Revision of the Atratus Group of Culex (Melanoconion) (Diptera: Culicidae)

Background Despite the importance of some species of Culex (Melanoconion) (Diptera: Culicidae) as vectors of several arboviruses that cause diseases in humans and other animals, there are few taxonomic studies focusing on species of the subgenus, especially providing morphological keys for species identification. Results Thirteen species of the Atratus Group of Culex (Melanoconion) were reviewed, five new species are described, and two taxonomic changes are proposed: Cx. (Mel.) exedrus Root, 1927 and Cx. (Mel.) loturus Dyar, 1925 are resurrected from synonymy with Cx. (Mel.) dunni Dyar, 1918 and Cx. (Mel.) zeteki Dyar, 1918, respectively. The Atratus Group now includes fourteen species: Cx. (Mel.) atratus Theobald, 1901; Cx. (Mel.) caribeanus Galindo & Blanton, 1954; Cx. (Mel.) columnaris Sá & Hutchings n. sp.; Cx. (Mel.) commevynensis Bonne-Wepster & Bonne, 1919; Cx. (Mel.) comptus Sá & Sallum n. sp.; Cx. (Mel.) dunni; Cx. (Mel.) ensiformis Bonne-Wepster & Bonne, 1919; Cx. (Mel.) exedrus; Cx. (Mel.) longisetosus Sá & Sallum n. sp.; Cx. (Mel.) longistylus Sá & Sallum n. sp.; Cx. (Mel.) loturus; Cx. (Mel.) spinifer Sá & Sallum n. sp.; Cx. (Mel.) trigeminatus Clastrier, 1970; and Cx. (Mel.) zeteki. Keys, descriptions and illustrations for the identification of the male, female, pupal and fourth-instar larval stages of each species are provided. The treatment of each species includes a complete synonymy, descriptions of available life stages, a taxonomic discussion, updated bionomics and geographical distribution, and a list of material examined. Conclusions The taxonomy of the Atratus Group of Culex (Melanoconion) is updated, including descriptions of five new species. The number of valid species is greater than the number recognized in the previous taxonomic study of the group, increasing from seven to 14 species. Distributional and bionomical data are updated. Morphology-based identification keys for females, males, fourth-instar larvae and pupae provided in this study will facilitate species identification.


Background
Species of the subgenus Melanoconion Theobald, 1903 of Culex Linnaeus, 1758 are considered to be of public health importance because they are vectors of several arboviruses, such as the West Nile virus, viruses of the Venezuelan equine encephalitis complex, and eastern equine encephalomyelitis virus [1][2][3][4][5]. Despite their medical importance, there are few taxonomic studies that focus on species of this subgenus, especially providing key characters for species identification [6][7][8][9]. Although some morphological characters of the fourth-instar larvae and pupae can be useful for species identification, there are few studies that focus primarily on identification of the immature stages. The most complete studies are those by Foote [7] and Sirivanakarn [8]. Thus, for accurate species identification, it is necessary to examine features of the dissected male genitalia, with the

Open Access
Parasites & Vectors *Correspondence: ivyluizi@usp.br 1 Departamento de Epidemiologia, Faculdade de Saúde Pública, Universidade de São Paulo, Av. Doutor Arnaldo 715, São Paulo, SP 01246-904, Brazil Full list of author information is available at the end of the article with a Leitz Wetzlar drawing tube. All measurements are in millimeters and are given as the range followed by the mean and the number of measurements in parentheses. The descriptions follow the morphological terminology in Harbach & Knight [25,26], with some modifications made by Harbach et al. [27]. Only the morphological characters that are unusual and diagnostic for each species are detailed. The Culex classification adopted is that proposed by Harbach [11]. The Anophelinae classification adopted is that proposed by Foster et al. [28].
Geographical distributions are based on both literatute records and material examined, including field collections and museum specimens examined. Distribution records of the material examined are listed in the following format: country, state, municipality and/or locality name, latitude and longitude.
Culex commevynensis is not included in this revision because the type specimen could not be examined.
To comply with the regulations set out in Article 8.5 of the amended 2012 version of the International Code of Zoological Nomenclature (ICZN) [29], details of all new taxa have been submitted to ZooBank. The Life Science Identifier (LSID) of the article is urn:lsid:zoobank. org:pub:837DA7C4-9E36-4E25-9F4E-17241FA79DAD. For each new taxon, the Life Science Identifier (LSID) is reported in the taxonomic summary.

