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Daphnia Strain and Culture Conditions 水生動物 第 2021 巻 令和 3 年 5 月 Morphological descriptions for late stage phyllosomas of furrow lobsters (Crustacea, Decapoda, Achelata, Palinuridae) collected off Okinawa Islands, Japan Kooichi Konishi*, Takashi Yanagimoto and Seinen Chow Fisheries Resources Institute, Japan Fisheries Research and Education Agency, 2-12-4 Fukuura, Yokohama, 236-8648 Japan. *Corresponding author; e-mail: [email protected] Abstract Two late stage phyllosomas collected from off Okinawa Islands were first determined to be Justitia or Nupalirus because of the chelate pereipods. DNA analysis indicated these to be Justitia longimana and Nupalirus japonicus. Morphological larval description with note on palinurid phyllosomas is given. Key words: Justitia; Nupalirus; phyllosoma; morphology; taxonomy Introduction planktons collected off Okinawa Islands. Seven palinurid genera, Justitia, Linuparus, Primary investigation of the morphology based Nupalirus, Palinustus, Panulirus, Puerulus, and on key characters from previous larval works Palinurellus have been recorded in Japanese and (Johnson and Robertson 1970; Sekiguchi 1986a, the adjacent waters, (Holthuis 1991; Chan and Yu b) indicated that these phyllosomas belonged to 1993; Miyake 1998; De Grave et al. 2009). the genera Justitia or Nupalirus, but the species Among them, furrow spiny lobsters of the genera could not be identified. In this study, we analysed Justitia and Nupalirus have been known as rare mtDNA 16S rDNA sequences of these species (Holthuis 1991), and unique appearance phyllosomas, then identified them to be Justitia among palinurids as “chelate Achelata”, at least longimana and Nupalirus japonicus. The aims of in the male of Justitia. The larval development of this study are to give morphological descriptions Justitia and Nupalirus has been described based of the phyllosomas and to compare their exclusively on plankton samples to date (Johnson morphological characters with those known for 1969; Robertson 1969; Baisre 1969; Johnson and the other genera of palinurid lobsters distributing Robertson 1970; Aoyama et al. 1984; Inoue et al. Japanese waters. 2004). Thus, no morphological descriptions of the phyllosomas of furrow spiny lobsters has Materials and Methods been done by more convincing method which Two late stage phyllosoma larvae were insure accurate parentage yet, i.e., laboratory- collected using MOHT (Matsuda, Oozeki & Hu rearing or DNA-barcording. Midwater Trawl) net towed stepwise at 50 m and During our research cruises in southern Japan 35 m depth for 10 min each in off Okinawa waters, two late stage phyllosomas, different Islands, of which one (designated by SP30) was from typical palinurid species in having chelate caught on 10 November 2004 at 22°33’N or subchelate pereiopods, were obtained from the 124°44’E and the other (designated by KY1404) Aquatic Animals | May 12, 2021 | Konishi et al. AA2021-7 on 19 November 2014 at 24°00’N 124°00’E. The (CW) and length (CL) of cephalic shield (CS), larval specimens were fixed with 80 % ethanol, and thorax width (TW) (see Fig. 1) were photographed and measured on board, and measured according to Higa and Shokita (2004) transferred to the laboratory for subsequent and Palero et al. (2008). molecular and morphological analyses. Total The voucher specimens are deposited at the DNA was extracted from a piece of pereiopod Hokkaido University Museum under the using a DNA extraction kit (Qiagen inc.). PCR accession No. ICHUM-6251-6252 primers to amplify partial mtDNA 16S rDNA region were 16Sar-L and 16Sbr-H by Palumbi et Results al. (2002). PCR was performed in 10 μl of total Molecular identification reaction mixture containing 5–10 ng template Nucleotide sequences determined are 508 bp DNA, 1 μl of 10 × buffer, 1 μl of dNTP (2.5 mM for SP30 and 505 bp for KY1404, which are each), 0.5 μl of each primer (10 μM), and 0.25 available in the International Nucleotide units of EX Taq HS DNA polymerase (Takara, Sequence Database Collection (INSDC) under Shiga, Japan) on an ABI 9700 Thermal cycler accession numbers of LC619699 and LC619700. (Applied Biosystems, Foster City, CA, USA). BLAST top hit sequence for SP30 was Justitia Reaction mixtures were preheated at 94°C for 2 longimana (AF502953) with K2P distance of min, followed by 35 amplification cycles (94°C 0.2 % and that for KY1404 was Nupalirus for 30 s, 55°C for 30 s, and 72°C for 50 s), with japonicus (KF828188) with K2P distance of a final extension at 72°C for 7min. The PCR 0.4%. These values are sufficiently small for products were treated with ExoSap-IT species identity (see Vences et al. 2005; (Amersham Biosciences) to remove primers and Lianming et al. 2014; Kannan et al. 2020). K2P used as template DNA for cycle sequencing distance between SP30 and KY1404 was 27.3 %. reactions using Big Dye Terminator Cycle Sequencing Kit (Ver.3.1, Applied Biosystems) Morphological description of the phyllosomas with PCR