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Pelodiscus cf. sinensis Wiegmann, 1834 — Non-native; Established. Trionyx (Aspidonectes) sinensis Wiegmann, 1834: 189–195. Lectotype: ZMB 38, designated by Stuckas & Fritz (2011: 337); ZMB 37–39 were recognised as syntypes, but ZMB 37 is apparently lost, according to Fritz et al. (1994: 171). Type locality: “kleinen Insel im Tigerflusse, dicht bei Macao” (= small island in the Tiger River near Macao), China. Chinese Softshell Turtle (Figure 10D) Singapore records. Landemania perocellata — Gray, 1873: 80. Trionyx ? sinensis —K. Lim & F. Lim, 1988a: 28 (Mandai). ? Trionyx sinensis —K. Lim & F. Lim, 1988b: 51 (Upper Bukit Timah Road). Trionyx sinensis —K. Lim, 1989b: 21 (Upper Peirce Reservoir).—L.M. Chou & Lam, 1989: 92.— Corlett, 1992: 418. Pelodiscus sinensis —K. Lim, 1989h: 65 (Kranji Reservoir).—K.K.P. Lim & L.M. Chou, 1990: 56.—Yong, 1990: 27.—K.K.P. Lim & F.L.K. Lim, 1992: 137, 151.—P.K.L. Ng et al., 1993: 20, 21.—L.M. Chou et al., 1994: 105.—R. Subaraj, 1994 (Seletar North Forest [USNF]; Seletar Reservoir).— R.C.H. Teo & Rajathurai, 1997: 393, 394.—Chan-ard et al., 1999: 41.—B.L. Lim & Das, 1999: 52.—Iskandar, 2000: 92.—B.P.L. Goh et al., 2002: 145.— Anonymous, 2003: 92 (Sungei Buloh Wetland Reserve).— Auliya, 2007: 90–91.—T.Y. Goh & O’Riordan, 2007: 98, 99.—Ramsay et al., 2007: 165.—H.T.W. Tan et al., 2007: 193.—N. Baker & K.P. Lim, 2008: 126, 165.—P.K.A. Ng, 2009: 20, 25, 26, 64, 143, 203.— Das, 2010: 177.—T.H. Ng & Lim, 2010: 121 (Jurong Lake).— D.C.J. Yeo & C.S.W. Chia, 2010: 35.—D.C.J. Yeo et al., 2010: 194.—H.T.W. Tan et al., 2010: 251.—P.K.L. Ng et al., 2011: 462.—N. Baker & K.P. Lim, 2012: 126, 165.—K.K.P. Lim, 2014e: 264 (Singapore Botanic Gardens).—D.L. Yong et al., 2014: 203.—H.H. Tan, 2015: 99.—W. Wong, 2017: 64.—W. Cheong, 2020: 104 (River Safari).—TTWG, 2021: 33. Pelodiscus (= Trionyx) sinensis —P.K.L. Ng, 1992a: 141.—P.K.L. Ng, 1992b: 141. Remarks. Pelodiscus sinensis is a species complex native to central and eastern China, Hainan, Taiwan, and northern Vietnam, but has been introduced to at least 16 countries (TTWG 2021, Gong et al. 2018). Found commonly in Singapore’s freshwater bodies throughout the country (Baker & Lim 2012), including the nature reserves (Teo & Rajathurai 1997), P. cf. sinensis was first reported from Singapore by Gray (1873) who examined two specimens collected by Wallace.Assuming that the locality of the Wallace specimens is accurate, these specimens represent an early introduction of P. sinensis into Singapore. These records were not recognised in subsequent publications (e.g., Boulenger 1889), except by Pope (1935) who mentioned that records from “ Singapore, Siam, the Batan Islands and even California” are “highly questionable records”. Present-day specimens of P. sinensis are believed to have been first introduced into Singapore by a farmer who ferried in hatchlings in the 1970s to farm for food (Chou & Jam 1989). Although Singapore has its own native softshell turtles, A. cartilaginea and D. subplana, farmers favour P. sinensis because of its higher fecundity and faster growth rate (Chou & Jam 1989). Thus, individuals in the wild are from escapees and intentional releases (Ng & Lim 2010). The next reported record after Gray (1873), and the first documented field observation of P. cf. sinensis, came 115 years later (Table 2) when a small individual was found hiding in a rusty tin can at Mandai on 28 April 1988 (Lim & Lim 1988a). As P. cf. sinensis is currently recognised as a species complex, and since individuals in Singapore likely originated from multiple locations, Singapore’s population may be composed of multiple species. Occurrence. Ubiquitous. Common. Singapore conservation status. Not Applicable. Conservation priority. None, non-native species. IUCN conservation status. Vulnerable [2000]. LKCNHM & NHMUK Museum specimens. Choa Chu Kang: ZRC.2.2525 (17-Mar-1986), ZRC.2.2526– ZRC.2.2527 (08-Dec-1979), ZRC.2.2549 (20-Jan-1987); Tampines Quarry Lake: ZRC.2.7305 (20-Jul-2014); Upper Seletar Reservoir: ZRC.2.6491 (14-Jun-2007). Additional Singapore museum specimens. Singapore (no locality): NMW. Singapore localities. Choa Chu Kang—Kranji Reservoir—Jurong Lake—Mandai—River Safari—Seletar Reservoir (not specified)—Singapore Botanic Gardens—Sungei Buloh Wetland Reserve—Tampines Quarry Lake—Upper Bukit Timah Road—Upper Peirce Reservoir—Upper Seletar North Forest—Upper Seletar Reservoir. Order Squamata Oppel, 1811 (117 species) Squamata Oppel, 1811: 5, 14. Lizards (40 species) Family Agamidae Gray, 1827 (7 species) Agamidae Gray, 1827: 57 (type genus Agama Daudin, 1802). Genus Aphaniotis Peters, 1864 (1 species) Otocryptis (Aphaniotis) W.C.H. Peters, 1864: 385 (type species: Otocryptis (Aphaniotis) fusca W.C.H. Peters, 1864, by monotypy; gender feminine)., Published as part of Figueroa, Alex, Low, Martyn E. Y. & Lim, Kelvin K. P., 2023, Singapore's herpetofauna: updated and annotated checklist, history, conservation, and distribution, pp. 1-378 in Zootaxa 5287 (1) on pages 85-87, DOI: 10.11646/zootaxa.5287.1.1, http://zenodo.org/record/7960319, {"references":["Fritz, U., Obst, F. & Gnther, R. (1994) Kritischer typen-katalog der schildkr ˆ tensammlung (Reptilia: Testudines) des Zoologischen Museums Berlin. Mitteilungen aus dem Museum f ¸ r Naturkunde in Berlin. Zoologisches Museum und Institut fur Spezielle Zoologie Berlin, 70 (1), 157 - 175. https: // doi. org / 10.1002 / mmnz. 19940700110","Gray, J. E. (1873) Hand-List of the Specimens of Shield Reptiles in the British Museum. Eighth Edition. British Museum (Natural History), London, iv + 124 pp.","Lim, K. & Lim, F. (1988 a) Reptiles. The Pangolin, 1 (2), 27 - 28.","Lim, K. & Lim, F. (1988 b) Reptiles. The Pangolin, 1 (3), 49 - 51.","Chou, L. M. & Lam, T. J. (1989) Introduction of exotic aquatic species in Singapore. In: DeSilva, S. S. (Ed.), Exotic Aquatic Organisms in Asia. Proceedings of the Workshop on Introduction of Exotic Aquatic Organisms in Asia. Asian Fisheries Society Special Publication. No. 3. Asian Fisheries Society, Manila, pp. 91 - 97.","Corlett, R. T. (1992) The ecological transformation of Singapore, 1819 - 1990. Journal of Biogeography, 19 (4), 411 - 420. https: // doi. org / 10.2307 / 2845569","Lim, K. (1989 h) Reptiles, Amphibians and Freshwater Fishes. The Pangolin, 2 (4), 63 - 66.","Lim, K. K. P. & Lim, F. L. K. (1992) A Guide to the Amphibians and Reptiles of Singapore. Singapore Science Centre, Singapore, 160 pp.","Chou L. M., Ng, P. K. L. & Lim, K. K. P. (1994) Animalia. In: Wee, Y. C. & Ng, P. K. L. (Eds.), A First Look at Biodiversity in Singapore. National Council on the Environment, Singapore, pp. 70 - 106.","Lim B. L. & Das, I. (1999) Turtles of Borneo and Peninsular Malaysia. Natural History Publications (Borneo), Kota Kinabalu, Malaysia, 151 pp.","Goh, B. P. L., Sodhi, N. S., Li, D., Ho, S. H. & Ng, P. K. L. (2002) A Guide to Urban Creatures. Singapore Science Centre, Singapore, 160 pp.","Anonymous (2003) Sungei Buloh Wetland Reserve - A Decade of Wetland Conservation. National Parks Board, Singapore, 98 pp.","Auliya, M. (2007) An Identification Guide to the Tortoises and Freshwater Turtles of Brunei Darussalam, Indonesia, Malaysia, Papua New Guinea, Philippines, Singapore, and Timor Leste. TRAFFIC Southeast Asia, s. n., 99 pp.","Goh, T. Y. & O'Riordan, R. M. (2007) Are tortoises and freshwater turtles still traded illegally as pets in Singapore? Oryx, 41 (1), 97 - 100. https: // doi. org / 10.1017 / S 003060530700155 X","Das, I. (2010) A Field Guide to the Reptiles of Thailand and South-East Asia. New Holland, London, 376 pp.","Lim, K. K. P. (2014 e) Chinese softshell turtle at Singapore Botanic Gardens. Singapore Biodiversity Records, 2014, 264.","Cheong, W. (2020) Consumption of a softshell turtle by Malayan water monitors. Singapore Biodiversity Records, 2020, 104 - 105.","Gong, S., Vamberger, M., Auer, M., Praschag, P. & Fritz, U. (2018) Millennium-old farm breeding of Chinese softshell turtles (Pelodiscus spp.) results in massive erosion of biodiversity. Science of Nature, 105, 34. https: // doi. org / 10.1007 / s 00114 - 018 - 1558 - 9","Baker, N. & Lim, K. P. (2012) Wild Animals of Singapore: A Photographic Guide to Mammals, Reptiles, Amphibians and Freshwater Fishes. Updated Edition. Draco Publishing and Distribution Pte Ltd and Nature Society, Singapore, 180 pp.","Gray, J. E. (1827) A synopsis of the genera of saurian reptiles, in which some new genera are indicated, and the others reviewed by actual examination. Philosophical Magazine, or Annals of Chemistry, Mathematics, Astronomy, Natural History, and General Science, 2, 54 - 58. https: // doi. org / 10.1080 / 14786442708675620"]}

