Descriptions of two new species of Culicoides Latreille from Sundarbans, India with an adult key to the ornatus species group of the Oriental region (Diptera, Ceratopogonidae)

Two new species of biting midges, Culicoides cornatus sp. nov. and Culicoides pileus sp. nov. are described based on adults. The new species are compared to their congeners with close similarity. All specimens were collected from the Indian Sundarban Mangrove Forests, few specimens were mounted in glass slides and few were prepared for SEM study. Total 78 species under the genus Culicoides Latreille including the two new ones (6 species from ornatus species group) are described from India. An adult key of the ornatus species group from the Oriental Region is provided.


Introduction
Culicoides Latreille, 1809 is one of the most species rich genera of the family Ceratopogonidae with approximately 1368 valid species, 32 subgenera, 38 species groups, and 176 unplaced species described worldwide (Borkent and Dominiak 2020); so far 76 species have been reported from India (Chatterjee et al. 2020). Most female members of this genus are haematophagous and many species act as important vectors of pathogenic viruses, protozoans and filarial nematodes (Mellor et al. 2000;Borkent 2005). These midges are of great concern because they transmit bluetongue (BT), Akabane and other viruses that cause disease in sheep, cattle and wild ruminants (Kettle 1977;Linley et al. 1983). The outbreak of the BT disease in Tamil Nadu (India) and its occurrence in many parts of India over the last few decades has affected millions of sheep and goats and other livestock (Ilango 2006). Immature stages of this genus can be found in a variety of aquatic or semi aquatic habitats, including tree holes, ponds, marshes, streams, various muddy and saturated organic materials, damp or rotting vegetation, and manure (Kettle and Lawson 1952;Jamnback 1965;Borkent 2014;Shults and Borkent 2018).
The ornatus species group of this genus comprises of 33 species worldwide (Nandi et al. 2013;Borkent and Dominiak 2020) including 27 reported from the Oriental region (Table 1). Before this study, four species of the ornatus group have been reported from India: C. peliliouensis Esaki 1936, C. aequalispinus Nandi, Mazumdar &Das Gupta, 2013, C. fuscitibialis Nandi, Mazumdar &Das Gupta, 2013, andC. pateli Nandi, Mazumdar &Das Gupta, 2013. Members of the ornatus group can be recognised by the following characteristics: Eyes usually bare, seldom hairy; contiguous to moderately separated; hind tibial comb with 4 (rarely 5) tibial spines, the one nearest the spur longest; wing with second radial cell usually dark to tip but pale distally in some species; presence of two large sclerotised spermathecae with a vestigial one, sclerotised ring present or absent; male genitalia with well developed apicolateral processes, aedeagus usually with low basal arch and long, tapering, rather blunt distal process; parameres usually fused at bases, the basal portion of each directed laterad with well developed anterior process, the distal portion usually straight, simple and slender with simple pointed tip.
In this paper we describe two new species of the genus Culicoides Latreille belonging to the ornatus species group from the Indian Sundarban Deltaic region and provide a key to the adults of the species of the ornatus group of the Oriental Region.

Materials and methods
Adults of both sexes of Culicoides pileus sp. nov. and C. cornatus sp. nov. were caught using ultraviolet light trap with an 8W UV light operated besides the cow shed adjoining to mangrove forest area in Sundarbans ( Fig. 1A-C). The adults were preserved in 70% ethanol. The insects were mounted on glass slides following Wirth and Marston (1968).
Morphology and terminology of adults follow Brown et al. (2009). The wing venation is after Szadziewski (1996). Measurements are in micrometres (μm), except length and width of wing, which are in millimetres (mm). All measurements are presented as ranges when sample size was more than one, with the mean value in parentheses. The illustrations were made using a compound microscope (Wild Leitz GMBH, Portugal) in combination with an attached camera lucida. The photographs were taken both with a trinocular compound microscope (Wild Leitz GMBH) and a Stereo zoom trinocular microscope (Olympus, model SZX16, Japan). For Scanning Electron Microscopy a Carl Zeiss Sigma 300 FESEM instrument was used.
Type specimens are now in the Entomological collections of the Department of Zoology, University of Burdwan (India) and will be deposited to the National Zoological Collections (NZCI), Kolkata (India) in due course.
Abbreviations of morphological terms used in the text and/or figures: Adult. AR -antennal ratio; SCh -sensilla chaetica; SCo -sensilla coeloconica; P/H -proboscis to head ratio; PR -palpal ratio; CR -costal ratio; WL -wing length; WW -wing width; TRn -tarsal ratio; BUENTD -Burdwan University Entomology Division; NZCI -National Zoological Collections of India.  Diagnosis. Eyes with interfacetal hair, wing with inconspicuous pale marking, pale spot on cell r 3 occupying greater than half of the entire cell, tips of veins pale; horn shaped appearance of anterior portion of parameres.
