Placobdelloides sirikanchanae, Trivalairat, Poramad, Chiangkul, Krittiya & Purivirojkul, Watchariya, 2019

Trivalairat, Poramad, Chiangkul, Krittiya & Purivirojkul, Watchariya, 2019, Placobdelloides sirikanchanae sp. nov., a new species of glossiphoniid leech and a parasite of turtles from lower southern Thailand (Hirudinea, Rhynchobdellida), ZooKeys 882, pp. 1-24 : 1

Placobdelloides sirikanchanae sp. nov. Figures 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9

Material examined.

Holotype (ZMKU-ANN-0006), puddle on rubber plantation, Sadao District, Songkhla Province, Thailand (6°62'57.7"N, 100°41'12.7"E), 21 October 2018. Paratypes (nine individuals, ZMKU-ANN-0007 to 0015), same locality data as the holotype. All collected specimens were kept in 70% alcohol and deposited at the Zoological Museum of Kasetsart University (ZMKU), Department of Zoology, Faculty of Science, Kasetsart University (13°50'53.6"N, 100°33'47.3"E) on 23 November 2018.

Diagnosis.

This species can be recognized from its elongated, narrow body, crimson median dorsal line, rich dark green pigmentation, 13-17 well-developed knob papillae on each annulus, symmetrical dorsal papillae between the left and right body sides, male gonopore on XIa1/a2, female gonopore on XIa3/XIIa1, amorphous salivary glands, smooth surface with random pits inside the anterior sucker, and rugged surface with randomly distributed pits inside the posterior sucker.

Description of holotype.

External morphology. A mature Placobdelloides sirikanchanae sp. nov. (ZMKU-ANN-0006) has an elongated, dorso-ventrally flattened, tri-annulate body ( Figure 2 ). The relaxed body length from the anterior tip to the posterior sucker is 20.83 mm. The widest point of the relaxed body (annuli 35; XV) is 4.21 mm. The cup-shaped anterior sucker diameter is 1.17 mm. The anterior sucker surface is smooth with numerous pits distributed inside ( Figure 3 ; paratype ZMKU-ANN-0009). One pair of dark spherical eyes touch each other on somite III ( Figure 4 ). The entire dorsal surface is quite rough, with 13-17 well-developed knob papillae present on each annulus ( Figure 5 ; paratype ZMKU-ANN-0010). The dorsal papillae present a symmetrical pattern between the left and right sides of the crimson median line. The dorsal color is dark brown to greenish. The numerous respiratory pores are randomly distributed on the dorsal surface. The ventral surface is transparent and smooth. Two gonopores are located around the neck region and separated by two annuli. The male gonopore is situated in a furrow of XIa1/a2, between annuli 23 and 24 ( Figure 6 ; paratype ZMKU-ANN-0009). The female pore lies in a furrow of XIa3/XIIa1, between annuli 25 and 26. The anus opening is on the dorsal furrow anterior to the last annulus (69; XXXIV). The posterior sucker diameter is 2.08 mm. The posterior sucker surface is rough with randomly distributed pits inside ( Figure 7 ; paratype ZMKU-ANN-0009).

Annulation . Somites I-III are uni-annulate, IV and V are biannulate (annuli 4-7), VI-XIV are tri-annulate (annuli 8-34), XV-XVIII are uni-annulate (annuli 35-38), XIX-XXV are tri-annulate (annuli 39-59), XXVI is biannulate (annuli 60-61), and XXVII-XXXIV are uni-annulate (annuli 62-69).

Internal morphology.

Digestive system: A cylindrical slender proboscis resides in a membranous sheath that protrudes through the lip of the posterior subterminal mouth ( Figure 9 ). The proboscis sheath line is on VIa1-Xa2 (annuli 8-21). Amorphous salivary glands are packed on Xa2-XIa3 (annuli 21-25), followed by the esophageal glands on XIa1-XIIa1 (annuli 23-26). Each esophageal gland has a salivary duct that joins it to each side of the esophagus. Seven pairs of crop cecae are on XIIIa2-XXIIIa1 (annuli 30-51) with the last pair on XXIIIa1-XXXI (annuli 51-66) being diverted and extended posteriorly into four post cecae. Four pairs of diverticulated intestine are on XXIIIa1-XXXIII (annuli 51-68). A simple narrow rectum resides on XXVIa2-XXXIV (annuli 61-69) and opens dorsally at the anus in a furrow anterior of the last somite (XXXIV, annulus 69).

