Culicoides paradoxalis Ramilo & Delécolle, 2013

Ramilo, David, Garros, Claire, Mathieu, Bruno, Benedet, Christophe, Allène, Xavier, Silva, Elisabete, Alexandre-Pires, Graça, Fonseca, Isabel Pereira Da, Carpenter, Simon, Rádrová, Jana & Delécolle, Jean-Claude, 2013, Description of Culicoides paradoxalis sp. nov. from France and Portugal (Diptera: Ceratopogonidae), Zootaxa 3745 (2), pp. 243-256 : 244-253

Culicoides paradoxalis Ramilo & Delécolle , sp. nov.

( Fig. 1–2 , 4 , 5B , 6B , 7B , 8B and 9C)

Holotype ♀: Corsica, Pietra Corbara, farm, light trap, 3/ 4-VI-2003, n.° 5. Deposited in Institut de Parasitologie et de Pathologie Tropicale de Strasbourg, Strasbourg, France.

Description of the holotype. Approximately total length: 1.6 mm.

Head. Eyes ( Fig. 1A ): fused, with a mean length of 19 µm (12 to 32), with a light circular suture above one median bristle of the vertex. Clypeus entirely pubescent, with 5 to 6 robust bristles in each side. Cibarium and posterior pharynx without armature. Antennae ( Fig. 1B ): light brown scape with eight robust bristles, dark brown pedicel with three robust bristles. Flagellomeres 1 to 13 are uniformly light brown; flagellomere 1 has a short pedicle; flagellomeres 2 to 8 are subcylindrical with a slight strangulation at the apex; flagellomeres 9 to 13 are subcylindrical; flagellomere 13 acuminate distally. Flagellomere 1 has six to eight sensilla chaetica; Flagellomeres 2 to 8 have six sensilla chaetica, two major sensilla trichodea and a shorter one. Sensilla chaetica are very thin, with a length shorter than the major sensilla trichodea; Flagellomeres 9 and 10 have two or three sensilla chaetica; flagellomere 11 has two sensilla chaetica; flagellomere 12 has one sensillum chaeticum and flagellomere 13 has one subapical sensillum chaeticum, with a median localization; flagellomeres 9 to 13 have also dispersed sensilla trichodea and basiconica. Two to four sensilla coeloconica are present on flagellomere 1 (observed using Composed Optical Microscopy (COM) and Scanning Electron Microscopy (SEM) techniques—see discussion section), one on flagellomeres 9 to 11 (occasionally with two present on the 10 and 11), three on flagellomeres 12 and 13 (rarely 1 or 4). Measures: 58, 41, 44, 48, 48, 48, 48, 48–67, 69, 74, 74, 90. Total length: 757 (683 to 806). Antennary index: 0.97 (0.94 to 1.03). Ratio 9/8: 1.39 (1.3 to 1.5). Palpi ( Fig. 1C ): uniformly light brown. Segment 3 is narrower on its base than on its tip, swollen in its median region and possesses several small sensorial depressions joined with each other, each one ornamented with multiple clavate organs. Measures: 97, 81, 35, 41. Total length: 254 (221 to 262). Maxillae and mandibles with twenty small teeth on average.

