Research Article |
Corresponding author: Ekgachai Jeratthitikul ( ekgachai.jer@mahidol.edu ) Academic editor: Fredric Govedich
© 2020 Ekgachai Jeratthitikul, Putita Jiranuntskul, Takafumi Nakano, Chirasak Sutcharit, Somsak Panha.
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:
Jeratthitikul E, Jiranuntskul P, Nakano T, Sutcharit C, Panha S (2020) A new species of buffalo leech in the genus Hirudinaria Whitman, 1886 (Arhynchobdellida, Hirudinidae) from Thailand. ZooKeys 933: 1-14. https://doi.org/10.3897/zookeys.933.49314
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Hirudinaria manillensis (Lesson, 1842), commonly known as the buffalo leech, shows a polymorphism of two ventral colorations. The green color morph has a plain green ventral surface and the red color morph has a brick-red ventral surface with two black submarginal stripes. Based on molecular and morphological evidence in the present study, these two color morphs were revealed as two different species. The red color morph fits well with the description of H. manillensis, while the green color morph showed some distinctions, and therefore is described herein as Hirudinaria thailandica Jeratthitikul & Panha, sp. nov. The new species can be distinguished from its congeners by the dark greenish or dark olive ventral surface and a round atrium with ventral insertion of ejaculatory ducts in the male reproductive organ. A phylogenetic tree based on concatenated data of COI and 28S genes supported the new species and further indicated it as a sister species to H. bpling Phillips, 2012.
Hirudinaria manillensis, Hirudinea, molecular phylogeny, new species, taxonomy, Thailand
“Buffalo leech” is a common name of obligatory blood-feeding ectoparasitic leeches in the genus Hirudinaria Whitman, 1886 and Poecilobdella Blanchard, 1893. Both genera belong to the subfamily Hirudinariinae Sawyer, 1986, and are characterized by the presence of a large female vaginal caecum, but are distinguishable from each other by characteristics of the female reproductive system. Poecilobdella species have a well-developed vaginal stalk, whereas Hirudinaria species are considered to lack this structure (
The genus Hirudinaria is widely distributed over tropical South and Southeast Asia (
In Thailand, the first scientific report of buffalo leeches was that of
In the present study, specimens of all valid species of the genus Hirudinaria, including the red and green color morphs of H. manillensis (Fig.
Hirudinaria leeches were collected from freshwater ponds, rice fields, and rivers in many locations in Thailand (Table
Morphological study and species identification of each specimen was based on
The syntypes of Hirudo maculosa Grube, 1868 (now synonymized with H. manillensis) deposited at the Museum für Naturkunde (ZMB: Zoologischen Museum Berlin), Berlin were examined to test whether the specific name maculosa would be resurrected: ZMB 1371, three individuals-the largest syntype (BL 57.95 mm, BW 7.66 mm; CL 8.27 mm, CW 8.66 mm), dissected; the next largest (BL 28.99 mm, BW 3.27 mm; CL 3.15 mm, CW 3.41 mm); and the smallest (BL 19.31 mm, BW 3.45 mm; CL 2.41 mm, CW 2.59 mm).
Locality with geographic coordinates and GenBank accession numbers for specimens used in phylogenetic analysis.