Reptilian skull morphology is highly diverse and broadly categorized into three architectural patterns: anapsid, synapsid, and diapsid. The anapsid condition where the temporal region is completely roofed by bones is observed in both basal reptiles and turtles. According to recent phylogenetic studies, turtles are derived from diapsid reptiles, implying that the temporal fenestrae were secondarily closed in turtles. Although the repeated evolution of dermatocranial bone reductions in the skull’s temporal region of the anapsid skull and the biomechanical factors driving the evolution of the anapsid skull in turtles have been actively discussed, its developmental basis for the anapsid skull remains poorly understood. To investigate the developmental mechanisms underlying the morphological evolution of turtle skulls, here we describe the cranial osteogenesis of three cryptodiran turtle species with distinct skull morphologies: Chelonia mydas (Cheloniidae), Mauremys reevesii (Geoemydidae), and Pelodiscus sinensis (Trionychidae). Our interspecific comparison of embryonic cranial osteogenesis suggests that the growth pattern of skull bone elements is presumed to be highly conserved during embryogenesis across cryptodiran turtles. We found that the growth pattern of the five dermatocranial elements that construct the temporal region of the skull (the parietal, postorbital, jugal, quadratojugal, and squamosal) is highly conserved among turtle species, and that the morphological diversity of turtle skulls arises from slight differences in growth patterns. Our study also suggests that interspecific morphological differences in the cheloniid adult skulls may become apparent in the post-hatching growth period rather than during embryogenesis.

Objectives Aeromonads cause severe diseases in farmed aquatic organisms. Herein, we examined 28 isolates causing disease in farmed aquatic organisms from India (n = 24) and Taiwan (n = 4) to gain insight of their genotypic and phenotypic properties. Results API 20NE biochemical phenotyping showed ≥ 90% similarity classifying all isolates as Aeromonas hydrophila. 16S rRNA genotyping showed ≥ 98% homology among all isolates with A. sobria (NR119044.1ATCC), A. veronii (MK990549.1), A. caviae (NR029252.1) and A. hydrophila (MG984625.1ATCC) and other reference strains. In contrast, gyrB showed a higher intraspecies diversity (≥ 96%) than 16S rRNA delineating the 28 isolates into three groups. Group-I consisted of seven Indian isolates clustered with A. sobria (MK484163.1ATCC), group-II comprised of five Indian and two Taiwanese isolates clustered with A. veronii AF417626.1ATCC while group-III had 11 Indian and three Taiwanese isolates grouped with A. hydrophila (AY987520.1 and DQ519366.1) reference strains. None of our isolates clustered with A. caviae (AJ868400.1ATCC) reference strain. These findings suggest that A. sobria, A. veronii and A. hydrophila could be the etiological agents of diseases observed in farmed fish and soft-shelled turtles (Pelodiscus sinensis) examined in this study. Overall, our findings accentuate the importance of combining phenotyping with genotyping for correct taxonomic classification of Aeromonas spp. in Aquaculture.

The aim of this study was to provide a baseline assessment of the turtle community in the coastal wetlands of the Greater Toronto Area. We documented turtle species diversity, abundance, reproductive classes, sex-ratios, and evidence of inter-wetland movement. Our study consisted of a series of mark-recapture surveys across eleven Lake Ontario coastal wetland complexes of the Greater Toronto Area performed between 2016 and 2019. We captured and marked 532 individual turtles of four native species (298 midland painted, Chrysemys picta marginata; 180 snapping, Chelydra serpentina; 7 Blanding’s, Emydoidea blandingii, and 5 map, Graptemys geographica) and three non-native species (40 red-eared slider, Trachemys scripta elegans; 1 false map, Graptemys pseudogeographica, and 1 Chinese softshell, Pelodiscus sinensis). Of note was the capture of an exceptionally large male snapping turtle, one of the largest recorded in Canada for both length and mass. The age classes of both snapping and midland painted species presented large proportions of breeding-sized adults, yet midland painted turtles showed a potential low recruitment with an underrepresentation of non-reproductive females. The sex ratios of both midland painted and snapping turtles across the whole waterfront did not differ from the expected 1:1 ratio. We also recaptured 198 turtles (135 midland painted, 53 snapping, 6 Blanding’s and 12 red-eared Sliders). The recaptured turtles revealed inter-wetland movements of 12 km over a two-year span for a midland painted turtle and an 8 km journey for a snapping turtle, potentially demonstrating some connectivity between geographically separate wetland complexes.

