https://orcid.org/0000-0002-4903-7168
Figures
Abstract
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.
Citation: He Y, Meng J, Li N, Li Z, Yu T, Zhang L, et al. (2023) Culicoides jiangchengensis, a new species of the subgenus Sinocoides (Diptera, Ceratopogonidae) based on integrative taxonomy from China. PLoS ONE 18(7): e0287266. https://doi.org/10.1371/journal.pone.0287266
Editor: Ulrike Gertrud Munderloh, University of Minnesota, UNITED STATES
Received: November 21, 2022; Accepted: May 31, 2023; Published: July 26, 2023
Copyright: © 2023 He et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All sequence are available from the accession number(s) OL471017-OL471027.
Funding: This work is supported by grants from the Basic Research Projects of Yunnan Province (grant no. 2019FA015 and 202201AS070062), National Natural Science Foundation of China (grant no. 32260896), Scientific and technological innovation team construction project of Kunming Medical University (grant no. CXTD202111), Projects funded by the central government to guide local scientific and Technological Development (202207AB110006), and Yunnan Chenggong expert workstation (202005AF150034). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Biting midges (Ceratopogonidae) are important vector insects that transmit arboviruses causing serious disease in human or animal, such as Oropouche virus, bluetongue virus (BTV), epizootic haemorrhagic disease virus (EHDV), African horse sickness virus (AHSV), Ibaraki disease virus (IBADV), Schmallenberg virus (SBV), Bovine ephemeral fever virus (BEFV), and vesicular stomatitis virus (VSV) by biting humans, livestock, poultry, and other animals [1–3]. Currently, 1764 species of bloodsucking midge are classified in four genera worldwide, of which Culicoides Latreile (Diptera, Ceratopogonidae) is the largest, including 1399 (79.31%) 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 [9–11]. In the last decade, Liu and Wu et al. described four new species in this subgenus [12–14]. 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 Ι 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 pMDTM19-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.
Results
Taxonomy
Culicoides (Sinocoides) jiangchengensis Wang et Liu sp. Nov, 2023. (Fig 1).
urn:lsid:zoobank.org:act:C059685C-7A56-4CD2-9A67-946CF68C2A67
A: Female; B: Male; C: Head; D: Front; E: Antenna; F: Maxillary palpus; G: Mandibles; H: Wing (Female); I: Wing (Male); J: Thorax; K: Hind tibial comb; L: Spermatheca; M: Hypopygium; N: Parameres.
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 scattered on the surface; a small, approximately oval, fuzzy pale spot at the distal end of r5 cell; m1 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.
Description
Female.
Body (Fig 1A) Small and medium-sized midges. Wing length 1.08 (1.0–1.22) mm, breadth 0.48 mm, CR 0.50 (0.48–0.53, n = 9).
Head (Fig 1C, 1D, 1E, 1F and 1G) Brown. Eyes bare, separated by distance approximately equal to the diameter of one ommatidium, and a transverse seam in the lower forehead. Antenna pedicel slightly pale brown; flagellomeres 1–8 short and stout, flagellomeres 9–13 longer, lengths of flagellomeres in the proportion of 19:19:19:19:19:19:18:19:26:28:34:35:55; AR 1.13 (1.08–1.18, n = 9); sensilla coeloconica on flagellomere 1, 9–13. Palpus five-segmented, lengths in the proportion of 10:20:22:12:13, segment 3 slightly swollen at apical 1/3, without sensory pit, with sensilla scattered on the surface; PR 2.63 (2.50–2.75, n = 9). Three bristles on each side of the frontoclypeus. Proboscis length 150 μm, head height 250 μm, P/H ratio 0.59 (0.53–0.64, n = 9); mandible with 15–17 teeth (n = 9), maxilla with 14 teeth (n = 9).
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 1st radial cell and r-m cross-vein in pale spot, and extension posterior over M1 to connect with narrow pale band of m2; distal 4/5 of 2nd radial cell covered by pale spot; apical pale spot in cell r5 cell faint, and reaching wing margin; m1 with two pale spots, proximal pale spot long band; m2 with two differently shaped pale spots: proximal pale spot long band extending from the base to distal, distal pale spot abutting the wing margin; m4 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 r5 cell and distal of m1, but no on the proximal of m1; hind tibial comb with four spines, 2nd spine longest, metatibial comb (mc) about 18 teeth, TR and F-T of legs are as in Table 1.
