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First Description of Developmental Processes in Sclerodactyla Multipes (Echinodermata: Holothuroidea: Dendrochirotida) from Misaki, Sagami Bay, Japan

First Description of Developmental Processes in Sclerodactyla Multipes (Echinodermata: Holothuroidea: Dendrochirotida) from Misaki, Sagami Bay, Japan

Plankton Benthos Res 16(3): 228–236, 2021 & Benthos Research © The Plankton Society of Japan

First description of developmental processes in Sclerodactyla multipes (Echinodermata: Holothuroidea: ) from Misaki, Sagami Bay, Japan

Hisanori Kohtsuka1, Kohei Oguchi2, Yusuke Yamana3 & Masanori Okanishi1,*

1 Misaki Marine Biological Station, The University of Tokyo, 1024 Koajiro, Misaki, Miura, Kanagawa 238–0225, Japan 2 National Institute of Advanced Industrial Science and Technology (AIST), 1–1–1 Higashi, Tsukuba, Ibaraki 305–8566, Japan 3 Wakayama Prefectural Museum of Natural History, 370–1 Funo, Kainan,Wakayama 642–0001, Japan Received 15 September 2020; Accepted 30 April 2021 Responsible Editor: Shinji Shimode doi: 10.3800/pbr.16.228

Abstract: More than 100 individuals of larvae were collected in the Japanese coastal sea of Moroiso, Sagami Bay, Kanagawa Prefecture, central-eastern Japan, in January 2018. Based on an obtained sequence of mitochon- drial 16S rRNA gene region of one juvenile, it was identified as Sclerodactyla multipes by BLAST search with 0.3% genetic distance. The developmental process of the S. multipes was observed for three months, in which time, they grew from 250 µm to about 4 mm in length; here they showed distinct tentacles and dermal ossicles. Detailed morphological features of this were described based on stereomicroscopic, fluorescence and SEM observations for the first time. This is the first description of life history through planktonic larva to juveniles in the family .