Atratus Group
According to Sirivanakarn [8], the following combination of morphological features diagnose the adults of the Atratus Group: head with narrow decumbent scales on central area of vertex and patch of broad decumbent scales laterally; pleural integument of thorax with pale and dark stripes across mesokatepisternum and mesepimeron; patch of numerous pale scales on upper corner of mesokatepisternum. Gonocoxite of male genitalia small, narrow, oblong; gonostylus narrow, simple, without a subapical crest; basal hook of lateral plate of aedeagus sclerotized, slender, in form of a curved arm. The morphological characteristics of the pupae are as follows: seta 9-VIII inserted before caudolateral angle of segment; seta 11-C usually single; trumpet long with index 10 or greater. Larvae can be recognized by the following combination of characteristics: margin of saddle with small spicules; seta 2-C absent; seta 14-C inserted at same level as 15-C or slightly anterior; siphon slender, long, with 4-6 pairs of posterolateral elements and with 3 or 4 pairs of dorsolateral elements.

Remarks
The male genitalia and adults of Cx. columnaris n. sp. bear more morphological similarities to Cx. zeteki than to other species of the Atratus Group. However, adult specimens of the new species differ from Cx. zeteki in having the mesepimeron with a slightly light stain on the median area, not divided into upper and lower areas. The male genitalia differ from those of Cx. zeteki in having the seta s without peduncle on base and with a broad apex, IX tergal lobes with bifid setae in the ventromedial region, and a lateral plate without undulations on the apical process. Furthermore, Cx. columnaris n. sp. has the proximal division of subapical lobe with an apically divided long columnar process which bears only two setae (a and b).

Remarks
Based on the original description of Bonne-Wepster & Bonne [58], and illustrations presented by Bonne & Wepster-Bonne [37], Sirivanakarn [8] and Pecor et al. [15], the male genitalia of Cx. comptus n. sp. are more morphologically similar to Cx. commevynensis than to any other species of the Atratus Group. These species have in common characteristics such as the shape and size of seta l of the distal division, the length of the columnar process of the distal division and the number and form of setae in the proximal division. However, the male genitalia of Cx. comptus n. sp. differ from those of Cx. commevynensis in possessing slightly conical, narrow and elongate tergum IX lobes. On the other hand, in the original description of the Cx. commevynensis adult, Bonne-Wepster & Bonne [58] mention the presence of pale golden, narrow and curved scales on the   Bionomics. Immature specimens of Cx. dunni were collected in permanent and semi-permanent partially shaded ground habitats, with slightly turbid water, associated with herbaceous vegetation such as Pistia sp. The larvae were collected in remnants of the Atlantic Forest in southeastern Brazil and in transition areas between the Cerrado and the Atlantic Forest biomes, in association with Cx. ensiformis and Cx. comptus n. sp.

Description
Male. [Fig. 12] Essentially similar to Cx. dunni, except as follows: Genitalia: gonocoxite with long, strong setae, aligned from base to apex on sternomesal surface.
Proximal division of subapical lobe with 3 parallel setae (a, b and c): seta a inserted basally, narrow; seta b long, spatulate, borne on salient tubercle; seta c filiform, long, spatulate, borne on small tubercle, apex blunt; and 1 saber-like, ribbed seta (seta s) with broad apex, arising Female. Not examined.

Pupa and larva. Unknown.
Bionomics. Immatures of Cx. exedrus were collected in ground water sites such as river margins, lagoons and ponds, associated with thick aquatic vegetation [72]. The name longisetosus is derived from a combination of the Latin noun saeta, meaning "seta, bristle" and with the Latin adjective lātus, meaning "extensive, broad". Culex longisetosus is named in reference to the four long and spatulate setae borne ventromesally between the proximal and distal divisions of the subapical lobe of the male genitalia.

