Culicoides Latreille, 1809 biting midges (Diptera, Ceratopogonidae) cause a significant biting nuisance to livestock and humans and are the biological vectors of a wide range of internationally important pathogens of both veterinary and medical importance (Harrup et al., 2015). Moreover, certain species have become notorious for this activity because of their vast population density and persistent biting attacks, shaping public perception of the genus in many regions including northern Europe (Carpenter et al., 2013). In Spain, outbreaks of African horse sickness (AHS) (1966, 1987-1990), bluetongue disease (1956-1960, 2000-today) and more recently Schmallenberg virus (SBV) (2012) have led to a substantial economic damage into the livestock industry in past decades (Pérez de Diego et al., 2014), with mortality, loss of production, trade restrictions and expensive surveillance and vaccination programs.

Since the first studies made by Strobl (1900) until today, there have been many contributions to the knowledge of these insects in our country. However, there was a turning point during the eighties, at which studies begun to be focused on the vectorial role (Mellor et al., 1983), leaving aside the purely faunal aspect of the past. Undoubtedly, the great veterinarian interest of Culicoides regarding the transmission of arbovirosis with an impact on livestock is the main reason why knowledge of these arthropods has advanced so much in recent years. The arrival of bluetongue virus (BTV) outbreaks in the new century contributed to an increased number of studies, and some incorporated the use of molecular techniques for the identification of certain closely related species (Ventura et al., 2005). To date, there are 81 species of Culicoides reported in Spain, divided in 9 subgenera, a miscellaneous group and a nomen dubium (Alarcón-Elbal & Lucientes, 2012). Thanks to this new finding the number of species rises to 82 and 28 in Spain and the Autonomous Community of Extremadura respectively (Sánchez Murillo et al., 2013).

Adult Culicoides are notable for their characteristic wing pigmentation pattern and distribution of wing macrotrichia, which can be used in most species as the main diagnostic features (Rawlings, 1996; Mathieu et al., 2012). Although the study of wing pattern is indeed the main tool in the taxonomy of the genus, identification based on morphological features is sometimes difficult because several cryptic species and species complexes are present (Pagès et al., 2009). In the particular case of subgenus Culicoides, taxonomy is still confused and observation of distinct intraspecific morphological variation has led to record of morphological cryptic diversity (Meiswinkel et al., 2004; Gomulski et al., 2006; Pagès et al., 2009; Muñoz-Muñoz et al., 2011). Therefore, the identification of these cryptic specimens to species level, or identification of other species, requires more detailed morphological study or even the use of molecular techniques (Nolan et al., 2007). Knowledge of which species could act as disease vectors, as well as their correct identification, remains essential to assess the real risk for disease transmission into disease-free areas (Augot et al., 2013).

In this contribution, we report the species Culicoides paradoxalis Ramilo & Delécolle, 2013, a new species recently described in France and Portugal (Ramilo et al., 2013), for the first time in Spain. At different localities, authors have collected significant numbers of C. paradoxalis specimens, which are morphologically close to Culicoides lupicaris and Culicoides newsteadi, but might be separated based on marked morphological observations. This work has as main objectives a) facilitate the reliable identification of C. paradoxalis based on morphological features observable under stereomicroscope (40x magnification) and b) to improve the knowledge of this species in terms of flight activity and gonotrophic status.

The Autonomous Community of Extremadura has been one of the regions in Spain most severely affected by bluetongue epidemics (Pascual-Linaza et al., 2014). Framed within a national surveillance program for BTV in Extremadura, CDC miniature light traps (Model 512; John W. Hock Company, Gainesville, Florida, USA) were used to capture adult Culicoides during 2014 in previously selected farms. A total of four locations (Alcollarín, Valencia de la Torres, Membrío and Villanueva del Fresno) were studied for Culicoides collection in the two provinces of Extremadura (mid-west, Spain). Only three locations were positive in capturing C. paradoxalis (Fig. 1A & 1B). Specimens of Culicoides were collected in a reusable plastic jar suspended below the fan of the trap, half-filled with water/ethanol (50%) and 1,2-propanodiol (50%) as an odorless collecting liquid. Light traps suspended near livestock farms were left overnight (set at dusk and retrieved at dawn), once a week. Adults were preserved in ethanol and subsequently cleaned, dissected and mounted on microscope glass slides using Hoyer's medium (50 ml of distilled water, 30 g of gum Arabic, 200 g chloral hydrate and 20 ml of glycerin) following the techniques described by González & Goldarazena (2011). Morphological identification was performed following the descriptions of Ramilo et al. (2013). Taxonomic features were photographed with a compound microscope Nikon Eclipse 80i coupled to a Canon Power Shot S-50 camera.

A total of 438 females and a single male were collected and voucher specimens were stored in the arthropods collection of the Parasitology Department, Regional Animal Health Laboratory of Extremadura (Spain).

