Update of the Culicoides (Diptera: Ceratopogonidae) species checklist from Algeria with 10 new records

The Culicoides fauna of Algeria has been historically investigated, leading to the description of many new species by Kieffer in the 1920s, Clastrier in the 1950s or Callot in the 1960s and to a comprehensive inventory by Szadziewski in the 1980s. The emergence of bluetongue in the late 1990s enhanced Culicoides collections made in the country over the last two decades, but information remained mostly unpublished. The aim of this study is therefore to provide a comprehensive and updated checklist of Culicoides biting midge species in Algeria. The literature (published and grey, in French and in English) from 1920 to date on Culicoides collections in Algeria was collected and analyzed in the light of the current taxonomic and systematic knowledge and methods. Fresh Culicoides material was also analyzed using light/suction trap collections carried out from November 2015 to September 2018 in nine localities of the ‘wilayah’ of Tiaret (northwestern Algeria). Slide mounted specimens were identified morphologically using the interactive identification key IIKC and original descriptions. Specimens were then compared with non-type material originating from different countries and partly with type material. A total of 13,709 Culicoides, belonging to at least 36 species within 10 subgenera, were examined leading to 10 new records in Algeria, including C. chiopterus, C. dewulfi, C. navaiae, C. grisescens, C. paradoxalis, C. shaklawensis, C. simulator, C. univittatus, C. achrayi and C. picturatus. These new records and all previous records provided by the literature review were discussed. We propose a Culicoides checklist for the Algerian fauna of 59 valid species, including species mainly with a large Palaearctic distribution and a specific Mediterranean distribution, and only a few species from the Afrotropical region. Among them, several species, mainly of the subgenera Avaritia and Culicoides, are confirmed or probable vectors of arboviruses important in animal health.


Background
Biting midges of the genus Culicoides Latreille (Diptera: Ceratopogonidae) are small hematophagous dipterans, subgenera and 38 species groups (not placed to a subgenus), whereas 13% of the world fauna are not placed in any subgenus or group [4].
The worldwide Culicoides fauna has been diversely investigated by a number of authors. In the western Palaearctic region, several monographs and catalogues are still considered as a reference for the Culicoides fauna [5][6][7], whereas an interactive identification key has been developed for Culicoides females [8]. The Afrotropical fauna has benefited from various contributions, including those of Khamala & Kettle [9] for eastern Africa and Glick [10] for Kenya in particular. Northern Africa is located in the southwestern part of the Palaearctic region and separated from the Afrotropical region by the Sahara Desert. The fauna of this region is composed mainly by species belonging to the Palaearctic fauna, along with some of the Afrotropical fauna.
From 2000 to 2011, three BTV serotypes were reported in Algeria, leading to 297 outbreaks in 2000 (BTV-2), 263 in 2006 (BTV-1) and six in 2011   [34,35]. A few years after these different episodes of BTV transmission, limited-scale surveys for Culicoides were carried out in northern and southern Algeria [36][37][38], before nationwide entomological surveillance was implemented in 2007 by the National Institute of Veterinary Medicine [39,40]. Moreover, extensive collections have been carried out in the eastern part of Algeria [41,42]. Altogether, these recent studies increased the total number of species recorded in Algeria to 52 by identifying 12 new species for the Algerian fauna.
In this paper, we aimed to produce a comprehensive checklist of Culicoides species in Algeria, including a list of confirmed or probable BTV and AHSV vectors. For this purpose, we compiled and discussed the existing known information from published and from grey literature and examined new material collected using UV light/suction traps carried out from November 2015 to September 2018 in the 'wilayah' of Tiaret (northwestern Algeria).

Methods
Published papers (in French and English) related to Culicoides collections in Algeria were collated following searches of classical bibliographic databases (PubMed ® and Google Scholar) and on institutional open access repositories (Agritrop from Cirad and Horizon from IRD). The unpublished 'grey' literature (field reports and theses) was collated from the two latter databases and from personal databases of authors, but also by contacting authors of the publications and the teams working on Culicoides in Algeria. The literature from 1920 to date was analyzed in light of the current taxonomic and systematic knowledge and methods.