Pelodiscus shipian sp. nov. (Figs. 4–6) Suggested English name: Chinese stone slab soft-shelled turtle Suggested Chinese name: 石片Dz (shi pian bie) Holotype. Jinan University, Guangzhou: JNU 20190011, adult female preserved in alcohol, Liaohe River, Fengxin County, Jiangxi Province, China (28°43’N, 115°25’E), leg. Shiping Gong, 18 June 2019, European Nucleotide Archive (ENA) accession numbers for DNA sequences: cyt b — OW235712, P26S4— OW235765, R35 — OW237755. Paratypes. Jinan University, Guangzhou: JNU 20190001–20190010, ten adult males preserved in alcohol, same data as the holotype; JNU 20190012–20190017, six adult females preserved in alcohol, same data as the holotype; JNU 20190019–20190022, four adult females preserved in alcohol, same data as the holotype; JNU 20190023, adult male preserved in alcohol, same data as the holotype; JNU 20190024, adult female preserved in alcohol, same data as the holotype; JNU 20210002, 20210003, two adult females preserved in alcohol, Shaoyang County, Hunan Province, China, leg. Shiping Gong, 7 May 2021; JNU 20210004, adult male preserved in alcohol, Taoyuan County, Hunan Province, China, leg. Shiping Gong, 7 May 2021; JNU 20210005, adult male preserved in alcohol, Yongzhou City, Hunan Province, China, leg. Shiping Gong, 7 May 2021; JNU 20210006 adult female preserved in alcohol, Yongzhou City, Hunan Province, China, leg. Shiping Gong, 7 May 2021. ENA accession numbers of the paratypes are for cyt b sequences: OW235702 – OW235711, OW235713 – OW235724, OW235726 – OW235730; for P26S4 sequences: OW235762 – OW235764, OW235766 – OW235772, OW235774, OW235775; for R35 sequences: OW237747 – OW237754, OW237756, OW237757, OW237760 – OW237762; and for TB01 sequences: OW235731 – OW235753, OW235756 – OW235761. Diagnosis. Besides its genetic distinctiveness (Gong et al. 2018), Pelodiscus shipian sp. nov. differs from its congeners by the following combination of characters: (1) small adult size, 90°. Pelodiscus shipian sp. nov. cannot be confused with any other species of Pelodiscus except for P. axenaria and, perhaps, P. huangshanensis, its closest relatives constituting the P. axenaria complex, which both have a dark chin and throat finely stippled with black or an indistinct paler colour. The remaining species (P. maackii, P. parviformis, P. sinensis, and P. variegatus) have a light grey chin and throat with white spots or larger white markings that are either or not edged with a darker colour. For additional features see Farkas et al. (2019: Table 4). Pelodiscus shipian sp. nov. differs from P. axenaria by having (1) a smaller adult size of 15 CL); (2) no plastral markings except, rarely, relatively small fuzzy-edged blotches behind the axillae and a slight black suffusion on the forelobe (vs. a dark grey central blotch usually present and blotches behind the axillae extending to the entoplastron); (3) usually unmarked inner thighs (vs. thighs usually carrying well-defined, rarely indistinct, black markings on either side of the tail); and (4) a head and snout with numerous black splotches but without clearly discernible pre-, sub- and postocular stripes (vs. a head with fine black specks and streaks and thin, incomplete pre-, sub- and postocular stripes). Pelodiscus shipian sp. nov. differs from P. huangshanensis by having (1) a larger adult size of> 12 cm CL (vs. In the cyt b gene, a frequently used marker for estimating interspecific divergence in turtles (e.g., Kindler et al. 2012; Iverson et al. 2013), P. shipian sp. nov. differs from other Pelodiscus species on average by 5.30–7.53% (Table 1). These values resemble those as observed between species of another softshell turtle genus (Nilssonia; Praschag et al. 2007). Description of the holotype. Carapace length (CL) 100.5 mm, carapace width (CW) 88.1 mm, plastron length (PL) 74.4 mm, head width (HW) 19.8 mm, eye diameter 6.6 mm, interorbital distance 4.1 mm, snout length (SL) 8.9 mm. Carapace oval, slightly domed but with a strong median keel, widest at level of the posterior buttress spurs of the hypoplastra. Marginal ridge low, central tubercle indistinct. Dorsal surface roughened by longitudinal ridging and smaller protuberances spread over the leathery margin. The olive clay-coloured carapace is adorned with an extremely complex greenish black pattern consisting of reticulations and stellate spots, some enclosed by incomplete rings of the same colour, that give it a marbled appearance. Even though the reticulations extend roughly from the outer (pleural) sutures of the neurals laterally and posteriorly to the periphery of the shell, the two halves of the “saddle” are interconnected by several blotches across the vertebral line. Black stellate spots are scattered over the carapace but are well camouflaged in their heavily patterned surroundings. Entoplastron boomerang-shaped, the amount of bending of the transverse bar between the two posteriolaterally directed rami>90°. Callosities are small and present on the ento-, hyo-hypo- and xiphiplastra. Ventral surfaces are yellowish white suffused with cream yellow on the extremities and the leathery margin. Bridge and underside of leathery margin bear some vague bruise-like grey marks that are best defined along the shell borders. Fore- and hindfeet are well-webbed, having five digits each, with claws on the first three digits only. Each forelimb with four antebranchial scales, three of them free-edged. Each hindlimb with two horny scales, one smooth on the posterodorsal surface while the other, which is free-edged, is located on the posteroventral surface. Tail short, barely extending beyond the rear margin of the carapace and indicating the female gender of the specimen. Head extended to posterior level of eyes, terminating in flexible snout. Jaws closed, each covered by fleshy lips except anteriorly where the horny beaks are exposed. Top of head and snout with black splotches on an olive clay-coloured ground. Pre-, sub- and postocular stripes are thick but incomplete, not well discernible among the other markings. The chin is grey brown with pale stipples, the throat dark grey, finely spotted with black; lips are olive clay-coloured with yellow and black dappling. Variation. CL ranges from 74.1 mm to 103.2 mm, PL from 54.6 mm to 76.5 mm in our type series consisting of 28 specimens. Measurements are given in Table 2. Males have a proportionally bulkier head (PL/HW = 3.37–3.99, mean = 3.60 in males, 3.18–4.20, mean = 3.77 in females; HW/SL = 1.80–2.30, mean = 2.00 in males, 1.69–2.44, mean = 1.95 in females) as well as a much longer and thicker tail. Sexual dimorphism is clearly apparent at a PL of 54.6 mm (JNU 20190010) or possibly even earlier. However, it is presently unknown at what size sexual maturity is reached in this species. According to our data, the two genders may attain similar sizes in Pelodiscus shipian sp. nov., unlike in P. axenaria, in which males seem to grow larger than females (Zhou et al. 1991; S. Gong pers. obs.). Pattern intensity varies considerably in Pelodiscus shipian sp. nov. Whereas the carapace of some individuals, such as the holotype (JNU 20190011; 74.4 mm PL) and one paratype (JNU 20190013; 64 mm PL), both females, has a decidedly marbled appearance, the pattern bears a more striking resemblance to leaf camouflage with more conspicuous stellate spots in others (e.g., paratypes JNU 20190001, 20190007, males; JNU 20190016, 20190017, 20210006, females) or is almost imperceptible due to a general darkening of the ground colour (e.g., JNU 20190002, 20190004, 20190008, 20190010, males; JNU 201900012, 20190019, females); see Figure 6 for examples. The plastron is typically unmarked except for some colour suffusion resembling bruising along its anterior perimeter and relatively small fuzzy-edged blotches behind the axillae. However, these markings may occasionally extend medially, covering much of the plastral forelobe but never actually contacting the entoplastron (JNU 201900005, male; JNU 201900012, 201900017, both females). The black markings on either side of the tail typical for P. axenaria are absent in P. shipian sp. nov., but melanistic specimens may display some dark pigmentation on their thighs (e.g., JNU 201900005, male; JNU 201900012, 20210006, both females). Two examples are shown in Figure 6. The lateral bands on the neck are bright yellow in smaller specimens, particularly males (e.g., JNU 201900010), and lose definition with increasing age/size but usually remain discernible in both sexes. Distribution. The exact geographical range of Pelodiscus shipian sp. nov. is unknown at present. Apart from the type locality, the Liaohe River in Fengxin County, Jiangxi, specimens have been obtained in the counties of Shaoyang and Taoyuan and the City of Yongzhou (Hunan Province), suggesting sympatry or confusion with P. axenaria. Originally, the distribution range of P. axenaria was believed to encompass the counties and cities of Taoyuan, Pingjiang, Rucheng, Lingling, and Shaoyang in Hunan Province, China (Zhou et al. 1991). However, all specimens that we could obtain from Taoyuan and Shaoyang Counties are P. shipian (see our paratype series); our neotype of P. axenaria comes from Tongdao County in Hunan Province. The records from Zhou et al. (1991) were not genetically verified, so that we cannot exclude that some of them referred to P. shipian. However, genetically verified records of P. axenaria (Chen et al. 2005, 2006; Yang et al. 2011; Gong et al. 2018) are known from Shaoguan (Guangdong), Longsheng and Quanzhou (Guangxi) and from Changde and Anren (Hunan). This suggests that P. axenaria and P. shipian at least occur in closest proximity, if not in sympatry, in Hunan (Fig. 7). Another small-bodied Pelodiscus species, P. parviformis, was described from Guangxi and Hunan (Tang 1997). Pelodiscus parviformis is morphologically and genetically distinctive (Gong et al. 2018; Farkas et al. 2019). Nevertheless, it is possible that some genetically unconfirmed records for P. parviformis refer to P. axenaria or P. shipian. Our map (Fig. 7) collates for the P. axenaria complex the currently known genetically verified records. Etymology. The name ‘ shipian’ is the romanisation of 石片 (shi pian, Chinese for “slab”), derived from the local (Fengxin, Jiangxi) name of the species 石片Dz (shi pian bie = stone slab soft-shelled turtle) that alludes to its resemblance to a flat stone. The suggested English common name refers to the same feature. The scientific species name is used as a noun in the nominative singular in apposition to the generic name (ICZN 1999: Article 11.9.1.2).

Interdigital cell death is an important mechanism employed by amniotes to shape their limbs; inhibiting this process leads to the formation of webbed fingers, as seen in bats and ducks. The Chinese softshell turtle Pelodiscus sinensis (Reptilia: Testudines: Trionychidae) has a distinctive limb morphology: the anterior side of the limbs has partially webbed fingers with claw‐like protrusions, while the posterior fingers are completely enclosed in webbings. Here, P. sinensis embryos were investigated to gain insights on the evolution of limb‐shaping mechanisms in amniotes. We found cell death and cell senescence in their interdigital webbings. Spatial or temporal modulation of these processes were correlated with the appearance of indentations in the webbings, but not a complete regression of this tissue. No differences in interdigital cell proliferation were found. In subsequent stages, differential growth of the finger cartilages led to a major difference in limb shape. While no asymmetry in bone morphogenetic protein signaling was evident during interdigital cell death stages, some components of this pathway were expressed exclusively in the clawed digit tips, which also had earlier ossification. In addition, a delay and/or truncation in the chondrogenesis of the posterior digits was found in comparison with the anterior digits of P. sinensis, and also when compared with the previously published pattern of digit skeletogenesis of turtles without posterior webbings. In conclusion, modulation of cell death, as well as a heterochrony in digit chondrogenesis, may contribute to the formation of the unique limbs of the Chinese softshell turtles., We show that multiple mechanisms regulate the unique limb shape of three‐clawed Chinese softshell turtle (Pelodiscus sinensis). Cell death and senescence appear to play a minor role in reducing the partial webbings in the anterior side. Delayed chondrogenesis/ossification and truncated tips are found in posterior digits. High expression of Bmps and Msx1 in the anterior clawed digits tips is correlated with faster ossification., Highlights Cell death and senescence shape the interdigital webbings of Pelodiscus sinensis.Delayed chondrogenesis/ossification and truncated tips are found in posterior digits, as well as differential expression of bone morphogenetic proteins and Msh homeobox 1 transcription factors.

Tumor necrosis factor receptor (TNFR)-associated factors (TRAFs) are a family of crucial signaling molecules that mediate the signal transduction of various immune signaling pathways. Extensive studies have demonstrated that TRAFs play vital roles in regulating cellular immune responses. However, the biological functions and expression profiling of TRAFs in Chinese soft-shelled turtle (Pelodiscus sinensis) remain unclear. In this study, the genes of the PsTRAF family at the genome-wide level were identified in P. sinensis, revealing six PsTRAF members that contained the conserved TRAF domain in the C-terminal regions. Molecular evolutionary analysis showed that PsTRAFs shared close evolutionary relationships and similar protein crystal structures with the TRAF homologs from other turtles, indicating the evolutionary conservation of PsTRAFs. Further expression analysis revealed the tissue-specific expression of PsTRAF genes. Obvious variations in the expression of PsTRAF genes were observed in the spleen in response to Aeromonas hydrophila infection. Three PsTRAF genes, PsTRAF2, PsTRAF3, and PsTRAF6, were significantly upregulated at the mRNA and protein levels post-infection, indicating their potential function in the immune response. Moreover, the protein-protein associations of PsTRAFs with several signaling receptors were predicted in P. sinensis. These results provide a basis for the investigation of the functional roles of PsTRAFs in immune defense against bacterial infection.

