Description of three new species of frog–biting midges (Diptera: Corethrellidae) from the Central Brazilian Amazon

Three species of Corethrella Coquillett, 1902 from the state of Amazonas, Brazil are described as new to science based on female adult specimens. Corethrella cabocla Feijó, Belchior, Marialva & Pessoa sp. nov. possesses four large setae on the frons between the ventromedial area of ommatidia, a FEIJÓ ALMEIDA J. et al., Three new species of Corethrellidae from Brazilian Amazon 149 wide clypeus with 1–4 setae, a wing with the apex of R2 basal to the apex of M2 and with a midlength band, and with the abdomen entirely dark brown. Corethrella ielemdei Feijó, Ramires, Lima & Pessoa sp. nov. possesses an elongated coronal suture, four large setae on the frons between the ventromedial area of ommatidia, a clypeus squarish with 42–43 setae, a wing with the apex of R2 basal to the apex of M1 and with a midlength band and dark scales on the basal and subbasal areas of the anterior margin, legs with dark scales, and with the abdomen entirely dark brown. Corethrella menini Feijó, Picelli, Ríos-Velásquez & Pessoa sp. nov. possesses wings with the apex of R2 basal to the apex of M2 and a midlength band, with darker basal scales along all veins, basal band dark scales on C, Sc, R, M, and Cu and the abdomen entirely dark brown. With the addition of the new species, the numbers of frog-biting midges described in the Amazon basin, Brazil and in Neotropical region are now 31, 49 and 80 species, respectively.


Introduction
The monogeneric family Corethrellidae Edwards,1932 is a widely distributed family in the tropical and subtropical areas (Borkent 2008) where species of the genus Corethrella Coquillett, 1902 locate their anuran hosts by sound instead of chemical cues (McKeever 1977;McKeever & Hartberg 1980;Toma et al. 2005;Bartlett-Healy et al. 2008;Borkent 2008;Bernal & de Silva 2015;Camp & Irby 2017). Female adults are ectoparasitic, feed on host blood and are documented vectors of trypanosomatids (Johnson et al. 1993;Bernal & Pinto 2016;Meuche et al. 2016). Recently, it was shown that members of this genus are involved in the transport of chytrid fungus, which is one of the main causes of the recent decline and extinction of amphibians (Toledo et al. 2021).
To date, the genus Corethrella includes 115 extant species and 10 extinct species. These insects have a semi cosmopolitan distribution with vast majority of extant species restricted to the tropics and subtropics (Kvifte & Bernal 2018;Amaral et al. 2019;Baranov et al. 2019). The Neotropical region possesses the highest richness of species of Corethrella with 77 registered species (Amaral 2018;Amaral et al. 2019), of which 28 species are from the Amazon basin (Borkent 2014;Amaral et al. 2019Amaral et al. ). 1998. The forest has a fairly dense canopy and an understory with low light, characterized by the abundance of palm trees (Guillaumet & Kahn 1982). Periodically, the extensive area of FAEXP-UFAM is flooded, due to the presence of large streams and an unregulated topography, with an altitude ranging from 42 to 130 m (Rojas-Ahumada et al. 2012).
Monthly air temperature variation is very low, with average temperatures between 24.6ºC and 26.9ºC. Daily relative air humidity ranges from 75% during relatively dry days up to 92% in the rainy season, and average annual rainfall is 2, 362 mm (Araújo et al. 2002).
The collections were carried out on two trails in the farm over two days, in the months of April, May, and July 2019. Frog-biting midges were collected using four HP model light traps, two with and two without light bulbs (Pugedo et al. 2005); with overnight 'frog call traps' speakers attached, as described by McKeever & Hartberg (1980) (Fig. 2). The recorded frog sounds were from the 'sapoteca' frog library (https://ppbio.inpa.gov.br/sapoteca/paginainicial) of the Instituto Nacional de Pesquisas da Amazônia (Lima et al. 2012) (Supp. file 1).
The samples were stored in 70% alcohol. The frog-biting midges were separated from the other families of dipterans, and morphologically identified using the identification key by Borkent (2008). Then, female samples were dissected and mounted on slides in phenol-balsam (Wirth & Marston 1968). The diagnostic characteristics were photomicrographed using a digital system (SynopticsTM, Cambridge, UK) coupled to an optical microscope (Leica DMTM1000, Frankfurt, Germany). The Helicon Soft High Assembly program was used to obtain the final images. For species descriptions, the morphological terms proposed by Borkent (2008)  We proposed an identification key for the new species, based on the species diagnoses in Borkent (2008), Amaral & Pinho (2015) and Amaral et al. (2019).
HEAD (Fig. 3b). Laterally expanded in dorsal view. Coronal suture elongate, reaching the area between ventral margin of eye bridge (Fig. 3c). Four large setae on frons between ventromedial area of the ommatidia. ANTENNA (Fig. 3b). Darker to light brown, with flagellomeres I and II darker brown, while others are light brown; flagellomere XIII apically bifurcated; sensilla coeloconica distributed according to Table  1.
LEgS (Fig. 4a). Dark to light brown with slender setae dark brown. Foreleg with dark brown femur to about 3/ 10 of total light brown structure in apex region, tibia dark brown, approximately 1/ 10 of the total light brown structure in the base region, tarsomeres 1 to 5 light brown. Midleg with dark brown femur, approximately 3/ 10 of total light brown apically, tibia light brown from base to about 5/ 10 of total length, other half dark brown, extending to apex, tarsomeres one to five light brown. Hindleg with dark brown femur from base to about 4/ 10 of length, apical 6/ 10 light brown, tibia mostly light brown, about apical 2/ 10 dark brown, tarsomeres 1 to 5 light brown. Tarsal claws equal, inserted apically; each claw without an inner tooth. Empodia ( Fig. 4b) with similar thickness to apical setae of last tarsomere, longer than average diameter of last tarsomere, with five or six bifid branches.