We examined Culicoides individuals collected using UV light/suction traps, from November 2015 to September 2018, from nine localities in the 'wilayah' of Tiaret. The collected Culicoides were transferred to 70% ethanol for preservation. The study area is located in the western part of the 'Hauts Plateaux' (Highlands or High plateaus), a steppe-like region between the Tell and Saharan Atlas ranges in northwestern Algeria. It is dominated by a continental and semi-arid climate, characterized by very dry summers (300-500 mm per year, with a drought of 6 to 8 months) and cold winters (average annual temperature ranging from 13 to 17 °C) [43]. Mainly dedicated to pastoral activities, the Tiaret region encompasses forested areas composed by cork and holm oaks, Aleppo pine and cypresses.
From the material available, we selected a series of specimens that exhibited a variety of wing patterns, and also those with plain wings. Before identification, the specimens were dissected and slide mounted following the Wirth & Martson [44] procedure. Slide mounted specimens were therefore identified morphologically to the species level using the interactive identification key IIKC of Mathieu et al. [8] and original descriptions. The specimens were then compared with non-type material from different countries and partly with type material available in the collection of the Institut de parasitologie et de pathologie tropicale de Strasbourg, France. To illustrate the new recordings, the wings were photographed using a Zeiss Standard 25 microscope (Zeiss, Oberkochen, Germany) equipped with a Nikon DS-Fi3 camera and Nikon NIS elements v4.6 software (Nikon corporation, Tokyo, Japan). The morphological terminology follows that of Mathieu et al. [8] and Ramilo et al. [45]. With the exception of the species composition of the subgenus Sensiculicoides, which follows Szadziewski et al. [46], the subgeneric placement of species other than the latter subgenus and distribution follow Szadziewski et al. [47].
To confirm morphological identification, a small number of specimens were sequenced for the cytochrome c oxidase subunit 1 (cox1) barcode region following the same procedure detailed in Bourquia et al. [19].

Results
A total of 13,709 Culicoides, belonging to at least 36 species within 10 subgenera, were collected in nine localities of the Tiaret region from November 2015 to September 2018. The results will be analyzed in detail in another publication. Here, we report the 10 new records from Algeria, with special emphasis on C. navaiae. The list of the examined material is presented in Table 1.
We report the presence of Culicoides (Avaritia) chiopterus (Meigen) (synonyms Culicoides amoenus Winnertz, 1852; Culicoides similis Goetghebuer, 1927; Culicoides dobyi Callot & Kremer, 1969). Females may be identified using the following criteria. General aspect of the wing light greyish with little contrast as represented in Fig. 1; pale spots poorly defined and barely visible; poststigmatic pale spot covering more than one-third of the 2nd radial cell; absence of pale spots in the distal part of the wing. Hairy eyes with scattered and short interfacetal hairs, and eyes united over a short distance. First abdominal tergite with 1-2 lateral hairs. Two functional spermathecae subequal and one rudimentary. This species is distributed in the Holarctic region, including Austria, Belgium, Czech Republic, Denmark, Estonia, France with Corsica, Germany, Hungary, Ireland, Lithuania, the Near East, the Netherlands, Poland, Romania, Russia, Spain, Slovakia, Switzerland, Turkey, Ukraine [48], the UK and in the Nearctic region. To date, only two species with hairy eyes have been recorded in the Palaearctic region, of which C. chiopterus is the only one described from North Africa. We produced a cox1 barcode sequence of the C. chiopterus female (GenBank: MT782149) to confirm the morphological identification.