Key words: Sea cucumber, 16S rRNA, SEM, fluorescence microscope, larvae

Sclerodactyla briareus (Lesueur, 1824) from the western Introduction Atlantic and S. multipes (Théel, 1886) from the western Although embryological studies on sea cucumbers Pacific (Théel 1886, Hendler et al. 1995, Imaoka 1995, (Echinodermata: Holothuroidea) have been provided for Kuramochi 2012). In these species, although a brief text- several commercially useful species, such as Apostichopus based description of development in S. briareus has been armata (Selenka, 1867), Athyonidium chilensis (Semper, published (Ohshima 1925), no embryological study accom- 1868), and Isostichopus fuscus (Ludwig, 1875) (Inaba & panied by photographs has been reported in this genus. Maruyama 1988, Hamel et al. 2003, Guisado et al. 2012, In this study, doliolaria and pentactula forms of the lar- Eguchi 2015, Huang et al. 2018), data for non-commercial vae of sea cucumbers were collected by towing plankton species are inadequate (Inaba & Maruyama 1988). The or- nets at the sea surface in Moroiso Bay, Miura Peninsula, der Dendrochirotida is a suspension-feeding sea cucumber Kanagawa Prefecture, Japan. We identified this species by world-widely distributed on the sea floor. The accumulated DNA sequences based on 16S rRNA gene region, and doc- biological knowledge of this order is important for fisheries umented the growth of its larvae using multiple observa- management and aquaculture of sea cucumbers, but its ear- tion methods. liest life stages are scarcely studied (Gianasi et al. 2018). Recently, studies on the earliest life stages of 61 species in Methods 32 genera of 5 families have been conducted, but only 15 species have been studied following the process of growth One hundred and ten individuals were collected on 5 from larvae to juvenile (Table 1). The genus Sclerodactyla January 2018, using a plankton net (diameter 60 cm, mesh (Sclerodactylidae: Dendrochirotoda: Actinopoda) includes size 0.33 mm, net length 150 cm) towed at the sea surface for 5 min at Moroiso Inlet (35°09.405′N.139°36.345′E), Mi- * Corresponding author: Masanori Okanishi; E-mail, mokanishi@ ura Peninsula, Kanagawa Prefecture (Fig. 1). tezuru-mozuru.com Larvae of sea cucumbers were housed in a cylindrical First description of developmental processes in Sclerodactyla multipes (Echinodermata: Holothuroidea:Dendrochirotida) 229 from Misaki, Sagami Bay, Japan 1925 1915 Reference Levin 1995 Miller 1985 Vaney 1925 Hyman 1955 Hyman 1955 Hyman 1955 So et al. 2011 Thorson 1946 McEuen 1987 McEuen 1987 McEuen 1987, Hickman 1978 Thomson 1878 Lo Bianco 1899 Rutherford 1977 Deichmann 1922 Deichmann 1941 Cherbonnier 1972 Gianasi et al. 2018, Guisado et al. 2012 McEuen 1987, 1988 Hyman 1955, Vaney Costelloe 1985, 1988, Hyman 1955, Ohshima Hamel & Mercier 1996, ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ Remarks Gonochoric. Gonochoric. hermaphroditic. Each individual casts 300,000 floating eggs. µ m µ m µ m µ m µ m µ m ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ Just 700– 875– 925– 689 21 days, Juvenile 46 days, observed 3,500 1,230 1,125 1,400 1,325 6–8 weeks, 29–30 days, 18–21 days, 22–25 days, µ m µ m µ m µ m µ m µ m ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ days, 7 days, 7 days, 629 700 1,300 9.5–10.75 Pentactula 8–13 days, 9–11 days, 8–9.5 days, 700–800 600–710 740–770 Just observed µ m, µ m, Time and maximum size Time µ m, µ m, µ m, µ m, ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ 5 days, 6 days, 8 days, 725 433 650 3.5 days, 3.5 days, Vitellaria Vitellaria Doliolaria Doliolaria Doliolaria Doliolaria 1,550 3.75 days, 675–765 675–765 Just observed Floating larva µ m µ m µ m µ m µ m µ m µ m ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ Size 400– 400– 480– 916– 500 360 650 500 900 601 1,237 ̶ ̶ ○ ̶ ̶ ○ ○ ○ ̶ ○ ̶ ○ ○ ○ ̶ ̶ ̶ ̶ Fertilized egg Observation µ m µ m µ m µ m µ m µ m µ m ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ Size 550 700 400 370 500 1,200 1,000 “̶” means no data. ̶ ○ ○ ○ ○ ○ ○ ○ ̶ ○ ̶ ○ ○ ○ ○ ○ ̶ ̶ ̶ ̶ ○ Unfertilized egg Observation ○ ̶ ○ ̶ ̶ ○ ̶ ○ ̶ ○ ̶ ○ ○ ○ ̶ ̶ ̶ ̶ Observation Spawning ̶ ̶ ̶ ̶ ̶ all ̶ ̶ ̶ Apr Dec Jan? May Period Jan–Apr Sep–Oct Jan–May Nov–Dec mid Dec– Mar, June early Janu Apr–Junu, ̶ ̶ ̶ larva larva larva larva larva larva Type of Type Brooding Brooding Brooding Brooding Brooding Brooding Brooding Brooding Brooding Brooding Brooding tion: fission reproduction Lecithotrophic Lecithotrophic Lecithotrophic Lecithotrophic Lecithotrophic Lecithotrophic Asexual reproduc -