Species | Voucher ID | Locality | Coordinates | COI | 28S |
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Hirudinaria manillensis (Lesson, 1842) | MUMNH-HIR014-01 | Thailand, Nong Khai Province, Phon Phisai District, Swamp near Chum Chang | 17°58.47'N, 103°05.06'E | MN882682 | MN882665 |
MUMNH-HIR015-16 | Thailand, Bueng Kan Province, Seka District, paddy fields near Sang | 17°50.13'N, 103°56.40'E | MN882683 | MN882666 | |
MUMNH-HIR012-01 | Thailand, Phitsanulok Province, Wat Bot District, daddy field near Tho Thae | 16°56.99'N, 100°19.73'E | MN882684 | MN882667 | |
CUMZ 3405 (NK1) | Thailand, Nakhon Phanom Province, Na Wa District, Ban Donsala, Nong Kok | 17°34.45'N, 104°07.31'E | MN882685 | MN882668 | |
CUMZ 3406 (NK2) | Thailand, Nakhon Phanom Province, Si Songkhram District, Ban Don Ma Chang | 17°36.88'N, 104°08.36'E | MN882686 | MN882669 | |
Hirudinaria javanica (Wahlberg, 1856) | MUMNH-HIR013-01 | Thailand, Nakhon Phanom Province, Si Songkhram District, Huai Khon Reservoir | 17°36.93'N, 104°17.57'E | MN882687 | MN882670 |
CUMZ 3422 (MD2) | Thailand, Mukdahan Province, Khamcha-I District, paddy fields near Ban Nonghai | 16°34.89'N, 104°29.48'E | MN882688 | MN882671 | |
CUMZ 3405(NK3) | Thailand, Nakhon Phanom Province, Na Wa District, Ban Donsala, Nong Kok | 17°34.45'N, 104°07.31'E | MN882689 | MN882672 | |
Hirudinaria bpling Phillips, 2012 | MUMNH-HIR016-04 MUMNH-HIR016-05 | Thailand, Phang Nga Province, Kapong District, swamp near 3004 Rd. | 8°46.08'N, 98°27.33'E | MN882690 MN882691 | MN882673 MN882674 |
MUMNH-HIR016-02 | Thailand, Satun Province, Mueang District, pond near Nong Plak Phra Ya | 6°44.48'N, 100°02.47'E | MN882692 | MN882675 | |
Hirudinaria thailandica sp. nov. | MUMNH-HIR008-28 (holotype) | Thailand, Chai Nat Province, Mueang District, Lotus pond near Ban Kluai | 15°10.65'N, 100°08.70'E | MN882693 | MN882676 |
MUMNH-HIR003-08 | Thailand, Phrae Province, Sung Men District, unnamed stream near Ban Kwang | 18°04.26'N, 100°11.26'E | MN882694 | MN882677 | |
MUMNH-HIR009-02 | Thailand, Buriram Province, Krasang District, Chi River at Nong Teng | 14°52.51'N, 103°22.72'E | MN882695 | MN882678 | |
MUMNH-HIR001-01 | Thailand, Mukdahan Province, Wan Yai District Chanot Stream at Wan Yai | 16°43.76'N, 104°43.77'E | MN882696 | MN882679 | |
MUMNH-HIR004-01 | Thailand, Nakhon Phanom Province, Tha Uthen District, Songkhram River | 17°39.10'N, 104°27.85'E | MN882697 | MN882680 | |
MUMNH-HIR010-04 | Thailand Ubon Ratchathani Province, Khemmarat District, Huai Na Muang stream | 16°01.75'N, 105°15.91'E | MN882698 | MN882681 | |
Hirudo verbena Carena, 1820 | – | – | – | HQ691223 a | HQ691219 a |
Aliolimnatis oligodonta (Johansson, 1913) | – | Tanzania | – | GQ368739 b | GQ368762 b |
Poecilobdella nanjingensis Yang, 1996 | KUZ Z1743 | Neidong Forest Trail, Wulai District, New Taipei City, Taiwan | 24°49.80'N, 121°31.92'E | LC145739 c | LC145738 c |
The total genomic DNA was extracted from the body-wall muscles to avoid contamination from any host DNA by using a NucleoSpin Tissue kit (MACHEREY-NAGEL), following the manufacturer’s standard protocol. Fragments of mitochondrial cytochrome c oxidase subunit I (COI) gene and the nuclear 28S ribosomal RNA large subunit gene (28S) were amplified and used to estimate molecular phylogeny. Primers used for polymerase chain reaction (PCR) and sequencing are presented in Table
Sequences of primers used for PCR amplification and sequencing gene fragments in this study. Those marked with ‘*’ were specifically modified for Hirudinaria species (F = Forward, R = Reverse).
