Placobdelloides sirikanchanae sp. nov., a new species of glossiphoniid leech and a parasite of turtles from lower southern Thailand (Hirudinea, Rhynchobdellida)

Abstract Abstract A new species of glossiphoniid leech, Placobdelloides sirikanchanaesp. nov., is reported in the Asian leaf turtle (Cyclemys dentata) and the dark-bellied leaf turtle (C. enigmatica) from Songkhla Province, southern Thailand. The examination of morphological characters revealed that this new species is similar to P. siamensis (Oka, 1917), a common turtle leech species found in Thailand. Placobdelloides sirikanchanaesp. nov. demonstrates distinct morphological characters, with an elongated, narrow body, 13–17 well-developed knob papillae on each annulus, dark brown to greenish dorsal color with a crimson median line, the absence of a scarlet dot, different male and female gonopore distributions, a rough posterior sucker with a random pit distribution, and 104–115 eggs per clutch. The phylogenetic relationships of COI-ND1 genes were clarified and shown to be distinct from those of P. siamensis. Additionally, habitat preferences tended toward low oxygen conditions such as puddles or water patches on rubber plantations.


Introduction
Glossiphoniid leeches are characterized as the only annelids that have parental care behavior by carrying cocoons and juveniles directly on the ventral surface for protection and feeding (Sawyer 1986;Siddall et al. 2005). Placobdelloides Sawyer, 1986 is a genus of jawless leech species in the most diverse family Glossiphoniidae, which are distributed in freshwater habitats on all continents except Antarctica (Grube 1866;Benham 1907;Johansson 1909;Harding 1920Harding , 1924Oka 1925;Ingram 1957;Baugh 1960;Cott 1961;Soós 1969;Mason 1974;Chandra 1977;Oosthuizen 1979;Govedich et al. 2002;Nesemann et al. 2004;McKenna et al. 2005). This genus has a protrusible proboscis for both blood-feeding and tissue meals on vertebrates (Soós 1969;Sawyer 1986;Govedich 2001;Govedich et al. 2002;Tucker et al. 2005). Glossiphoniid leeches can be used as alkalinity stress indicators of their ecosystems and they are also vectors of apicomplexan blood parasites of aquatic vertebrates and are therefore very important in both ecology and the environment (Grantham and Hann 1994;Siddall and Burreson 1994).
This study presents the first report of the use of a combination of morphological and molecular techniques to describe a new leech species that parasitizes Asian leaf turtles, Cyclemys dentata (Gray, 1831) and dark-bellied leaf turtles, C. enigmatica Fritz et al., 2008. This newly discovered turtle leech is here presented along with new information about its identification and geographic distribution in Thailand.

Leech collection and preservation
Leech specimens were collected from two different turtle species at six different collecting sites. Seven leaf turtles (three individuals of C. dentata and four individuals of C. enigmatica) were collected from the bottom of small muddy puddles or patches of approximately 20-30 cm depth in rubber plantations in Sadao District, Songkhla Province (6°62'57.7"N, 100°41'12.7"E) on 21 October 2018. Leeches were removed from the body and shell of each turtle using forceps and then stored in sealed bottles with water from the capture sites to keep them alive. The carapace length was measured for all turtles, after which they were released back into their capture sites when finished.
Leeches were maintained in a glass container (10×12×8 cm 3 ) half full of puddle water and fitted with an oxygen-pumping machine for behavioral study in the laboratory. Afterward, some individuals were preserved in absolute ethanol in a relaxed stage for scanning electron microscopy (SEM) and molecular techniques, while still others were preserved in 70% ethanol in a relaxed stage for identification.

Morphological study
Each specimen was examined for eye number and placement, annulation, digestive system (including the number and structure of gastric ceca), and reproductive system, following Sawyer (1986) under an MVX10 Research Macro Zoom microscope (Olympus) at 250× magnification. For scanning electron microscopy (SEM), leeches were preserved in absolute alcohol, dried using the critical point drying technique (CPD), and coated in gold, and their morphology was studied using a Quanta 450 Scanning Electron Microscope equipped with an Oxford Instrument X-Max (Kruger and Du Preez 2015).

