Finding the Missing Puzzle Piece of the Nisto Stage in the Larval Cycle of the Slipper Lobster Scyllarides squammosus: A Molecular and Morphological Approach

© 2022 Academia Sinica, Taiwan Open Access Finding the Missing Puzzle Piece of the Nisto Stage in the Larval Cycle of the Slipper Lobster Scyllarides squammosus: A Molecular and Morphological Approach Chiho Hidaka1, Chien-Hui Yang2, and Kaori Wakabayashi3,* 1School of Applied Biological Science, Hiroshima University, Kagamiyama 1-4-4, Higashi-Hiroshima, Hiroshima 739-8528, Japan. E-mail: [email protected] (Hidaka) 2Institute of Marine Biology and Centre of Excellence for the Oceans, National Taiwan Ocean University, 2 Pei-Ning Road, Keelung 202301, Taiwan. E-mail: [email protected] (Yang) 3Graduate School of Integrated Sciences for Life, Hiroshima University, Kagamiyama 1-4-4, Higashi-Hiroshima, Hiroshima 739-8528, Japan. *Correspondence: E-mail: [email protected] (Wakabayashi) Received 18 June 2021 / Accepted 2 September 2022 / Published 14 December 2022 Communicated by Benny K.K. Chan Slipper and spiny lobsters are crustaceans that are in high demand and possess great commercial potential as valuable foods. The early life stages are important to understand the distribution and resource ecology of those lobsters. However, much less information is available about slipper lobsters than spiny lobsters. Biological information concerning the transition stage from the planktonic to the benthic phase, the so-called nisto stage, is limited probably due to its short duration. An individual scyllarid nisto was discovered while scuba diving off Chichijima Island. DNA analyses using mitochondrial 16S rRNA and cytochrome c oxidase subunit 1 (COI) genes confirmed this specimen to be Scyllarides squammosus (H. Milne Edwards, 1837). Detailed morphological observations of this specimen and its comparison with previous reports on Scyllarides nistos suggest that the diagnostic character of S. squammosus nisto is the pleura of the second to fifth pleonites possessing prominent teeth entirely on the lateral margin. Other morphological characteristics are the carapace with the widest distance in the middle and the second to fifth pleonites bearing two tubercles on each side. This report describes the identification of the first worldwide record of a Scyllarides nisto, confirmed by molecular barcoding. Key words: Decapoda, Taxonomy, Larva, Settlement, DNA barcoding. BACKGROUND Scyllaridae, a family of the infraorder Achelata (Decapoda: Crustacea), consists of 91 species and two subspecies assigned to 19 genera in four subfamilies (Holthuis 1991; WoRMS 2021). The family includes approximately 20 lobster species of commercial interest in the following four genera: (1) Scyllarides, (2) Parribacus, (3) Ibacus, and (4) Thenus (Holthuis 1991; Spanier and Lavalli 2013). Despite its commercial importance, much fewer studies are available for Scyllaridae, especially in contrast to another family of Achelata, Palinuridae, which includes many highly valuable lobster species (Spanier and Lavalli 2007; Ohta and Uehara 2017). Larval phases are particularly important for understanding the distribution and reproductive efficiency of those lobster species. The scyllarid zoea, called phyllosoma, grows through a series of molting stages and then metamorphoses into the decapodid phase, called the nisto (Martin 2014; Lavalli et al. 2019). A nisto provides the link between the pelagic and benthic phases and then becomes a Citation: Hidaka C, Yang CH, Wakabayashi K. 2022. Finding the missing puzzle piece of the nisto stage in the larval cycle of the slipper lobster Scyllarides squammosus: a molecular and morphological approach. Zool Stud 61:73. doi:10.6620/ZS.2022.61-73. Zoological Studies 61:73 (2022) doi:10.6620/ZS.2022.61-73 1 © 2022 Academia Sinica, Taiwan juvenile with a single molting (Booth et al. 2005; Sekiguchi et al. 2007). Puerulus, the decapodid phase of palinulids, is known to swim towards the shore from or beyond the continental shelf break (Booth and Phillips 1994), whereas the precise role of the nisto likely varies among the species (Booth et al. 