Thorax. Not observed in fresh material. Mesonotum more or less uniformly dark-brown with the suggestion of very dark spots at its top, extending rearward from the body axis and laterally below humeral impressions. Six lateral bristles present, one of them localized in front of the suture and five behind. A major portion of the median area of the scutellum is brown, becoming gradually lighter on the lateral edges, with four robust bristles, two median and one lateral on each side and two to three shorter bristles between the lateral and the median ones. Wings ( Fig. 2 , 3 and 9C): one pale spot begins at the distal edge of the wing and covers a large proportion of the first radial cell (r 1) and the crossvein r-m (r-m). Poststigmatic pale spot (Pst) in cell r 3, including the distal half of the second radial cell (r 2); it does not reach the M 1 vein. The distal spot of r 3 cell is well developed, limiting with the precedent one a large dark spot with a poorly delineated hour glass shape. The distal edge of this dark spot does not have a significant notch at its base. The median light spot of the m 1 is small and joined with the M 2; this spot sometimes extends to the cell base. The pale spot of the m 2 is extremely small and can be joined to the M 2 or be absent. One pale spot is present in the apex of the m 1 and m 2 cells and the pale spot of the cua 1 has a centered dark spot which is well marked and joined with the dark part of the CuA 1. Two pale spots are clear and separate in the distal part of the anal cell (a), one localized in the apex and the other in the distal edge of the cell. This last one is prolonged with a narrow light band bordering the posterior edge of the cell, joining with the light spot largely localized at the distal part of the same cell. Measures—length: 1170 (773 to 1215); width: 540 (465 to 540); costa: 690 (600 to 735); cubital: 525 (450 to 540). Wing ratio (WR): 0.58 (0.57 to 0.61). Uniform light balancers. Legs: all segments are narrow and cylindrical, with a few sparse and small hairs. Femora are uniform brown, with a discrete basal enlightening; fore and mid tibiae are brown with a discrete basal enlightening; hind tibiae possess one pale ring at the base and a distal enlightening; tarsi are uniform beige. The apex of fore tibia possesses a welldeveloped spur and one small comb; apex of the mid tibia without notable particularities and apex of the hind tibia with one short and pectinated spur and two combs. The larger of these combs comprises 5 teeth whose sizes are: 42 (42 to 58), 55 (55 to 69), 44 (39 to 64), 39 (35 to 53), 39 (35 to 46). Two tarsal spines are present in the apex of tarsomeres 1 and 2, and one at the apex of tarsomere 3 of mid legs. The claws of the three pairs of legs are small, equal and simple at their extremity. A rudimentary empodium is present. Measures of the three pairs of legs (fore, mid and hind respectively):

398 – 413 – 218 – 110 – 74 – 51 – 51

488 – 495 – 285 – 113 – 76 – 51 – 51

458 – 465 – 233 – 143 – 87 – 51 – 53

Abdomen. Sternite 8 is brown and indented in the middle of its posterior edge. Sternite 9 is narrow and brown. Sternite 10 is round and light, having 2 robust bristles and 2 shorter localized in front of the precedent ones. Two functioning pyriform spermathecae, subequals (63 x 45 and 59 x 42) with a small neck and 1 rudimentary spermatheca with a digital form; the sclerified ring is small and cylindrical ( Fig. 1D ).

Morphometrics for holotype. Resumed in Table 1 .

Allotype ♂: Corse, Poggiale ( Commune de Figari ), farm, light trap, 19/ 20-VIII-2003, n.° 1. Deposited in Institut de Parasitologie et de Pathologie Tropicale de Strasbourg, Strasbourg, France.

Description of the allotype. Approximate total length: 1.5 mm.

Head. Eyes fused, without pubescence. Antennae: light brown scape, dark brown pedicel; flagellomeres 1 to 13 are uniform light beige. Flagellomere 1 has a long pedicle; flagellomeres 2 to 10 are approximately the same size, with flagellomere 10 marginally longer; flagellomeres 11 to 13 are large and cylindrical, the last one acuminating distally. Long plumes of sensilla chaetica are present and implanted in the median whorl of flagellomere 10; flagellomere 11 has four robust sensilla chaetica arranged on the base, including one slightly isolated from the others; flagellomere 13 with one subapical sensillum chaeticum. Flagellomere 1 with two major sensilla trichodea; flagellomeres 2 to 4 with two major sensilla trichodea and one minor; flagellomeres 5 to 8 with one major and one minor sensillum; flagellomeres 11 to 13 with dispersed sensilla trichodea and basiconica. Presence of two sensilla coeloconica on flagellomere 1; three and two sensilla coeloconica in the flagellomeres 12 and 13, respectively. Measures: 97, 39, 41, 39, 37, 37, 39, 37, 35, 46–113, 90, 90. Total length: 740. Antennary index: 0.65. Ratio 11/10: 2.45. Palpi: uniform light beige. Segment 3 is softly bulging and has on its distal part one to many small sensorial depressions filled with claviform organs. Measures: 69, 62, 28, 44. Total length: 203.