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Paw - (Sem - Semper, Vaney, Ludwig, Cherbon - Ohshima, (Barrois, 1882) 1868 1914 1915 1875 1835) Species son, 1967 nier, 1949 per, 1868) son, 1864) pert, 1886) nerus, 1767) (Grube, 1840) (Vaney, 1914) bonnier, 1972) (Lesson, 1830) (Lesson, 1830) Hickman, 1962 Deichmann, 1941 Deichmann, 1938 Echinopsolus koehleri egum (Pourtalès, 1868) Neoamphicyclus lividus Hemiocnus syracusanus panamica pseudocurata Cucumaria fallax Cucumaria joubini Cucumaria frondosa (Gun - Cucumaria ijimai Lissothuria antillensis Cladodactyla crocea Cladodactyla crocea Incubocnus incubans (Cher - (Brandt, Cucumaria vaneyi Cucumaria piperata (Stimp - Athyonidium chilensis Cucumaria georgiana (Lam - Benthophyllophorus conchil Aslia lefevrei Cucumaria japonica - idae Summaruy of developmental studies dendrochirotid sea cucumbers. Family Cucumari rotida Order Dendrochi - Table 1. Table 230 H. Kohtsuka et al. 1925 1946 1925 1982 1915 1946 1915 1982 1898 Reference Hyman 1955 Hyman 1955 Ludwig 1900 McEuen 1988 McEuen 1987 McEuen 1987 Simpson 1982 Wootton 1949 Ohshima 1921 Lo Bianco 1899 Lo Bianco 1899 Mortensen 1894, Deichmann 1941 Hyman 1955, Clark Hyman 1955, Vaney Orton 1914, Thorson Hyman 1955, Ekman Hyman 1955, Ludwig Hyman 1955, Simpson Hyman 1955, Ohshima Hyman 1955, Ohshima Chia & Buchanan 1969 McEuen 1987, Engstrom ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ tentacles Remarks Gonochoric. Hermaphroditic. Hermaphroditic. In vitro observatipn. The pentactula has 5 The pentactula has 5 A record of juvenile. tentacles. Gonochoric. µ m µ m ̶ ̶ ̶ ̶ ○ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ○ ̶ ̶ ̶ 925– Juvenile 17 days? 9,000 1,325 22–25 days, µ m ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ 7 days 7 days Pentactula 6–7.5 days, 490–600 Just observed Time and maximum size Time µ m µ m, µ m, ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ 2 days, 2 days, 500 500 550 Doliolaria Doliolaria Doliolaria Doliolaria 2.25 days, Floating larva µ m µ m µ m µ m µ m µ m ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ Size 877– 250– 339– 300– 500 350 400 400 439 1,078 ̶ ̶ ̶ ̶ ̶ ○ ̶ ̶ ○ ̶ ̶ ̶ ̶ ̶ ○ ○ ̶ ̶ ̶ ̶ Fertilized egg Observation µ m µ m µ m µ m µ m µ m µ m µ m µ m µ m ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ Size 512– 700 200 800 560 600 640 1,050 1,200 1,500 1,800 “̶” means no data. ̶ ○ ̶ ○ ○ ○ ̶ ○ ○ ̶ ̶ ̶ ○ ○ ○ ○ ̶ ̶ ○ ○ ̶ ○ Unfertilized egg Observation ̶ ̶ ̶ ̶ ○ ○ ̶ ̶ ̶ ̶ ○ ̶ ̶ ○ ̶ ̶ ̶ ̶ ̶ Observation Spawning ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ all ̶ ̶ Jan Period Winter Feb–Mar Oct–Nov Mar–Apr Nov–Dec Nov–Dec May–Aug June–Aug May–June ̶ ̶ ̶ larva larva larva larva larva larva? Type of Type Brooding Brooding Brooding Brooding Brooding Brooding Brooding Brooding Brooding Brooding Brooding Brooding reproduction Lecithotrophic Lecithotrophic Lecithotrophic Lecithotrophic Lecithotrophic Lecithotrophic