2005; Sekiguchi et al. 2007). Nisto can be easily distinguished from other decapod planktonic phases by the presence of extremely flattened carapace and antennae. The nisto of each four subfamilies shows specific carapace characteristics, which can also be seen in the adult (Webber and Booth 2007). These characteristics include orbits at extreme anterolateral angles, V-shaped in Theninae (Barnett et al. 1984; Wakabayashi and Phillips 2016), incision at the middle of posterior margin in Scyllarinae (Wakabayashi et al. 2017b 2020), and cervical incision deep in Ibacinae (Takahashi and Saisho 1978; Marinovic et al. 1994; Yoneyama and Takeda 1998), whereas none of those characteristics are found in Arctidinae. The nisto of Arctidinae has another diagnostic character, namely, a strong median carina with a sharp tip on pleonite terga 4 and 5 (Chace Jr 1966; Lyons 1970). Both biological and ecological studies on the scyllarid nisto should begin with the identification of wild-caught specimens at the species level, but the identification is still in the preliminary stages due to a serious lack of information (Johnson 1975; Wakabayashi et al. 2017b 2020). This issue arises from the short duration of the nisto phase (about a week under culture) and the nisto’s unknown settling ground preference, which limits researchers’ collection abilities (Sekiguchi et al. 2007). In Arctidinae, complete larval development from hatching to metamorphosis has not been achieved yet in captive rearing, but the duration of the entire planktonic phase is estimated to be 8–9 months, with stages occurring prior to metamorphosis into the nisto stage (Robertson 1969a b; Crosnier 1972; Sekiguchi et al. 2007). Twenty-four nisto specimens from the wild were recorded and assigned to seven species of Scyllarides based on how their collection localities accorded with the adult distributions (Guérin-Méneville 1855; Pfeffer 1881; Miers 1882; Bouvier 1913; Barnard 1950; Chace Jr 1966; Michel 1968; Robertson 1968; Lyons 1970; Crosnier 1972; Johnson 1975). However, considering the overlapping distributions among some species of Scyllarides (Holthuis, 1991) and the potential of wide dispersal of phyllosoma larvae with a long planktonic life in this genus, the species assignment for the 24 nistos are not fully reliable. Such uncertainty can be solved nowadays by DNA barcoding, which has contribute to correct species identifications in many animals, including marine crustacean larvae (see Chen et al. 2013; Palero et al. 2016; Wakabayashi et al. 2017b 2020; Li et al. 2019; Wong et al. 2021; Ueda et al. 2021; Chow et al. 2022). We recently collected an Arctidinae nisto specimen from an area off the Japanese Ogasawara Islands. Based on molecular evidence, the nisto was confirmed to belong to Scyllarides squammosus (H. Milne Edwards, 1837). It is worthwhile to present detailed morphological descriptions of the nisto with a correct identification. MATERIALS AND METHODS Sampling An individual scyllarid nisto was found off Chichijima Island, part of the Ogasawara Islands, Japan (27°04'29.7"N, 142°11'06.3"E) in May 2017 during a scuba diving trip. It was trapped underwater in a plastic bottle after being photographed (Fig. 1) and then fixed with 99% ethanol on land. The depth at which it was found was 8 m, and the water temperature and salinity at the site were 20°C and 35, respectively. DNA analyses The sequences of the mitochondrial COI gene (COI) and 16S rRNA gene (16S) regions were used for molecular identification (Hebert et al. 2003; Hajibabaei et al. 2007; Vogler and Monaghan 2007; Bucklin et al. 2011). Genomic DNA was extracted from the fifth pereopod of the nisto specimen (NSMT-Cr 30950) using NucleoSpin Tissue XS (Macherey-Nagel, Germany) according to the manufacturer’s protocol. Abdominal muscles of S. squammosus (NTOU M00972 and NTOU M02303) were also used for genomic DNA extraction using the QIAGEN® DNeasy Blood and Tissue Kit (Cat. No. 69504, Valencia, CA). The target regions were amplified via a polymerase chain reaction (PCR) with the universal primer pairs, LCO1490/ HCO2198 (Folmer et al. 1994) for COI and 16SarL/16Sbr-H (Palumbi et al. 2002) for 16S PCR. Cycle sequencings were conducted following the methods described by Yoshimoto et al. (2020) and Yang et al. (2012). The nucleotide sequences of the studied species were deposited through the International Nucleotide Sequence Database Collaboration at the National Center for Biotechnology Information, Bethesda, MD, USA. Multiple sequence alignment was conducted using MUSCLE with default parameters on MEGA 7 (Kumar et al. 2016). Poorly aligned and non-informative sites on 16S were removed using Gblocks 0.91b (Castresana 2000). The final lengths of aligned COI and 16S were 500 bp and 413 bp, respectively. MEGA 7 was also used to compute the genetic distances as estimated page 2 of 12Zoological Studies 61:73 (2022) © 2022 Academia Sinica, Taiwan with the K2P model (Kimura 1980) among 10 species of Scyllaridae, including all nine species of Arctidinae with available sequences and Parribacus antarcticus (Ibacinae) as the outgroup for the phylogenetic tree analysis (Tables 1 and Table S1). The two sequences of COI and 16S were combined, and a phylogenetic tree was reconstructed using Bayesian inference (BI) based on the combined sequence dataset (COI+16S) with a total of 33 sequences (Table 1). Missing data were treated as ‘N’. The BI analysis was conducted using MrBayes 3.2.6 (Ronquist et al. 2012). Models of nucleotide evolution for the sequence of 16S and each codon position of COI were determined based on the smallest Akaike Information Criterion (AIC) score on MEGA 7 under default parameters using a neighbor-joining tree. For COI, a general time-reversible model with gamma distribution (GTR+G), Hasegawa-Kishino-Yano model (HKY), and GTR+G were determined to be the best-fit models for the first, second, and third codon positions, respectively. For 16S, GTR with gamma distribution having invariant sites (GTR+G+I) was the best-fit model. A tree search was run for 500,000 generations with trees every 1,000 generations, and the first 125,000 trees were discarded as “burn-in.” Support was given as posterior probabilities (Pp) calculated under default parameters. The reconstructed trees were visualized in Figtree v. 1.4.4 (Rambaut 2018). Morphological description Appendages were separated from the body under a stereo microscope (SZX-7, Olympus, Japan). The appendages were soaked in lactophenol for 5 to 10 min and then preserved on glass slides in CMCP10 (Polysciences, Inc., USA), which is a highly viscous mounting medium. Measurements and body drawings were made under the same stereo microscope while using a drawing tube. Observations and drawings of each appendage were made using a compound microscope (BX-51, Olympus, Japan) equipped with a drawing tube. The body length, cephalic length, carapace width, and telson length (BL, CL, CW, and TL, respectively) were defined from the anterior end of the antenna to the posterior end of the telson, from the anterior end to the posterior end of the carapace, the widest distance of the carapace, and from the anterior end to the posterior end of the telson, respectively. Terminology for the scyllarid nisto was based on studies by Wakabayashi et al. (2017b 2020). The drawings were edited using a drawing application ibis Paint X (Ibis inc., Japan). RESULTS DNA analyses The K2P genetic distances of the COI within species of the genus Scyllarides were 2.5% or lower (0%–0.6% in S. squammosus, 0.4% in S. brasiliensis, and 0%–2.5% in S. latus) except in S. haanii (0.6%– 11.3%). Distances between species of this genus ranged from 4.4% to 21.1% (Table S1). For 16S, the K2P distances within the Scyllarides species were 0%–0.7% in S. squammosus and 0%–1.0% in both S. latus and S. Fig. 1. Scyllarides squammosus (H. Milne Edwards, 1837), nisto. A: in situ, live (photographed by Hideki Abe, reproduced with permission from Wakabayashi et al. 2017a, p. 59 in which the nisto was named as Scyllarides sp.); B: fixed specimen (NSMT-Cr 30950) in 99% ethanol. Scale bar = 5 mm. page 3 of 12 Zoological Studies 61:73 (2022) © 2022 Academia Sinica, Taiwan haanii. Our nisto specimen showed the K2P distances of 0.2%–0.6% for COI and 0.2%–1.0% for 