Remarks
Adults of Cx. longisetosus n. sp. differ from Cx. atratus in possessing an inconspicuous basal patch of whitish scales on palpomere III and a small basal patch of whitish scales on palpomere II. The male genitalia of Cx. longisetosus n. sp. can be distinguished from those of other species of the Atratus Group in having a long columnar process in the distal division, elongate and slightly widened from the base to the apex of the ninth tergal lobe, and 4 long, spatulate setae on the ventromesal surface of the gonocoxite. . Occiput with dark brown erect forked scales. Thorax: scutum covered with narrow, dark brown falcate scales, except prescutellar area with whitish scales. Median scutellar lobe w ith 6 large, dark setae; lateral lobes each with 4 setae. Pleural setae with 2 types of colouring: dark brown with bronzy reflections: 3-6 antepronotal; 3-5 prealar; and pleural setae golden, hyaline: 4 or 5 upper mesokatepisternal, 4 or 5 lower mesokatepisternal; 5 upper mesepimeral; lower mesepimeron with 1 long, strong seta. Pleura with patch of broad, white scales on upper mesokatepisternum; lower mesokatepisternum with few scales, extending dorsally on posterior margin. Wing: mostly dark-scaled, sometimes with minute patch of white scales at proximal end of vein C; length 2.08-2.45 (2.27) (n = 6). Halter: scabellum and pedicel whitish; capitellum pale brown with few golden scales. Legs: coxae pale; ventral surface of fore-and midfemur with longitudinal stripe of white scales; tibiae dark-scaled; joints of fermur-tibia and tibia-tarsomere I with ring of pale scales; tarsi entirely dark-scaled. Abdomen: tergum I with dark scales, terga III-VII dark-scaled with white basal bands. Genitalia: tergum IX as illustrated (Fig. 14d), tergal lobes each with 15-18 slender, simple, apically bifid setae arising from median portion; apex glabrous; distance between lobes as long as basal width of 1 lobe. Gonocoxite oblong, narrow, small; subapical lobe divided into 2 columnar divisions; proximal division with 2 parallel, apically pointed setae (a and b); seta a short, slender, inserted basal to seta b; seta b spatulate; gonocoxite with 3 short filiform setae with pointed apices on ventromesal surface; distal division with long columnar process, with 5 setae: 3 narrow, filiform, apically pointed setae, subequal in size (seta f), 1 long seta with hook-like apex (seta h), and 1 large, broad, ribbed asymmetrical seta arising subapically (seta l); 1 saber-like seta (seta s) arising apically. Gonostylus slender, slightly curved, tapering towards apex, apex moderately blunt, ventral surface with 2 apical hyaline setae; 1 short leaf-like gonostylar claw. Aedeagus with sclerotized, slightly pointed, dorsolaterally directed lateral process; ventral process straight; apical process convex. Proctiger with tergum X somewhat triangular in outline, inner process pointed. Paraproct elongate, crown with 9 or 10 simple blades. Cercal sclerite with 1 seta.

Remarks
Adults of Culex longistylus n. sp. differ from the adults of Cx. atratus in having dark-scaled wings, occasionally with an inconspicuous patch of white scales on the base of vein C, and dark-scaled terga III-VII with white basal bands. Based on male genitalia, Cx. longistylus n. sp. can be distinguished from the other species of the Atratus Group in possessing fine, subapically bifid setae interspersed with simple setae on tergum IX lobes, a long columnar process of the distal division with a large, broad and ribbed seta, lateral plate of the phallosome with a straight ventral process, and 3 short filiform setae on the ventromesal surface of the gonocoxite.  [19].

Description
Male. [Fig. 15] Essentially similar to Cx. zeteki, except as follows: Genitalia: distal division of subapical lobe with median columnar process; seta l large, long, asymmetrical, ribbed; aedeagus with apical process of lateral plate pointed, without ripples.  a and b); seta a shorter, slender, inserted basal to seta b; seta b spatulate, robust and stronger than seta a; gonocoxite with 2 or 3 short, pointed, hyaline setae on ventromesal surface; distal division with short columnar process, with 5 setae: 3 narrow filiform, apically pointed setae of different in sizes (seta f), 1 longer hook-like seta (seta h), and 1 short, broad, asymmetrical seta arising subapically (seta l); 1 saber-like seta (seta s) arising apically. Gonostylus with broad leaf-like gonostylar claw with pointed apex, arising apically. Aedeagus with ventral process slightly convex and with spicules. Proctiger with tergum X with slightly pointed inner process.    a, b, c and d): seta a more basal, spoonshaped; seta b robust, spatulate, inserted on tubercle; seta c thin, slender, filiform, inserted between setae b and d; seta d borne on tubercle apical to seta b, filiform, long, with slightly narrowed apex. Distal division with mediumsized, elongate columnar process, with 5 setae: 3 filiform, narrow, pointed, apically inserted, subequal sized (seta f), 1 filiform, hook-like apex (seta h), and 1 large, broad, asymmetrical ribbed seta with apex slightly pointed on median portion, arising subapically (seta l); and 1 saber-like, ribbed seta (seta s) arising apically. Gonocoxite with slender, hyaline, short, inconspicuous setae on ventromesal surface. Gonostylus as in Cx. atratus, except for dorsal surface of the apex which may bear 2 or 3 superficial, inconspicuous emarginations. Lateral plate of aedeagus with rounded apical process, ventral process with short pointed projection directed ventrobasally. Proctiger with tergum X somewhat triangular in outline, inner process pointed and short.