primers. Sequencing was conducted on Justitia longimana an ABI Prism 3730XL (Applied Biosystems). Stage VII (SP30, Figs. 1, 2) Nucleotide sequences determined were subjected Dimensions: TL = 17.2 mm, CL = 12.4 mm, to BLAST homology searches (Altshul et al. CW = 10.9 mm, TW = 10.3 mm. 1990) in GenBank to find identical or similar Cephalothorax (Fig. 1A): CS nearly circular in sequences. Calculation of Kimura’s two outline, slightly longer than wide 1.14 in parameter distance (K2P) between sequences CL/CW ratio, and 1.06 in CW/TW ratio. was performed using MEGA6 (Tamura et al. Posterolateral margin of the CS not covering 2013). the base of maxilliped 3. Posterior margin of After the molecular analysis, the appendages thorax concave between the coxae of were dissected using fine insect pins. pereiopod 3. Eyes stalked, stalk longer than Observations and drawings were made with an antennule and antenna. aid of drawing tube attached to an Olympus Antennule (Fig. 2A): Biramous, peduncle 3- BX51 microscope and a SZX10 segmented, each segment without setae. Inner stereomicroscope. Total body length (TL), width flagellum shorter than outer flagellum. Aquatic Animals | May 12, 2021 | Konishi et al. AA2021-7 Antenna (Fig. 2B): Uniramous, 2-segmented, Maxilla (Fig. 2E): Basis with 2 thin anterior setae. slightly shorter than antennule. Scaphognathite with 37 marginal thin Mandibles (Fig. 2C): Slightly flattened dorso- plumose setae. ventrally, asymmetrical in dentition. Incisor Maxilliped 1 (Fig. 2F): Small bud, 3 setae on process and medial gnathal edge with a series basal part. Exopod short process without setae. of teeth. Molar process crowned by many Maxilliped 2 (Fig. 2G, 2H): Endopod 3- denticules and minute papillae. Labrum and segmented, with a stout long seta and 2 short paragnath well-developed, covers distal inner setae on its distal part. Exopod unsegmented half portion of mandible. with 4 distal plumose setae. Maxillule (Fig. 2D): Basal endite with 2 stout Maxilliped 3 (Fig. 1A): Endopod 3-segmented, cuspidate spines and 4 subterminal setae many setae on distal segment. Exopod with 19 while coxal endite with 2 stout setae and 3 annulations, eeach eannulation ewith ea pair of short setae. Two setae on the presumptive natatory plumose setae. endopod area (Fig. 2D, arrow). Fig. 1. Justitia longimana, phyllosoma larva, stage VII. A: whole animal in dorsal view, B-C: enlarged distal part of pereiopod 1 and 3, D: pleon. TL: total body length, CW: cephalic shield width, CL: cephalic shield length, TW: thorax width. Scale bars: A = 5.0 mm; B-D = 1.0 mm. Aquatic Animals | May 12, 2021 | Konishi et al. AA2021-7 Pereiopod 1 (Fig. 1A, 1B): Endopod 3- distally and terminating in 2 stout setae segmented, chelate in distal part. Exopod with between which the dactylus close (Fig. 1C). 17 annulations, each annulation with a pair of Exopod as in pereiopod 1. natatory plumose setae. No conspicuous coxal Pereiopod 4 (Fig. 1A): Endopod 2-segmented. and subexopodal spines on all pereiopods. Exopod with 16 annulations, otherwise as in Pereiopod 2 (Fig. 1A): Longest among pereiopod 1. pereiopods. Dactylus of endopod long sickle- Pereiopod 5 (Fig. 1D): Two-segmented, shaped with setae and spines on its inner uniramous projection, reaching posterior margin, resembling raptorial claw. Exopod margin of pleonal somite 4. with 18 annulations, otherwise as in pereiopod 1. g Pleon (Fig. 1D): Somites segmented, rudiments Pereiopod 3 (Fig. 1A): g Distal gpart of g endopod of pleopods and uropod as biramous buds. subchelate ggwith ggthe gpropodus ggextending Fig. 2. Justitia longimana, phyllosoma larva, stage VII. A: antennule, B: antenna, C: mandibles in dorsal view, D: maxillule, arrow indicating presumptive endopod, E: maxilla, F: maxilliped 1, G: maxilliped 2, H: enlarged distal part of endopod of maxilliped 2. Scale bars: A, B, G = 1.0 mm; C-F, H = 0.1 mm. Aquatic Animals | May 12, 2021 | Konishi et al. AA2021-7 Nupalirus japonicus Antennule (Fig. 4A): Biramous, peduncle 3- Stage VIII (KY1404, Figs. 3-5) segmented, each segment without seate. Inner Dimensions: TL= 17.4 mm, CL = 12.8 mm, CW flagellum shorter than outer flagellum. = 9.5 mm, TW = 10.4 mm. Antenna (Fig. 4B): Three-segmented, longer Cephalothorax (Fig. 3A): CS oval in outline, than antennule, but distal part missing. longer than wide, 1.35 in CL/CW ratio and Mandibles (Fig. 4C): Slightly flattened dorso- 0.98 in CW/TW ratio. Posterolateral margin ventrally, asymmetrical in dentition. Incisor of the CS covering the base of maxilliped 3. process and medial gnathal edge with a series Posterior margin of thorax concave from the of teeth which densely in left mandible. Molar coxae of the pereiopod 3. Eyes stalked, stalk process crowned by many denticules and longer than antennule and antenna. minute papillae. Fig. 3. Nupalirus japonicus, phyllosoma larva, stage VIII. A: whole animal in dorsal view, B-E: enlarged distal part of pereiopod 1-4, F: abdomen in ventral view.
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