16S from S. squammosus, respectively (Table S1). Two Hawaiian and Chilean specimens of Arctides regalis showed a K2P distance of 0%–0.6% for both gene regions, but the specimen from Réunion had a distance of 5.3% to 5.5% for COI and 3.3% for 16S from the former specimens. The K2P distances between our nisto and the species of Arctides were greater than 20% and 12% for COI and 16S, respectively. The BI tree demonstrated that the conspecific sequences were monophyletic (Fig. 2). The nisto specimen examined in this study was placed in a clade with S. squammosus with strong support (Pp = 1). TAXONOMY Order DECAPODA Latreille, 1802 Family SCYLLARIDAE Latreille, 1825 Table 1. List of arctinid species and outgroup lobster used for DNA analyses Species Voucher Locality GenBank Accession Nos. Source COΙ 16S Scyllaridae Arctidinae Scyllarides squammosus NTOU M02303 Hepingdao, Taiwan OP379522 OP379972 this study NTOU M00972 Market in Taiwan JN701654 JN701689.2 Yang et al. (2012) n.a. China MK783265 * MK783265 * Liu et al. (2019) n.a. South Africa KX275388 #1 - Singh et al. (2017) (nisto) NSMT-Cr 30950 Ogasawara, Japan OP379523 OP379973 this study (phyllosoma) NHMUK-2015.3285 Coral Sea KX373667 #2 - Palero et al. (2016) (phyllosoma) n.a. East Australian Sea MK371349 #3 - Woodings et al. (2019) (phyllosoma) n.a. East Australian Sea MK371352 #4 - Woodings et al. (2019) Scyllarides brasiliensis KC6292 n.a. KF827966 KF828186 Bracken-Grissom et al. (2014) n.a. Brazil JX896701 #5 - Rodríguez-Rey et al. (2014) n.a. Brazil JX896724 #6 - Rodríguez-Rey et al. (2014) Scyllarides haanii n.a. n.a. MN817127 * MN817127 * Liu et al. (unpubl.) KC6018 Hawaii, USA JN701656 JN701690 Yang et al. (2012) KC6019 Hawaii, USA - JN701691Δ1 Yang et al. (2012) NTOU M00973 Taiwan JN701655 #7 - Yang et al. (2012) n.a. Easter Island, Chile MW699539 #8 - Báez et al. (2022) Scyllarides deceptor CCDB BRA 4190 Brazil MF490045 MF490148 Mantelatto et al. (2017) Scyllarides latus n.a. n.a. KC107814 * KC107814 * Shen et al. (2013) FP0014 n.a. FJ174947 FJ174907 Palero et al. (2009) JSDAz37 Portugal JQ306108 #9 - Matzen da Silva et al. (2011) n.a. n.a. JQ623990 #10 - Keskin (unpubl.) n.a. Izmir Bay, Turkey KC311420 #11 - Keskin and Atar (2013) n.a. Portugal JF928188 #12 - Froufe et al. (2019) n.a. n.a. - DQ377974Δ2 Cannas et al. (unpubl.) Scyllarides herklotsii ICMD 2301998 Eastern Atlantic Ocean FJ174946 FJ174906 Palero et al. (2009) Scyllarides nodifer ULLZ7845 Northern Gulf of Mexico, USA JN701657 JN701692 Yang et al. (2012) n.a. Gulf of Mexico, USA - U96088Δ3 Tam and Kornfield (1998) Arctides antipodarum KC6076 Australia - JN701688Δ4 Yang et al. (2012) Arctides regalis MNHN-IU-2009-462 Réunion JN701651 JN701685 Yang et al. (2012) KC6004 Hawaii, USA JN701653 JN701687 Yang et al. (2012) KC6003 Hawaii, USA JN701652 JN701686 Yang et al. (2012) n.a. Chile MW699538 #13 - Araneda et al. (unpubl.) Ibacinae Parribacus antarcticus NTOU M00975 Taiwan JN701666 JN701701 Yang et al. (2012) n.a.: not available. See figure 2 caption for the meaning of hash (#), delta (Δ), and asterisk (*) symbols. page 4 of 12Zoological Studies 61:73 (2022) © 2022 Academia Sinica, Taiwan Genus Scyllarides Gill, 1898 Scyllarides squammosus (H. Milne Edwards, 1837) Nisto Material examined: Nisto, NSMT-Cr 30950, BL = 39 mm, CL = 14.5 mm, CW = 17 mm, TL = 9 mm. Description: Carapace (Fig. 3A, 3B): Wider than longer (CW/CL 1.17) with maximum width in the middle; surface with few tubercules, entirely furnished with fine setae; anterior margin smooth, a prominent horn present at both angles; lateral margin possessing a distinct post-cervical incision, with 8 (right) to 9 (left) anterolateral and 14 (right) to 12 (left) post-lateral teeth; rostral short with blunt end; 2 prominent tubercules and 1 small tubercule on the inner and posterior edges of orbits; gastric tooth possessing 2 prominent tubercles anteriorly and 4 small setae posteriorly; cardiac tooth bearing 2 rows of prominent tubercules anteriorly and 2 rows of small setae posteriorly, cardiac ridge with small setae; 10 small setae (8 each on the right and left sides, 2 in the middle) present along the post-cervical