Remarks
Culex trigeminatus was described by Clastrier [23] based on an adult male from French Guiana. Culex trigeminatus is more closely related to Cx. caribeanus within the Atratus Group, especially regarding adult specimens. However, Cx. trigeminatus differs from Cx. caribeanus in having palpomeres I and II dark-scaled, palpomere III with small basal whitish patch, palpomeres IV and V with inconspicuous whitish basal patches, and wings with proximal patches of white scales on veins C and R. The male genitalia of Cx. trigeminatus differ from those of Cx. caribeanus by having the median portion of the apex of seta l (distal division of subapical lobe) slightly pointed, robust and with blunt apex and proximal division with seta b strong, the lateral plate of the aedeagus having a rounded apical process and a ventral process with short, pointed projection directed ventrobasally. Fourth-instar larvae of Cx. trigeminatus differ from those of the other species of the Atratus Group by having seta 5-C with short branches that do not reach the insertion of seta 6-C and a siphon with only two pairs of dorsal setae. Furthermore, Cx. trigeminatus differs from Cx. ensiformis in possessing strongly serrated comb scales and short pecten spines. With respect to pupae, Cx. trigeminatus can be distinguished from the other species by having a slender trumpet and with the pinna small and appearing heartshaped in dorsal view.

Discussion
The Atratus Group proposed by Sirivanakarn [8] included seven valid species, namely Cx. atratus, Cx. caribeanus, Cx. commevynensis, Cx. dunni, Cx. ensiformis, Cx. trigeminatus, and Cx. zeteki. Adults of the group can be readily identified by having the vertex with narrow, decumbent scales restricted to the central area, a pleural integument with a striking pattern of dark and pale areas, and a patch of white scales on upper mesokatepisternum. In the male genitalia, the aedeagal sclerite is slender and curved in lateral view, the gonocoxite is oblong and narrow, and the gonostylus is narrow, simple and tapering to the apex. The trumpet of pupae is thin, long with a length/width ratio of 10 or higher. The fourth-instar larvae have a long, slender and tapering to apex siphon, with 3 or 4 pairs of small dorsolateral setae 1-S. Thus, according to Sirivanakarn [8], immatures of species of the Atratus Group should have a trumpet index greater than 7.0 in pupae and possess 3 or 4 dorsolateral seta 1-S in larvae. Based on the data we have in hand, contrasting with Sirivanakarn, the pupal trumpet index of Cx. atratus is 7.0 and Cx. trigeminatus larvae possess only 2 pairs of dorsolateral setae on the siphon. These characteristics disagree with the diagnosis for the group provided by Sirivanakarn [8] but they do not invalidate the Atratus Group; they facilitate the differentiation of Cx. atratus and Cx. trigeminatus from the other species of the Group, being diagnostic of these two species.
In addition, there are several characters that are useful for identification of species of the Atratus Group. Our comparative observations of adults of all examined species clearly indicate that the presence of a ring of white scales on all of the femoro-tibial joints, represents a character that helps recognize species of the group. However, other species of the Melanoconion Section also possess white scales on the knees, such as Cx. theobaldi (Lutz).
Culex exedrus can be distinguished from Cx. dunni by having several long setae visibly lined up on the sternomesal surface of the gonocoxite, seta s of gonocoxite with a large apex, and a proctiger with a large inner process of tergum X. Therefore, these features justify the resurrection of Cx. exedrus from synonymy with Cx. dunni. Similarly, Cx. loturus was resurrected from synonymy with Cx. zeteki based on the possession of a large seta l borne subapically on the distal division of the subapical lobe and a slender apical process without ripples of the lateral plate of the aedeagus.
Furthermore, examination of the available material resulted in the discovery of five new species of the Atratus Group, based on morphological characters of adults, male genitalia and, where possible, of immature stages.

Conclusions
The Atratus Group of Culex (Melanoconion) currently comprises 14 species and it has been markedly updated with respect to the number of known species, their bionomics and distribution, providing tools to facilitate the identification of the adult and immature stages of the species in the group. As a result, the current knowledge leads us to suggest the following composition of the Atratus Group: Cx. atratus (syns Cx. advieri and Cx. falsificator), Cx. caribeanus, Cx. commevynensis, Cx. columnaris n. sp., Cx. comptus n. sp., Cx. dunni (syn. Cx. ruffinis), Cx. ensiformis, Cx. exedrus, Cx. longisetosus n. sp., Cx. longistylus n. sp., Culex loturus, Culex spinifer n. sp., Culex trigeminatus and Culex zeteki. Additional studies utilizing molecular methods, particularly to investigate phylogenetic relationships within the Atratus Group, and to determine the placement of the group within the genus Melanoconion, are necessary.