Distribution
This species is known only from its type locality in the experimental farm of the Universidade Federal do Amazonas FAEXP-UFAM, Manaus, Amazonas State, Brazil.

Taxonomic comments
In the Neotropical key by Borkent (2008) C. cabocla sp. nov. keys to C. lepida Borkent, 2008. However, it is distinguished from this species by having the antennal flagellomeres dark brown, whereas in C. lepida the antennal flagellomeres I-IV are light brown. Furthermore, C. cabocla sp. nov. has the first flagellomere with four sensilla coeloconica, whereas in C. lepida, the first flagellomere has been described with two sensilla, and the scutum of C. cabocla sp. nov. is dark brown and the scutellum light brown, distinct from C. lepida, in which the color of the scutum and scutellum ranges from medium to light brown. The posterior portion of the dorsocentral thorax in C. cabocla sp. nov. has a group of six elongate setae, whereas in C. lepida five elongate setae are present. The wing of C. cabocla sp. nov. has the apices of R 2 and M 2 at approximately the same level, whereas in C. lepida R 2 and M 1 are nearly at the same level. In C. lepida, a pale brown band has been described on each of tarsomeres 2-4 of the mid and hind leg; tarsomeres are uniformly pigmented in C. cabocla sp. nov. The abdomen of C. cabocla sp. nov. is entirely dark brown, whereas in C. lepida only segments VIII and IX are dark brown. Corethrella caribbeana Borkent, 2008 shares some characteristics with C. cabocla sp. nov. and C. lepida, but C. caribbeana is distinguished from C. cabocla sp. nov. based on several features. In C. caribbeana the flagellomeres I-III are short, whereas in C. cabocla sp. nov. they are elongate. In addition, Corethrella caribbeana has two sensilla distributed on each of flagellomeres IX-XII, whereas in C. cabocla sp. nov. there is only one on each of flagellomeres VIII-XIII. The clypeus of C. caribbeana is squarish, different from that of C. cabocla sp. nov., which is wide. The abdomen of C. caribbeana is light to dark brown, with sternites I-II slightly darker and segment IX dark brown, whereas in C. cabocla sp. nov. the abdomen is entirely dark brown.
The holotype has a set of characteristics that do not fit within the existing species groups of Corethrella (Borkent 2008). The only apparent synapomorphy that C. cabocla sp. nov. shares with the groups fulva, appendiculata, brakeleyi and wirthi is found on the posterior dorsocentral region of the thorax, where a group of more than three elongated setae is present. The lack of knowledge of the immature stages and adult males of C. cabocla sp. nov., makes it difficult to associate the new species with a specific species group as defined by Borkent (2008).
We suggest to incorporate the following emendation to the Corethrella. Key by Borkent (2008)

Diagnosis
The species can be distinguished from other species of the genus by the following characteristics of the female adult. Coronal suture elongate, four large setae on frons between ventromedial area of ommatidia, clypeus squarish, with 42-43 setae; antenna uniformly medium brown, flagellomere I with two sensilla coeloconica, flagellomere II with one sensillum coeloconicum, flagellomeres IX-XIII with two sensilla coeloconica; posterior portion of dorsocentral row with a group of about eight elongate setae; wing with apex of R 2 basal to apex of M 1 and with midlength band and dark scales on basal and subbasal areas of anterior margin; foreleg, midleg, and hindleg with femora and foretibia with dark scales.

