Culicoides jiangchengensis, a new species of the subgenus Sinocoides (Diptera, Ceratopogonidae) based on integrative taxonomy from China

Biting midges of the genus Culicoides are important in both medicine and veterinary medicine because their blood-feeding regime enable them to transmit a variety of pathogens. In this study, the morphological characteristics of the new species of Culicoides (Sinocoides) jiangchengensis Wang et Liu sp. nov are described and compared with the other species of female Culicoides in the subgenus Sinocoides. Three morphological characteristics of C. jiangchengensis, such as without sensory pit in 3rd palpus segment, sensilla coeloconica on flagellomeres 1,9–13, and m1 and m2 cell of the wings with pale spots, were different from the other nine species of culicoides in subgenus Sinocoides. Genetically, C. jiangchengensis are most closely related to C. malipoensis, but they were located in different branches and the minimum interspecific distance between them was 12.6%. In addition, a checklist of 10 species in the subgenus Sinocoides Chu, 1983 (Diptera: Ceratopogonidae: Culicoides) in China, including the new species C. jiangchengensis Wang et Liu sp. nov., is provided, and an updated key to species of the subgenus Sinocoides Chu, 1983 was presented.


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PLOS ONE | https://doi.org/10.1371/journal.pone.0287266 July 26, 2023 1 / 18 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 species in 38 species groups in 33 subgenera [4]. In China, there are 451 species of bloodsucking midges in three genera, and Culicoides includes 336 species in 12 subgenera [5,6]. Qu (1983) established the subgenus Sinocoides Chu, 1983 in genus Culicoides [7]. At that time, only one species of this subgenus was described in China, Culicoides hamiensis [8]. Subsequently, eight new species in the subgenus were described by Liu, Yu et al [9][10][11]. In the last decade, Liu and Wu et al. described four new species in this subgenus [12][13][14]. Here, a new species of the subgenus Sinocoides is described in an investigation on blood-sucking midges in Yunnan Province, China. Cytochrome c oxidase subunit gene (COI) sequences were obtained to identify the species of Culicoides. The main distinguishing characteristics, geographical distribution, and a species checklist of the 10 species of this subgenus found in China are reported.

Materials and methods
Biting midges were collected overnight from 7:00 pm to 7:00 am the next morning in domestic animal pens using light traps (12 V, 300 mA; Wuhan Lucky Star Environmental Protection, Hubei, China) in Jiangcheng County in September 2015 and Gongshan County in August 2017 of Yunnan Province, China. The midges were stored in 70% ethanol at 4˚C and immersed in 250 μL tissue digestive solution with 1% proteinase K (TIANGEN DNA extraction kit) for non-destructive tissue digestion [15]. The midges were slide-mounted in Canada balsam, as described by Yu et al [5]. Midge genomic DNA was extracted from the digestive supernatant using Micro DNA Kit (TIANGEN, Beijing, China) according to the manufacturer's instructions. Partial mitochondrial cytochrome c oxidase I (COI) gene sequence was obtained by PCR amplification using forward primer C1-J-1718 (5'-GGAGGATTTGGAAATTGATTAGT-3') and reverse primer C1-N-2191 (5'-CAGGTAAAATTAAAATATAAACTTCTGG-3') [16]. The PCR reaction volume was 50 μL, and contained Takara Ex Taq (5 U/μL) 0.25 μL, 10 × Ex Taq Buffer 5 μL, dNTP Mixture (2.5 mM) 4 μL, 0.5 μM of each primer and 4 μL of midge genomic DNA. The DNA amplification program was: 94˚C 3 min, 30 cycles of 94˚C 30 s, 55˚C 30 s, 72˚C 30 s, followed by 72˚C 5 min. Purified amplicon of the COI gene was cloned into the pMD TM 19-T vector (Takara, Dalian, China). Recombinant plasmids were transformed into Escherichia coli DH5α competent cells. Positive clones were identified through PCR using M13 universal primers and sequenced using an automated ABI 3730 DNA Sequencer (Applied Biosystems). The COI gene sequence was submitted to GenBank under accession number OL471017-OL471027. Sequence alignments were performed using Clustal X (version 2.0) [17] and MAFFT [18] to ensure proper alignment. Phylogenetic trees were constructed by the neighbour-joining method using distance matrices generated by the p-distance determination algorithm in MEGA-X with 1000 bootstrap replicates.