We report the presence of Culicoides (Avaritia) dewulfi Goetghebuer (synonym Culicoides pseudochiopterus Downes & Kettle, 1952). Females may be identified using the following criteria. General aspect of the wing greyish and well contrasted with the darker part between the 1st and the 2nd radial cells as represented in Fig. 2; pale spots poorly defined; poststigmatic pale spot covering more than one third of the 2nd radial cell; the distal pale spot/ area of r3 larger than the pale spot/area in the distal part of m1. Eyes bare and joined over a short distance. First abdominal tergite with 8-12 lateral hairs. Two functional spermathecae unequal and one rudimentary. This Palaearctic species is widely distributed in Europe, including Belgium, Czech Republic, Denmark, Estonia, France with Corsica, Germany, Italy, Poland, Romania, Russia, Slovakia, Spain, Switzerland and the UK [48]. While phylogenetically well separated from species of the Obsoletus group [49], the wing pattern of C. dewulfi shares similarities with the latter group. By prior confirmation of slide mounted specimens, the combination of (i) the contrasted wing pattern, (ii) the pale spot/area in the distal part of r3 larger than that of m1 and (iii) the numerous lateral hairs on the first abdominal tergite are good indicators of the species under stereomicroscopy.
We report the presence of Culicoides (Beltranmyia) navaiae Lane. Females may be identified using the following criteria. Wing with a few very faint spots as represented in Fig. 3; no spots in the distal part of r3; absence of macrotrichia in the basal cell. Eyes bare and closely separated (Fig. 4). Sensilla coeloconica present on flagellomeres 1, 9-12. Absence of a postpharyngal armature. Third palpal segment triangular and moderately swollen with single, wide and shallow sensory pit. One functional spermathecae without a pigmented neck (Fig. 5). Males may be identified using the following criteria. Wings pale with fainter spots than in females and barely visible. Ninth tergite broad with a median deep notch, short and divergent apicolateral processes; ninth sternite with bare ventral membrane. Aedeagus with rounded basal arch extending more than two-thirds of the total length, slender and curved-ended basal arms, distal process moderately long (one third of the total length) and bluntended (Fig. 6). Parameres separated, swollen at the base and tapering distally to a slender point. This species is reported in the Arabian Peninsula especially in Bahrain, Saudi Arabia, and in the Sinai [50][51][52]. Culicoides navaiae is the only Beltranmyia species from the Palaearctic and the Afrotropical regions exhibiting the combination of the following characters: very faint wing pattern close to the plain wing aspect, no spots on the distal part of r3 and sensilla coeloconia distribution on flagellomeres 1,    9-12. The wing pattern of C. navaiae may appear similar to the unspotted wing of C. homochrous Remm, but the latter has a sensilla coeloconica distribution on flagellomeres 1-2, 7, 9, 11-12 [53]. However, Glukhova [54] reported for C. homochrous the sensilla distribution on flagellomeres 1-3, (7), 9-12 and thus the morphological characteristics of this species should be clarified. We report the presence of Culicoides (Culicoides) grisescens Edwards (synonyms Culicoides remmi Damian-Georgescu, 1972; Culicoides arschanicus Mirzaeva, 1984). Females may be identified using the following criteria. Spotted wing as represented in Fig. 7; cua1 cell pale. Eyes bare, joined over a short distance or separated but connected by a suture. Third palpal segment slender or slightly swollen with multiple and irregular pits. Two functional spermathecae subequal; short pigmented neck and one rudimentary spermathecae. Foreleg and hind leg with spines on first and second tarsomeres; middle leg with spines from first to fourth tarsomeres. This species is distributed in the northern and eastern parts of the Palaearctic region, including Belgium, Denmark [55], Estonia, France, Germany, Poland, Romania, Russia, Slovakia, Switzerland and Ukraine [48]. A phylogenetic study based on ITS2 markers and morphological observations led their authors to suggest that C. remmi Damian-Georgescu should be raised from synonymy with C. grisescens [56,57]. We produced a cox1 barcode sequence of a C. grisescens female (GenBank: MT782148) to confirm the morphological identification.