Molecular analysis
The leech specimens in absolute ethanol were sectioned into two equal pieces. The posterior part was used for DNA extraction with TIANamp Genomic DNA Kit (catalog number DP304-02; TIANGEN Biotech (Beijing) Co., Ltd., Beijing) while the anterior part was stored in absolute ethanol to be used later for a DNA sample stock. For the proteinase K treatment step, tissue samples were lysed for two hours at 58°C. The DNA was eluted from the spin column with 200 µl of buffer.

Statistical analysis
The DNA sequences were aligned using ClustalW v. 1.83 (Thompson et al. 1994) and analyzed using MEGA6 v. 6 (Tamura et al. 2013) for maximum likelihood analysis and MrBayes v. 3.1.2 (Ronquist and Huelsenbeck 2003) for Bayesian analysis.
The maximum likelihood analysis consisted of 2000 tree search replicates, with 25 initial GAMMA rate categories and final optimization using four GAMMA shape categories. Bootstrap values were calculated using 2000 pseudoreplicates of the rapid bootstrap algorithm. Bayesian analysis was run for 20 million generations with trees sampled every 100 generations with a general time reversible (GTR) model and GAM-MA distribution of nucleotide rates for all partitions. Burn-in was set to 10%. Bootstrap values ≥70% for maximum likelihood analysis and Bayesian posterior probabilities of ≥95% were considered a priori as being indicators of highly supported nodes (Felsenstein 2004).

Turtle body size and prevalence
In total, six muddy puddles on rubber plantations (6°62'57.7"N, 100°41'12.7"E) were inhabited by two turtle species: Cyclemys dentata and C. enigmatica ( Figure 1). Three individuals of C. dentata had a mean carapace length of 19.20 ± 2.36 cm (minmax: 17.50-21.90 cm), and four individuals of C. enigmatica had a mean carapace length of 24.02 ± 0.66 cm (min-max: 22.7-26.3 cm). Each leaf turtle had 2-3 individuals of Placobdelloides sirikanchanae sp. nov. attached to it. In total, twenty individuals of P. sirikanchanae were removed, mostly from the carapace or plastron surfaces. The turtles at the collecting sites were seen to be predating on small fishes and Rhacophorus tadpoles.
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).
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 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 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 (4 th 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).
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.   (Chiangkul et al. 2018) A dorsal papillae (arrow) B smooth surface with randomly scattered pits (arrows).