Reproductive system. The male gonopore rim is thick and curled. The ejaculatory bulb on XIa2-XIIa2 (annuli 24-27) is an apple-like sac opening into the vas deferens. Two vas deferens extend posteriorly and recurve in front of post ceca anteriorly to connect to the testisacs. Six pairs of ovoid testisacs are present, and each is located in the space between a pair of crop cecae. The female gonopore rim is thinner and smoother than that of the male. The spermatheca is a rectangular sac on XIIa2-XIIIa3 (annuli 27-31), which opens into bifurcated ovisacs.

Variation.

External morphology. The average relaxed body length is 10.77 mm long (range 7.62-40.39 mm, N = 20), and the average relaxed body width at the widest point (annuli 35, XV) is 3.96 mm (range 3.52-4.89 mm, N = 20). The average anterior sucker diameter is 1.08 mm (range 0.93-1.42 mm, N = 20). The average posterior sucker diameter is 1.94 mm (range 1.70-2.60 mm, N = 20), half the size of the maximum body width.

Color in life is uniformly dark brown to greenish, with randomly distributed dark brown, red, yellow, and especially rich dark green pigments. There is a crimson median line present dorsally from the neck region to the posterior sucker ( Figure 8 ). On the margin of the body, brown, dark green and yellow spots are present along the posterior sucker. The ventral surface is transparent.

Reproductive system. The length of the ovisacs depends on the reproductive stage. During the normal, non-reproductive period, ovisacs are present on XIIIa1-XIVa1 (annuli 29-32), but they can extend from XIIIa1 to XXa1 (annuli 29 to 42 (4th pair of crop cecae)) during the gestational period.

Molecular description.

Molecular comparisons based on p -distances among five specimens of P. sirikanchanae sp. nov. from a rubber plantation in the Sadao District, Songkhla Province, Thailand revealed a difference of 2.5-6.2% for 518 nucleotides of COI (GenBank MK282428-MK282432) and 1.3-3.3% for 555 nucleotides of ND1 (GenBank MK282433-MK282437) (see Tables 2 , 3 ). The five specimens of P. sirikanchanae revealed differences based on p -distances of 10.4-27.7% for the COI gene and 5.4-6.9% for ND1 compared to ten specimens of P. siamensis (GenBank AY962449, MH777415-MH777420, MN221458-MN221460 for COI, and AY962462, MH777409-MH777414, XX123456-XX13456 for ND1) collected from Bangkok and Udon Thani Province, Thailand; differences of 19.3-21.7% for the COI gene and 15.1-15.8% for ND1 compared to a specimens of P. multistriatus (GenBank DQ414338 for the COI gene, and DQ414383 for the ND1 gene) collected from Louisiana, USA; and differences of 21.0-23.5% for the COI gene and 15.1-16.0% for ND1 compared to a specimen of P. jaegerskioeldi (GenBank AY692463 for COI, and AY962450 for ND1) collected from Sudan, South Africa. The Bayesian inference and maximum-likelihood trees of the COI and ND1 genes of the glossiphoniid leeches indicated high posterior probabilities and bootstrap support values for divergence between P. sirikanchanae and P. siamensis ( Figure 10 ).

Type host.

Dark-bellied leaf turtles ( Cyclemys enigmatica ).

Additional host.

Asian leaf turtles ( C. dentata ).

Habitat.

Placobdelloides sirikanchanae sp. nov. can be found attached on the shell surface, both the carapace and plastron, of C. dentata and C. enigmatica , which inhabit the bottom of enclosed shallow muddy puddles on rubber plantations. In the rainy season, several puddles will be connected due to an increase in the water level. Numerous small vertebrates are present in these puddles, such as small fishes or tadpoles. In the dry season, the puddles will be disconnected as the shallower waters disappear from evaporation. These aquatic ecosystems usually have low oxygen due to decomposition of leaf litter and nonflowing water.

Laboratory observations.

Ten individuals of P. sirikanchanae sp. nov. were released into a tank with water from the type locality and equipped with an oxygen pump. All ten died almost immediately. The ten remaining specimens survived in a sealed bottle under low dissolved oxygen conditions. No ventilation (undulating movement display) was observed. After three days, they initiated copulation and deposited eggs in the sealed bottles.