groove; 10 small setae in a row along post-lateral teeth; 5 small setae present at middle of posterior margin. Antennule (Fig. 4A): Peduncle 3-segmented with flagellated distal end; proximal segment bearing dense long setae on ventral side; second segment possessing small setae scattered; third segment almost naked; accessory flagellum 18-articulated as long as the primary flagellum; primary flagellum thicker than accessory flagellum, 15-articulated, group of aesthetascs present on articles 5–12. Antenna (Fig. 3A): 4-segmented, biramous, broad, flat; proximal segment inverted triangle, anterior margin with a ridge at middle; second segment with serrated lateral margin, prominent horn at anterolateral angle, a small notch on anteromedial margin, 2 small teeth on medial margin; third segment bearing a prominent tooth on anteromedial angle; distal segment surrounded with finely serrated margins. Mandible (Fig. 4B): Poorly developed, slightly Fig. 2. Bayesian phylogenetic tree of the subfamily Arctidinae with partial sequences of COI and 16S. Posterior probability (Pp) values are shown when above 0.8. The tree was constructed basically with both the COI and 16S sequences, but the operational taxonomic units (OTUs) with hash (#) were inferred only from the COI and those with delta (Δ) were inferred only from the 16S, due to a lack of information. Asterisks (*) indicate the OTUs with sequences resulting from the mitogenome. 0.02 1.0 1.0 1.0 0.97 1.0 1.0 0.82 0.94 0.91 1.0 1.0 1.0 1.0 0.85 1.0 1.0 1.0 1.0 S. squammosus nisto_Japan [NSMT-Cr 30950] S. squammosus_Taiwan [NTOU M00972] S. squammosus_Taiwan [NTOU M02303] S. squammosus phyllosoma_East Australian Sea #3 S. squammosus_South Africa #1 S. squammosus phyllosoma_East Australian Sea #4 S. squammosus phyllosoma_Coral Sea [NHMUK-2015.3285] #2 S. squammosus_China * S. brasiliensis [KC6292] S. brasiliensis_Brazil #5 S. brasiliensis_Brazil #6 S. haanii_Hawaii [KC6018] S. haanii_Hawaii [KC6019] Δ1 S. haanii_Taiwan [NTOU M00973] #7 S. haanii * S. haanii_Chile #8 S. deceptor_Brazil [CCDB BRA 4190] S. latus * S. latus [FP0014] S. latus_Portugal [JSDAz37] #9 S. latus #10 S. latus_Portugal #12 S. latus Δ2 S. herklotsii_Eastern Atlantic Ocean [ICMD 230 1998] S. nodifer_Northern Gulf of Mexico [ULLZ 7845] S. nodifer Δ3 Parribacus antarcticus A. regalis_Chile #13 A. regalis_Hawaii [KC6003] A. regalis_Hawaii [KC6004] A. regalis_Réunion [MNHN-IU-2009-462] A. antipodarum_Australia [KC6076] S. latus_Turkey #11 page 5 of 12Zoological Studies 61:73 (2022) © 2022 Academia Sinica, Taiwan asymmetrical; palp present. Maxillule (Fig. 4C): Uniramous; poorly developed; basal endite with 5 small tubercules on anterior margin; coxal endite unarmed on anterior margin, bearing 3 small setae on basal part. Maxilla (Fig. 4D): Flattened; endopod slightly differentiated with 8 setae and 3 small spines on the medial margin; endites differentiated, 4 (2+2) and 7 (5+2) setae on anterior ends of coxal and basal endites, 1 and 2 setae present on the inner proximal parts of coxal and basal endites, respectively; hair-like setae lining the outer margin of scaphognathite. First maxilliped (Fig. 4E): Biramous, coxa and basis undifferentiated, flattened; endopod unsegmented, smooth; exopod with 15 marginal setae on proximal part, small setae on distal end; epipod membranous, expanding posteriorly, bearing 5 long setae; 2 small setae present at the distal end of basal endite. Second maxilliped (Fig. 4F): Biramous, coxa and basis undifferentiated; endopod 4-segmented with 0,1,13,0 setae; exopod incompletely segmented, longer than endopod, 2 small setae at distal end. Third maxilliped (Fig. 4G): Biramous, coxa and basis differentiated; endopod 5-segmented, carpus with dense setae (~30), propodus with 14 setae, dactylus with 4 long setae on inner margin; exopod segmented, poorly developed with 7 small setae on distal end. Pereiopods (Fig. 5A): Pereiopods 1–4 biramous, coxa and basis differentiated, endopods 5-segmented with almost equal length, exopods degenerated, merus and carpus bearing a prominent horn at distal end, ischium of pereiopod 2–4 with 4 small spines; Pereiopod 5 