Chinese soft-shelled turtle (Pelodiscus sinensis) adults exhibit sexual dimorphism, but sex identification during early life stages (hatchlings and juveniles) is difficult because of the ab...

HIGHLIGHTSArtificial sunlight with the main components of UVB, UVA, and visible light was used to investigate the influence of photoperiod on large-scale farming of juvenile Chinese soft-shelled turtles in a greenhouse.The effects of photoperiod on digestive enzyme activity, stress, and oxidative status of juvenile Chinese soft-shelled turtles were revealed.The optimal photoperiod for juvenile Chinese soft-shelled turtles reared in a greenhouse was 0 h light:24 h dark.Abstract. To improve light conditions for soft-shelled turtles in a greenhouse, the effects of photoperiod on growth, digestive enzyme activity, stress, and oxidative status of juvenile Chinese soft-shelled turtles were investigated. In this study, juvenile Chinese soft-shelled turtles were reared under three photoperiods (hours light:hours dark), i.e., 0L:24D, 6L:18D, and 12L:12D) for four months. Results demonstrated that differences in growth were insignificant during the experiment, but the growth parameters of final weight, weight gain rate (WGR), and feed conversation ratio (FCR) were better at 0L:24D, and the survival rate was significantly higher at 0L:24D than at 12L:12D. Regarding digestive function, serum trypsin activity was significantly higher at 0L:24D than at 6L:18D, while lipase activity was significantly higher at 0L:24D than at 12L:12D. For the stress response parameters, serum cortisol level was significantly lower in turtles exposed to 0L:24D compared to the other treatments. With regard to antioxidant enzyme activities, the total antioxidant capacity (T-AOC) was significantly lower at 0L:24D compared to the other treatments, while superoxide dismutase (SOD) activity was significantly higher in turtles exposed to 0L:24D. The activity of glutathione peroxidase (GSH-PX) was significantly lower at 0L:24D than at 12L:12D. The malonaldehyde (MDA) activity was a little lower at 0L:24D than at 6L:18D; however, it significantly decreased in turtles exposed to 12L:12D. On the whole, the results indicated that the photoperiod treatments had no significant effects on the growth of juvenile Chinese soft-shelled turtles, while extended light exposure may cause physiological stress in turtles. Considering the survival rate, digestive function, cortisol level, and oxidative status, it is suggested that juvenile soft-shelled turtles should be reared under a 0L:24D photoperiod. Keywords: Chinese soft-shelled turtle, Digestive enzyme activity, Growth, Oxidative status, Photoperiod, Stress.

Pelodiscus parviformis Tang 1997: 13 Holotype: GXUA 95012., Published as part of Iverson, John B., 2022, A review of Chelonian type specimens (order Testudines), pp. 1-85 in Megataxa 7 (1) on page 65, DOI: 10.11646/megataxa.7.1.1, http://zenodo.org/record/5964441

Pelodiscus sinensis (Wiegmann 1835:189) Syntypes (3; Fritz et al. 1994:171): ZMB 38 and 39 (extant) and ZMB 37 (lost); ZMB 38 designated lectotype by Stuckas & Fritz (2011:336–37, and photographed and sampled genetically therein). The type of Testudo rostrata (Thunberg, 1787:179) in the UPSZTY (= UUZM) 128) is not Amyda cartilaginea as usually recorded (e.g., Wermuth & Mertens, 1977:107), but rather Pelodiscus sinensis (Webb 1985:84; see also Deraniyagala 1961). The International Commission on Zoological Nomenclature was petitioned (Case 2693; Webb, 1990) to suppress the earlier name and conserve Wiegmann’s name; conservation of the latter was mandated by ICZN Opinion 1659 (in 1991). Testudo rostrata Thunberg 1787:179 Holotype: UPSZTY 128 (formerly UUZM 128 and UUZM 2); Thunberg (1828:49), Lönnberg (1896), Webb (1985, 1990), and Wallin (2001). See comment under Pelodiscus sinensis. Testudo semimembranacea Herman 1804: 219 Holotype: Not located; possibly ZMUS. Trionyx japonica Temminck & Schlegel 1838:139 Syntypes (number uncertain): only two locatable; RMNH 3259 and 3264 (Hoogmoed et al. 2010:22; illustrated in the original description; Pl. VII, Figs. 1–2, and Pl. V, Fig. 7); RMNH 3264 photographed in Hoogmoed et al. (2010:22). Trionyx tuberculatus Cantor 1842:482 Syntypes (3): BMNH 1946.1.22.87-89. Tyrse perocellata Gray 1844:48 Syntypes (3): BMNH 1946.1.22.87-89 (same as Trionyx tuberculatus). Trionyx schlegelii Brandt 1857:111 Holotype: Not located; possibly BMNH. Landemania irrorata Gray 1869:216 Holotype: BMNH 1946.1.22.9 (formerly 1862.11.1.299). Gymnopus simonii David 1875:214 Holotype: MNHN 835; Bour et al. (1995:88). Psilognathus laevis Heude 1880:24 Syntypes: (number uncertain): Not located; illustrated in the original description (Pl. II). Temnognathus mordax Heude 1880:26 Syntypes: (number uncertain): Not located; illustrated in the original description (Pl. III). Gomphopelta officinae Heude 1880:27 Syntypes: (number uncertain): Not located; illustrated in the original description (Pl. IV). Coelognathus novemcostatus Heude 1880: 29 Syntypes: (number uncertain): Not located; illustrated in the original description (Pl. V). Tortisternum novemcostatum Heude 1880: 31 Syntypes: (number uncertain): Not located; illustrated in the original description (Pl. VI). Ceramopelta latirostris Heude 1880: 33 Syntypes: (number uncertain): Not located; illustrated in the original description (Pl. VII). Coptopelta septemcostata Heude 1880: 35 Syntypes: (number uncertain): Not located; illustrated in the original description (Pl. VIII). Cincisternum bicinctum Heude 1880: 37 Syntypes: (number uncertain): Not located; illustrated in the original description (Pl. IX). Trionyx cartilaginous newtoni Ferreira 1897: 114 Holotype: Not located; possibly MUHNAC. Amyda schlegelii haseri Pavlov 1932:27 Holotype: TNZ unnumbered. Amyda schlegelii licenti Pavlov 1932:28 Holotype: TNZ 14. Amyda schlegelii laoshanica Pavlov 1933: 3 Holotype: TNZ 638. Pelodiscus variegatus Farkas, Ziegler, Pham, Ong, & Fritz 2019:72 Holotype: IEBR (Hanoi) 4480 (photographed in the original description, p. 73, Fig. 1); paratypes (14),AMNH 30125 (photographed in the original description, p. 80, Fig. 4d; HNHM 2018.111.1 (photographed in the original description, p. 76, Fig. 3a), and 2018.112.1; MTD 42534, 42834 (photographed in the original description, p. 74, Fig. 2b, d), and 44045 (photographed in the original description, p. 74, Fig. 2a, c); NMW 30221:1-6; VUM AQT001 - HTVN2018 (photographed in the original description, p. 76, Fig. 3b, and p. 80, Fig. 4a); ZFMK 101820., Published as part of Iverson, John B., 2022, A review of Chelonian type specimens (order Testudines), pp. 1-85 in Megataxa 7 (1) on pages 65-66, DOI: 10.11646/megataxa.7.1.1, http://zenodo.org/record/5964441, {"references":["Fritz, U., Obst, F. & Gunther, R. (1994) Kritischer Typen- Katalog der Schildkrotensammlung (Reptilia: Testudines) des Zoologischen Museums Berlin. Mitteilungen aus dem Museum fur Naturkunde in Berlin. Zoologisches Museum und Institut fur Spezielle Zoologie Berlin, 70 (1), 157 - 175. https: // doi. org / 10.1002 / mmnz. 19940700110","Stuckas, H. & Fritz, U. (2011) Identity of Pelodiscus sinensis revealed by DNA sequences of an approximately 180 - yearold type specimen and a taxonomic reappraisal of Pelodiscus species (Testudines: Trionychidae). Zoological Systematics and Evolution Research, 49, 335 - 339. https: // doi. org / 10.1111 / j. 1439 - 0469.2011.00632. x","Thunberg, C. P. (1787) Beskrifning pa trenne skold-paddor. [Descriptionofthreeturtles]. KongligaVetenskapsAcademiens Nya Handlingar, Stockholm, (2) 8, 178 - 180. [in Swedish]","Wermuth, H. & Mertens, R. (1977) Liste der rezenten Amphibian und Reptilien, Testudines, Crocodylia, Rhynchocephalia. Das Tierreich, 100, 1 - 174.","Webb, R. G. (1985) Taxonomic status of Testudo rostrata Thunberg, 1782 (Testudines, Trionychidae). Herpetologica, 41 (1), 84 - 88.","Deraniyagala, P. E. P. (1961) Some of Linne's reptiles and a mammal from Ceylon and elsewhere that are in Sweden. Spolia Zeylanica, 29, 195.","Webb, R. G. (1990) Trionyx sinensis Wiegmann, 1834 (Reptilia, Testudines): proposed conservation of the specific name. Bulletin of Zoological Nomenclature, 47 (2), 122 - 123. https: // doi. org / 10.5962 / bhl. part. 2677","Thunberg, C. P. (1828) General-Catalogue pa Kongl. Akademiens i Upsala, Natural-Samlingar. Unpublished manuscript, Uppsala University Zoological Museum, 311 pp. [in Swedish]","Lonnberg, E. (1896) Linnean type-specimens of birds, reptiles, batrachians and fishes in the Zoological Museum of the R. University in Upsala. Bihang till Kongl. Svenska vetenskapsakademiens handlingar, Band 22, 4 (1), 1 - 45.","Wallin, L. (2001) Catalogue of type specimens. 4. Linnaean specimens. Museum of Evolution, Zoology Section, Uppsala University, 128 pp.","Hoogmoed, M. S., Gasso Miracle, M. E. & van den Hoek Ostende, L. W. (2010) Type specimens of recent and fossil Testudines and Crocodylia in the collections of the Netherlands Centre for Biodiversity Naturalis, Leiden, the Netherlands. Zoologische Mededelingen Leiden, 84, 1 - 41.","Bour, R., Dubois, A. & Webb, R. G. (1995) Types of recent trionychid turtles in the Museum national d'Histoire naturelle, Paris. Dumerilia, 2, 73 - 92."]}