(St - (von (Lud - (Lud - Verrill, Ekman, (Brady & (Vaney, (Ljungman, Deichmann, 1876 1937 1867 1925 1879) 1901) 1908) 1901) Species wig, 1886) wig, 1875) Ayres, 1852 impson, 1864) (Dendy, 1897) (Ohshima, 1915) (Ohshima, 1915) Robertson, 1871) urna Grube, 1840 Marenzeller, 1882) Marenzeller, 1874) lamperti Stereoderma imbricata Pseudocnus grubii (von Pentamera pulcherrima (Düben & Koren, 1846) Psolicrux coatsi benti Pentamera populifera parva Lissothuria ornata Verrill, Pseudocnus echinatus Lissothuria nutriens (Clark, Pentamera chiloensis Paraleptopentacta elongata glacialis Psolidium incubans Pseudocnus lubricus (Clark, (Brandt, 1835) Thyone inermis Heller, 1868 Pseudopsolus macquariensis Pentactella laevigata Phyllophorus (Phyllophorus) Summaruy of developmental studies dendrochirotid sea cucumbers. Continued. Family Phyllo - phoridae Order Table 1. 1. Table Table First description of developmental processes in Sclerodactyla multipes (Echinodermata: Holothuroidea:Dendrochirotida) 231 from Misaki, Sagami Bay, Japan 2017 2010 1925 1909 1898 1993 1925 1925 1886 Reference This study & Chia 1991 & Chia 1991 Hyman 1955 Chaffee 1982 Oshima 1925 Hyman 1955, Runnstrôm & McEuen 1988 McEuen 1988 Runnstrôm 1919, Deichmann 1941, Smiley et al. 1991, Tyler & Gage 1983 Hyman 1955, Théel Dolmatov & Yushin Hyman 1955, Vaney Hyman 1955, Ekman Hyman 1955, Ekman Thorson 1946, Pearse Hyman 1955, Ludwig & Chia 1982, McEuen McEuen 1987, Johnson McEuen 1987, Colwin & 1956, & Johnson 1950, Young Martinez & Penchazadeh Gimenez & Penchazadeh ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ sole. Remarks activation. Hermaphroditic. Brooding under the In vitro observation. In vitro observation. The settled juveniles survived for 50 weeks. Observation of sperma µ m µ m µ m µ m ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ 670 Juvenile 22 days, 4,000 1,941 7 months, 26–32 days, 730–855 15–194 days, µ m µ m ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ 700 445 Pentactula 2–15 days, 11–12 days 10–11 days Just observed 7.75–8.5 days, µ m, Time and maximum size Time µ m, µ m, ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ 4 days, 4 days, 2 days, 600 925 Vitellaria doliolaria At least 1 Doliolaria Doliolaria day, 250 Just observed Floating larva µ m µ m µ m µ m µ m ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ Size 293– 570– 354 400 708 350 887 ̶ ̶ ̶ ̶ ̶ ○ ̶ ̶ ̶ ○ ○ ̶ ○ ̶ ̶ ○ ̶ Fertilized egg Observation µ m µ m µ m µ m µ m µ m ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ Size 350 640 700 250 500 640 “̶” means no data. ○ ̶ ̶ ○ ̶ ○ ○ ○ ○ ○ ̶ ○ ̶ ̶ ○ ○ ̶ Unfertilized egg Observation ̶ ○ ̶ ○ ̶ ̶ ̶ ̶ ○ ̶ ̶ ̶ ○ ̶ ̶ ̶ ̶ Observation Spawning ̶ ̶ ̶ ̶ ̶ all ̶ ̶ ̶ ̶ ̶ Dec Jan? Aug? Period Feb–Mar Feb–Mar Janu–Feb Mar–May ̶ ̶ ̶ larva larva larva larva larva? Type of Type Brooding Brooding Brooding Brooding Brooding Brooding Brooding Brooding Brooding Brooding reproduction Lecithotrophic Lecithotrophic Lecithotrophic Lecithotrophic Lecithotrophic

(De - talis - Clark, (Clark, Massin, (Philippi, Thomson, Martinez & 1925 1901 1996 1876 1925 1906 1901) 1857) Species felt, 1765) (Théel, 1886) Ohshima, 1915 ichmann, 1939) (Selenka, 1867) (Lesueur, 1824) Savel ’ eva, 1958) Psolidium bullatum Penchaszadeh, 2017 mani E. Perrier, 1886 fraudatrix Sclerodactyla multipes (D ’ yakonov & Baranova punctatus Ekman, Psolus granulosus Vaney, in D ’ yakonov, Baranova & Pachythyone rubra Pachythyone lugubris Psolus patagonicus Ekman, Psolus figulus Ekman, 1925 Ypsilothuria talismani Psolus ephippifer (Strussen - stracki Psolus antarcticus Psolus lawrencei - Ypsi - Summaruy of developmental studies dendrochirotid sea cucumbers. Continued. tylidae Family Sclerodac lothuriidae Order Table 1. Table 232 H. Kohtsuka et al.