uniramous, shortest, merus possessing a prominent horn at the distal end, ischium bearing a conspicuous spine pointing posteriorly. Pleon (Fig. 3A, 3B): Tergum of pleonite 1 smooth; median carina distinct on tergites of pleonites 2–5; terga of pleonites 2–3 with a few spicules, small spines on median carina, a small tooth at center of posterior margin; terga of pleonites 4–5 smooth, a large tooth present at center of posterior margin; pleura of pleonites 2–5 serrated with large tooth, 2 small spines on the medial margins of pleura. Pleopods (Fig. 5B): Present on pleonites 2–5; biramous; 2 setae at medial distal angle of protopod; exopod surrounded with 35 (pleopod 1), 37 (2), 38 (3), 33 (4) long plumose natatory setae, each possessing 6 (pleopod 1), 12 (2), 14 (3), 12 (4) small plumose setae at distal end; endopod surrounded with 48 (pleopod 1), 52 (2), 48 (3), 48 (4) long plumose natatory setae, each possessing 0 (pleopod 1), 2 (2), 5 (3), 4 (4) small plumose setae at distal end, appendix interna not reaching distal end of endopod, with approximately 12 cincinnuli at the tip, a plumose seta present just beside the cincinnuli for pleopods 2 and 4. Uropod (Fig. 5C): A tooth present at lateral Fig. 3. Scyllarides squammosus (H. Milne Edwards, 1837), nisto. A: Body, dorsal, a pair of antennules omitted; B: Body, lateral (left), ventral side of telson in gray. Scale bar = 5 mm. page 6 of 12Zoological Studies 61:73 (2022) © 2022 Academia Sinica, Taiwan Fig. 4. Scyllarides squammosus (H. Milne Edwards, 1837), nisto. A: Antennule, right, ventrolateral; B: Mandibles, ventral; C: Maxillule, right, ventral; D: Maxilla, right, ventral; E: First maxilliped, right, dorsal; F: Second maxilliped, right, ventral; G: Third maxilliped, right, dorsal. Scale bars: A, E, G = 1 mm; B, F = 0.5 mm; C, D = 0.1 mm. page 7 of 12Zoological Studies 61:73 (2022) © 2022 Academia Sinica, Taiwan margins of exopod and endopod; medial margin of endopod serrated. Telson (Fig. 5D): A tooth present at the proximal, lateral margins. Color (Fig. 1): Body entirely transparent in live specimen except a yellow line along the anterior margin of carapace and gills in pink. Specimen preserved in ethanol entirely white with transparent body margins. Fig. 5. Scyllarides squammosus (H. Milne Edwards, 1837), nisto. A: Pereiopods, left, ventral, degenerated exopods shown in shaded; B: Pleopod 3, right, anterior, with enlarged distal ends of pleopods 1 and 3 (a) and pleopods 2 and 4 (b); C: Telson, dorsal; D: Uropod, right, dorsal. Scale bars: A, D = 3 mm; C = 2 mm, B = 1 mm. page 8 of 12Zoological Studies 61:73 (2022) © 2022 Academia Sinica, Taiwan DISCUSSION The nisto specimen showing the morphological features of the subfamily Arctidinae collected off the coast of the Ogasawara Islands was confirmed to be S. squammosus based on DNA analyses. Although the larval development of the planktonic phase has been well documented in this species, the transition phase has scarcely been recognized. Mid to late development from stages VI to XII (final) was illustrated based on wild-caught specimens (Michel 1968; Johnson 1971 1977; Palero et al. 2016; Chow et al. 2022), and the identities of larval specimens examined by Palero et al. (2016) and Chow et al. (2022) were confirmed based on molecular evidence. This study finally found the missing puzzle piece in the early life history of S. squammosus with the description of the nisto stage, which plays a role in the species’ transition from planktonic to benthic life. Captive breeding of this species was attempted by Saisho and Sone (1971) using Artemia as food, and they described the early larval development from newly hatched phyllosoma to instar 6. The youngest larva of S. squammosus collected from the wild assessed as stage VI (Palero et al. 2016) was much more developed than the larva at instar 6 (Saisho and Sone 1971), suggesting that several stages in the early development of this species are still missing. The adult S. squammosus inhabits oceanic reefs and is widely distributed in the Indo-West Pacific from the Hawaiian Islands to the east coast of Africa (Holthuis 1991). In Japan, this