Abdomen Light brown. Three subequal-size ovoid spermathecae (Fig 1L), each measuring 25.00×23.00 μm.
Male (Fig 1B) Similar to female with usual sexual differences.
Head The lengths of Antenna flagellomeres in the proportion 26:13:13:14:14:14:12:11:10:9:34:33:38, sensilla coeloconica on flagellomeres 1, 11–13; palpus with segments in proportions of 8:10:11:7:9, PR 1.57 (n = 1); one bristles on each side of the frontoclypeus.
Thorax Wing with pattern of pale spots as in Fig 1B, wing length 0.82 (n = 1), width 0.33 (n = 1); CR 0.63 (n = 1); wings light dark, with eight pale spot; wing costal with three obvious dark spots; basal 1/3 of 1st radial cell and r-m cross-vein in pale spot; distal 2/3 of 2nd 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 9th 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.
Type material.
Holotype female, Qiaotouhe Village, Menglie Town, Jiangcheng County, Yunnan Province, China (22°54′49″N, 101°88′70″E), September 2015, light trap. Male, Maxidang Village, Bangdang Town, Gongshan County, Yunnan Province, China (27°49′54.8328″N, 98°41′46.0536″E), Augest 2017, light trap.
Paratypes: 9 females and 1 male, same data as holotype.
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 3rd 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 3rd 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 3rd palpus segment and the sensilla coeloconica on flagellomere, but there was no pale spot in the wings m1 and m4 of C. nanniwanensis, while the m1 and m4 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 m1 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 m1 and a pale spot at the proximal in the anal cell.
ant, antenna; f, front; map, maxillary palpus; md, mandibles; spt, spermatheca; w, wing.
ant, antenna; f, front; map, maxillary palpus; md, mandibles; spt, spermatheca; w, wing.
ant, antenna; f, front; map, maxillary palpus; md, mandibles; spt, spermatheca; w, wing.
Ant, antenna; f, front; map, maxillary palpus; md, mandibles; spt, spermatheca; w, wing.
ant, antenna; f, front; map, maxillary palpus; md, mandibles; spt, spermatheca; w, wing.
ant, antenna; f, front; map, maxillary palpus; md, mandibles; spt, spermatheca; w, wing.
ant, antenna; f, front; map, maxillary palpus; md, mandibles; spt, spermatheca; w, wing.
ant, antenna; f, front; map, maxillary palpus; md, mandibles; spt, spermatheca; w, wing.
ant, antenna; f, front; map, maxillary palpus; md, mandibles; spt, spermatheca; w, wing.
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%.
The best DNA substitution model is GTR+G+I, and the trees were drawn using njplot 2.4 with the bootstrap value based on 1,000 replications.
Checklist of Sinocoides in China and their geographical distribution
There are 10 species in the subgenus Sinocoides in China, distributed in eight provinces and autonomous regions of China.
- (1) Culicoides (Sinocoides) jiangchengensis Wang et Liu sp. nov. (Fig 1) Type locality: China: Yunnan (Jiangcheng).
Distribution: Yunnan (Jiangcheng, Gongshan).
Culicoides (Sinocoides) anthropophygas Yu et Liu, 2005 [5]:892; Type locality: China: Sichuan (Jiulong).
Distribution: Sichuan (Jiulong).
Culicoides (Sinocoides) hamiensis Chu, Qian et Ma, 1982 [8]:105; Type locality: China: Xinjiang (Hami).
Distribution: Xinjiang (Hami).
Culicoides (Sinocoides) jinghongensis Wu et Liu, 2018 [14]:290; Type locality: China: Yunnan (Jinghong).
Distribution: Yunnan (Jinghong).
Culicoides (Sinocoides) kongmiaoensis Liu et Zhou, 2006 [12]: 467; Type locality: China: Shandong (Qiuhu).
Distribution: Shandong (Qiuhu).
Culicoides (Sinocoides) multifarious Liu, Gong et Zhang, 2003 [11]:359; Type locality: China: Gansu (Tianshui).
Distribution: Gansu (Tianshui).
Culicoides (Sinocoides) opertus Liu et Yu, 1990 [9]:15; Type locality: China: Heilongjiang (Hulin).