Thorax. Coloration similar to female. Five lateral robust bristles present, one of them prior to the suture. Scutellum with four bristles disposed in a 1-2-1 arrangement and one shorter bristle on each side between the lateral and the median. Wings, coloration and distribution of the light spots similar to those of females. Abundant macrotrichia, but limited in the distal part by r 3, m 1 and m 2 cells. Measures—length: 1035; width: 360; costa: 555; cubital: 495. Wing ratio (WR): 0.53. Light balancers. Legs: coloration similar to female. Measures of the three pairs of legs (fore, mid and hind respectively):

366 – 375 – 188 – 99 – 71 – 46 – 46

450 – 443 – 270 – 106 – 71 – 46 – 46

405 – 413 – 210 – 127 – 83 – 51 – 44

Abdomen. Genitalia ( Fig. 4 ): Sternite 9 wide with a slight indentation in the central posterior edge and slightly pubescent in the lateral edges. Tergite 9 barely wider than long, with a flat posterior edge and a clear medial incision; on each side there is one well developed appendix with a rose spine form converging with its homolog of the opposite side. The gonocoxite is slightly swollen in its basal part; internal edge is lined with small spines that are reduced towards the joint with the gonostylus. The ventral apodeme is small and narrow, usually forming a small hook and the dorsal apodeme is short, cylindrical and robust. The gonostylus is slightly swollen at the base, twisted and arched in its medial part and slightly enlarged at its apex with pubescence at its base. Parameres are separated, slightly curved outwardly at their base and gradually narrow, becoming thin and bent to a fine point ornamented with some elements of pubescence. The aedeagus is Y shaped with long lateral cylindrical arms, curved sideways at the base extremity and joined at the base of the body, which is more or less triangular in shape, being round at the tip.

Morphometrics for allotype. Resumed in Table 2 .

Other specimens defined as PARATYPES: FRANCE, CORSICA: 1 ♂: Cargèse , sheepfold, light trap, 29/ 30-VI-2005 ; 1 ♂: Pietra Corbara , sheepfold, light trap, 17/ 18-VIII-2004 ; 1 ♂: Porto-Vecchio , lieu-dit Bala, sheepfold, light trap, 19/ 20-VI-2002 ; 1 ♂: idem, 25/ 26-VI-2003 ; 2 ♀: Pietra Corbara , sheepfold, light trap, 3/ 4-VI- 2003 ; 1 ♀: idem, 6/ 7-VII-2004 ; 2 ♀: Porto-Vecchio , lieu-dit Bala, sheepfold, light trap, 22/ 23-IX-2005 ; 1 ♀: Sarrola-Carcopino , sheepfold, light trap, 26/ 27-VI-2003 ; 2 ♀: Sartène , sheepfold, light trap, 27/ 28-IX-2005 ; 1 ♀: Sartène , LEGTA, barn, light trap, 12/ 13-VI-2002 ; 2 ♀: idem, 20/ 21-VI-2002 ; 2 ♀: Porto-Vecchio , lieu-dit Bala, sheepfold, light trap, 4/ 5-X-2000 ; 2 ♀: idem, 25/ 26-VI-2003 ; 10 ♀: idem, 19/ 20-VIII-2003 ; 1 ♀: idem, 8/ 9-IX- 2003 ; 1 ♀: Sartène , sheepfold, light trap, 9-IX-2002 ; 1 ♀: Sartène , LEGTA, light trap, 11-VI-2002 ; 11 ♀: idem, 12/ 13-VI-2002 ; 1 ♀: idem, 20/ 21-VI-2002 ; 3 ♀: San-Giuliano , sheepfold, light trap, 11/ 12-VI-2001 ; 13 ♀: Pietra Corbara , sheepfold, light trap, 3/ 4-VI-2003 1 ♀: idem, 5/ 6-VIII-2003 ; 6 ♀: idem, 6/ 7-IX-2004 ; idem, 6/ 7-VII- 2004 ; 1 ♀: Poggiale (commune de Figari), sheepfold, light trap, 19/ 20-VI-2003 ; 1 ♀: idem, 8/ 9-X-2003 . Specimens are deposited in Institut de Parasitologie et de Pathologie Tropicale de Strasbourg, Strasbourg, France .

FRANCE: 10 ♀: Department of the Var, Roquebrune-sur-Argens , Campagne de l’Orme , Sciarafa , light trap, 18/ 19-VI-2012. Specimens are deposited in Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad), Montpellier, France .