species has been found south of the Boso Peninsula, Chiba Prefecture, and the Ryukyu and Ogasawara Archipelagoes (Miyake 1982; Nishikiori and Sekiguchi 2001). Indeed, the presence of the berried females of this species have been confirmed off the coast of Chichijima Island (Nishikiori and Sekiguchi 2001) from which our nisto specimen was found. This particular nisto probably reached the coastal area of Chichijima Island to seek ground for settlement. Since another species, S. haanii, is also present off Chichijima Island (Nishikiori and Sekiguchi 2001), the nisto found off the Ogasawara Islands could either be S. squammosus or S. haanii. Genetic distances between the two species are greater than 15.7% for COI and 10.8% for 16S; thus, the two species could be clearly distinguished using the K2P genetic distance. The species with the closest genetic distance from our nisto specimen (S. squammosus) is S. brasiliensis. The adult S. brasiliensis is distributed in the Southern Atlantic from Brazil (the States of Maranhão to Bahia) to the Dominican Republic (Holthuis 1991). It is hard to believe that a nisto of S. brasiliensis could be found off the Ogasawara Islands. Nisto has been described for seven out of 14 species of the genus Scyllarides, although all previous descriptions had been based on wild-caught specimens that had not undergone molecular identification. Our nisto specimen agreed well with the description of a nisto identified as S. squammosus recorded from off the coast of New Caledonia (Michel 1968), as both nistos possess pleurae 2 to 5 surrounded by large sharp teeth on both the anterior and posterior margins, though minor differences between them are also present in body length and CW/CL ratio. Michel’s nisto is probably S. squammosus, after considering the morphological similarity to our nisto. Nevertheless, we cannot eliminate the possibility of other species with similar morphology and minor size differences, since the existence of undescribed species in the Pacific Ocean that are closely related to S. squammosus has been suggested (Chow et al. 2022). Pseudibacus pfefferi (Miers, 1882) collected from off Mauritius is considered the nisto stage of S. squammosus (Bouvier 1913). However, it is hard to assess whether P. pfefferi is likely this species due to an ambiguous description for the pleurae. Pleura 2 of the nisto of S. nodifer is distally subacute with a small spine at the posterior angle and a blunt spine at the anterior angle (Lyons 1970). The posterior margin of pleura 2 is smooth in the nistos of S. herklosti (Chace Jr 1960; Crosnier 1972) and S. astori (Johnson 1975). In the nisto of S. aequinoctialis, six rounded teeth are present on the anterior margin of pleura 2 with an acute spine at the lower extremity, and the upper third of posterior margin bears seven rounded or smooth teeth (Lyons 1970). Pseudibacus gerstaeckeri (Pfeffer 1881) was assigned to the nisto stage of S. aequinoctialis (Bouvier 1913) after which its certainty was morphologically confirmed (Lyons 1970). Guérin-Méneville (1855) described that all pleurae of Pseudibacus veranyi [identified as S. latus by Bouvier (1913)] are scalloped and prickly with teeth of different sizes and arrangements. The CW/CL is 1.5 in S. aequinoctialis (calculated based on Lyons’s illustration), whereas that value is 1.2 to 1.3 in other species, including our nisto specimen. The nisto of S. nodifer has a low blunt tubercle on the medial margins of pleura, while our nisto presented two lows of blunt tubercles. The nisto described by Barnard (1950) that was later identified as S. elisabethae by Chace Jr (1966) has a carapace with its widest distance on the anterior margin. The nistos of other species show the widest distance on the middle part of carapace. Several diagnostic characteristics are known to distinguish the adult specimens of Scyllarides from those of Arctides: (1) ornamental patterns on carapace and pleonites (tubercules in Scyllarides versus spines in Arctides), (2) transverse groove on tergum of pleonite 1 (absent in Scyllarides versus present in Arctides), page 9 of 12Zoological Studies 61:73 (2022)