In order to facilitate a quick identification of C. paradoxalis without needing to prepare slide-mounted specimens, authors suggest three anatomical characteristics which are species-specific for C. paradoxalis (Fig. 2A) to be distinguished from other sibling species of Culicoides subgenus, a) based initially on their unique wing pattern (Ramilo et al., 2013). The pigmentation of C. paradoxalis wing covers a large area of the wing (Fig. 2B) in comparison to morphologically related species (C. lupicaris and C. newsteadi). Anal cell and central part of the wing (medial cells) are quite covered by pigmentation, characteristics that usually occur in some members of subgenus Oecacta (e.g. Culicoides cataneii). Other features are required for a clear separation from C. lupicaris and C. newsteadi, such as b) absence of spine on the fourth tarsomere of mid legs (Fig. 2C), and c) sensory pits (clavate organs) of third segment of palp (Fig. 2D). As Ramilo et al. (2013) illustrated, the third segment of palpus shows a unique large, moderately deep depression with limited borders, which is markedly different from C. newsteadi and C. lupicaris.

Later species having individual depressions, giving an appearance of irregular borders that corresponds to small groups of shadow sensory pits. There are other additional anatomical features that might be diagnostic. However, they are quite similar across other species of the subgenus. Eyes contiguously joined for a short distance (1-2 facets), a characteristic with high intraspecific variation across specimens (Fig. 2E). Four sensilla coeloconica on flagellomere 1 were observed in most of the specimens studied (Fig. 2F). Female genitalia have two functional pyriform spermathecae, subequal with a small neck, plus one rudimentary spermatheca with digital shape (Fig. 2G).

Males are typically rare in field collections (González, 2014), and their identification is usually based on genitalia supported by wing pattern. A single male of this species was collected with genitalia characteristics compatible with C. paradoxalis (Fig. 2H), as Ramilo et al. (2013) pointed out. Additionally, sensilla coeloconica distribution differs between species: C. paradoxalis has sensilla coeloconica on flagellomeres 1, 12 and 13 (Fig. 2F); C. newsteadi and C. lupicaris on flagellomeres 1, 11-13. The presence of sensilla coeloconica on flagellomere 11 is polymorphic for C. newsteadi and C. lupicaris (Delécolle, 1985).

Regarding seasonal collections, most European species produce between one generation (univoltine) and several generations annually (multivoltine) (Sarto i Monteys & Saiz-Ardanaz, 2003; Miranda et al., 2003; González et al., 2013a, 2013b). Seasonal dynamics of Culicoides are determined by climatic factors, mainly rainfall and temperature, depending on each species, but also by chemical characteristics of breeding sites (Uslu & Dik, 2010; Venail et al., 2012). Culicoides paradoxalis displayed a high peak of activity with maximum abundance in July and declined during early autumn (Fig. 3). This suggests a univoltine pattern for this species in contrast to C. lupicaris, which is a bivoltine species with peak activity from May to August and from October to November in the Basque Country region (González et al., 2013a). These seasonal dynamics have also been observed for C. pulicaris in Sardinia (Foxi et al., 2011) and the Basque Country (González, 2014), where the species shows a first generational peak (late April/early May), on average larger than the second one (late summer/early autumn). In Extremadura, in contrast, C. pulicaris shows a single peak of activity in June and July, according to current and historical data (Sánchez Murillo et al., 2011).

Gonotrophic status examination revealed that 74,5% were nulliparous and 25,5% parous, with no specimens belonging to gravid or blood-engorged stages. The high number of nulliparous females is characteristic of the summer season when more midges emerge (Lysyk, 2007). The estimation of the physiological stages in a population is important for various reasons, particularly because the determination of the parous gonotrophic stage in epidemiological studies serves to give an idea of the potential capacity of these midges for transmitting virus (EFSA, 2008) as well as an indicator of the population age structure (Mullens & Schmidtmann, 1982).

Culicoides species of subgenus Avaritia (Culicoides obsoletus, Culicoides scoticus, Culicoides imicola, Culicoides chiopterus and Culicoides dewulfi) are thought to be the primary vectors of BTV and SBV in the Mediterranean region, based on abundance and host preference, vector competence studies, and isolation or detection of virus in field-collected midges (see Garros et al., 2014). Similarly, species of subgenus Culicoides are implicated as vectors of BTV (Caracappa et al., 2003; Vanbinst et al., 2009; Romón et al., 2012) and SBV (De Regee et al., 2012; Elbers et al., 2013; Balenghien et al., 2014). In Europe, more recently, some members of subgenus Culicoides are increasingly being implicated as suspected vectors, such as Culicoides punctatus (Larska et al., 2013) and Culicoides nubeculosus (Balenghien et al., 2014), while C. imicola is considered to be the main vector of BTV and AHS in Spain (Calvete et al., 2008).

Considering that C. paradoxalis is a member of the pulicaris group, closely related to species that play a role in BTV and SBV, their accurate identification to species level is crucial in epidemiological investigations, as great differences in vectorial capacity are found even between these species. However, morphological identification of Culicoides is considered challenging, requiring significant expertise and is restricted to relatively few entomologists in Europe (Koenraadt et al., 2014). Unfortunately, due to the large number of samples that taxonomists and specialists have to face daily, this species has most probably been notoriously overlooked in field-based studies, because of its resemblance to other members of subgenus Culicoides. Re-examination of stored specimens in preparations and/or those preserved in alcohol could provide more details about its presence in other regions of Spain.