Discussion
Placobdelloides sirikanchanae sp. nov. was identified as a new leech species based on morphological and genetic characteristics and was shown to be distinct from other members of its genus. Comparison of P. sirikanchanae with other species of Placobdelloides that parasitize crocodiles and turtles revealed the following: P. bancrofti is distinguished from P. sirikanchanae by having one annulus separating the male and female Table 4. Comparison of morphological characters, egg number per clutch, host, and distribution of Placobdelloides sirikanchanae sp. nov. and P. siamensis (Oka, 1917)  gonopores and an absence of dorsal papillae; P. emydae has a slightly dilated head and three pairs of metameric papillae on the dorsum; P. fimbriata has a unique gill-like marginal fringe; P. multistriata has two pairs of salivary glands and the absence of dorsal papillae; the original description of P. siamensis from the description by Oka (1917) has an elongated oval shape, 22-27 cone papillae, and a different gonopore distribution; P. siamensis based on the description by Chiangkul et al. (2018), has an elongated oval shape, yellow median line, numerous scattered yellow pigments on dorsal, 5-9 welldeveloped rod papillae, a different gonopore distribution, and smooth posterior sucker with random pits (Figures 8, 12, clarified from previous study); and P. stellapapillosa Govedich et al. 2002 has a proboscis opening on the anterior subterminal mouth and unique star-shaped papillae (Oka 1917;Harding and Moore 1927;Best 1931;Sawyer 1986;Govedich et al. 2002;McKenna et al. 2005).
The phylogenies (Fig. 10) obtained in this study revealed the monophyletic relationship of Placobdelloides species that inhabit Thailand. The phylogenetic trees clearly indicated the divergence between P. sirikanchanae and P. siamensis (Bangkok and Udon Thani population) by having a high percentage of differences between the species for both the COI and ND1 gene. However, after several attempts, we were unable to retrieve the topotype of P. siamensis from Pattalung and could not conduct the sequence comparisons, but the morphological characters of P. siamensis from the other localities are clear and easily differentiate it from P. sirikanchanae. According to the phylogenetic analysis, P. sirikanchanae is the sister taxon of P. siamensis (Bangkok population). This is the first report of the reproductive biology of P. sirikanchanae. This hermaphroditic leech displayed monandrous copulation and exchanged pseudospermatophores with other leeches a few hours before separation . The gestational period after copulation through egg deposition was approximately 5-7 days, which began in the ovisacs beginning 2-3 days after copulation. The family Glossiphoniidae is unique in that members of this family exhibit parental care of their eggs and juveniles (Sawyer 1971). Compared to other glossiphoniid leeches, P. sirikanchanae had more eggs per clutch (104-115 eggs per clutch) than Glossiphonia complanata (60 eggs per clutch) or Helobdella stagnalis (Linnaeus, 1758) (50 eggs per clutch) but fewer than P. stellapapillosa (100-200 eggs per clutch) and P. siamensis (173-412 eggs per clutch) (Kutschera and Wirtz 2001;Chiangkul et al. 2018). For the incubation period, P. sirikanchanae had a shorter period from egg deposition through juvenile hatching (3-4 days) compared to H. robusta (9 days and 13 hr) (Weisblat and Huang 2001). For the parental care period, it had a shorter period from egg deposition through separation of juveniles from the parent (13-19 days) than G. complanata (30 days) and H. stagnalis (45-50 days) (Sawyer 1986;Kutschera 1992). Therefore, P. sirikanchanae might currently have the smallest number of eggs per clutch in the genus Placobdelloides and the shortest periods of incubation and parental care in the family Glossiphoniidae. This is the first report of P. sirikanchanae parasitizing Asian leaf turtles (C. dentata) and dark-bellied leaf turtles (C. enigmatica). In the field surveys of this study, both the leech and the turtles inhabited the bottom of enclosed shallow muddy puddles or patches in rubber plantations. Small puddles and patches are a temporary aquatic system that usually occurs after rain and disappears within a few weeks or months from evaporation or seeping into the ground. In addition, this aquatic system usually has low dissolved oxygen conditions from leaf decomposition and the absence of flowing water, but despite this, there were numerous small vertebrates living there, such as fishes and Rhacophorus tadpoles (Shahriza et al. 2010).
For P. sirikanchanae, its small clutch size and faster development times might be an adaptation to living in these temporary ponds. Moreover, the observed behavior in the laboratory combined with water conditions in the field indicated that P. sirikanchanae is a leech that can tolerate low dissolved oxygen conditions.
Cyclemys dentata and C. enigmatica are members of the family Geoemydidae, the main freshwater turtle family found in Thailand, along with Cuora amboinensis, Heosemys annandalii, Malayemys macrocephala, M. subtrijuga, M. khoratensis, and Siebenrockiella crassicollis, all of which are the hosts of P. siamensis (Oka 1917;Chiangkul et al. 2018). However, most host turtles of P. siamensis usually inhabit ponds, lakes, or rivers that have flowing water and differ from the habitats of C. dentata and C. enigmatica (Das 2010;Fritz et al. 2008). Accordingly, the habitat preferences of host turtles also support the identification of Placobdelloides leech parasites in Thailand.