Nomenclatural acts
The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix "http://zoobank.org/". The LSID for this publication is: urn:lsid:zoobank.org:pub:E8649BF5-471C-4812-A61E-360486C59946. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS.

Ethics statement
Authorization for the collection of Culicoides has been obtained from Institute for Yunnan Animal Science and Veterinary Institute, Kunming, China (protocol approval number: 2019FA015 and 202005AF150034). No specifc permits were required for the field studies. After explanation of the purposes and activities of the study, oral consent was obtained from the local participating residents prior to Culicoides collection. No sites were protected by law and this study did not involve endangered or protected species.

Taxonomy
Culicoides (Sinocoides) jiangchengensis Wang et Liu sp. Nov, 2023. (Fig 1). urn:lsid:zoobank.org:act:C059685C-7A56-4CD2-9A67-946CF68C2A67 Diagnosis. Females: only for Culicoides species with the following combination of features: palpus third segment slightly swollen at distal 1/3, without sensory pit, with sensilla PLOS ONE scattered on the surface; a small, approximately oval, fuzzy pale spot at the distal end of r 5 cell; m 1 with two pale spots, anal cell with a wide pale band extended from its base to distal; three equally sized spermathecae. Male: only for Culicoides species in Yunnan with the following combination of features: palpus third segment without sensory pit; anal cell with a big, long irregular pale spot shaped like a pocket; the arch of aedeagus with deep bow type, the middle of aedeagus reflexed in apex, parameres separate, the apex part bends in a hook shape. Thorax (Fig 1J and 1K) Scutum light brown, scutellum dark brown; wing with contrasting pattern of pale/dark spots; wing base with a large pale spot and connected to the wide pale band of the anal cell; basal 2/3 of 1 st radial cell and r-m cross-vein in pale spot, and extension posterior over M1 to connect with narrow pale band of m 2 ; distal 4/5 of 2 nd radial cell covered by pale spot; apical pale spot in cell r 5 cell faint, and reaching wing margin; m 1 with two pale spots, proximal pale spot long band; m 2 with two differently shaped pale spots: proximal pale spot long band extending from the base to distal, distal pale spot abutting the wing margin; m 4 with a pale spot abutting wing margin; anal cell with a wide pale band extending from base to distal; macrotrichia on the costal of the proximal of r 5 cell and distal of m 1 , but no on the proximal of m 1 ; hind tibial comb with four spines, 2 nd spine longest, metatibial comb (mc) about 18 teeth, TR and F-T of legs are as in Table 1.

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Culicoides jiangchengensis, a new species of the subgenus Sinocoides dark spots; basal 1/3 of 1 st radial cell and r-m cross-vein in pale spot; distal 2/3 of 2 nd radial cell covered by pale spot; anal cell with a big irregular pale spot. Metatibial comb (mc) about 18 teeth, TR and F-T of legs are given as Table 1. Genitalia (Fig 1M and 1N): the middle of the posterior margin of the 9 th genitalia sternite concave, wide, arc and the membrane free of microhairs. The posterior margin 9th basal tergite flat, a small V-shaped concave was observed in the middle part, parameres robust, sharp Angle shape. Gonocoxite slender in its basal part, the dorsal ankle digitation, mucro part bend to the medial side. The middle aedeagus is nearly tapered, aedeagus with deep bow type, the arch height of the aedeagus is about 1/2 of the total length of the aedeagus, reflexed in apex. Parameres separated, the middle part thick, diminution, curve and with a hook shape in apex.
Etymology. The name jiangchengensis refers to the collecting location of the species. Remarks. C. jiangchengensis collected in Jiangcheng County in this study have three spermathecae, which were similar to those of the subgenus Pontoculicoides, Sinocoides, Jilinocoides and Trithecides. Two eyes of C. jiangchengensis were separated, excluding the subgenus Jilinocoides and Trithecides, and the wings have pale spots and dark spots excluding pontoculicoides, indicating that C. jiangchengensis is a member of the subgenus Sinocoides. At present, there are 9 species of Culicoides in subgenus Sinocoides, among which C. anthropophygas (Fig 2), C. hamiensis (Fig 3), C. jinghongensis (Fig 4), C. kongmiaoensis (Fig 5), C. multifarious (Fig 6), C. opertus (Fig 7) had an obvious sensory pit in the 3 rd segment of palpus, while C. jiangchengensis had no an obvious sensory pit, but with capitate sensilla scattered on the surface, which had significant differences between them. Although C. pungobovis (Fig 8) and C. jiangchengensis have similar morphological characteristics in the 3 rd segment of palpus, their sensilla coeloconica on flagellomere are significantly different, the former is 1,6-8, while the latter is 1,9-13. C. malipoensis (Fig 9), C. nanniwanensis (Fig 10) and C. jiangchengensis had similar morphological characteristics in the 3 rd palpus segment and the sensilla coeloconica on flagellomere, but there was no pale spot in the wings m 1 and m 4 of C. nanniwanensis, while the m 1 and m 4 of C. malipoensis and C. jiangchengensis with pale spot. C. jiangchengensis is the most similar to C. malipoensis in morphological characteristics, but the former has 2 pale spots in the m 1 and a wide pale band extended from the basal to the distal in the anal cell, while the latter has a pale band extended from base to the distal in the m 1 and a pale spot at the proximal in the anal cell.
In addition, the main characteristics distinguishing Culicoides in China are wing length, costal ratio (CR), antennal ratio (AR), proboscis ratio (PR), mandible teeth, and sensilla coeloconica on flagellomere. Table 2 provides detailed measurements.

