Egg Capsule of Phymorhynchus Buccinoides (Gastropoda: Turridae) in a Deep-Sea Methane Seep Site in Sagami Bay, Japan
Total Page：16
File Type：pdf, Size：1020Kb
VENUS 67 (3-4): 181-188, 2009 Egg Capsule of Phymorhynchus buccinoides (Gastropoda: Turridae) in a Deep-Sea Methane Seep Site in Sagami Bay, Japan Hiromi Watanabe1, 2*, Katsunori Fujikura1, Gin Kinoshita1, 3, Hiroyuki Yamamoto1 and Takashi Okutani1 1 Japan Agency for Marine-Earth Science and Technology, 2-15 Natsushima, Yokosuka, Kanagawa 237-0061, Japan; *[email protected] 2 Research Fellow of the Japan Society for the Promotion of Science 3 Department of Environmental Dynamics and Management, Hiroshima University Graduate School of Biosphere Science, 1-4-4 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8528, Japan Abstract: Among deep-sea gastropods, the characteristics of their egg capsules can reveal important clues regarding early life history traits. The egg capsule of P. buccinoides in a deep-sea methane seep site in Sagami Bay, Japan was identified from partial mitochondrial DNA sequences. DNA sequence comparison supported the suggestion that co-occurring egg capsules belong to P. buccinoides. The egg capsule is translucent and semicircular in shape. Its dimensions average 10.8 mm in length, 5.3 mm in width and 4.2 mm in height. Capsules contained an average of 1098 eggs or larvae. P. buccinoides deposits larger egg capsules that contain a larger number of eggs and larvae than other known cold water turrids. The distribution pattern of the egg capsules was discontinuous and irregular but biased towards the shells of Bathymodiolus. Keywords: chemosynthetic biological communities, egg capsule, Turridae Introduction The study of life-history traits such as adult fecundity and egg and larval size and number provide important clues for discerning the mode of larval development in marine invertebrates (Ramirez-Llodra, 2002). These studies normally require year-round sampling to determine reproductive seasonality; however, the relative inaccessibility of deep-sea chemosynthetic biological communities (CBCs) makes this difficult. CBCs are an important component of deep- sea ecosystems, because of their relatively large biomass (up to 22.19 kg / m2; cf. Fujikura et al., 1995). Because laboratory culture of deep-sea organisms has proved difficult, much of what we know about the mode of reproduction in deep-sea invertebrates in CBCs has come from the study of their life-history traits based on preserved specimens. Gastropods are a major component of CBCs, accounting for more than 50% of observed animals in some cases (Bouchet & Warén, 1991). The most advanced gastropods lay eggs in the form of benthic egg masses or capsules in various shapes (Amio, 1963) and in general, the capsules are left without any parental care after deposition. Some gastropod egg capsules in CBCs have been identified and observed (Berg, 1985; Gustafson et al., 1991; Bouchet & Warén, 1993; Warén & Bouchet, 2001). However, in most cases, identification of a deposited egg capsule is based on its morphology and the co-occurrence of adult gastropods. Sometimes the species originally identified as having deposited certain egg capsules has been modified later based on additional evidence (e.g. Warén & Bouchet, 2001). Thus, the determination of DNA sequences, which do not vary during deve lopment, from eggs and larvae contained in egg capsules has become the most reliable way to correctly identify the egg capsule to species. The turrid gastropod Phymorhynchus buccinoides Okutani, Fujikura & Sasaki 1993 lives in a 182 H. Watanabe et al. deep-sea methane seep area at a site off Hatsushima Island, Sagami Bay, Japan. P. buccinoides is a large species (approximately 6 cm in shell height) and its population density is estimated at up to 63 individuals / m2 (Okutani et al., 1993; Fujikura et al., 2002) indicating that it is one of the more important components of the CBC in the site off Hatsushima Island. Egg capsules found at this site were assigned to P. buccinoides because adult whelks are frequently sighted nearby (Fujikura et al., 2002; Sasaki et al., 2007), but the characteristics of the egg capsule have not been reported to date. The present study confirms the parent gastropod of the egg capsule based on DNA sequence analysis, and describes characteristics of the egg capsule. Materials and Methods During R/V Natsushima and ROV Hyper-Dolphin cruise NT06-04 (March 11-18, 2006) in Sagami Bay, the distribution of adult Phymorhynchus buccinoides and an egg capsule that had been assumed to be that of P. buccinoides were observed using an ultra-sensitive HDTV camera (SS-HD3500, Hitachi Electronics Services Co., Ltd., Tokyo, Japan) and a digital still camera (DPC-7000, Deep Sea Systems International, Inc. Falmo uth, MA, USA) installed on the ROV. Both adult shells and the egg capsules were observed on only four outcrops in a 25 m2 area (Fig. 1A). The egg capsules and adult P. buccinoides were