 22 with three new ones: C. paolae, C. imicola and C. newsteadi [5]. In 2008, Hammami et al. identified 14 species with one new for the fauna: C. punctatus. Finally, Sghaier et al. [7] identified 25 species of which 7 were identified for the first time: Culicoides obsoletus, Culicoides fascipennis, Culicoides subfasciipennis, Culicoides santonicus, C. submaritimus, Culicoides univittatus and Culicoides indistinctus.
Despite the reports of severe outbreaks of arboviral diseases in domestic animals in Center of Tunisia, the studies interested in vector potential of Culicoides are disparate and sparse. Thus updated information on species composition and distribution is required to assess the economic losses due to this serious hematophagous pest.
The present study aimed to improve the knowledge of the Culicoides fauna in Tunisia by studying the biodiversity using light trapping and breeding site sampling.

Study area
An entomological investigation was carried out in both Eastern and Western Centre of Tunisia including Monastir, Mahdia, Kairouan and Sidi Bouzid region (Table 1 and Figure 1).

Regions
Surface (

Field capture of biting midges
Light traps: Biting midges were collected between 2009 and 2012 using two light traps models: home-made miniature CDC (Centre of Disease Control, Atlanta, USA) and OVI (Onderstepoort Veterinary Institute). All collections were done in human-inhabited biotopes where domestic animals (i.e., cattle, horses, dogs, goats, and chicken) are present ( Table 2). The traps were installed no more than 1 m from the ground near to animals, either outside or inside shelters (Table 2). Traps were set before sunset and collected the next morning.  Breeding sites: Mud samples were collected from different breeding sites ( Figure 2). The dominant vegetation is Juncus and Salicornia. Mud samples of 750 cm 3 and were scraped from the soil surface using a flat trowel in a line parallel to the water's edge. Samples containing the Culicoides larvae at different instars were transported to the laboratory and placed in crystallizers closed by a glass plate to prevent the escape of adults. Morphological identification: The head, wings and genitalia of individual biting midges were cut off within a drop of ethanol and slide-mounted in Canada balsam. Culicoides spp were morphologically identified and separated using their wing patterns according to the key of [8,6,9,10].

Period of Collections
Statistical analysis: The analysis of the faunistic data was conducted according to the methods of standard community analysis described by [11] using "PRIMER 6" (Plymouth Routines in Multivariate Ecological Research) package. The multivariate analysis of the faunistic sites affinity was carried out by non-parametric multidimensional scaling (NMDS) ordination on the basis of Bray-Curtis similarity. For estimation of similarity and differences in the Ceratopogonidae community composition, cluster analysis was used. Similarity among sites was determined using the Bray-Curtis similarity index.  Several indexes were calculated for each site in order to assess the species assemblages: species richness (number of species: S), diversity (Shannon-Wiener index H' = -å (Pi × Log2 Pi)) and evenness (Pielou index J'=H'/ Log2S), where H' is the diversity index and RS is the species richness (Table 3).

Results
Culicoides spp presence and abundance in Central Tunisia. Between 2009 and 2012 a total of 5325 specimens of biting midges (3589 females and 1736 males) were collected using light traps. Thus, 19 species of Culicoides were identified. Diversity of Culicoides varied depending on sites, reflecting the environmental differences in Center of Tunisia. A summary of the frequency of each of these species and their abundance in the study area is provided Table 4.    Table 6: Ecological data and characterization of the sampling place. Table 5 shows the results of the environmental surveys for the investigated sites. The analysis of the faunistic data using according to table 6, Culicoides spp frequent biotope represented by vegetation, water, human housing and farms. An updated checklist of all 34 species of the genus Culicoides recorded from Tunisia is provided in Table 7 and including the references of the first report of each species.   Moreover, the distribution of Culicoides spp based on geographic characteristics of the studied region is shown in Figure 3.