DNA analyses
The phylogenetic tree based on the COI gene sequences of C. jiangchengensis, C. malipoensis from Jiangcheng County and Gongshan County, Yunnan Province and another 24 species of Culicoides shows that nine female C. jiangchengensis from Jiangcheng and two male C. jiangchengensis from Gongshan formed a separate branch (Fig 11). The mean intraspecific distance was 1%, and the maximum was 2.36%. Although C. jiangchengensis and C. malipoensis are on different branches, their genetic relationship is closest among other Culicoides; the minimum interspecific distance was 12.6%.               Distribution: Shanxi (Nanniwan).

Discussion
The three main morphological characteristics that identified C. jiangchengensis as subgenus Sinocoides are the separation of two eyes, capitate sensilla scattered on the surface of 3 rd segment palpus, the contrasting pattern of pale/dark spots in the wing, and three subequal-size ovoid spermathecae, which is similar to that of other species of subgenus Sinocoides. According to the following three morphological characteristics of C. jiangchengensis: 1) without sensory pit in 3 rd segment of palpus; 2) sensilla coeloconica on flagellomere 1, 9-13; 3) PLOS ONE m 1 and m 2 cell of the wings with pale spots, which were different from the other eight species of culicoides in subgenus Sinocoides, such as C. anthropophygas, C. hamiensis, C. jinghongensis, C. kongmiaoensis, C. miaoensis, C. opertus, C. pungobovis and C. nanniwanensis. C. jiangchengensis is morphologically similar to C. malipoensis, but C. malipoensis is a medium-sized Culicoides, with a wing length of 1.35 mm, mandible with nine teeth, P/H ratio 0.64, the distal of r 3 with a large fuzzy pale spot, m 1 cell with a wide pale band extending from the base to distal, and distal of anal cell with a pale spot, which is obviously different from C. jiangchengensis.
Molecular biology is one of the methods for rapid and accurate identification of vector insect species [19]. COI gene with moderate evolutionary rate is the most commonly used molecular target for mosquito and midges identification [19,20]. In this study, the COI gene was used as a molecular target to identify C. jiangchengensis and C. malipoensis collected in Jiangcheng and Gongshan. The results showed that C. jiangchengensis was mostly related to, but distinct from, C.malipoensis in subgenus Sinocoides. The minimum interspecific distance between them was 12.6%, higher than between C. selandicus and C. kalix (5.9%) [21] and C. fagineus F1 and C. subfagineus (sensu stricto) (12%) within the subgenus Culicoides [22] and C. bolitinos and C. tutti-frutti (9.5%) within the subgenus Avaritia [23] (Augot et al. 2016). These data indicated that C. jiangchengensis is a new species in subgenus Sinocoides based on its

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compiled. Fortunately, in this study, we used molecular biology methods to identify the males of C. jiangchengensis, and systematically described the morphological characteristics of the males, providing data for the classification and identification of C. jiangchengensis in the future. However, due to the lack of morphological description of other subgenus Sinocoides male, failed to compile a key to the males of 10 Culicoides in this subgenus, and needs further investigation.