Research Article |
Corresponding author: Zi-Chao Liu ( abclzc@aliyun.com ) Academic editor: Fredric Govedich
© 2022 Hao Wang, Fan-Ming Meng, Si-Jie Jin, Jiang-Wei Gao, Xiang-Rong Tong, Zi-Chao Liu.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Wang H, Meng F-M, Jin S-J, Gao J-W, Tong X-R, Liu Z-C (2022) A new species of medicinal leech in the genus Hirudo Linnaeus, 1758 (Hirudiniformes, Hirudinidae) from Tianjin City, China. ZooKeys 1095: 83-96. https://doi.org/10.3897/zookeys.1095.74071
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Medicinal leeches in the genus Hirudo have been utilized for therapeutic procedures for thousands of years. To date, six known species of Hirudo are widely distributed in different regions of the Eurasian continent. In this study, a new medicinal leech species Hirudo tianjinensis Liu, sp. nov. is described based upon specimens collected from Tianjin City, China. The new species can be distinguished from its congeners by a combination of characters: blackish green dorsum with five continuous yellow longitudinal stripes; six sensillae on dorsal annulus a2 of segments VIII–XXV; greyish green ventrum with irregular bilateral dark brown spots; dorsum and abdomen separated by a pair of pale yellow stripes; front half atrium wrapped by white prostate; apparent albumen gland; epididymis massive in relation to ejaculatory bulb. The phylogenetic tree based upon COI implies a sister relationship to H. nipponia Whitman, 1886. A key to the known species is provided.
Blood-feeding leech, COI, molecular phylogeny, new species, taxonomy
Leeches are carnivorous, hermaphroditic, and wormlike invertebrates with a sucker at each end of their bodies, belonging to the class Hirudinea of phylum Annelida. Approximately 650–680 leech species belonging to four subclasses, five orders, 13 families, and 149 genera have been nominated worldwide (
“Medicinal leech” is a common name referring to a group of aquatic blood-feeding ectoparasitic leech species traditionally employed for treating such a large variety of human diseases as amenorrhea, osteoarthritis, trauma, and blood stasis syndrome (
As a well-known medicinal leech species, H. nipponia has been reported in China for many decades (
Located in North China, Tianjin City is one of the four major municipalities directly under the central Chinese government. It is one of the major areas producing medicinal leeches such as H. nipponia and W. pigra (Whitman, 1884) in China. In a recent collection, a total of 30 medicinal leech specimens differed significantly in morphology from the other Hirudo species collected from Tianjin City. These specimens represent a new species of Hirudo.
On 19 August 2020, a total of 30 specimens described herein was captured in Caobai River, Haogezhuang Town, Baodi District, Tianjin City, China (39°36'40"N, 117°23'13"E). After relaxing in 15% ethanol, leeches were fixed with 95% ethanol for preservation, measurement, dissection, and molecular analysis. All type specimens (one holotype and 16 paratypes) were measured with a digital caliper with an accuracy of 0.1 mm. Six specimens were used for anatomical observation. The female and male reproductive systems were observed after dissecting along dorsal midline and fixing with insect needles on a wax tray. Jaws and teeth inside the anterior sucker were observed after cutting along the ventral midline of the anterior sucker. Morphological traits of the holotype specimen were observed and photographed by a stereomicroscopy with digital camera. To determine the taxonomy, seven specimens of the new species, two specimens of its sister species H. nipponia from Tianjin City (39°28'04"N, 117°28'48"E), and one specimen of the outgroup species Haemadipsa yanyuanensis Liu et Song, 1977 from Yunnan Province (26°02'35"N, 102°49'34"E) were utilized for molecular studies (Fig.
Caudal suckers were removed with a scalpel and immediately ground to powder in liquid nitrogen. The objective of selecting caudal sucker tissue was to avoid contamination from the gut contents of DNA from unknown host blood and various microorganisms. According to the manufacturer’s instructions, genomic DNA was extracted with Universal DNA kit (Mei5bio, China).
Mitochondrial cytochrome c oxidase subunit I (COI) fragments were amplified by polymerase chain reaction (PCR) with primers LCO 1490 and HCO 2198 (
Prior to further analysis, DNA sequences generated here were deposited into the database of GenBank with the following accession numbers: MZ820656–MZ820659 for H. tianjinensis; MZ820661 and MZ820662 for its sister species H. nipponia; MZ820660 for Haemadipsa yanyuanensis as outgroup. In addition, three sequences of Whitmania were also included here for its close relationship with Hirudo, which were reported in previous studies (
Locality with geographic coordinates and GenBank accession numbers of specimens for phylogenetic analysis.