Distribution: Heilongjiang (Hulin, Raohe, Suifenhe).
Culicoides (Sinocoides) pungobovis Liu, Yan et Liu, 1996 [10]:35; Type locality: China: Hainan (Qiongzhong).
Distribution: Hainan (Qiongzhong).
Culicoides (Sinocoides) malipoensis Liu et Ren, 2011 [13]:257; Type locality: China: Yunnan (Malipo).
Distribution: Yunnan (Malipo)
Culicoides (Sinocoides) nanniwanensis Liu et Wang, 2011 [13]:257; Type locality: China: Shanxi (Nanniwan).
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 3rd 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 3rdsegment of palpus; 2) sensilla coeloconica on flagellomere 1, 9–13; 3) m1 and m2 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 r3 with a large fuzzy pale spot, m1 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 morphology and molecular biology, increasing the number of Culicoides species in China. Meanwhile, this is the first time to obtain the sequence of Culicoides in subgenus Sinocoides, which provides sequence information for rapid molecular identification and genetic evolution of Culicoides species in China.
Since subgenus Sinocoides was established in China in 1983, females of nine Culicoides species in this subgenus have been recorded in different regions of China. This survey discovered a new Culicoides species in Sinocoides in Yunnan, increasing the species in this subgenus to 10. Based on the literature, a key to the females of 10 Culicoides in subgenus Sinocoides was 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.
An updated key to females in the subgenus Sinocoides of Culicoides
- 1 Eyes hairy, Wing without pale and dark spot (Fig 2)...... C. (S.) anthropophygas
Eyes bare, Wing with pale and dark spot................................................................ 2
- 2 (1) m1,m2,m4 cell with pale......................................................................................... 3
m1,m2,m4 cell without pale.................................................................................... 7
- 3 (2) r5 cell with 2~3 pale spots except the distal pale.................................................. 4
r5 cell with 1 pale spot except the distal pale........................................................ 5
- 4 (3) r5 cell with 3 pale spots except the distal pale, m1 cell with 2 pale spots (Fig 6) .......................................................................... C. (S.) multifarious
r5 cell with 2 pale spots except the distal pale, m1 cell with 3 pale spots (Fig 8)........................................................................... C. (S.) pungobovis
- 5 (3) m2 cell with a pale band and a pale spot................................................................ 6
m2 cell with 4 pale spots (Fig 4)........................................... C. (S.) jinghongensis
- 6 (5) m1 cell with pale band extended from base to the distal, anal cell with a pale spot at the Proximal (Fig 9)...................................................... C. (S.) malipoensis
m1 cell with 2 pale spots, anal cell with a wide pale band extended from the basal to the distal (Fig 1)..................................................... C. (S.) jiangchengensis
- 7(2) sensilla coeloconica on flagellomeres 1~12 or 1~13.............................................. 8
sensilla coeloconica on flagellomeres 1,9~12 or 1,9~13........................................ 9
- 8 (7) sensilla coeloconica on flagellomeres 1~12; mandible 18 teeth (Fig 3)................................................................................................... .. C. (S.) hamiensis
sensilla coeloconica on flagellomeres 1~13; mandible 13 teeth(Fig 5)................................................................................................ C. (S.) kongmiaoensis
- 9 (7) sensilla coeloconica on flagellomeres 1,9~12; m2 and anal cell without pale spot (Fig 7)........................................................................................... C. (S.) opertus
sensilla coeloconica on flagellomeres 1,9~13; m2 and anal cell with a pale spot respectively (Fig 10).............................................................. C. (S.) nanniwanensis
References
- 1. Carpenter S, Groschup MH, Garros C, Felippe-Bauer ML, & Purse BV. Culicoides biting midges, arboviruses and public health in Europe. Antiviral research. 2013,100(1):102–113. pmid:23933421
- 2. Sakkas H, Bozidis P, Franks A, Papadopoulou C. Oropouche fever: A review. Viruses. 2018,10(4):175. pmid:29617280
- 3. Rozo-Lopez P, Londono-Renteria B, Drolet BS. Venereal transmission of vesicular stomatitis virus by Culicoides sonorensis midges. Pathogens. 2020, 9(4):316.
- 4. Borkent A, Dominiak P. Catalog of the biting midges of the world (Diptera: Ceratopogonidae) (Zootaxa). Auckland New Zealand: Magnol Pre. 2020, 4787 (1):1–377.