Genes | Amplicon length (bp) | Primer name | Primer sequence (5’ to 3’) | Reference |
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COI | ||||
1 | 720 | LCO1490hiru (F) | ATT CTA CTA GTC ATA AAG ATA TTG G | This study |
HCO2198hiru (R) | AAA ATC AAA ATA TAT ACT TCT GGA TG | This study | ||
2 | 805 | LCOinHiru (F) | GAA ATG AGC GAG TCC TTT ATT TG | This study |
HCO-out* (F) | TCT GGA TAG TCT GAA TAT CG |
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28S | ||||
1 | 802 | 28F2-2 (F) | GCA GAA CTG GCG CTG AGG GAT GAA C |
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28R2 (R) | GAG GCT GTK CAC CTT GGA GAC CTG CTG CG |
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2 | 824 | 28F2-3 (F) | ATC GAA AGG GAA TCG GGT TAA TAT TCC |
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28R3 (R) | GAT GAC GAG GCA TTT GGC TAC C |
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Sequences were aligned and edited using ClustalW as implemented in MEGA7 (
Genetic divergences based on the COI sequences were also calculated to depict evolutionary divergence between Hirudinaria species using uncorrected p-distances as implemented in MEGA7 (
Subfamily Hirudinariinae Sawyer, 1986
Genus Hirudinaria Whitman, 1886
Holotype. Thailand • Chai Nat Province, Mueang Chai Nat District, Ban Kluai Subdistrict; 15°10.65'N, 100°08.70'E; 17 Mar. 2018; ASME members leg.; Lotus pond; MUMNH-HIR008-28; dissected (BL 111.70 mm, BW 15.50 mm; CL 12.45 mm, CW 12.85 mm). Paratype. Thailand • 2 specimens; same collection data as for holotype; MUMNH-HIR008-01; dissected (BL 129.15 mm, BW 13.35 mm; CL 11.90 mm, CW 11.95 mm), MUMNH-HIR008-02; dissected (BL 117.25 mm, BW 13.05 mm; CL 11.70 mm, CW 12.30 mm).
Live specimens of Hirudinaria species A dorsal and B ventral views of H. manillensis from Sakon Nakhon Province C dorsal and D ventral views of H. bpling from Satun Province E dorsal and F ventral views of H. thailandica sp. nov. MUMNH-HIR008-28 (holotype) from Chai Nat Province. Scale bar: 10 mm.
Thailand • 25 specimens; same collection data as for holotype; MUMNH-HIR008-03 to HIR008-27 • 1 specimen; Mukdahan Province, Wan Yai District, Wan Yai Subdistrict, Chanot Stream; 16°43.76'N, 104°43.77'E; 1 Apr. 2018; ASME members leg.; MUMNH-HIR001-01 • 1 specimen; Nakhon Phanom Province, Tha Uthen District, Songkhram River; 10 Apr. 2018; E. Jeratthitikul leg.; MUMNH-HIR004-01 • 8 specimens; Phrae Province, Sung Men District, Ban Kwang Subdistrict; 18°04.26'N, 100°11.26'E; 13 May. 2018; Local peoples leg.; MUMNH-HIR003-01 to HIR003-08 • 3 specimens; Buriram Province, Krasang District, Nong Teng Subdistrict, Chi River; 14°52.51'N, 103°22.72'E; 18 Sep. 2018; E. Jeratthitikul and C. Sutcharit leg.; MUMNH-HIR009-01 to HIR009-03 • 4 specimens; Ubon Ratchathani Province, Khemmarat District, Huai Na Muang stream; 16°01.75'N, 105°15.91'E; 12 May. 2018; E. Jeratthitikul leg.; MUMNH-HIR010-01 to HIR010-04.
External morphology of the holotype of Hirudinaria thailandica sp. nov. in preserved state A dorsal and B ventral views of the whole body C dorsal views of somites XIX–XXIII D ventral views of somites X–XV E dorsal and F ventral views of somites I–VIII G dorsal and H ventral views of somites XXIV–XXVII and caudal sucker. Abbreviations: an, anus; fg, female gonopore; mg, male gonopore; np, nephridiopores; and ss, sensillae. Scale bar: 10 mm (A, B), 5 mm (C–H).
In life, dorsal surface dark brown to green. Lateral spots on annuli b2 and b5, black, squared, not prominent, reduced to small spots on anterior somites. Ventral surface green to dark greenish or dark olive. Male gonopore in somite XI b5/b6. Female gonopore in somite XII b5/b6. Gonopores separated by 5 annuli. Atrium of male reproductive organ moderate-sized, bulbous, rather round. Penis sheath short. Ejaculatory ducts ventrally inserted into middle of atrium. Common oviducts open into the female bursa near the insertion point of vaginal caecum to female bursa.