Fig. 1. Map of collecting sites. Moroiso (circled area). Misaki Marine Biological Station (Black dot). polystyrene water tank with a diameter of 300 mm and a height of 150 mm, and with a water volume of 10 L. The larvae were kept in a water bath with natural seawater. The water was changed every other day. Water temperatures were adjusted to that of the natural seawater, ranging from 11.8°C to 24.0°C, during 5 January 2018 to 17 July 2018 (194 days). The temperature of the surface seawater where the larvae were collected was 12°C. Since this was the same as the water temperature in the laboratory, the larvae Fig. 2. Schematic of water tank for rearing of larvae. were reared in natural seawater. A small synchronous mo- tor for agitating the water was attached to the tank (Fig. 2). with phosphate-buffered saline for 15 minutes×3 times. The food provided for planktonic larvae of the sea cucum- The nuclei (DNA) and cytoskeletons (F-actin) were stained ber was mainly the planktonic Chaetoceros calci- with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma, St. trans (Paulsen) Takano, 1968, and after the larvae settled Louis, MO, USA) and rhodamine-phalloidin (Invitrogen, on the bottom, biological films collected in the intertidal Paisley, UK), respectively, for 1 h at room temperature, zone of Moroiso Inletat the laboratory were also provided and then washed with Phosphate-buffered Saline+Tween as food. 20 (PBT). After staining, samples were washed three times For molecular analysis, DNA was extracted from the with PBT. The samples were observed with a fluorescence whole body of one individual (fixed in 99% ethanol) us- microscope BZ-9000E, KEYENCE, Japan. Image pro- ing the DNeasy Blood and Tissue extraction kit (Qiagen) cessing was done using the image analysis software BZ-II according to manufacturer’s protocol. A partial sequence Analyzer, KEYENCE, Japan. For SEM observations, min- of the mitochondrial 16S rRNA gene region was amplified ute dermal ossicles were extracted by immersing the tis- by PCR using primers employed by Palumbi (1996). PCR sue in domestic liquid bleach (approximately 5% sodium conditions followed Okanishi & Fujita (2013). The PCR hypochlorite solution) and dissolving the soft parts. After products were separated from excess primers and oligonu- air-drying, the ossicles were fixed to SEM stubs with dou- cleotides using Exo-SAP-IT (GE Healthcare), following the ble-sided tape, their surfaces sputter-coated with platinum- manufacturer’s protocol. All samples were sequenced bi- palladium, and observed with a JSM 5200LV SEM. directionally and sequence products were run on a 3730xI DNA Analyzer (Thermo Fisher Scientific). The accession Results number of the sequence deposited in the DNA Data Bank of Japan (DDBJ) is LC577887. For molecular identification, we obtained 528 bp of a mi- Living larvae were photographed with a digital camera tochondrial 16S rRNA gene region for the examined larva. (OLYMPUS XZ7) mounted on a dissecting microscope. A BLAST search of the sequence on the DDBJ website For fluorescence observations, samples were fixed with (http://blast.ddbj.nig.ac.jp/blastn?lang=ja) yielded Sclero- 4% Paraformaldehyde (PEA) for 30 minutes and washed dactyla multipes (accession no. MG586798) as the closest First description of developmental processes in Sclerodactyla multipes (Echinodermata: Holothuroidea:Dendrochirotida) 233 from Misaki, Sagami Bay, Japan