Discussion
The objective of this study was to update knowledge on the Culicoides fauna present in Center of Tunisia. In fact, few data were found describing the Culicoides population composition in Tunisia [6,12,13,7]. Ceratopogonidae in Tunisia have received modest attention from collectors and their biology is poorly understood. Only restricted geographical areas have been intensively surveyed and In the present study, 22 Culicoides spp were identified in Central Tunisia including two newly described species: C. sergenti (district of Kairouan) and C. semimaculatus (region of Sidi Bouzid), which is less than the previously reported study. In fact, [6] identified 10 species newly introduced for the fauna. Evenly, Chaker and co-workers identified three new ones [12]. Moreover, [7] identified 7 for the first time. This may be due to the trap type, position and the period of study. Long-term trapping is recommended for a better assessment of the number of Culicoides spp present in the country.
C. sergenti was reported from Algeria and Maroc [5]. Therefore, it is important to assess the potential role and the host preference of C. sergenti.
C. semimaculatus has previously been reported to be geographically limited to southern Europe and detected in proximity to cattle, goats and sheep [14]. So it's important to capture this species and to analyze its potential role to transmit disease.
The most abundant species collected were C. imicola, C. paolae and C. jumineri. This finding agrees with those of previous surveys in Tunisia [12,15] in which these species, were found to represent >90% of all biting midges captured. Even more interesting, C. imicola was trapped in high numbers in the governorate of Monastir. The high abundance of this species is can be ascribed to: (1) the use of the Onderstepoort blacklight (UV) suction traps [16]. In fact, [17] have demonstrated that Blacklight was more attractive to vector insects than white light and increases monitoring sensitivity in areas where vector abundances are low; (2) the biotic and abiotic factors linked to the availability of suitable breeding habitats which did not occur locally and equally for all species. Indeed, in Sardinia, C. imicola and C. newsteadi occur more frequently in coastal areas at lowers altitudes, whereas C. obsoletus and Culicoides pulicaris are restricted to more mountainous central areas [14]. Previous studies have suggested that the high abundance of C. imicola is most likely linked to its preference for breeding in areas where soil is moist and nutrient-rich and with full exposure to sunlight, characteristics which are more commonly found in the centre [18]. Statistical analysis of our data suggested that C. imicola was significantly more abundant in the region of Monastir than in other regions. We therefore assume that C. imicola populations is likely to be settled in Tunisia and seem to have a major role in BT outbreaks. It's necessary to verify the presence of other potential vector than C. imicola in Center of Tunisia.
The second abundant species was C. paolae. This species was reported from Malta, Sardinia, Corsia, Tunisia, France, Algeria, Greece and Spain in chronological order [19]. In Tunisia, it was reported for the first time in the governate of Monastir from Central Tunisia [12]. Previous studies, have proposed that C. paolae from southern Italy feed on horses [20]. Although its antennal and palpal morphology have led other authors to speculate that it feeds preferentially on birds [2]. Even so, the establish of the host preference and larval habitat of C. paolae is crucial, since this species is very widespread and abundant in the Center of Tunisia.
C. jumineri was caught in light traps. However, only low numbers were reared from mud near an irrigation channel. This finding corroborates earlier observations made by [21] who reported that C. jumineri emerged from mud near irrigation channel. Also, [22] suppose that C. jumineri was reared in the reach environment with vegetation like the gross-covered pool.
C. circumscriptus were the most abundant species obtained by emergence. As previously reported, this species prefers puddles of water contaminated with animal excreta [23] and in the absence of surface water occurs in wet soil rich in organic matter [24]. In our study, C. circumscriptus were reared in almost all collected mud samples and seems to be plastic in its tolerance for the range of environmental conditions that existed. Even more interesting is that C. circumscriptus sharing the same biotope with C. puncticollis, C. cataneii and C. sahariensis. The current result corroborates the findings of [22] who demonstrated the association of C. circumscriptus with C. cataneii and C. sahariensis.
Information on the species composition and distribution of Culicoides vectors is a necessary prerequisite to understand the epidemiology of Culicoides-borne pathogens.
The results of the survey described here therefore have the potential to significantly improve our understanding of the epidemiology of BT in Tunisia in recent years. Culicoides are important vectors for a range of pathogens causing diseases with veterinary and public health importance including BTV, AHSV, epizootic haemorrhagic disease (EHDV), filarial diseases and the recently discovered SBV. It is important to note that the high abundance of C. imicola in the Center Tunisia underlines the real risk of spreading new disease. Thus, it is important to improve our understanding of climatic factors in C. imicola activity influencing their distribution and seasonal pattern.
Further study, are needed to continue the monitoring of other potential vectors in an attempt to limit the potential incursion and spread of the disease in other region of Tunisia like southern Tunisia.