We report the presence of Culicoides (Culicoides) paradoxalis Ramilo & Delécolle. Females may be identified using the following criteria. Spotted wing as represented in Fig. 8; pale spot in the proximal part of m2 smaller than the one in m1 or absent; poststigmatic pale spot covering half of the 2nd radial cell; cua1 cell centered by a dark spot. Eyes bare and joined over a short distance. Two functional spermathecae subequal and one rudimentary. Middle leg with spines on tarsal segments 1 to 3. This species is distributed in France and Portugal [45]. In recent years, the morphological and molecular diversities within the subgenus Culicoides have been studied, leading to the description of several new species [58][59][60].   We report the presence of Culicoides (Sensiculicoides) shaklawensis Khalaf (synonym Culicoides caspius Gutsevich, 1959). Females may be identified using the following criteria. Spotted wing as represented in Fig. 9; second radial cell completely dark with the R3 vein slightly covered by the poststigmatic pale spot; most of the veins M1, M2 and CuA1 pale with the exception of the distal tip dark. Eyes bare and separated narrowly. Sensilla coeloconica present on flagellomeres 1, 9-13. Two functional spermathecae unequal and one rudimentary. Middle leg with spines on tarsal segments 1 to 3. Described from Iraq, this species is widespread in the Middle East [61], and reported from Bulgaria, Cyprus [61], Czech Republic, France with Corsica [62], Italy, Morocco, Slovakia, Spain, Tunisia, Turkey and Ukraine. The very characteristic wing pattern of C. shaklawensis can be used to identify this species with certainty in the Palaearctic region. In Central Asia, C. kugitangi Ataev described in Turkmenistan has a wing pattern similar to that of C. shaklawanesis and females can be easily separated from the latter species by the sensilla coeloconica distribution present on all flagellomeres [54].
We report the presence of Culicoides (Sensiculicoides) simulator Edwards. Females may be identified using the following criteria. Spotted wing as represented in Fig. 10; poststigmatic pale spot covering one third of the 2nd radial cell; pale spot on the r-m cross vein extending to the proximal part of m2 which layers and crosses the vein M2; distal pale spot in r3 larger than the distal pale spot in m1. Eyes bare and separated narrowly. Sensilla coeloconica present on all flagellomeres 1-13. Two functional spermathecae unequal and one rudimentary; absence of sclerotized ring at the end of the spermathecal duct. Middle leg with spines on tarsal segments 1 to 4. This species is reported from the Balkans [63], Czech Republic, Denmark, Estonia, France, Germany, Hungary, Italy, Lithuania, Morocco, the Netherlands, Poland, Portugal, Russia, Slovakia, Spain, Turkey, Ukraine [64] and the UK. It is the only Palaearctic Culicoides with the pale spot on the R-M cross vein extending to the proximal part of m2 cell, which layers and crosses the M2 vein and therefore reaches the proximal part of m1 cell.
We report the presence of Culicoides (Sensiculicoides) univittatus Vimmer (synonym Culicoides agathensis Callot, Kremer & Rioux, 1963). Females may be identified using the following criteria. Spotted wing as represented in Fig. 11; second radial cell entirely dark with the R3 vein slightly covered by the poststigmatic pale spot; pale spots on the distal part of r3 and m1 rounded and separated from the wing margin. Eyes bare and separated narrowly. Sensilla coeloconica present on all flagellomeres. Two functional spermathecae unequal and one rudimentary. Male wing similar to female with the usual sexual differences. Aedeagus with rounded basal arch extending about one-third of total length, distal process long (twothirds of total length), triangular with blunt rounded tip. Parameres separated and tapering to a fine point; ninth sternite with ventral membrane bare or rarely bearing few spicules. This species is reported from Albania, Cyprus [61], France (southern mainland and Corsica), Italy (mainland and Sardinia), Morocco [11,12], Portugal, Spain, Tunisia [20] and Turkey. This is the only species of Sensiculicoides with the pale spots on the distal part of r3 and m1 separated from the wing margin. Very close to C. pictipennis (Staeger), the male can be distinguished by the wing pattern and the distal process of the aedeagus, which is triangular for C. univittatus and rectangular for C. pictipennis. We report the presence of Culicoides (Silvaticulicoides) achrayi Kettle & Lawson. Females may be identified using the following criteria. Greyish wing with few spots as represented in Fig. 12; second radial cell completely dark with the R3 vein slightly covered by the post-stigmatic pale spot. Eyes bare and separated narrowly. Sensilla coeloconica present on flagellomeres 1, 9-13. Third palpal segment triangular and moderately swollen with multiple irregular sensory pits. Two functional spermathecae subequal with a long, pigmented neck and one rudimentary spermathecae; parallel sclerotized ring at the end of the spermathecal duct. Middle leg with spines on tarsal segments 1 to 4. This species is reported from the Balkans [63], Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, France with Corsica, Germany, Hungary, Italy, Ireland, Lithuania, Morocco [65], the Near East, Poland, Portugal, Russia, Slovakia, Switzerland, Ukraine [64] and the UK.