Pelodiscus maackii (Brandt 1857:110) Holotype: Not located., Published as part of Iverson, John B., 2022, A review of Chelonian type specimens (order Testudines), pp. 1-85 in Megataxa 7 (1) on page 65, DOI: 10.11646/megataxa.7.1.1, http://zenodo.org/record/5964441

Pelodiscus huangshanensis Gong, Peng, Huang, & Nie, in Gong, Peng, Huang, Lin, Huang, Xu, Yang, & Nie 2021:140 Holotype: ANU 20210001 (photographed in the original description, p. 141, Fig. 2 and p. 142, Fig. 4); paratypes (6), ANU 20210002 (photographed in the original description, p. 142, Fig. 3), ANU 20210003, ANU 20210004 (photographed in the original description, p. 142, Fig. 4), ANU 20210005, CIB 116368, and SNHM 5414., Published as part of Iverson, John B., 2022, A review of Chelonian type specimens (order Testudines), pp. 1-85 in Megataxa 7 (1) on page 65, DOI: 10.11646/megataxa.7.1.1, http://zenodo.org/record/5964441

Pelodiscus axenaria (Zhou, Zhang, & Fang 1991:382) Holotype: None located; illustrated in the original description (Figs. 1–2)., Published as part of Iverson, John B., 2022, A review of Chelonian type specimens (order Testudines), pp. 1-85 in Megataxa 7 (1) on page 65, DOI: 10.11646/megataxa.7.1.1, http://zenodo.org/record/5964441

Pelodiscus huangshanensis sp. nov. Gong, Peng, Huang & Nie, 2021 (Figs. 2���5) Suggested English name: Huangshan soft-shelled turtle Suggested Chinese name: ������������ (Hu��ng Shān M�� t�� Biē) Holotype. ANU20210001, an adult male (Fig. 2), was found in a stream from Xikou Town, Xiuning County, Huangshan City, Anhui Province, China (118.02��N, 29.69��E, and 170 m A. S. L.). The coordinates are given only with a precision of 0.01 degrees to avoid private or commercial sampling. The specimen was collected on 10 June 2017 and deposited at Anhui Normal University Museum (voucher: ANU20210001). Paratypes. ANU20210002, adult female (Fig 3); ANU20210003, adult female; ANU20210004, juvenile; ANU20210005, juvenile; CIB116368, juvenile; SNHM5414, juvenile. They were sampled from May to June, 2017 around the site of holotype. Diagnosis. Pelodiscus huangshanensis sp. nov. differs from the five known congeners by a combination of the following characters: (1) Small size; maximum carapace length of 84.0 mm and maximum body length of 190 mm; (2) keel high; (3) tiny yellowish-white spots on the throat; (4) no black pinstripes around the eyes; (5) white longitudinal bands on both sides of the neck in juveniles, absent in adults (Fig. 4); (6) plastron yellowish-white, and only a dark patch on each side of the armpit; (7) many tubercles on the dorsal surface, but indistinct in the center; and (8) entoplastron ���⌒��� shaped. Description of holotype and Variation. Where they differ from the holotype, features of the paratypes are listed in parentheses (ANU20210002���5, CIB116368, SNHM5414). Holotype of an adult male (the first two are adult females and the last four are juveniles); carapace length 82.5 mm (adult females 73.4���84.0 mm, juvenile 40.5���55.9 mm); plastron length 79.3 mm (adult females 75.7���86.1 mm, juvenile 39.9���55.8 mm); body length 190.0 mm (adult females 144.9���160.0 mm, juvenile 88.2���114.7 mm) (Table 3). Back and side of head have many fine dark brown to black spots; no black pinstripes around the eyes. The neck shows no white longitudinal striae bilaterally (there are white longitudinal bands on both sides of the neck in juveniles). The throat has many tiny yellowish-white spots. The carapace slightly oval with a high keel. The carapace is olive brown to dark brown. The dorsal surface has many tubercles, but it is indistinct in the center. The plastron yellowish-white, and only a dark patch on each side of the armpit. Entoplastron ���⌒��� shaped. Ecological notes. On June 20, 2017, a female individual (raised in Xiuning Institute of Rare Aquatic Animals) laid five eggs with a postpartum weight of 134 g. The eggs are white, nearly spherical; average length 15.95 �� 0.11 (15.79���16.01) mm; average short diameter 15.6 �� 0.16 (15.56���15.89) mm; and average weight 2.12 �� 0.23 (1.84���2.4) g. Hatching occurs at natural temperatures after 60���65 days. The average weight of neonates was 1.30 �� 0.08 (1.20���1.40) g. Three months old horse-hoof soft-shelled turtles are shown in Fig. 5. Etymology. The specific name refers to the type locality of this species, Huangshan City, Anhui Province. We suggest the following common names: horse-hoof soft-shelled turtle in English and ���Hu��ng Shān M�� t�� Biē (������������)��� in Chinese. The native people of Huangshan have called it ���M�� t�� Biē (������)��� for more than a thousand years. Distribution. This species is currently known only from the type locality, Xiuning County, Huangshan City, Anhui Province, China. Comparisons. Pelodiscus huangshanensis sp. nov. can be distinguished from P. axenaria by the following morphological characters: (1) Many tubercles on the dorsal surface, but indistinct in the center (vs. smooth carapace, no tubercles); (2) plastron yellowish-white, and only a dark patch on each side of the armpit (vs. a single dark-gray central figure enclosed by the hypoplastra and xiphiplastra); and (3) white longitudinal bands on both sides of the neck in juveniles, absent in adults (vs. absence of spots on the neck). Pelodiscus huangshanensis sp. nov. can be further distinguished from P. sinensis by the following morphological characters: (1) Small size; maximum carapace length 115.6 mm (vs. medium size; maximum carapace length 230 mm); (2) plastron yellowish-white, and only a dark patch on each side of the armpit (vs. plastron yellowishwhite with many dark spots); (3) keel high (vs. keel low); (4) no black pinstripes around the eyes (vs. black pinstripes around the eyes); (5) the throat has many tiny yellowish-white spots (vs. large yellowish-white spots with dark edges); and (6) entoplastron ���⌒��� shaped (vs. entoplastron �������� shaped). Pelodiscus huangshanensis sp. nov. can be further distinguished from P. maackii by the following morphological characters: (1) Small size; maximum carapace length of 115.6 mm (vs. larger size; maximum carapace length more than 350 mm); (2) plastron yellowish-white, and only a dark patch on each side of the armpit (vs. plastron yellowish-white with no markings or relatively small, faint round to oval dark markings); (3) the throat has many tiny yellowish-white spots (vs. large yellowish-white spots with dark edges); (4) keel high (vs. keel low); and (5) no black stripes around the eyes (vs. black pinstripes around the eyes). Pelodiscus huangshanensis sp. nov. can be further distinguished from P. parviformis by the following morphological characters: (1) Plastron yellowish-white, and only a dark patch on each side of the armpit (vs. plastron yellowish-white with no markings); (2) many tubercles on the dorsal surface, but indistinct in the center (vs. dorsal tubercles in longitudinal series more or less discrete; central tubercle in front of marginal ridge of carapace small); and (3) entoplastron ���⌒��� shaped (vs. entoplastron �������� shaped). Pelodiscus huangshanensis sp. nov. can be further distinguished from P. variegatus by the following morphological characters: (1) Many tubercles on the dorsal surface, but indistinct in the center (vs. dorsal surface roughened by longitudinal ridging and smaller protuberances spread over the leathery margin); (2) plastron yellowish-white, and only a dark patch on each side of the armpit (vs. plastron pinkish white to pale reddish orange distinct, large dark blotches, underside of leathery margin of carapace pigmented); (3) no black pinstripes around the eyes (vs. black pinstripes around the eyes); and (4) the throat has many tiny yellowish-white spots (vs. large yellowish-white spots with dark edges)., Published as part of Gong, Yan-An, Peng, Li-Fang, Huang, Song, Lin, Yan-Feng, Huang, Ru-Yi, Xu, Yu-Hao, Yang, Dian-Cheng & Nie, Liu-Wang, 2021, A new species of the Genus Pelodiscus Fitzinger, 1835 (Testudines: Trionychidae) from Huangshan, Anhui, China, pp. 137-145 in Zootaxa 5060 (1) on pages 140-143, DOI: 10.11646/zootaxa.5060.1.7, http://zenodo.org/record/5606848