Description
Adult female (n = 10) HABITUS. As shown in Fig. 5a. HEAD (Fig. 5b). Laterally expanded in dorsal view. Coronal suture elongate, reaching the area between ventral margin of eye bridge (Fig. 5c). Four large setae on the frons between the ventromedial area of ommatidia.
ANTENNA (Fig. 5b). Uniformly medium brown; flagellomere XIII apically bifurcated; sensilla coeloconica distributed according to Table 1.  THORAx. Dark brown, scutum, scutellum, pale sclerites around the base of the wing. Posterior portion of dorsocentral row with a group of approximately eight elongate setae. Prescutal suture short, not extending to dorsocentral row of setae. Anterior anepisternum dark brown, divided by diagonal suture, dorsal portion about equal to ventral portion. Posterior anepisternum dark brown, divided by transversal suture, with ventral portion triangular. WINg ( Fig. 6c; Table 2). Apex of R 2 basal to apex of M 1 . Anterior margin with differently and discretely pigmented scales, with midlength band, with darker more basal scales along all veins (except A 1 ); some scattered dark scales on basal and subbasal areas of anterior margin; veins with well-developed scales. Halter dark brown, darker than scutellum.
LEgS (Fig. 6a). Dark to light brown and slender setae light brown. Foreleg with femur entirely dark brown and thin dark scales on upper margin of medial portion extending near apex of femur; tibia dark brown, with thin dark scales on lower margin extending from the base to middle of the structure, first tarsomere dark brown and about 2/ 10 of the total light brown structure in base region, and other tarsomeres dark brown. Midleg with dark brown femur with a tuft of thin dark scales near apex region of the structure, tibia, and tarsomeres entirely dark brown. Hindleg with entirely light brown femur with a tuft of dark scales on posterior margin near apex of the structure, tibia predominantly light brown but about 3/ 10 dark brown in the apex region, tarsomeres 1 to 5 dark brown; tarsal claws equal, inserted apically; each claw without inner tooth. Empodia (Fig. 6b). With thickness larger than apical bristles of last tarsomere, longer than average diameter of last tarsomere, with seven bifid branches. ABDOMEN (Fig. 6d). Uniformly dark brown. One ovoid spermatheca. Cercus dark brown.

Distribution
The species is known only from its type locality in the experimental farm of the Universidade Federal do Amazonas FAEXP-UFAM, Manaus, Amazonas State, Brazil.

Taxonomic comments
The characters present in C. ielemdei sp. nov., as distribution of sensilla coeloconica, eight setae in the dorsocentral region of the thorax, and dark brown cercus and halter, are shared with C. squamifemora Borkent, 2008 andC. hirta Borkent, 2008. However, they can be distinguished from these species by the presence of an elongated coronal suture (in C. squamifemora and in C. hirta, they are shorter), four long setae between the ommatidia (in C. squamifemora and in C. hirta, there are just two), and squarish clypeus (in C. squamifemora and in C. hirta, they are broadly roundish). The presence of an elongate coronal suture, a dark antenna, and the wing with the apex of R 2 basal to apex of M 1 in C. ielemdei sp. nov. are also shared with C. albicoxa Borkent, 2008, C. appendiculata Grabham, 1906, C. calathicola Edwards, 1930, and C. melanica Lane & Aitken, 1956. However, the new species can be distinguished from these by the dark brown palpus in comparison to the pale palpus in C. albicoxa, C. appendiculata, C. calathicola, and C. melanica. The third palpal segment is evenly broad throughout its length in C. ielemdei sp. nov., whereas in C. albicoxa, C. appendiculata, C. calathicola, and C. melanica it is somewhat swollen in the middle of this segment. In addition, the pattern and distribution of dark bands and scales along the wing are different in C. ielemdei sp. nov., C. albicoxa, C. appendiculata, C. calathicola, and C. melanica.
Corethrella ielemdei sp. nov. shares characteristics with species in the appendiculata group (Borkent 2008), including scales on the femora and tibiae, and having a group of more than three elongated setae in posterior dorsocentral region of the thorax. The color of the medial and posterior femur distinguishes C. ielemdei sp. nov. from the other species of this group. The description of males and immature stages will probably confirm the inclusion of this species in the appendiculata group.
We suggest to incorporate the following couplets in the Corethrella. Key by (Borkent 2008

Diagnosis
The species can be distinguished from other species of the genus by the following characteristics of the female adult. Coronal suture elongate, two large setae on the frons between ventromedial area of ommatidia, clypeus squarish, with three setae; antenna dark brown, flagellomere I with four sensilla coeloconica, flagellomere II with one sensillum coeloconicum, flagellomeres IX-XIII with one sensillum coeloconicum; posterior portion of dorsocentral row with a group of about six elongate setae; wing with apex of R 2 basal to apex of M 2 and midlength band, with darker more basal scales along all on veins, basal band dark scales on C, Sc, R, M, and Cu; abdomen, completely dark brown.