We report the presence of Culicoides (Silvaticulicoides) picturatus Kremer & Deduit. Females may be identified using the following criteria. Spotted wing as represented in Fig. 13; second radial cell completely dark, small and faint pale spots in the distal part of r3, m1 and m2 cells. Eyes bare and separated narrowly. Sensilla coeloconica present on flagellomeres 1, 9-13. Two functional spermathecae subequal and one rudimentary. Middle leg with spines on tarsal segments 1 to 3. Males may be identified using the following criteria. Wing similar to female with the usual sexual differences. Ninth sternite with broad and deep arch-shape caudomedian excavation, ventral membrane densely spiculate; basistyle with ventral root long and slender. This species was first described in France (Normandy region) by Kremer & Deduit [66], and reported from Corsica, Denmark, Italy (mainland, Sardinia and Sicily), Morocco [11,12], Portugal, Romania, Slovakia [67], Spain, Switzerland, Turkey, and the UK. Culicoides alazanicus Dzhafarov has a wing pattern similar to that of C. picturatus but can be easily separated from the latter under stereomiscrocopy by the ratio of the length of flagellomere 9 to that of flagellomere 8 on the female antenna being greater than 2. On the contrary, C. picturatus has such a ratio around 1.4 [67].

Discussion
We report ten new records of Culicoides species in Algeria (Table 2) by examining fresh material collected from November 2015 to September 2018 using light/suction traps in nine localities of the Tiaret region (northwestern Algeria). Among these ten species new to Algeria, C. shaklawensis, C. simulator and C. picturatus were already known from Morocco and C. univittatus from both Morocco and Tunisia [19,20].
We also recorded the presence of C. chiopterus (one female) and C. dewulfi (13 females). Both species are widespread in Europe, but rare in the Mediterranean area. This is the first record of these species in North Africa, which underlines the need for further investigations to clarify their distribution in Algeria and more widely in the Maghreb. Currently, six species of the subgenus Avaritia are reported in Algeria (Table 2).
Among these species, C. imicola is a proven BTV and AHSV vector species: it is a livestock and equid biting species; numerous isolations of both viruses have been made from field-collected individuals; and the entire cycle of transmission of both viruses has been experimentally reproduced [68,69]. Culicoides imicola can be considered a probable SBV vector, as the viral genome was recovered from field-collected females [70,71]. Culicoides obsoletus, C. scoticus, C. chiopterus and C. dewulfi are probable BTV and SBV vectors because of their ecological habits, and virus isolation or viral genome detection from field-collected individuals and experimental infections. BTV has been isolated from field-collected females, reported as 'C. obsoletus' taxon [72][73][74]; however, it was not clear whether this taxon referred to a species or an assemblage of species. BTV-8 genome has been identified from C. dewulfi and C. chiopterus fieldcollected individuals by real-time RT-PCR in the Netherlands [75,76] and France [77]. BTV-1 genome was detected by real-time RT-PCR in C. obsoletus/C. scoticus parous females in the Basque country [78], and in C. obsoletus and C. scoticus parous females in Sardinia [79]. SBV genome has been identified in field-collected individuals: entire females of C. obsoletus/C. scoticus in Italy, the Netherlands and Poland [80][81][82] and of C. obsoletus, C. scoticus, C. dewulfi and C. chiopterus in France [70], and heads of C. obsoletus, C. scoticus, C. chiopterus and C. dewulfi in Belgium, Denmark and/or the Netherlands [83][84][85]. Moreover, C. obsoletus and C. scoticus from the UK have been experimentally infected with BTV-8 and BTV-9; C. scoticus exhibiting higher viral titers [86]. Dissemination efficiency has been estimated to be about 20% for BTV-1 in C. scoticus populations in Switzerland [32].