Species | Voucher ID | Locality | Coordinates | GenBank Acc. No. | References |
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H. tianjinensis | 20200251 20200254 20200256 | Tianjin, CN | 39°36'40"N, 117°23'13"E | MZ820656 | This study |
20200252 20200253 | Tianjin, CN | 39°36'40"N, 117°23'13"E | MZ820657 | This study | |
20200255 | Tianjin, CN | 39°36'40"N, 117°23'13"E | MZ820658 | This study | |
20200257 | Tianjin, CN | 39°36'40"N, 117°23'13"E | MZ820659 | This study | |
H. nipponia | 20200301 | Tianjin, CN | 39°28'04"N, 117°28'48"E | MZ820661 | This study |
20200302 | Tianjin, CN | 39°28'04"N, 117°28'48"E | MZ820662 | This study | |
KR | AY763153 | ( |
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KR | AY425450 | ( |
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KR | GQ368749 | ( |
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H. medicinalis | Halle, DE | AY763148 | ( |
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SMNH111543 | Gasavartar, SE | 57°40'52"N, 18°35'35"E | HQ333519 | ( |
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HR20 | Hrast, SI | EF446712 | ( |
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FR | EU100093 | ( |
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Saratov, RU | 51°91'03"N, 47°34'90"E | KU672396 | ( |
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H. orientalis | COIH03 | Urmia, IR | 37°32'51.09"N, 56°22'56.72"E | KY989464 | ( |
Agdam, AZ | AY763154 | ( |
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Samarkand, UZ | EF405599 | ( |
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AZ | GQ368750 | ( |
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H. verbana | Stavropol, RU | 49°01'09"N, 43°48'21"E | KU672397 | ( |
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HV3 | Bursa, TR | 40°10'23"N, 28°37'26"E | KU216244 | ( |
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HV19 | Samsun, TR | 41°34'48"N, 36°04'31"E | KT692947 | ( |
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H2P | Galicia, ES | MT797290 | ( |
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17(1) | Kherson, UA | JN104644 | ( |
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13(1) | Izmir, TR | JN083804 | ( |
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Giessen, DE | EF125043 | ( |
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Ohrid, MK | AY763150 | ( |
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KA46 | Povir, SI | EF446701 | ( |
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H. troctina | Marrakech, MA | AY763155 | ( |
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HT56 | Lebna Dam, TN | JQ364946 | ( |
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H28b | AZ | GQ368751 | ( |
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H. sulukii | HS1 | Adiyaman, TR | 37°59'35"N, 38°48'52"E | KU216239 | ( |
HS2 | Adiyaman, TR | 37°59'35"N, 38°48'52"E | KU216240 | ( |
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HS5 | Gaziantep, TR | 37°18'12"N, 37°14'53"E | KU216241 | ( |
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HS6 | Batman, TR | 37°51'46"N, 41°01'00"E | KU216242 | ( |
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HS7 | Batman, TR | 37°51'46"N, 41°01'00"E | KU216243 | ( |
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W. acranulata | NC023928 | ||||
W. pigra | MN729556 | ||||
W. laevis | Shaanxi, CN | 32°43"N, 108°46"E | KM655839 | ( |
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Haemadipsa yanyuanensis | 20200351 | Yunnan, CN | 26°02'35"N, 102°49'34"E | MZ820660 | This study |
Sequences were aligned and edited using ClustalW implemented in MEGA7 (
Family Hirudinidae Whitman, 1886
Holotype. 20200231; Engineering Research Center for Exploitation & Utilization of Leech Resources in Universities of Yunnan Province, School of Agriculture & Life Sciences, Kunming University, Kunming; Body length 31.2 mm, maximal body width 3.6 mm, width of anterior sucker 1.8 mm, width of posterior sucker 3.3 mm; Caobai River, Haogezhuang Town, Baodi District, Tianjin City, China; 39°36'40"N, 117°23'13"E, 5 m; collected by Zichao Liu, 19 Aug. 2020 (Figs
External morphology of H. tianjinensis holotype A dorsal and B ventral view of whole body C dorsal and D ventral view of somites X–XIV E dorsal and F ventral view of somites I–VIII G dorsal and H ventral view of somites XXIV–XXVII and caudal sucker. Abbreviations: an, anus; e, eye; fg, female gomopore; mg, male gomopore; np, nephridiopores. Scale bars: 5 mm (A, B), 2.5 mm (C–H).
Internal characters of H. tianjinensis A dorsal view of reproductive system B ventral view of dissected anterior sucker C ventral view of jaw. Abbreviations: a, atrium; ag, albumen gland; co, common oviduct; e, epididymis; eb, ejaculatory bulb; g, ganglion; j, jaw; o, ovisac; p, prostate; ps, penis sheath; s, sulcus; te, testisac; to, tooth; v, vagina; vd, vas deferens.