Fig. 3. Larval and juvenile development of Sclerodactyla multipes. A: doliolaria larva (dissecting microscope). B: doliolaria larva (SEM). The 5 primary tentacles and primary podia are emerging. (pt) primary tentacles. (ptf) primary tube feet. C: tentacles. * 5 tentacles. D: settled pentactula. (ten) tentacles. (tf) tube feet. E–H: pentactula. same individual. E: stereo microscope image. F: SEM. G: fluorescence microscope image (blue: DAPI, red: phalloidin). H: light microscope image. (oss) ossicle. I: 15-day pentactula. J: ossicle of 15-day pentactula. K: 30-day juvenile. L: 60-day juvenile. M: ossicles of 60-day juvenile. overlap in a tiled manner. N: 90-day juvenile. O: ossicles of 90-day juvenile also appears inside podia. P: 150-day juvenile. Scale bars: A–B, D, 50 µm; E–I, 100 µm; K, L, N, P, 1 mm. sequence with 0.3% K2P genetic distance. appeared, and a pair of early primary podia (Fig. 3B) and The larvae of S. multipes examined in this study were five primary tentacles (Fig. 3C) had developed. Therefore, about 250 µm in body length when collected (Fig. 3A–C). the larvae were identified as doliolaria larvae (late stage) SEM observations showed that the cilia had already dis- because they were actively swimming, and had not yet 234 H. Kohtsuka et al. settled to the seafloor. On the second day after collection study, McEdward & Miner (2001) speculate that this spe- (d.a.c.), the larvae became pentaculae (Fig. 3D–H). The cies has pelagic, lecithotrophic and direct development. length of the body was 300 to 350 µm, and the body was This study is the first documentation of the growth process equipped with one layer of reticulated micro-ossicles. One of larvae of S. multipes. primary podia and five primary tentacles were extended In this study, larvae of S. multipes were successfully and the larvae began an active benthic life (Fig. 3D, E, reared for approximately 200 days and grown to a maxi- G). At this stage, phalloidin signals (i.e., well-developed mum size of 4 mm in length. Regarding their growth rates, actin filaments) were observed in the podia and primary the body lengths reach about 700 µm in 15 days, 1 mm in tentacles (Fig. 3G). On the 15th d.a.c., the body length was 30 days, 2 mm in 60 days, and 3.5 mm in 90 days. When over 700 µm and some branching was observed on the five compared to other species within the order Dendrochi- primary tentacles (Fig. 3I). The ossicles in the body were rotida, these values are similar to other species with leci- flatter and more prominent (Fig. 3J). By the 30th d.a.c., the thotrophic larvae, which reached about 700 µm to 1 mm body length had reached more than 1 mm and the ossicles in 22–45 days, i.e., Athyonidium chilensis (Semper, 1868), on the dorsal surface had become multi-layered; the num- Cucumaria fallax Ludwig, 1875, Cucumaria frondosa ber of tentacles was still five at this stage (Fig. 3K). At the (Gunnerus, 1767), Cucumaria miniata (Brandt, 1835), Cu- 60th d.a.c., the body length was about 2 mm and the num- cumaria piperata (Stimpson, 1864), Paraleptopentacta ber of podia had increased to 10 (Fig. 3L). The multi-lay- elongata (Düben & Koren, 1846), Psolidium bullatum Oh- ered structure of the dorsal ossicles was more pronounced shima, 1915, and Psolus chitonoides Clark, 1901 (Table 1). (Fig. 3M). At the 90th d.a.c., the 20 surviving individuals In Apostichopus japonicus (Selenka, 1867) of the order had reached a body length of 3.5 mm (Fig. 3N). The dorsal Aspidochirotida, the larvae grow to 4 mm in length after ossicles were single-layered and were scattered. The ten 60 days of rearing (Inaba & Maruyama 1988), suggesting tentacles began to have distinct ossicles (Fig. 3O). Despite that the growth of the order Dendrochirotida is relatively continuous observation, no significant growth of the os- slow. This might correlate to the ecological difference be- sicles was observed. At the 150th d.a.c., the body length tween Aspidochiroitida (deposit-feeding) and Dendrochi- exceeded 4 mm (Fig. 3P). The podia became longer and rotida (suspension-feeding) (Massin 1982). increased in number (16 on ventral and 18 on dorsal side). Sclerodactyla briareus has a larval stage that is an egg Although these individuals were kept alive for 194 days, yolk-nutrient lecithotrophic form (Oshima 1925), while the we could not identify them to species using morphological current species S. multipes, has a different developmental criteria. pattern with respect to its planktonic larva stage. How- ever, with regard to the reproduction types of the 61 den- drochirotid