PORTUGAL: 1 ♀: Termas de Monfortinho, Nave da Barca, Salvaterra do Extremo , Idanha-a-Nova , Castelo Branco, cattle, light trap, 28/ 29-VIII-2008 ; 1 ♀: Almojanda de Baixo , Fortios, Portalegre, cattle, light trap, 25/ 26- VI-2008 ; 5 ♀: Casas Altas, Avis, Portalegre, sheepfold, light trap, 19/ 20-V-2008 ; 1 ♀: idem, 29/ 30-V-2008 ; 1 ♀: Torrinha, Santa Maria, Odemira, Alcácer do Sal , cattle, sheepfold, goats, light trap, 12/ 13-VIII-2008 ; 1 ♀: idem, 23/ 24-IX-2008 . Specimens are deposited in Faculty of Veterinary Medicine, Technical University , Lisbon .

Diagnostic characters with closely related species. The wing pattern of Culicoides paradoxalis is very similar to Culicoides newsteadi and Culicoides lupicaris . The differences and similarities between the three species are summarized in Table 3 and represented in Figures 5–9 .

COI barcode and molecular phylogeny. Using COI barcode sequences, we reconstructed phylogenetic relationships of sequences of C. paradoxalis from Corsica and Portugal, together with closely related species from other geographical areas ( Table 4).

Although the between-clade relationships are not resolved, the dendogram showed 5 separate clades, each grouping individuals representing a single species ( Fig. 10 ). The clade clustering the C. paradoxalis individuals is well separated from the morphologically related species, C. newsteadi and C. lupicaris . The lowest genetic distance was between C. paradoxalis and C. lupicaris (0.166) and the highest distance was between C. paradoxalis and C. newsteadi (0.210). This level of genetic divergence is in favour of C. paradoxalis to be confirmed as a new species ( Table 5). The mean distance within species reveals cryptic diversity within the C. pulicaris clade as also mentioned in a previous study ( Pagès et al. 2009). Indeed, the dendogram showed two different groups within this clade, with one grouping 2 sequences from Spain and one from Corsica, France ( Fig. 10 ).

Legend: 1— Culicoides lupicaris ; 2— Culicoides pulicaris ; 3— Culicoides punctatus ; 4— Culicoides paradoxalis ; 5— Culicoides newsteadi .

Figures

FIGURE 1. Schematic illustrations of: A) eyes; B) flagellomeres 1–2, 8–9 and 13; C) palpus; D) spermathecae of Culicoides paradoxalis sp. nov. female.

FIGURE 2. Schematic illustration of the wing pattern of Culicoides paradoxalis, sp. nov.

FIGURE 3. Schematic illustration of wing spots, venation and cells in biting midges of Culicoides genus (Diptera: Ceratopogonidae): r-m—crossvein r-m; r1—first radial cell; r2—second radial cell; r3—third radial cell; Pst—Poststigmatic pale spot; M1—first medial vein; m1—first medial cell; M2—second medial vein; m2—second medial cell; cua1—anterior cubital cell; CuA1—first branch of anterior cubital vein; a—anal cell.

FIGURE 4. Schematic illustration of genitalia of Culicoides paradoxalis, sp. nov. male.

FIGURE 5. Segment 3 of palpus: A) C. newsteadi; B) C. paradoxalis; C) C. lupicaris.

FIGURE 6. Sensilla coeloconica on the flagellomere 1: A) C. newsteadi; B) C. paradoxalis; C) C. lupicaris.

FIGURE 7. Segment 3 of palpus: A) C. newsteadi; B) C. paradoxalis; C) C. lupicaris. Bar = 10 µm.

FIGURE 8. Tarsomere 4 of the mid legs, pointed with a white star: A) C. newsteadi; B) C. paradoxalis; C) C. lupicaris. White arrow—presence of spine; Black Arrow—absence of spine. Bar = 10 µm.

FIGURE 10. Phylogenetic tree for Culicoides inferred from COI sequences. The tree was unrooted. Bootstrap support values from maximum likelihood values are displayed on the nodes. Values lower than 70% are not shown.