An updated list of the Culicoides (Diptera, Ceratopogonidae) fauna from Ecuador

An updated list of biting midges of the genus Culicoides inhabiting Ecuador is provided. Entomological investigations were carried out from July 2010 to May 2019 using CDC light traps in three Ecuadorian regions (Amazon basin, Andean (foothills and highlands) and Pacific Coast). A total of 12,073 Culicoides specimens from seven subgenera and nine species groups were collected. More species and higher variation were found in the Amazon basin than in either of the Andes regions or coastal sites. A total of 53 species were identified. Of these, 15 are herein reported as new species records for Ecuador: Culicoides acotylus Lutz, C. aitkeni Wirth & Blanton, C. benarrochi Ortiz & Mirsa, C. carvalhoi Wirth & Blanton, C. freitasi Wirth & Blanton, C. ginesi Ortíz, C. lopesi Barretto, C. lyrinotatus Wirth & Blanton, C. profundus Santarém, Felippe-Bauer & Trindade, C. pseudoreticulatus Santarém, Felippe-Bauer & Castellón, C. quasiparaensis Clastrier, C. vernoni Wirth & Blanton, C. youngi Wirth & Barreto and two new species. Our results show that the updated list of the Ecuadorian Culicoides fauna comprises 70 species. This inventory highlights the presence of species that have been incriminated as vectors of disease elsewhere in animals and humans, mainly C. insignis and C. paraensis.

Both BTV and OROV are encountered in South America [28]. Based on vector distribution and environmental conditions, the epidemiology of BTV can be categorized into three zones: endemic (South America north of the Tropic of Capricorn), epidemic (south of the Tropic of Capricorn to Uruguay) and incursion zone (South of Uruguay) [37]. In the Neotropical region, Culicoides insignis Lutz and to a lesser extent C. filarifer Hoffman, C. pusillus Lutz and C. trilineatus Fox [24] are suspected to transmit BTV, while C. paraensis (Goeldi) is a proven vector of OROV [28].
In Ecuador, OROV has been reported in the Pastaza province in the Ecuadorian Amazon basin [26] and in the Esmeraldas province in the coastal region [58]. Similarly, BTV has been reported: in cattle in El Oro Province in southern Ecuador [23], as well as in Pichincha, Napo, Esmeraldas and Manabi Provinces [11,50]; in sheep in West of Ecuador [27] and in Pichincha Province [30]. EHDV is present in Pichincha, Napo, Esmeraldas Provinces [50]. In contrast, BTV and epizootic hemorrhagic disease virus (EHDV) are not present in cattle in the Galapagos Islands [51].
Inventories of Culicoides have been carried out in the Neotropical region since the middle of the 20th century. The studies carried out by O.P. Forattini and W.W. Wirth and collaborators, and the standardization of morphological characters used to describe Culicoides species led to the production of identification aids and dichotomous keys [17,21,35,43,53,56].
Moreover, in the list of the biting midges of the World [7], more than 300 new species were described from the Neotropical region between 1853 and 2016.
In Ecuador, biting midges are not monitored as part of a national vector surveillance programme in contrast to European countries that have implemented extensive control strategies in response to BTV emergence (Regulation Commission (EC) No. 1266/2007). Nevertheless, in the last decade, a few local academic studies have contributed to better knowledge of biting midges (particularly in taxonomy and distribution) [19,30,31]. We aimed to revise the current inventory of Culicoides species present in Ecuador in the light of the recent detection of BTV and OROV. The purpose of this study was to produce a comprehensive list of Culicoides species in Ecuador, to provide information that may contribute to implement adequate surveillance and vector control strategies in the future. . This last collection of 2019 was carried out due to the recent report of Oropouche cases in this coastal province. In Orellana Province, research was conducted at Tiputini Biodiversity Station (TBS). TBS is located on the north bank of the Tiputini River, bordering Yasuní National Park and within Yasuní Biosphere Reserve, one of the most diverse regions of the world [4]. Most of the collections were carried out during the dry season (from July to February) since these sites are more accessible.

Materials and methods
The collection sites were selected based on the known ubiquitous capture sites of Culicoides spp., like animal shelters, forests, and mud rich sites with organic matter [6,52]. Seven-night catches per site were performed monthly. Traps were set approximately 1 h before sunset until 1 h after sunrise under favorable climatic conditions (absence of heavy rain and/or wind) [1]. All these specimens were stored in 70-100% ethanol. In the laboratory, Culicoides specimens were separated from other arthropods using a stereo microscope (Olympus SZ51), placed in 1.5 mL Eppendorf Ò tubes containing 70% ethanol and stored at À20°C. Specimens were identified using different morphological keys [13, 15-17, 44, 47, 52, 54, 56, 57]. The Culicoides species were classified into subgenera, species groups according to Borkent and Dominiak [7].
Specimen identification was performed, using a microscope, after mounting the head, wings, genitalia and spermathecae on microscope slides with Gum Chloral [1] or Euparal medium [20]. Digital images of the wings were obtained using an Olympus (BX41 or BX53) microscope equipped with an Olympus SC100 camera and software (cellSens or stream). All voucher specimens were deposited in the entomological collections of San Francisco de Quito University, Ecuador.   ¥ Specimens from Ecuador previously considered to be C. verecundus were restored from synonymy by Felippe-Bauer et al. [15] and are now known as C. contubernalis Ortíz & Léon.