collected for DNA sequence analyses and subsequent observations. Another turrid gastropod, Oenopota sagamiana, at the same site was also collected for DNA sequence analyses for reference. DNA from each of the above mentioned samples was extracted using the DNeasy Tissue Extraction Kit (Qiagen, Hilden, Germany). Based on the extracted DNA, partial sequences of the genes encoding mitochondrial cytochrome oxidase c subunit I (COI) and 16SrRNA (16S) were amplified using the ExTaq kit (TaKaRa, Kyoto, Japan) with the previously designed universal primer sets COI-3 and COI-6 (Watanabe & Yokobori, 1993) and 16Sar and 16S br (Palumbi, 1996), respectively. PCR products were purified with Wizard SV Gel and the PCR Clean-Up System (Promega, Madison, WI, USA), and cycle sequencing was performed using the BigDye Terminator Kit ver. 3.1 (Applied Biosystems, Foster City, CA, USA). The products were purified with Performa DTR gel filtration cartridges (EdgeBiosystems, Gaithersburg, MD, USA) before sequencing. The DNA fragmen ts were analyzed using an ABI3130 automated sequencer (Applied Biosystems). All the procedures above were performed in accordance with the manufacturers’ instructions. The DNA sequences of the egg capsule and adult whelks were compared and phylogenetic trees based on these data were constructed with MEGA ver. 3.1 (Kumer et al., 2004). To describe the distribution of the egg capsule, we examined the p reserved molluscan shells (Bathymodiolus platifrons, Bathymodiolus japonicus, and P. buccinoides) that are used as substrata for the egg capsule deposition, because no egg capsule was found on abiotic substrata by ROV observation. All the animals subjected to this examination were collected from the same outcrop on which adult P. buccinoides were observed. The numbers of the shells with or without egg capsules were counted. Morphology of 21 egg capsules was carefully examined. The lengths, width and height of egg capsules were measured using a light microscope (MZ16F, Leica Microsystems, GmbH, Germany) equipped with a video measuring system (ACT2-U ver. 1.62, Nikon, Tokyo, Japan). The number of eggs in a single egg capsule could be counted for only seven out of 21 egg capsules, because of bad fixation conditions, and the lengths of the major and minor axes of 20 eggs in each of the seven capsules (140 total eggs) were measured. Egg Capsule of Phymorhynchus buccinoides 183 A B C D E F G Fig. 1. Adult P. buccinoides and egg capsules. A. In situ observation. Yellow arrowheads indicate a patch of egg capsules; red arrowheads aggregation of adults. B. Egg capsule on adult P. buccinoides shell. C. Egg capsules on B. japonicus shell. D. Egg capsules on B. platifrons shell. E. Frontal view of egg capsule. F. Lateral view of egg capsule. G. Overhead view of egg capsule. Scale bars indicate 1 cm in B, C and D, and 5 mm in E, F and G. 184 H. Watanabe et al. Egg 3 53 Egg 2 66 Egg 1 A 100 P. buccinoides 2 P. buccinoides 3 P. buccinoides 1 O. sagamiana 1 99 O. sagamiana 2 71 O. sagamiana 3 L. cerithiformis 0.02 P. buccinoides 2 P. buccinoides 3 97 Egg 1 B P. buccinoides 1 Egg 3 Egg 2 O. sagamiana 3 O. sagamiana 1 100 O. sagamiana 2 L. cerithiformis 0.02 Fig. 2. Molecular phylogenetic trees of egg capsules and turrid gastropods at the site off Hatsushima Island. A. Phylogenetic tree based on 357bp of partial sequence of mitochondrial COI gene. B. Phylogenetic tree based on 543 bp of partial sequence of mitochondrial 16S gene. As an outgroup, the turrid gastropod Lophiotoma cerithiformis (DQ284754) was used. Results Partial sequences of the COI (357-bp) and 16S (545-bp) regions were obtained. These sequences were submitted to the DDBJ, GenBank, and EMBL databases under accession numbers AB365483-AB365486 and AB453969-AB453975. The sequences obtained from egg capsule contents were identical to those from one of the adult Phymorhynchus buccinoides and were placed in a different clade from Oenopota sagamiana (Fig. 2). Thus, DNA sequence analysis showed that the egg capsule belongs to P. buccinoides. The egg capsules were not distributed in regular manner on a single substratum (Figs. 1B-D). Except for a single egg capsule observed on the shell of a P. buccinoides specimen (Fig. 1B) and two on a Bathymodiolus japonicus shell (Fig. 1C), all of the remaining egg capsules were found on the shells of Bathymodiolus platifrons (Fig. 1D; Table 1). The egg capsules are semicircular in shape and translucent white or yellowish-white in color (Fig. 1E-G). Egg capsules are attached to the substratum with a membranous base. A prominent axial ridge runs along the major axis of the capsule. Most capsules have a hole at the top of the axial ridge (Fig. 1F). The axial ridge is easily detached when the specimens are dried. The measurements of capsules and eggs are shown in Egg Capsule of Phymorhynchus buccinoides 185 Table 1. Numbers of examined substrata with or without egg capsules. Substrata With egg capsule Without egg capsule B. platifrons 15 28 B.