Description. Female (n = 3). Head. Brown, eyes with interfacetal hair (Fig. 3C), separated by distance of 1-2 ommatidia; frontovertex with 29-31 SCh; antenna pale; length ratio of antennal segments (I-XIII): 21-22  Thorax (Fig. 3B). Brown in colour. Wing (Fig. 3A). Wing with inconspicuous pale marking; costa moderately long; cell r 2 moderately broad with distinct lumen; macrotrichia sparse; wing length 0.90-0.92 (0.91) mm, width 0.44-0.46 (0.45) mm; CR 0.68-0.69 (0.685); second radial cell entirely in dark region; pale spot over r-m cross vein moderately large, extending from vein M 1 to costal margin; post stigmatic pale spot obliquely placed with slight medial constriction; cell r 3 with distal large pale spot occupying greater than half of cell r 3 and broadly touching wing margin; cell M 1 with two pale spots, distal one touching wing margin; anal cell with one medially constricted distal pale spot, proximal one extended to pale spot at arculus, ends of veins pale at wing margin.
Leg (Fig. 2C). Brown in colour. Fore femora and tibia dark throughout with proximal small pale region, mid tibia and femora dark throughout; hind femora moderately dark all over and hind tibia with distal pale region; hind tibial comb (Figs 2D, 3E) with 4 spines, one nearest to spur longest, spur tip frayed.
Abdomen. Brown Male (n = 1). Same as female with the usual sexual differences.
Wing. Similar with female except morphometric characters. Distribution of macrotrichia in cells; wing length 0.89 mm, width 0.34 mm.
Leg. Brown in colour, all legs uniformly dark without any pale region; hind tibial comb with 4 spines and nearest to spur longest, spur tip frayed.
Remarks. New species shows similarities with C. aequalispinus and C. fuscitibialis but differs from the former in wing pattern and spine structure of hind tibial comb (second nearest the spur longer in C. aequalispinus). Culicoides fuscitibialis differs from the new species in disposition of some pale spots of wing and structure of third palpal segment. New species shows similarities with C. circumbasalis Tokunaga, 1959 andC. cordiger Macfie, 1934 in wing pattern but differs in many attributes such as eyes with interfacetal hair and pale spots on wing inconspicuous in Culicoides cornatus sp. nov. Culicoides cornatus shows similarities with C. cordiger Macfie, 1934 in wing pattern but differs in attributes like eyes (eyes with interfacetal hair in new species but eyes are bare in C. cordiger), antennal ratio, mandibular teeth number and distribution of SCo on antennal segments. The new species shows similarities with C. corti Causey, 1938 in some characters like eyes with interfacetal hair, disposition of some pale spots on wing but differs in characters like palpal ratio, distribution of SCo, and on antennal segments (SCo are present on I-XII in C. cornatus but SCo on I and XI-XIII in C. corti), P/H ratio, shape of the aedeagus and paramere. Distribution of SCo on antennal segments of Culicoides cornatus are similar with C. damnosus Delfinado, 1961 and C. flumineus Macfie, 1937 but differs in characters like eyes with interfacetal hair, antennal ratio, mandibular teeth, and number and disposition of pale spots on wing. Culicoides cornatus shows similarities with C. palawanensis Delfinado, 1961 in SCo distribution and hairy eyes but differs in disposition of pale spots on wing, shape of the parameres and aedeagus, P/H ratio (much less in C. cornatus), mandibular teeth number (17 in C. cornatus but 11-13 in C. palawanensis), and antennal ratio. Culicoides cornatus also shares similarities with C. papuensis Tokunaga, 1962 in disposition of some pale spots on wing but differs in the presence of interfacetal hair on eyes in Culicoides cornatus (bare in C. papuensis), distribution of SCo on the antennal segments, mandibular teeth number, antennal ratio and the shape of parameres. Culicoides cornatus differs with C. hollandiensis Tokunaga, 1959 in pale marking of wing (inconspicuous pale marking in C. cornatus whereas prominent pale marking present in C. hollandiensis) and shape of the aedeagus (terminally rounded in C. hollandiensis). Culicoides cornatus differs from C. pampangensis Delfinado, 1961 in structure of paramere and pale marking of wing. Culicoides cornatus is different from C. paragarciai Dyce, 1996 in pale marking on wing (pale marking absent in C. paragarciai) and structure of hind tibial comb (hind tibial comb with 5 spines and 2 nd from the spur is longest in C. paragarciai whereas 4 spines and 1 st from the spur longest in C. cornatus). Culicoides quaterifasciatus Tokunaga, 1959 differs in presence of densely spread macrotrichia on wing and also some pale marking is different from C. cornatus. Etymology. The name 'cornatus' refers to Latinised version of cow horn shaped appearance of anterior portion of parameres.
Distribution and bionomics. The species was collected in adjacent to Indian Sundarbans Deltaic region, a world heritage site (West Bengal state). Adult midges were collected from a cowshed using an ultraviolet light trap in the type locality, situated in the vicinity of Datta River. The type locality is at an altitude of 7.50 m above sea level. Diagnosis. Eyes bare; wing with one distal pale spot in cell r 3 separated from wing margin; cell m 1 with two pale spots, distal one away from wing margin; anal cell with one distal pale spot with slight medial constriction; SCo present on flagellomeres I-VII and IX-XII; parameres basally fused, cap shaped basal knob, mid portion wide, distally slender, abruptly bent laterad with pointed end.