Reproduction.

Ten individuals of P. sirikanchanae sp. nov. displayed reproductive activity in a sealed bottle (low oxygen condition). One copulated with another individual for a few hours before they separated. The beginning of gestation was observed inside the ovisacs of both individuals (seen through the ventral surface) 2-3 days after copulation and gestation continued for approximately 3-4 days more before deposition of eggs. Round creamy-colored eggs, approximately 104-115 eggs per individual, were deposited and aggregated inside the transparent membrane beneath the venter groove of the parent ( Figure 11 ). Eggs were incubated for 3-4 days before hatching. Juveniles remained beneath the ventral groove of the parent for 10-15 additional days before leaving the parent and living on their own.

Etymology.

The species is named in honor of Associate Professor Prapaisiri Sirikanchana , the pioneer aquatic parasitologist of Thailand. The following common names, Sirikanchana 's leech (English), Pling Arjan Prapaisiri (Thai), and Sirikanchanas Plattegel (German) are suggested.

Remarks.

Placobdelloides sirikanchanae sp. nov. was distinguished from P. siamensis (based on the original description by Oka (1917) and the re-description by Chiangkul et al. (2018)) based on the following combination of characteristics (Table 4 ): elongated narrow body, smooth anterior sucker surface with numerous pits inside, 13-17 well-developed knob papillae on each annulus, 69 total annuli, dark brown to greenish color when live with a crimson median line, male gonopore between XIa1/a2 (annuli 23 and 24), female gonopore between XIa3/XIIa1 (annuli 25-26), anus opening between the last annulus and the posterior sucker, rough posterior sucker surface with random pits, and 104-115 eggs per clutch. In addition, P. sirikanchanae was found on C. dentata and C. enigmatica , which inhabit the bottom of enclosed shallow muddy puddles on rubber plantations, differing from P. siamensis , in that it is found on Cuora amboinensis , Heosemys annandalii , Malayemys macrocephala , M. subtrijuga , M. khoratensis , and Siebenrockiella crassicollis inhabiting larger, more open ponds.

Figures

Figure 2. Dorsal surface (upper) and ventral surface (lower) of the live holotype of Placobdelloides sirikanchanae sp. nov.

Figure 3. Scanning electron micrograph of anterior sucker of the paratype ZMKU-ANN- 0009 of Placobdelloides sirikanchanae sp. nov. showing smooth surface with numerous pits.

Figure 4. Anterior part of the live holotype of Placobdelloides sirikanchanae sp. nov. (A) Dorsal surface showing eyes touching on somite III (white arrow) and crimson red dorsal median line (yellow arrow), (B) Ventral surface showing rolled anterior lip (yellow arrow) and eyes (white arrow).

Figure 5. Scanning electron micrograph of dorsal surface of the paratype ZMKU-ANN- 0010 of Placobdelloides sirikanchanae sp. nov. A Dorsal surface of the complete body B Dorsal papillae (arrows) C Respiratory pores on dorsal surface (arrows).

Figure 6. Scanning electron micrograph of ventral surface of the paratype ZMKU-ANN- 0009 of Placobdelloides sirikanchanae sp. nov. showing gonopore arrangement. Upper arrow points to the male gonopore and lower arrow to the female gonopore.

Figure 7. Scanning electron micrograph of posterior sucker of the paratype ZMUKU-ANN- 0009 of Placobdelloides sirikanchanae sp. nov. showing rough surface with random pit distribution (white arrows).

Figure 8. Posterior part of live specimens A holotype Placobdelloides sirikanchanae sp. nov. showing dorsal crimson median line (yellow arrow) with numerous scattered dark green pigments. Bubbles are emerging from the respiratory pores (white arrows) B Placobdelloides siamensis (Oka, 1917) from Bangkok, Thailand showing yellow median line (yellow arrow) with numerous scattered yellow pigments.

Figure 9. Internal anatomy of Placobdelloides sirikanchanae sp. nov.

Figure 10. Phylogenetic analysis of the COI-ND 1 genes of glossiphoniid leeches. The upper diagram is from the Bayesian analysis; the lower is from the maximum likelihood analysis.

Figure 11. Two-day old creamy whitish coloured eggs of Placobdelloides sirikanchanae sp. nov. after deposition.