Hirudo tianjinensis can be distinguished from its congeners by the following combination of characters: blackish green dorsum with five continuous yellow longitudinal stripes; six sensillae on dorsal annulus a2 of segments VIII–XXV making dorsal golden midline notched, and two lateral blackish green dorsal line rosary; greyish green ventrum with irregular dark brown spots bilaterally; dorsum and abdomen separated by a pair of pale yellow stripes; front half of atrium wrapped by white prostate; apparent albumen gland; epididymis massive in relation to ejaculatory bulb.
Blood-feeding aquatic leech, medium body size, length 34.8 ± 3.5 mm (n = 17), maximum body width 3.7 ± 0.4 mm, width of anterior sucker 1.8 ± 0.2 mm, width of caudal sucker 3.6 ± 0.4 mm. Caudal sucker diameter slightly narrower than maximal body breadth.
In relaxed state, dorsum and abdomen flat and willow-leaf like. Blackish green dorsum with five continuous yellow stripes. Dorsal midline widest, extending from the first to the last somite. Yellow stripes separate dorsum into six blackish green longitudinal stripes of which the middle two are the widest and the lateral four are narrower. Six sensillae on middle a2 of segments VIII–XXV making middle golden stripe notched and two lateral blackish green stripes rosary. Venter greyish green with irregular dark brown spots bilaterally edged by a pair of pale yellow stripes. No visible sensillae in abdomen. Caudal sucker reddish brown, with dark dorsum and pale-colored abdomen.
Complete somite five-annulate, numbers of annuli per somite: I–III: one, IV–V: two, VI–VII: three, VIII: four, IX–XXIII: five (b1, b2, a2, b5, b6), XXIV: four, XXV: three, XXVI–XXVII: two, in total: 27 somites and 103 annuli.
Wide mouth in white anterior sucker. Jaws trignathous, one in the middle and one on each side, 55–67 horny teeth in each jaw. Five pairs of eyes almost circular or irregular shaped on annulus 2, 3, 4, 6, and 9. Fifth pair of eyes smallest and sometimes difficult to observe. Two nephridiopores in submarginal annulus b2 of each complete somite. Gonopores situated in furrow between annuli, separated by five annuli, male pore in furrow XI b5/b6 (annulus 31/32), female pore in furrow XII b5/b6 (annulus 35/36). Anus in middle dorsum between the last two annuli.
Male reproductive system: pyriform atrium large, located at venter of ganglion in segment XI; prostate white and developed, covering front half of atrium, strikingly different from its sister species H. nipponia whose prostate is almost invisible; penis sheath with a duct bent anteriorly in segment XI; epididymis massive in relation to ejaculatory bulb, tightly packed masses of ducting standing upright on either atrial side; ejaculatory bulb tapering sharply and connected to dorsal epididymis; testisacs ovoid, 11 pairs, in segments XIII–XXIII.
Female reproductive system: composed usually of one vagina, two ovisacs, and a curved duct in segments XII–XIII; vagina long, upright, and ellipsoidal with apparent albumen gland on middle surface, no vaginal duct; ovisacs ovoid, smaller than testisacs, connected to vagina via a curved common oviduct.
This species is frequently confused with H. nipponia due to their morphological similarities and overlapping distributions. Local villagers often mistook it for H. nipponia for therapeutic usage. However, they can be distinguished using a series of morphological characteristics such as color pattern, number of sensillae, and reproductive system morphology (Table
Traits | H. tianjinensis | H. nipponia |
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Sensillae | six sensillae in dorsum, absent in abdomen | six sensillae both in dorsum and abdomen |
Stripes on dorsal surface | five yellow continuous longitudinal stripes | five yellow dotted longitudinal stripes, yellow spots on a2 weak or absent |
Prostate | developed, wrapping front-half of atrium | absent |
Ejaculatory bulb | tapering sharply | tapering gradually |
The specific name tianjinensis is derived from Tianjin City, a municipality directly under the central government in China, where type specimens were collected.
Hirudo tianjinensis was collected from Caobai River, Haogezhuang Town, Baodi District, Tianjin City, China, which flows into Bohai Sea. Water was moderately polluted. Aquatic plants and irregular pumice stones were abundant along the riverbed. This species normally hid under pumice stones. Whenever people and animals pass by, it quickly adsorbs on their ankles for sucking blood.
Phylogenetic analysis was performed based upon available COI sequences of Hirudo from NCBI. Generally, sequences of each species were all well clustered. The results of ML and BI trees were largely similar to each other with the exception of H. sulukii. The newly reported species H. tianjinensis was clustered with H. nipponia and species of Whitmania and formed a single branch separated from the other species. This result was consistent with previous phylogenetic analysis on Hirudo and revealed the Hirudo a paraphyletic group (Fig.