A new species of the soft-shelled turtle genus Pelodiscus is described based on seven specimens from Huangshan, southern Anhui Province, China. The new species, Pelodiscus huangshanensis sp. nov., is distinguished from other species in the genus Pelodiscus by the following characteristics: (1) Small size (maximum carapace length of 101.16 mm and maximum body length of 190 mm); (2) keel high; (3) tiny yellowish-white spots on the throat; (4) no black pinstripes around the eyes; (5) white longitudinal bands on both sides of the neck in juveniles, absent in adults; (6) plastron yellowish-white, and only a dark patch on each side of the armpit; (7) many tubercles on the dorsal surface, but indistinct in the center; and (8) entoplastron “⌒” shaped. The phylogenetic relationships of the species in Pelodiscus were reconstructed using the sequences of cytochrome b (cyt b) and NADH dehydrogenase subunit 4 (ND4) genes. The new species formed a monophyletic clade with strong support. The uncorrected pairwise distances between the new species and other representatives of Pelodiscus ranged from 5.4% to 9.2% for cyt b and 4.1% to 7.6% for ND4. The new species brings the number of species of the genus Pelodiscus to six; five species are distributed in China, with three species endemic to China.

The Chinese soft-shelled (Pelodiscus sinensis) turtle exhibits obvious sex dimorphism, which leads to the higher economic and nutritional value of male individuals. Exogenous hormones can cause the transformation from male to female phenotype during gonadal differentiation. However, the molecular mechanism related to the sexual reversal process is unclear. In this study, we compared the difference between the small RNAs of male, female, and pseudo-female turtles by small RNA-seq to understand the sexual reversal process of Chinese soft-shelled turtles. A certain dose of estrogen can cause the transformation of Chinese soft-shelled turtles from male to female, which are called pseudo-female individuals. The result of small RNA-seq has revealed that the characteristics of pseudo-females are very similar to females, but are strikingly different from males. The number of the microRNAs (miRNAs) of male individuals was significantly less than the number of female individuals or pseudo-female individuals, while the expression level of miRNAs of male individuals were significantly higher than the other two types. Furthermore, we found 533 differentially expressed miRNAs, including 173 up-regulated miRNAs and 360 down-regulated miRNAs, in the process of transformation from male to female phenotype. Cluster analysis of the total 602 differential miRNAs among females, males, and pseudo-females showed that miRNAs played a crucial role during the sexual differentiation. Among these differential miRNAs, we found 12 miRNAs related to gonadal development and verified their expression by qPCR. The TR-qPCR results confirmed the differential expression of 6 of the 12 miRNAs: miR-26a-5p, miR-212-5p, miR-202-5p, miR-301a, miR-181b-3p and miR-96-5p were involved in sexual reversal process, which was consistent with the results of omics. Using these six miRNAs and some of their target genes, we constructed a network diagram related to gonadal development. We suggest that these miRNAs may play an important role in the process of effective sex reversal, which would contribute to the breeding of all male strains of Chinese soft-shelled turtles.

The Chinese soft-shelled turtle (Pelodiscus sinensis) is one of the most important freshwater aquaculture reptiles in East and Southeast Asia. In contrast to the other strains of this species, the Qingxi black turtle has black skin. In this study, three Qingxi black turtles (WBF) and three Dongting Lake turtles (DTF) were investigated to compare and analyze their differences in skin color by mRNA sequencing and miRNA sequencing. Total of 1,772 differentially expressed mRNAs were identified including 1,079 upregulated and 693 downregulated in WBF. Through miRNA sequencing, 1,071 miRNAs were identified. Three- hundred nineteen miRNAs were differentially expressed, including 187 upregulated and 132 downregulated, which corresponded to 38,563 target mRNAs. Nine differentially expressed mRNAs and twelve differentially expressed miRNAs were validated by qRT-PCR. Integrated analysis of the profiles of differentially expressed miRNAs and mRNAs predicted 2,262 miRNA-mRNA interaction sites. Several mRNAs (such as tyr, mitf, gpnmb and kit) and miRNAs (such as miR-138, miR-383-3p, miR-1388b-5p and miR-18a-3p-3) involved in turtle skin pigmentation were identified. The results obtained in this study concerning mRNA-miRNA regulatory networks may help to establish a foundation for future research on the mechanisms underlying pigmentation in P. sinensis.

Genetic diversity is an important component of biodiversity and investigating and protecting the wild genetic diversity of species has always been an important research topic in conservation biolog...

Experiments were performed to investigate the effects of stocking density (6, 8, 10, and 12 turtles/m²) of Chinese soft‐shelled turtles (Pelodiscus sinensis) and coculture interactions on growth performance and environment in a turtle–rice coculture system. The average yields of rice grain and rice straw in four turtle–rice coculture (TR) groups were significantly higher than those in a rice monoculture with fertilizer (RMF) group and a rice monoculture (RM) group (p

The objective of the present study was to estimate the dietary thiamine (vitamin B₁) requirement of juvenile soft‐shelled turtles, Pelodiscus sinensis. Eight experimental diets containing 0, 1.90, 3.63, 5.65, 7.51, 9.62, 11.37 and 13.64 mg thiamine/kg diet were fed to 160 soft‐shelled turtles reared individually for 10 weeks. The turtles had an average weight of 5.33 ± 0.21 g. Among all the dietary groups, weight gain, feed utilization and tissue thiamine were the lowest in the turtles fed with a thiamine‐free diet. The variables increased when dietary thiamine increased and then levelled off beyond 3.63 mg/kg diet. In contrast, plasma pyruvate and lactate concentrations in turtles decreased when dietary thiamine increased and then levelled off beyond 3.63 and 5.65 mg/kg diet respectively. Estimation using a broken‐line regression model, the vitamin B₁ requirement of soft‐shelled turtles was 3.2 mg thiamine/kg diet based on weight gain and plasma lactate concentration. When tissue thiamine and plasma pyruvate concentrations were used as the indicators, the estimated requirement was 5.4–6.5 mg/kg diet.

Interferon regulatory factors (IRFs) were originally identified as transcriptional regulators of type I interferon (IFN) expression. Recent studies have widely identified the roles of IRFs as central mediators in immune defence against pathogen infection. However, the functional roles and expression profiles of IRFs are still unclear in Chinese soft-shelled turtle (Pelodiscus sinensis). In this study, eight members of the PsIRF family were identified in P. sinensis through a genome-wide search. These PsIRF genes contained the conserved domains of this group of proteins, including the N-terminal DNA-binding domain and C-terminal IRF-associated domain. Phylogenetic analyses among IRF homologs showed that the PsIRFs shared the closest phylogenetic relationships with IRFs of other turtle species. Further molecular evolutionary analyses revealed evolutionary conservation of the PsIRF genes. Moreover, expression profiling demonstrated that eight PsIRF genes exhibited constitutive expression in different tissues of P. sinensis. Several genes, such as PsIRF1, PsIRF2 and PsIRF4, showed predominant expression in the spleen and were significantly upregulated upon Aeromonas hydrophila infection. Remarkably, PsIRF1, PsIRF2 and PsIRF4 exhibited rapid increases in their protein expression levels post-infection and were mainly expressed in the splenic red pulp according to immunohistochemistry analysis. These results provide rich resources for further exploration of the roles of PsIRFs in immune regulation in P. sinensis and other turtles.

Rapid evaporative ionization mass spectrometry (REIMS) is a recently developed technology for real-time lipidomics profiling of biological tissues without time-consuming sample preparation. In this study, a REIMS method was established for the differentiation of Pelodiscus sinensis cultured in two distinct modes, the organic ones cultured ecologically in an outdoor pond, and the greenhouse ones cultured in the greenhouse. The tissue samples were analyzed directly using an electrosurgical monopolar cutting handpiece, and the aerosol generated was simultaneously transferred into the mass spectrometer by a Venturi pump. Finally, two turtle groups were successfully differentiated with the REIMS method, and the markers for identification were detected. The results of method validation and the successful application of this method indicated that the well-established real-time REIMS technique is rapid, high-throughput, efficient, and reliable in detecting the food fraud of P. sinensis.

The Chinese soft-shelled turtle ( Pelodiscus sinensis) has become one of the leading cultured organisms in China and South East Asia. The objectives of the present study were to use next generation sequencing to identify viral genomes present in liver tissues from Chinese soft-shelled turtle in China. BLAST analysis of viral sequences from liver samples showed high homology with the human adenovirus (HAdV) penton base and encapsidation proteins. This homology points to possible existence of HAdV in freshwater environments used for the culture of soft-shelled turtles. Therefore, our findings merit further investigations to determine possible contamination of HAdV in aquaculture environments and the possible role of the Chinese soft-shelled turtle in transmitting HAdV to humans.