Results
Fifty-three species of the genus Culicoides were identified morphologically from a total of 12,073 captured Culicoides specimens. The locality with the highest abundance was Yasuni-Tiputini in Orellana Province in both collection periods, with 7,691 specimens collected. We also found 73 specimens in  [1] and the other belongs to the limai group according to morphological characters.
Culicoides insignis, C. pachymerus, and C. paraensis have medical and/or veterinary importance because they have been reported as possible causative agents of dermatozoonosis [42,46]. Intriguingly, three species identified in this study (C. foxi, C. insignis, and C. filarifer) have been reported to carry DNA from Leishmania brasiliensis and Le. Amazonensis [39]. Additionally, C. foxi is a causative agent of allergic dermatitis and C. pifanoi is known as a causative agent of filariasis [8]. Moreover, C. acotylus, C. fluvialis, and C. leopoldoi are suspected to cause allergic dermatitis in humans [8]. Finally, C. guttatus may play a role as a vector of mansonellosis, a neglected tropical disease [38] and of BTV [8]. Further investigations are needed to determine the role of these species in dermatological disorders.
According to [6,10,45], the reported distribution of the species newly cited herein for Ecuador are: i) C. acotylus Culicoides paraensis is distributed from the USA to Argentina [49,56], and it is abundant in the humid tropics in Panama [52] and in urban areas with banana crops in the Amazon region and in the southern states of Brazil [14]. It was collected in South Carolina zoos [33]. In Peru, populations of C. paraensis seemed to be highest from October to December, with numbers fluctuating depending on the year and collection site [29]. Interestingly, the presence of C. paraensis, one of the known vectors of OROV [40], was also confirmed herein in Ecuador. An estimated half-million people have been affected by OROV since it was first isolated in Brazil [9]. In Ecuador, OROV was detected in the Esmeraldas Province (Coast) [58] and there is serological evidence in febrile patients from the Amazon basin (Pastaza Province) [26]. In our study, C. paraensis was found in Orellana province, located north of Pastaza province, suggesting that this vector may be transmitting OROV in Pastaza province where serologic evidence was detected [26]. In contrast, C. paraensis was absent in the Coastal region, (Esmeraldas and Manabi Provinces). Future investigations will address the distribution of this species particularly in forested and urban areas in Ecuador. In this study, C. neoparaensis, C. paraensis, and C. quasiparaensis were collected in the same area (Yasuní-Tiputini -Amazon basin). These three species belong to the paraensis group that includes seven species (C. diversus Felippe-Bauer, C. filiductus Wirth, C. peruvianus Felippe-Bauer, and C. austroparaensis Spinelli) [13,48]. It is worth remarking that our identifications were based only on morphological characters; thus, further molecular analysis would be needed to explore the boundaries of these closely related/similar species.
Finally, C. insignis is considered the main candidate vector species of BTV in South America [5], an arbovirus that infects livestock worldwide and is responsible for global losses of agriculture of up to US$3 billion [41]. Bluetongue virus has been detected in blood samples from Ecuadorian cattle [11,50]; however, its vector species in Ecuador have not been confirmed yet. Culicoides insignis is distributed in the USA (Alabama, Georgia, and Florida states), Mexico (Chiapas, Yucatán), Central America and Caribbean to central Argentina [6,49,56]. It is abundant in the arid tropics and absent in humid tropics in Panama [52]. Populations of C. insignis were highest from July to December at most sites in the Caribbean, and from June to October in Central America [18]. Therefore, in this region, nulliparous and parous females of this species were captured throughout the year [18]. In this study, C. insignis was collected in the Amazon basin and in the foothills (from north to south) of the Andean region until 2013, but we also found specimens in 2019 in the coastal region of Esmeraldas province; thus, it is possible that this species has favorable adaptation capacities due to its broad distribution throughout South America [3,12].
In conclusion, our examination of fresh material revealed 15 new species records for the country, and confirmed 13 species previously reported in Ecuador. Herein, we present an updated list of the Ecuadorian Culicoides that comprises 70 species, including possible vectors of human and veterinary interest. This species inventory is a prerequisite for the future development of a barcode library and the construction of an image database of Culicoides wings. Both may be useful for further ecological studies and to establish risk maps for Culicoides-borne diseases in Ecuador.