Phylogenetic analysis of H. tianjinensis with other species of Hirudo A maximum-likelihood tree was constructed using MEGA 7 with 1000 ML bootstrap replications B bayesian inference tree was constructed using MrBayes with four Markov chains. Chain length was set as 1,500,000 generations and sampled every 1000 generations.
The average genetic divergence based upon uncorrected p-distances was also performed. In general, the distance of intra-species was < 1.5% except for sequences of H. verbena (range 0.2% to 6.4%). The intraspecific distance of the new species H. tianjinensis ranged from 0.2% to 0.9%. And the interspecific distance between H. tianjinensis and H. nipponia was 16.7% (Table
Interspecific genetic divergence between seven Hirudo species and intraspecific difference within each species based upon analyses of their COI sequences (uncorrected p-distance: %±SD).
Species | Intraspecific range & SD | Interspecific mean ±SD | |||||
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H. tianjinensis | 0.2–0.9±0.2 | ||||||
H. nipponia | 0–0.6±0.4 | 16.7±0.2 | |||||
H. medicinalis | 0–0.6±0.2 | 18.1±0.2 | 19.3±0.2 | ||||
H. orientalis | 0–0.2±0.1 | 16.5±0.1 | 20.7±0.0 | 8±0.2 | |||
H. verbana | 0.2–6.4±2 | 19.3±0.6 | 19.3±0.5 | 9.3±0.5 | 8.7±0.4 | ||
H. troctina | 0–0.9±0.5 | 17.7±0.2 | 18.5±0.1 | 8.9±0.1 | 8.7±0.1 | 9.5±0.5 | |
H. sulukii | 0–1.5±0.6 | 17.2±0.2 | 19.6±0.5 | 9.6±0.4 | 10.6±0.3 | 11.3±0.4 | 11.3±0.3 |
1 | Dorsum with five longitudinal yellow or greenish yellow stripes | 2 |
– | Dorsum with two or four reddish or orange stripes | 3 |
2 | Dorsum with five continuous yellow or greenish yellow stripes; prostate apparent | H. tianjinensis sp. nov. |
– | Dorsum with five yellow dotted stripes; prostate absent | H. nipponia |
3 | Epidimymis massive in relation to ejaculatory bulb | 4 |
– | Epidimymis small/medium-sized in relation to ejaculatory bulb | 6 |
4 | Vagina terminally folded | H. medicinalis |
– | Vagina long and upright | 5 |
5 | Dorsum green, two orange paramedian stripes thin; venter black, irregularly arranged and sized black markings | H. troctina |
– | Dorsum olive green, two orange paramedian stripes thick; venter pale greenish, and covered with small number of irregular black markings | H. sulukii |
6 | Dorsum with broad, pale orange diffuse paramedian stripes; vagina sharply folded; epidimysis small, not much larger than eiaculatory bulb | H. verbana |
– | Dorsum with thin, deep orange colored paramedian stripes; vagina evenly curved; epidimysis medium-sized, somewhat larger than ejaculatory bulb | H. orientalis |
Six species of Hirudo have been identified globally. Except H. nipponia, the other five species are distributed in Europe and the Middle East, usually referred to as the European leeches. Their diagnostic features and species differences were described in detail (
Hirudo tianjinensis has been collected from Tianjin City, a northern Chinese metropolis. It belongs to Hirudo based upon a series of morphological characteristics (
Although most medical leeches can bite people, causing continuous bleeding, inflammation, itching, and even bacterial infection, the major reason for attracting people’s attention lies in their important medical values. In China, medicinal application of leeches was first recorded in Shennong’s Herbal Classic 2,000 years ago. In the modern era, Chinese Pharmacopoeia recommends three leech species, H. nipponia, W. pigra, and W. acranulata (Whitman, 1886) for treating arthritis, stroke, myocardial infarction, and amenorrhea (
The medical leech has important clinical applications since it contains many of bioactive components with the functions of anticoagulant, thrombolytic, matrix degradation analgesic, and anti-inflammatory, such as hirudin, antistasin, and hyaluronidase (
Although the medical leech has important medical value, its taxonomic research is rather behind. Traditional practitioners tend to refer to H. nipponia and H. tianjinensis as the linear and dotted leeches respectively in Tianjin City. In this work, H. tianjinensis is formally named as a new species and its medical value needs to be further studied in the future.
This work was supported by the grants from National Natural Science Foundation of China (31360516), Joint Special Project of Universities in Yunnan Province (2017FH001-004), Yunnan Provincial Training Program of Youth Leaders in Academic & Technical Reserve Talent (2018HB101), and Yunnan Provincial Ten Thousand People Plan.