A 10‐week feeding trial was conducted to estimate the dietary vitamin K requirements of juvenile Chinese soft‐shelled turtles, Pelodiscus sinensis, using menadione sodium bisulphite (MSB) as the vitamin K source. Juvenile soft‐shelled turtles with 6.1 ± 0.1 g body weight were individually reared and fed diets containing seven levels of MSB (0.0, 5.0, 8.9, 12.3, 16.6, 20.7 and 41.0 mg/kg). Turtles fed with an MSB‐free diet exhibited the lowest feed utilization, carapace Ca concentrations, carapace strength or liver menaquinone (MK‐4) concentrations among all dietary groups. Weight gain (WG) in the turtles tended to increase with increase in dietary MSB until a dietary MSB level of 16.6 mg/kg, where WG levelled off. Total plasma prothrombin concentrations, an indicator for blood clotting function, in the turtles fed on MSB supplemented diets were significantly (p

Heat‐shock proteins 70/110 (Hsp70/110) are vital molecular chaperones and stress proteins whose expression and production are generally induced by extreme temperatures or external stresses. The Hsp70/110 family is largely conserved in diverse animals. Although many reports have studied and elaborated on the characteristics of Hsp70/110 in various species, the systematic identification and analysis of Hsp70/110 are still poor in turtles. In this study, a genomewide search was performed, and 18 candidate PsHSP70/110 family genes were identified in Chinese soft‐shelled turtle, Pelodiscus sinensis. These PsHSP70/110 proteins contained the conserved “heat shock protein 70” domain. Phylogenetic analysis of PsHSP70/110 and their homologs revealed evolutionary conservation of Hsp70/110 across different species. Tissue‐specific expression analysis showed that these PsHSP70/110 genes were differentially expressed in different tissues of P. sinensis. Furthermore, to examine the putative biological functions of PsHSP70/110, the dynamic expression of PsHSP70/110 genes was analyzed in the testis of P. sinensis during seasonal spermatogenesis following germ cell apoptosis. Notably, genes such as PsHSPA1B‐L, PsHSPA2, and PsHSPA8 were significantly upregulated in P. sinensis testes along with a seasonal decrease in apoptosis. Protein interaction prediction revealed that PsHSPA1B‐L, PsHSPA2, and PsHSPA8 may interact with each other and participate in the MAPK signaling pathway. Moreover, immunohistochemical analysis showed that PsHSPA1B‐L, PsHSPA2, and PsHSPA8 protein expression was associated with seasonal temperature variation. The expression profiling and interaction relationships of the PsHSPA1B‐L, PsHSPA2, and PsHSPA8 proteins implied their potential roles in inhibiting the apoptosis of germ cells in P. sinensis. These results provide insights into PsHSP70/110 functions and will serve as a rich resource for further investigation of HSP70/110 family genes in P. sinensis and other turtles.

Environmental DNA (eDNA) techniques show promise in facilitating the study and conservation of threatened species. The advantages of high sensitivity and high efficiency have been demonstrated by studies across various taxa. Asian turtles, one of the most threatened vertebrate groups, are facing extinction due to overexploitation for use as traditional Chinese medicine, food, and pets. Most species are extirpated across their ranges, but Hong Kong is unique because wild turtle populations persist. Due to the rarity and secretiveness of turtles, data on the location and size of populations are incomplete, even for a small place like Hong Kong. The result is that research and conservation are hindered. In this study, quantitative PCR eDNA assays were developed for three turtle species: two native [big-headed turtle (Platysternon megacephalum) and Chinese softshell turtle (Pelodiscus sinensis)] and one invasive [red-eared slider (Trachemys scripta elegans)]. For each assay, we tested species specificity, optimized reactions, and benchmarked analytical sensitivity for eDNA surveys. The assays will help in the conservation of Hong Kong turtles by identify priority sites for protection and research, provide another tool for population monitoring, and clarify the impact of the invasive T. s. elegans on native species.

Background The relationships among energy metabolic levels, behavioral and other physiological traits help to determine the trade-off of energy allocation between different traits and the evolution of life-history driven by natural selection. However, these relationships may be distinctive in selected animal taxa because of their unique traits. In the present study, the relationships among energy metabolic levels, behavioral defense strategies, and antioxidant capacity were explored in three freshwater turtle species with different shell morphologies, by assessing responses to attack, righting time, shell morphology, whole-organism metabolic rates, tissue metabolic enzyme activities and antioxidant levels. Results The Chinese three-keeled pond turtles, Chinemys reevesii, showed a passive defense strategy, relatively larger shells, a higher resting metabolic rate (RMR) and higher antioxidant levels compared to the snapping turtle, Chelydra serpentina, or the Chinese soft-shelled turtle, Pelodiscus sinensis. These latter two species both showed an active defense strategy, a higher factorial aerobic scope and better muscle anaerobic metabolic capacity but relatively smaller shells, lower RMR and antioxidant capacity. Conclusion Our results indicate a negative relationship between RMR and activity levels in behavioral defense strategies along small-big shell continuum among the three turtle species. We also found a positive relationship between antioxidant capacity and energy metabolism but a negative one between antioxidant capacity and activity levels in defense strategies. The present study indicated a role of turtle shell in forming unique relationship between energy metabolic levels and behaviors in freshwater turtle taxa and a possible trade-off between the maintenance of physiological homeostasis and activity levels in energy allocation. Electronic supplementary material The online version of this article (10.1186/s12983-019-0301-5) contains supplementary material, which is available to authorized users.

Island ecosystems provide habitat for many endemic species that may be threatened by nonnative species introductions. We examined nonnative freshwater turtle occurrences and diets to examine potential predation effects on native species in Kawai Nui Marsh, Oahu, Hawaii. No freshwater turtles are native to the Hawaiian Archipelago. The Pond Slider (Trachemys scripta) and Chinese Softshell (Pelodiscus sinensis) were the only turtles found in the marsh after 767 trap days. Trachemys scripta stomachs (n = 50) contained mostly the nonnative plant Commelina diffusa and nonnative snails (Pomacea sp.), whereas Pelodiscus sinensis stomachs (n = 5) contained mostly snails. Interspecific dietary overlap was low and intersexual dietary overlap in the sliders was high, with more diverse female diets. Small, medium, and large size classes of T. scripta stomachs contained different proportions of plant and animal matter, with the small size class containing less plant matter than the medium size class, and the large size class containing a greater volume of animal than plant matter. No native species were found in the stomach contents of the turtles sampled except a freshwater sponge (Heteromyenia baileyi). This lack of native species in their diets may have more to do with the degraded state of the marsh and lack of native taxa than with a preference for nonnative taxa. A potential concern could be nonnative freshwater turtle presence in pristine wetland habitats in Hawaii, because of the higher abundances of native species in those areas.

Background Chinese soft-shell turtle (Pelodiscus sinensis) is an important commercial species for their high nutritional value and unique taste, but it has been a vulnerable species due to habitat loss. In this study, homologous juvenile turtles were allocated to lake, pond and paddy field to investigate the habitat effects on turtles. Results The growth, morphology and gut microbial communities were monitored during the 4 months cultural period. It showed higher growth rate of turtles in paddy field and pond. The appearance, visceral coefficients, gut morphology and microbial communities in turtles were distinct among different habitats. The microbial community richness on Chao1 was obviously lower in initial turtle guts from greenhouses, whereas it was relative higher in turtle guts sampled from paddy fields than ponds and lake. Significant differences on dominant microbes were found among initial and subsequent samples from different habitats. Firmicutes was the most abundant phylum in the guts of turtles sampled from the greenhouse initially, while Proteobacteria was the most abundant phylum after cultivation in different habitats, followed by Bacteroidetes. The microbial composition were distinct in different habitats at 60d, and the appearance of dominant phyla and genera was more driven by sampling time than habitats at 120d. Both the sampling time and habitats affected the appearance of dominant phyla and genera during the cultivation. The functional predictions indicated that both habitat type and sampling time had significant effects on metabolic pathways, especially amino acid and carbohydrate metabolism. Conclusions The turtles could adapt to natural lakes, artificial ponds and paddy fields. The gut microbial abundance was different among the habitats and sampling time. The species of microbes were significantly more diverse in paddy field specimens than in those from ponds and lakes. Rice-turtle coculture is a potential ecological and economic farming mode that plays important roles in wild turtle protection and food security.

The Chinese soft-shelled turtle (Pelodiscus sinensis) is among the most primitive amphibians and reptiles in nature. On account of its environmental suitability and unique hibernation habit, the peculiar physiological phenomenon in P. sinensis attracted the attention of researchers in the field of marine science. The present study aimed to investigate the molecular mechanisms underlying the periodic variation of lipid droplet (LD) in the liver of P. sinensis. Histological results indicated that accumulated LD in the liver of P. sinensis during non-hibernation was gradually consumed during hibernation. RNA-Seq results revealed that genes responsible for carbohydrate catabolism were down-regulated during hibernation, while genes involved in lipid oxidation were up-regulated. These results suggest that energy metabolism in the liver of P. sinensis changes during hibernation, i.e., the energy generation mode shifted from carbohydrate catabolism to lipid oxidation. Further analysis of RNA-Seq results indicated that both lipolysis and autophagy could promote the degradation of hepatic LD during hibernation. To further determine the relationship between lipolysis and autophagy in the process of LD breakdown, we applied the inhibitors of lipolysis and autophagy (diethylumbelliferyl phosphate and 3-Methyladenine) in cultured primary hepatocytes of P. sinensis. The results indicated that lipolysis is the main way for LD degradation in the hepatocyte of P. sinensis. These data provide clear evidence about the seasonal changes in hepatocytes, corresponding with the different energy generation mode in the liver of P. sinensis.