Description
Adult female (n = 10) HABITUS. As shown in Fig. 7a. HEAD (Fig. 7b). Laterally expanded in dorsal view. Coronal suture elongate, reaching the area between ventral margin of eye bridge (Fig. 7c). Two large setae on frons between ventromedial area of ommatidia.

Distribution
This species is known only from its type locality in the experimental farm of the Universidade Federal do Amazonas FAEXP-UFAM, Manaus, Amazonas State, Brazil.

Taxonomic comments
The structures present in C. menini sp. nov., such as the elongated coronal suture, the presence of two long frontal bristles between ommatidia, the third palpal segment being almost constant in width, and the group of six bristles in the thoracic dorsocentral region are characteristic for species in the brakeleyi group: C. hispaniolensis Borkent, 2008, C. puella Shannon & Del Ponte, 1928, C. condita Borkent, 2008, C. brakeleyi (Coquillett, 1902, and C. longituba Belkin, Heinemann & Page, 1970. However, C. menini sp. nov. can be distinguished from these based on the color of the palpus (dark brown in C. menini sp. nov., pale in C. hispaniolensis and C. longituba, and medium brown in C. puella, C. condita, and C. brakeleyi), the number of sensilla coeloconica on the flagellomeres, the band pattern and scales along the wing, and the color of the halter (dark brown in C. menini sp. nov. and pale in C. hispaniolensis, C. puella, C. condita, C. brakeleyi, and C. longituba). The squarish clypeus and the dark brown color of the antenna, halter, and cercus are characteristics shared by C. menini sp. nov., C. curta Borkent, 2008, andC. ranapungens Borkent, 2008. However, the thoracic dorsocentral region of C. curta and C. ranapungens have a group of three and four elongated bristles, respectively as opposed to six setae in C. menini sp. nov.; in addition, C. menini sp. nov. has the apex of R 2 basal to the apex of M 2 , whereas in C. curta and C. ranapungens, the apex of R 2 is basal to the apex of M 1 . Other characters, such as the color pattern of the thorax and palpus, can also be used to distinguish the species C. menini sp. nov. from C. curta and C. ranapungens. The new species likely belongs to the brakeleyi group, which includes eight species. However, male characters are still necessary to support this hypothesis because the occurrence of wings with apical bands, a characteristic that defines the group, is more commonly found in adult males.
We suggest to add the following couplets to the Corethrella key by Borkent (2008)   Identification key to female Amazon basin species of Corethrella.
The species C. maculata Lane, 1939, C. shannoni Lane, 1939and C. munteantaroku Amaral, Mariano & Pinho, 2019 are not included as females of these species have not been described yet.

Discussion
The possible preference for the calls of certain frog species seems to be a relevant factor that influences the diversity of frog-biting midges captured (Ambrozio-Assis et al. 2019). In our study, it was not possible to evaluate the preference of species of Corethrella to a specific type of frog call, because we used a database with more than 80 frog sounds. Most of the individuals were collected in the modified trap, with speakers; similar results were also found in studies of Caldart et al. (2016). Some other studies carried out in Brazil (Amaral & Pinho 2015;Amaral et al. 2019;Caldart et al. 2016), using same collection methods, captured less samples. Partly, this can be explained by the differences in collection methods, frog and toad mosquito fauna, and biomes.
Of the four species of frog-biting midges collected at FAEXP-UFAM, three are new to science. Collections of Corethrella are being carried out in several regions of the Brazilian Amazon and soon we hope to obtain data in order to expand the distribution of frog-biting midges, especially in areas with no existing records, but with favorable conditions for their establishment. The uncertainty of associating the new species to the groups described by Borkent (2008), exemplified the need to collect immature stages and males to robustly evaluate the phylogenetic position of Corethrellidae species.
The Amazon Basin is a vast heterogeneous area that constitutes half of the Brazilian territory, but very few surveys have been conducted for Corethrella in this region. Extensive ecological and biodiversity studies, especially focussing on areas with geographical barriers, e.g., areas between large rivers, are necessary to understand the overall distribution and abundance of these species in Brazil.