- 5.
Yu YX, Liu JH, Liu GP. Ceratopogonida of China. Vol. Ⅰ Ⅱ. Beijing: Milit Med Sci Pre. 2005, 43–114,692–802,816–1322.
- 6.
Yang D, Li Z, Liu QF. Species catalogue of China, Vol 2 Animals, Insecta (V). Beijing: Science Press, 2020,119–221.
- 7. Qu FY.Two new subgenera and two new species of Culicoides from China (Diptera: Ceratopogonidae). Entomotaxonoia, 1983, 5(1):25–32.
- 8. Qu FY, Qian JQ, Ma DX. Records of biting midges collected from Xinjiang, China, with descriptions of two new species (Diptera: Ceratopogonidae). Acta Entomology Sinica, 1982, 25(1):105–110.
- 9.
Liu GP, Yu YX. Three new species of blood-sucking in Northeastern China. Contr blood-sucking Dipt. Ins. Shanghai: Shanghai Science Press, 1990,(2):14–18.
- 10.
Liu JH, Yan G, Liu GP. The biting midge from Hainan Island. Beijing: Milit Med Sci Pre, 1996,14–184.
- 11. Liu ZJ, Gong ZW, Zhang JJ, Shi SZ. Two new species of Culicoides (Diptera: Ceratopogonidae). Acta Zootaxonomica Sinica, 2003,28(2):359–361.
- 12. Liu GP, Zhou X. Culicoides (Sinocoides) and A new species from China (Diptera: Ceratopogonidae). Chin J Vector Biol & Control, 2006,17(6):467–469.
- 13. Liu GP, Ren QM, Wang X. Culicoides (Sinocoides) and two new species from China (Diptera: Ceratopogonidae). Chin J Vector Biol & Control, 2011,22(3):257–259.
- 14. Wu CG, Liu GP. Classification of Culicoides (Sinocoides) and a new species from China (Diptera: Ceratopogonidae). Chin J Vector Biol & Control, 2018,29(3):290–292.
- 15. Yuan ZJ, Bai F, Li Zhao, He YW, Meng JX, Li N, et al. () Morphological and molecular biological identification of a new species of Allohelea from Yunnan border area. Chinese Frontier Health Quarantine, 2019,42(1):29–32.
- 16. Dallas JF, Cruickshank RH, Linton YM, Nolan DV, Patakakis M, Braverman Y, et al. Phylogenetic status and matrilineal structure of the biting midge, Culicoides imicola, in Portugal, Rhodes and Israel. Med Vet Entomol, 2003, 17(4): 379–87. pmid:14651651
- 17. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, et al. Clustal W and Clustal X version 2.0. Bioinformatics. Nov 2007,23(21):2947–8. pmid:17846036
- 18. Katoh K, Asimenos G, Toh H. Multiple alignment of DNA sequences with MAFFT. Methods Mol Biol, 2009,537:39–64. pmid:19378139
- 19. Wang G. Molecular identification and Phylogeny of China main mosquito species based on DNA Barcoding. Academy of Military Sciences. 2011, Doctoral Dissertation.
- 20. Monaghan MT, Balke M, Gregory TR, Vogler AP. DNA-based species delineation in tropical beetles using mitochondrial and nuclear markers. Phil. Trams. R. Soc.B, 2005,360: 1925–1933. pmid:16214750
- 21. Nielsen SA, Kristensen M. Delineation of Culicoides species by morphology and barcode exemplified by three new species of the subgenus Culicoides (Diptera: Ceratopogonidae) from Scandinavia. Parasit Vectors, 2015,8:151. pmid:25889579
- 22. Pagès N, Munoz-Munoz F, Talavera S, Sarto V, Lorca C, Nunez JI. Identification of cryptic species of Culicoides (Diptera: Ceratopogonidae) in the subgenus Culicoides and development of species-specific PCR assays based on barcode regions. Vet Parasitol, 2009,165:298–310. pmid:19682796
- 23. Augot D, Hadj-Henni L, Millot C, Lehrter V, Cousinat M, Depaquit J. First report of Culicoides boyi (Diptera: Ceratopogonidae) in France. Ann Société Entomol Fr NS, 2016, 0:1–8.