A total of 287 females and 1 male collected from the 'wilahya' of Tiaret were identified as C. grisescens. This species has been reported to be widely distributed in the northern part of the western Palaearctic region, but has never been recorded in North Africa. This species does not seem an efficient experimental vector for BTV [32]. We reported the presence of C. paradoxalis (1 female). This species has recently been described in southeastern France, Corsica and Portugal [45]. This new record confirms the Mediterranean distribution of C. paradoxalis. Currently, seven species of the subgenus Culicoides are reported in Algeria (Table 1). Among these, C newsteadi, C. pulicaris and C. punctatus are considered possible BTV and SBV vectors, due to their feeding habits and to virus isolation or viral genome detections from field-collected individuals. In Italy, BTV has been isolated from C. pulicaris [87]; however, it was not clear whether this taxon referred to a species or an assemblage of species. BTV genome has been detected in field-collected individuals of C. newsteadi, C. pulicaris and C. punctatus in Spain and Italy [30,33,78,79], while SBV genome has been detected in C. punctatus in Poland [82] and C. newsteadi and C. pulicaris in France [70].
We reported the presence of C. navaiae (286 females and 1 male), first described in Saudi Arabia: females by Lane [50] and males by Boorman [51]. This species of the subgenus Beltranmyia was relatively common in Saudi Arabia [88,89], reported in the Sinai, Egypt [52], and recorded for the first time in our study in the Maghreb. When studying specimens from Algeria, Szadziewski [28] described a single female under the label C. (Beltranmyia) sp. indet. aff. homochrous because identification remained unsatisfactory. With the exception of the presence of sensilla coeloconica on the flagellomere 2 of the latter unidentified female [28], all characters are in agreement with the descriptions of C. navaiae females [50,52] and with our observations. Consequently, we agreed with Boorman's remark [51] that the female C. (Beltranmyia) sp. indet. aff. homochrous from Szadziewski [28] referred probably to C. navaiae.  We collected three females identified as C. achrayi. This species has a wide distribution in the western Palaearctic region, including Mediterranean areas. This species has been collected in 2017 in Morocco as the first record in North Africa [65].
Kabbout [42] reported the first records of C. jurensis, C. sphagnumensis and C. truncorum from Algeria. As these species are rare and exhibit a non-Mediterranean Palaearctic distribution, we suggest not validating these species in the Culicoides checklist of Algeria until confirmation of identification by experts and molecular confirmation ( Table 2).
The proposed checklist includes 59 Culicoides species in Algeria, while 54 species are recorded in Morocco [19] and 35 in Tunisia [20]. Among these species, C. kingi is considered a probable vector of Onchocerca gutturosa affecting cattle in the Sahelian region [90,91] and a potential vector of epizootic hemorrhagic disease virus [92,93]. Recently, the detection of BTV genome in the head of C. circumscriptus and C. paoale in Sardinia has raised the question of a Reported as Culicoides kabyliensis n. sp. Kieffer, 1922 in [22] b Reported as Culicoides halophilus Kieffer, 1924 in [40] c Reported as Culicoides distigma n. sp. Kieffer, 1922 and Culicoides donatieni n. sp. Kieffer, 1922 in [22] d Reported as Culicoides coluzzii Callot, Kremer & Bailly-Choumara, 1970 in [42] e Reported as Culicoides citrinellus n. sp. Kieffer, 1923 in [23] f Reported as Culicoides cubitalis Edwards, 1939 in [27] the possible involvement of these species in BTV transmission in the Mediterranean basin [33].

Conclusions
The examination of fresh material resulted in the recording of ten new species for the Algerian fauna, although this material was collected in a small part of the country, namely in the 'wilahya' of Tiaret (northwestern Algeria). We have combined this information with existing published and grey literature to produce a comprehensive Culicoides checklist for Algeria of 59 species, including potential and probable vectors of arboviruses of veterinary interest. This is a prerequisite for the development of a barcode library and an atlas of diagnostic characters. Both may be useful for further ecological studies, in order to establish risk mapping for Culicoides-borne diseases in Algeria.