species, including S. multipes, lecithotrophic Discussion larvae (17 species) and brooding (33 species) predominate, Although the larvae could not be reared to the adult and these types occur in a polyphyletic manner within in stage in this study, molecular analysis showed that the ge- each lineage (Table 1). Thus, the reproduction types are netic distance between larvae and Sclerodactyla multipes highly plastic within this order, and it is not surprising (MG586798) was 0.3%. This falls within the range of in- that the types were different even within the genus Sclero- traspecific differences (less than 0.5%) for 16S in holothu- dactyla. In this study, we were unable to directly observe rians (e.g., Kim et al. 2013, Vergara et al. 2018). Because the exact spawning date of S. multipes, but we assume the morphology of the S. multipes recorded in Genbank that the spawning occurred probably in early January, has not been reported, the possibility of misidentification which was near the collection date of the floating larvae. cannot be ruled out. However, it is reasonable to assume When compared with the 30 dendrochirotid species for that our larvae were indeed S. multipes as registered in which the spawning periods are known, there is no simi- Genbank (MG586798, sampling locality unidentified). larity between the spawning periods and lineages. Assum- Currently, Sclerodactyla briareus and S. multipes, from ing spawning of S. multipes occurs in January, this differs the western Atlantic and western Pacific respectively from other species of the family Sclerodactylidae (Table 1). (Théel 1886, Hendler et al. 1995, Imaoka 1995, Kuramo- The lack of knowledge on the development of sea cu- chi 2012) are known in this genus. S. multipes has been cumbers, especially in the order Dendrochirotida, is prob- recorded from Yokohama in Tokyo Bay and Monbetsu in ably due to difficulty in encouraging spawning, and to the Hokkaido (Imaoka 1995), and this study is the first to re- difficulty in rearing larvae. Additionally, the lack of read- port this species from the vicinity of Misaki in Sagami ily visible taxonomic characters in larvae makes it diffi- Bay, assuming that the identification according to Genbank cult to identify the species. In the order Dendrochirotida, is correct. only 13 species have been described through the planktonic The only known embryological record of this genus is a larval stage to juveniles, and in the family Sclerodactyli- brief text-based study for S. briareus (Lesueur 1824) which dae, planktonic larva to doliolaria of Eupentacta fraudatrix documented that this species is lecithotrophic without a (D’yakonov & Baranova, 1958) has been observed, but the planktonic larval stage (Ohshima 1925). Referring to that juveniles of sclerodactylids have never been observed (Ta- First description of developmental processes in Sclerodactyla multipes (Echinodermata: Holothuroidea:Dendrochirotida) 235 from Misaki, Sagami Bay, Japan ble 1). In this study, we reared large numbers of the larvae Norstedt & Söner, Stockholm, pp. 1–194. of S. briareus, obtained from the plankton and we identi- Engstrom NA (1982) Brooding behavior and reproductive biol- fied the species based on sequences of the 16S mitochon- ogy of a subtidal Puget Sound sea cucumber, Cucumaria lu- drial DNA region, rearing them for a long period of time in brica (Clark, 1901) (Echinodermata: Holothuroidea) In: Inter- a waterfront laboratory to understand their developmental national Conference, Tampa Bay (ed Lawrence process. This is the first description of life history through JM). A.A. Balkema, Rotterdam, pp. 447–450. planktonic larva to juveniles in the family Sclerodactylidae. Gianasi BL, Hamel J-F, Mercier A (2018) Morphometric and be- havioural changes in the early life stages of the sea cucumber Cucumaria frondosa. Aquaculture 490: 5–18. Acknowledgements Gimenez J, Penchazadeh PE (2010) Brooding in Psolus pata- gonicus (Echinodermata: Holothuroidea) from Argentina, SW We thank Dr. David L. Pawson of the National Museum Atlantic Ocean. Helgol Mar Res 64: 21–26. of Natural History, Smithsonian Institution, and Mr. James Guisado C, Carrasco SA, Guisado DD, Maltrain R, Rojas H Scott Walker for their careful and critical reading of the (2012) Embryonic development, larval morphology and juve- manuscript. We take this opportunity to express our grati- nile growth of the sea cucumber Athyonidium chilensis (Ho- tude to Michiyo Kawabata (MMBS), for her cooperation in lothuroidea: Dendrochirotida). 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