Circular RNA (circRNA) is a noncoding RNA that can regulate a variety of biological processes. CircRNAs can regulate gene expression posttranscriptionally by acting as microRNA sponges. Many turtle species are remarkable organisms due to their reproductive processes. However, information on circRNA in the gonads of turtles is limited. In this study, 6, 121 circRNAs were identified in the testes and ovaries of Chinese soft-shelled turtles (Pelodiscus sinensis) using the Illumina platform, and 710 circRNAs were significantly differentially expressed (DE). The DE circRNAs included 541 upregulated and 169 downregulated circRNAs in the testes. GO and KEGG pathway analysis indicated that the DE circRNAs were enriched in several signaling pathways, including GnRH, Wnt, FoxO, Progesterone mediated oocyte maturation, and mTOR signaling pathways. Five DE circRNAs were randomly selected, and their relative expression levels in ovaries and testes were detected by quantitative real-time PCR. All of these circRNAs were differentially expressed. In addition, 9, 883 interactions between circRNAs and miRNAs were predicted in the turtles. Target genes of the miRNAs include a range of genes regulating gonadal development. Seven ceRNA networks (DE circRNAs-DE miRNAs-DE mRNAs), including 7 DE circRNAs, 11 DE miRNAs and 20 DE mRNAs, were constructed. The networks included Cdc6, the miR-1 family, the miR-203 family, and the miR-302 family. The expression profile of gonadal circRNAs might help to elucidate the roles of nonprotein coding RNAs in turtle gonadal development.

In this study, Restriction site-associated DNA sequencing (RAD-seq) was used to develop single-nucleotide polymorphism (SNP) markers for Pelodiscus sinensis, which is a commercially important species in China. We collected 15 individuals from Anhui Province and RAD-seq data of 20 individuals from NCBI: PRJNA530350 to screen high quality SNP maker through genome-wide. A total of 105 high-quality SNP markers were identified. The population genetic statistics showed that the minor allele frequency varied from 0.0571 to 0.3382, and the observed heterozygosity and expected heterozygosity ranged from 0.1143 to 0.6765 and 0.1078 to 0.4477, respectively. These novel SNP markers will serve as a valuable genetic tool for future conservation and population genetics studies in P. sinensis.

Background Chinese soft-shell turtle (Pelodiscus sinensis) is an important commercial species for its high nutritive and medicinal value, but it has been a vulnerable species due to habitat loss. In this study, homologous juvenile turtles were allocated to lake, pond and paddy field to investigate the habitat effects on turtles. Results The growth, morphology and gut microbial communities were monitored during the 4 months cultural period. It showed higher growth rate of turtles in paddy field and pond. The appearance, visceral coefficients, gut morphology and microbial communities in turtles were distinct among different habitats, which was mainly attributed to food abundance and living space. The diversity of gut microbial community was obviously higher in turtles from paddy field than pond and lake. Significant differences on dominant phyla, genera and species were found among initial samples and subsequent samples from different habitats. Firmicutes was the most abundant phylum in turtle gut sampled from greenhouse at initial and Proteobacteria was the most abundant phylum after cultivated in different habitats. The functional predictions indicated that both habitat type and sampling time had significant effects on metabolic pathways, especially amino acid and carbohydrate metabolism. Conclusion The microbial abundance was different among the habitats and varied within cultural periods. Gut microbes in turtles from paddy field presented high diversity and richness. Rice-turtle coculture was a potential ecological and economic farming mode for its high production and quality of both turtles and rice, which would play important roles in wild turtle protection, food security and paddy field environment improvement.

Pelodiscus sinensis is rich in a variety of nutrients and possesses a broad range of edible and medicinal properties, consequently, this species has a long history of culture in China and other Asian countries. A strain of P. sinensis named the ‘Yongzhang golden turtle’ was created by selective breeding methods over a period of 16 years and was approved as a new strain by Chinese government. In contrast to P. sinensis with atrovirens coloration, which belongs to the Yellow River wild type strain, the body coloration of the Yongzhang golden turtle is yellow. The especial yellow coloration will possess a high ornamental value. However, the specific mechanisms underlying this coloration change have yet to be investigated. In our present research, we measured the calipash hue of the Yongzhang golden turtle and atrovirens wild type turtle using indices defined by the International Commission on Illumination lightness, red/green and yellow/blue (CIE-LAB), this analysis showed that the calipash hue of atrovirens wild type turtle was atrovirens, while, that of Yongzhang golden turtle was yellow. Compared with atrovirens wild type turtle, there was no obvious accumulation of melanin granules in the epidermis of the Yongzhang golden turtle, as determined by hematoxylin-eosin (HE) staining and transmission electron microscopy (TEM). We also determined that the levels of melanin (P

Vitamin C (Vc) is an important antioxidant that helps turtles tolerating stressful environment. This work quantified changes in tissue Vc levels during winter hibernation in the soft-shelled turtle Pelodiscus sinensis, to reveal the stress response pattern of tissue Vc during hibernation and contribute basic data for turtle culture. We sampled juvenile soft-shelled turtles at pre-hibernation (17.0°C mud temperature; MT), during hibernation (5.8°C MT) and after arousal (20.1°C MT) in the field. The transcript levels of the gene encoding L-gulonolactone oxidase (GLO), the key enzyme for Vc synthesis, decreased significantly during hibernation. However, GLO activity did not match the GLO transcription patterns and remained stable during hibernation, and showed temperature-dependent kinetic characteristics. Vitamin C levels in the brain, liver, kidney, and spleen (but not muscle) all decreased significantly during hibernation, but recovered to pre-hibernation levels or even higher levels after arousal. The soft-shelled turtle endured 5 months of hibernation with no significant oxidative damage in most tissues, except in the spleen. Splenic Vc was nearly exhausted during hibernation, accompanied by a significantly elevated malonaldehyde (MDA) level. Although the high level of oxidative damage quickly decreased after arousal, the potential tissue damage in the spleen during hibernation might account for the weakened immune capacity of turtles after hibernation.

The nasal cavity of turtles is composed of the upper and lower chambers, lined by the upper and lower chamber epithelia, respectively. In many turtles including the Reeve's turtle Mauremys reevesii, the upper chamber epithelium contains ciliated olfactory receptor neurons (ORNs) and the lower chamber epithelium contains microvillous ORNs. However, in the olfactory organ of the Chinese soft-shelled turtle Pelodiscus sinensis, both the upper and lower chamber epithelia contain ciliated ORNs. In the present study, we immunohistochemically examined the developmental process of olfactory organs in soft-shelled turtle and the Reeve's turtle to clarify the developmental origins of the lower chamber epithelium in these turtles. Obtained data indicate that olfactory organs of these turtles have identical origin and follow similar process of development, suggesting that, in the lower chamber epithelium of the nasal cavity, ciliated ORNs differentiate in soft-shelled turtle whereas microvillous ORNs differentiate in the Reeve's turtle.

We conducted an 8-week feeding trial to determine the effects of dietary starch levels on growth performance, body composition, and digestive enzyme activities of juvenile soft-shelled turtles. Six fish meal-based diets containing 120, 180, 240, 300, 360 and 420 g kg−1 cornstarch were formulated. Body weight gain (BWG), crude protein concentration in whole body, as well as protease activity increased with increasing dietary starch levels until the highest at 240–300 g kg−1, but it was decreased at higher dietary starch content. In contrast, the feed conversion ratio, moisture content and hepatosomatic index were the lowest at 240–300 g kg−1 of cornstarch. The weight gain correlated significantly with the survival rate, the crude protein composition and the protease activity, but correlated negatively to the moisture content and the hepatosomatic index. The optimal dietary starch levels for the maximum BWG and maximum protease activity were 267.25 g kg−1 and 266.79 g kg−1, respectively. The α-amylase activity was the lowest at 120 g · kg−1 of cornstarch, and increased with increasing cornstrach content. The lipase activity and the amount of crude lipid in the turtles were not influenced by dietary cornstarch. Our results suggest that around 300 g kg−1 of cornstarch in diets is optimal for juvenile soft-shelled turtles.

The Chinese soft-shelled turtle (Pelodiscus sinensis) is one of the most important aquatic animals in China. The low growth rate is the main factor which hinders the development of the turtle’s industry. Therefore, it is urgent to development available molecular marker associated with genetic variation in growth for P. sinensis. The aim of this study was to identify and validate SNPs from growth-related gene of P. sinensis. The Illumina Hiseq4000 sequencing was used to develop single nucleotide polymorphism (SNP) markers in P. sinensis. Among them, 38 SNPs from growth-related genes were randomly selected and 24 loci exhibited significant polymorphism in genotyping by ARMS-PCR. The observed and expected heterozygosity ranged from 0.0625 to 0.5208 and 0.0807 to 0.4974, respectively. Significant deviation from HWE was observed for 3 loci. These findings provide molecular basis and candidate markers for P. sinensis breeding.