Wing. Wing pattern as in figure 5A, costa moderately long; cell r 2 moderately broad with distinct lumen, macrotrichia sparse, arranged from middle to distal most portion of wing; wing length 0.77-0.80 (0.78) mm; width 0.38-0.42 (0.40) mm; CR 0.64-0.65 (0.645). wing spots moderately distinct, second radial cell completely in dark region; pale spot over r-m cross vein moderately large, extending from vein M 1 to costal margin; post stigmatic pale spot obliquely placed with slight medial constriction; cell r 3 with distal large pale spot away from wing margin; cell M 1 with two pale spots, distal one separated from wing margin; cell m 2 with narrow pale streak connecting pale spot at basal arculus, pale spot behind medial fork and distal pale spot; distal pale spot touching wing margin; cell cua 1 with moderately large, roughly round pale spot touching CuA 1 vein and broadly touching wing margin; anal cell with one medially constricted distal pale spot, proximal one extended to pale spot at arculus. Leg (Fig. 4C). Brown in colour. Fore and mid femora pale throughout, fore and mid tibia with proximal pale region; hind femora moderately dark throughout and hind tibia also with proximal pale region; hind tibial comb with 4 spines, one nearest to spur longest, spur tip frayed (Figs 4D, 5D).  (Fig. 4E).
Male (n = 3). Same as female with the usual sexual differences.
Leg. Light brown in colour, hind tibial comb with 4 spines and nearest to spur longest, spur tip frayed.
Remarks. New species shows similarities with C. aequalispinus in bare eyes and mandibular teeth number but differs in attributes like distribution of SCo on antennal segments, and presence of two equal length spines nearest to the spur in C. aequalispinus. The new species shares a few similarities with C. peliliouensis Tokunaga in Tokunaga and Esaki 1936 like bare eyes, disposition of some pale spots on wing but differs in characters like distribution of SCo on antennal segments (SCo present on I, V-VII and IX-XII in C. peliliouensis but in C. pileus sp. nov. these are on I-VII and IX-XII), pale spot on cell r 3 broadly touching the wing margin but in new species it is separated by dark region; pale spot over r-m cross vein is different in shape, post stigmatic pale spot medially constricted in C. pileus but not in C. peliliouensis. Culicoides pileus shows some similarities with C. circumbasalis Tokunaga, 1959 andC. ornatus Taylor, 1913  but differs in characters like distal pale spot in anal cell of wing (two separate distal pale spots present in both C. circumbasalis and C. ornatus but in C. pileus a single pale spot present with little medially constricted), cell r 3 with three pale spots and most distal one touching the wing margin in C. circumbasalis but two in C. pileus sp. nov. and most distal one away from the wing margin. It is quite similar in some attributes with C. cordiger Macfie, 1934 but differs in distribution of SCo on antennal segments (SCo are present on antennal segments I-XIII in C. cordiger) and mandibular teeth number (7-12 in C. cordiger but 16-17 in C. pileus). Culicoides pileus differs from C. damnosus Delfinado, 1961 andC. quatei Wirth &Hubert, 1989 in the shape of pale spot over r-m cross vein and distribution of SCo on antennal segments. It shares similarities with C. mcdowelli Delfinado, 1961 but differs in the shape of pale spot over r-m cross vein, distal pale spot on cell r 3 (touching the wing margin in C. mcdowelli) and mandibular teeth number (11-13 in C. mcdowelli). It also shows some similarities with C. hollandiensis Tokunaga, 1959 in wing pattern but pale spot is absent at the base of CuA 1 in C. pileus and anal cell with two separated distal pale spots in C. hollandiensis. Culicoides pileus differs from C. hollandiensis in pale marking of wing (anal cell with two separate distal pale spots in C. hollandiensis) and shape of the aedeagus. Culicoides pileus differs from C. pampangensis Delfinado, 1961 in structure of paramere and pale marking of wing (cell r 3 with distal pale spot not prominent in C. pampangensis Delfinado). Culicoides paragarciai Dyce, 1996 differs from C. pileus in pale marking on wing (pale marking absent in C. paragarciai) and structure of hind tibial comb (hind tibial comb with 5 spines and 2 nd from the spur is longest in C. paragarciai whereas 4 spines and 1 st from the spur longest in C. cornatus).
Etymology. The name 'pileus' refers to Latinised version of cap shaped basal knob of parameres.
Distribution and bionomics. The species was collected from Sundarbans Mangrove Forest in India (West Bengal state). Adult midges were collected from a cowshed using an ultraviolet light trap in the type locality situated in the vicinity of Datta River. The type locality is at an altitude of 7.50 m above sea level.
An inventory of ornatus group of the genus Culicoides Latreille from the Oriental region Distribution of Oriental species of ornatus group has been presented in Table 1 and Fig. 6.  Table 1. A key to the ornatus group of the genus Culicoides Latreille from the Oriental region    Chu, 1986 21 SCo present on antennal segments I, IX-XII, sometimes also on II, III, IV and VI ..