Illustration of reproductive system of the holotype of Hirudinaria thailandica sp. nov. A dorsal view of male and female reproductive organs including positions of ganglia XII and XIII of the ventral nervous system. Lateral views of B male and C female reproductive organs. Scale bar: 2 mm. Abbreviations: atr, male atrium; cod, common oviduct; ej, ejaculatory bulb; ep, epididymis; fg, female gonopore; mg, male gonopore; ov, ovary; ps, penis sheath; ts, testisac. vas, vas deferens; vc, vaginal caecum; and vg, vagina.
Body firm, muscular, with constant width posteriorly. In life, dorsal surface dark brown to green in background color. Dorsal median line black, distinct, somewhat narrow on annuli b1 and b6 of each mid body somite, and very narrow or broken between somites V and VI, VI and VII, and VII and VIII. Two longitudinal, inconspicuous, and broken stripes on each side of median stripe, each stripe present in b2–b5 and absent in b1 and b6 of each mid body somite. Lateral spots on annuli b2 and b5, black, squared, not prominent, reduced to small spots on some anterior somites. Ventral surface green to dark greenish or dark olive, with narrow marginal pale-yellow stripe. Color faded in preservative, except the dorsal median line and other dorsal markers.
Number of annuli 103. Somites I–III uniannulate. Somites IV and V biannulate, (a1+a2)=a3. Somite VI dorsally triannulate, a1=a2<a3; ventrally biannulate, (a1+a2)=a3. Somite VII triannulate, a1=a2<a3. Somite VIII quadrannulate, a1>a2=b5>b6. Somites IX–XXIII quinquannulate, b1=b2=a2=b5=b6. Somite XXIV quadrannulate, b1=b2=a2=a3. Somite XXV being last complete annulus ventrally; triannulate, a1=a2=a3. Somite XXVI biannulate, (a1+a2)>a3. Somite XXVII biannulate, (a1+a2)>a3. Anus on somite XXVII a3. Clitellum between X b5 and XIII a2.
Eyespots 5 pairs, arranged dorsally in parabolic arch: first pair anterior on somite II, remaining eyespots situated laterally; second pair on somite III; third pair on somite IV (a1+a2); fourth pair on somite V (a1+a2); and fifth pair on somite VI a2. Sensillae on annulus a2 of complete somites; 3 bilateral rows dorsally, one proximal to midline, 2 paired distal to midline; and 2 bilateral rows ventrally. Nephridiopores 17 pairs, positioned on ventral surface, situated bilaterally at posterior margin of VIII a1, and at posterior margin of b2 of somites IX to XXIV.
Oral sucker, dorsal lip prominent, with several furrows. One median longitudinal furrow on ventral surface deep, extending from margin of lip to pharynx. Two deep furrows on each side, and one or two shallow furrows between deep furrows. Three jaws in oral cavity, 1 dorsal and 2 ventrolateral. Small salivary papillae circular, placed along denticular ridge. Large salivary papillae circular to ovate; irregularly placed laterally on each jaw; minimum of 30 visible on one side of right ventro-lateral jaw. Teeth on median jaw 105–125 per jaw. Pharynx muscular and tubular reaching to somite IX b2/a2. Crop reaching to somite XIX b5. Crop caeca10 pairs: Intestine tubular, acaecate, extending from somite XIX b6 to somite XXIII. Rectum thin-walled, tubular, extending from somite XXIII to somite XXVII. CL 11.9 mm, 13.0 mm wide, round, with eight rays of sensillae on dorsal surface radiating out from center.
Male gonopore in somite XI b5/b6. Female gonopore in somite XII b5/b6. Gonopores separated by 5 annuli. Nerve cord passing along the left side of reproductive system. Male reproductive system in somites XI and XII. Atrium moderate-sized, bulbous, rather round. Penis sheath short, recurved anteriorly to atrium. Epididymes round, paired, lateral to atrium. Ejaculatory bulbs present, muscular, and thick. Ejaculatory ducts short, ventrally inserted into middle of atrium. Testisacs 11 pairs. Female reproductive system in somites XII and XIII.
Ovaries paired, small, droplet-shaped. Oviduct paired, narrow, short, join to form common oviduct. Common oviduct moderate in length, slightly bent distally, then descending to vagina. Albumin gland at point of intersection of two oviducts and common oviduct. Common oviduct opens into female bursa near the insertion point of vaginal caecum to female bursa. Vaginal caecum elongated, two times as long as wide at widest point. Vaginal caecum anteroventral end connected to female bursa.
The specific name “thailandica” refers to the country in which specimens of the new species were collected.
This species is known from several river basins in Thailand, including the Chao Phraya and the Middle Mekong river basins (Fig.
Phylogenetic trees estimated by ML and BI gave equivalent topologies. Therefore, only a BI tree is shown in Fig.
The average interspecific divergence based on uncorrected p-distances for COI sequences between H. thailandica and other congeners was 9.21% (4.51%–12.12%; Table
Phylogenetic analysis of Hirudinaria species and related taxa A bayesian inference tree based on 2533 bp alignment dataset of nuclear 28S rRNA and mitochondrial COI genes. Numbers at nodes indicate bootstrap values from maximum likelihood (ML) and bpp from Bayesian inference analysis (BI). Nodes with black circles are sufficiently supported by both BI and ML, while nodes with grey circles are supported only by ML B map showing the sampling locations for the specimens used for DNA sequence analysis.
Hirudinaria thailandica belongs in the genus Hirudinaria based on the large vaginal caecum and the lack of a vaginal stalk (
Morphological comparison among the known species of the genus Hirudinaria.
Characters | H. manillensis (Lesson, 1842) | H. thailandica sp. nov. | H. bpling Phillips, 2012 | H. javanica (Wahlberg, 1856) |
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Ventral surface color | brick-red to brown | green to dark greenish or dark olive | dark green | green |
Submarginal stripe on ventral surface | present | absent | absent | absent |
Number of annuli between gonopores | 5 | 5 | 5 | 7 |
Atrium | bulbous | long | bulbous | short |
Ejaculatory ducts | inserted anteroventrally into atrium | inserted medially into atrium | inserted anteroventrally into atrium | inserted laterally into atrium |
Average interspecific genetic divergence (uncorrected p-distance: %±SE) matrix for the 658 bp barcoding region of COI gene between Hirudinaria species (below diagonal) and average intraspecific distances within each taxon (in bold).
Taxa | 1. | 2. | 3. | 4. |
1. Hirudinaria manillensis | 0.66±0.22 | |||
2. Hirudinaria javanica | 10.16 ±1.10 | 0.40±0.20 | ||
3. Hirudinaria bpling | 12.75±1.23 | 11.41±1.11 | 0.50±0.21 | |
4. Hirudinaria thailandica sp. nov. | 12.12±1.18 | 11.01±1.23 | 4.51±0.74 | 0.80±0.23 |
The present new species can be clearly distinguished from the Singaporean Hirudo maculosa by its male ejaculatory ducts reaching to the midventral part of the male atrium, while the latter possesses ejaculatory ducts running toward the anteroventral part of the male atrium. In fact, morphological characteristics of H. maculosa resemble those of the recently described, Hirudinaria bpling, by
Genital characteristics of H. thailandica are most similar to that of H. manillensis. This is the reason that some authors included this species as a variant of H. manillensis (
The authors thank members of Animal Systematics and Molecular Ecology, Mahidol University; and Animal Systematics Research Unit, Chulalongkorn University for collecting samples. We also thank Dr Birger Neuhaus (Museum für Naturkunde) for the loan of the type materials of Hirudo maculosa, Mr. Kongkit Macharoenboon for photography, and Mr David John Anderson for grammar checking. Animal use in this study was approved by the Mahidol University-Institute Animal Care and Use Committee (MU-IACUC) under the approval number MU-IACUC 2018/003. This project was supported by the Center of Excellence on Biodiversity, Thailand (No. BDC-PG4-160021), and also JSPS KAKENHI Grants (No. JP18K14780 and JP17K20064).