Report of Systematic Zoology Lab Practicum, August, 2011

Laboratory Course in Systematic Zoology 2011

Hiroshi Kajihara

Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan


Introduction
Oshoro Bay, a small inlet on the Japan Sea coast of western Hokkaido, harbours rich marine fauna and flora. Waters around the bay are influenced by the warm Tsushima Current in summer and autumn, while in other seasons the cold water of the Liman Current is mixed with coastal water, giving a characteristic composition of organisms in the bay (Motoda 1971). Although Okada et al. (1971) listed 389 species of invertebrates that had been recorded from the bay, little is known about the biology of each species. To facilitate understanding of the marine invertebrate fauna in Oshoro Bay, a practicum was designed in which students collect various marine animals, identify the taxa as far as possible, and determine a barcoding sequence for each species.
      Thirty-five 3rd-year undergraduate students who take Laboratory Course in Systematic Zoology (Division of Biology, Department of Biological Sciences, School of Science, Hokkaido University), made collection of marine invertebrates at Oshoro Marine Biological Station on 23 May, 30 May, 6 June, and 13 June 2011 (Figs 1–4). Each student selected a specimen ad libitum for further examination. The specimens were photographed in living state before fixed in 99% EtOH.

Fig. 1. Students sampling at a rocky shore in Oshoro Bay, on 23 May 2011.



Fig. 2. Sampling at a rocky shore in Oshoro Bay, on 30 May 2011.



Fig. 3. Wading along the shore, on 6 June 2011.



Fig. 4. Sorting the samples in the laboratory of the marine station, on 13 June 2011.



      After bringing the specimens back to the Sapporo campus, the students extracted DNA from the sample, amplified mitochondrial cytochrome c oxidase subunit I gene (COI) using Folmer et al.'s (1994) universal primer pair (HCO2198 and LCO1490). For specimens in which COI amplification was unsuccessful, an attempt was made to amplify nuclear 28S rDNA gene using the primer set LSU5 and LSU3 (Littlewood 1994). The same primer pair were used for sequencing.


Results
Thirty-three students submitted their reports (see below), of which four succeeded in determining COI sequence for an unidentified gammaridean amphipod, the colonial ascidian Botrylloides violaceus Oka, 1927, the peanut-worm Phascolosoma scolops (Selenka & de Man, 1883), and the chiton Acanthochitona achates (Gould, 1859). Partial sequences of 28S rDNA were determined for 16 species. For various reasons, neither COI nor 28S rDNA sequences were determined for 12 samples.
      Morphological voucher specimens of 30 species have been deposited in the Hokkaido University Museum. These are: Annelida (6 specimens), Arthropoda (15 ),Chordata (3), Echinodermata (2), Mollusca (2), Nemertea (1), Platyhelminthes (2), and Sipuncula (2). These specimens can be offered for future researchers by request (contact: Dr. Hiroshi Kajihara, kazi@mail.sci.hokudai.ac.jp). Five specimens (the sea-anemone Actinia equina (Linnaeus, 1758), the flatworm Notocomplana humilis (Stimpson, 1857), the polychaete Amphicorina mobilis (Rouse, 1990), and an unidentified species of gammariean amphipod) were completely destroyed for DNA extraction and mo morphological voucher remains.


Students' reports

Phylum Cnidaria

Anthopleura pacifica Uchida, 1938 Actinia equina (Linnaeus, 1758): 28S rDNA partial sequence of Actinia equina (Cnidaria: Anthozoa: Actiniaria: Actiniidae) by Ryutaro Kimura.


Phylum Platyhelminthes

Notocomplana humilis (Stimpson, 1857): Partial sequences of 28S rDNA from the flat worm Notocomplana humilis (Turbellaria: Polycladida: Notoplanidae) by Nanako Matsuzawa.

Notocomplana humilis (Stimpson, 1857): 28S rDNA partial sequence of Notoplana humilis (Turbellaria: Polycladida: Leptoplanidae) by Run Minoura.

cf. Pseudoceros sp.: A partial sequence of 28S rDNA from an unidentified species of flatworm, likely belonging to the genus Pseudoceros (Platyhelminthes: Polycladida: Pseudocerotidae) by Yusuke Obinata.


Phylum Nemertea

Emplectonema gracile (Johnston, 1837): 28S rDNA partial sequence of Emplectonema gracile (Nemertea: Hoplonemerta: Monostilifera) by Toshio Kubotsu.


Phylum Annelida

Hydroides ezoensis Okuda, 1934: 28S rDNA partial sequence of Hydroides ezoensis (Polychaeta: Sabellida: Serpulidae) by Takuto Kishikawa.

Arabella iricolor (Montagu, 1804): Observation of Arabella iricolor (Polychaeta: Eunicida: Arabellidae) by Takashi Kumada.

Terebellidae sp.: 28S ribosomal RNA partial sequence of Terebellidae sp. (Annelida: Polychaeta) by Shinichiro Honma.

Amphicorina mobilis (Rouse, 1990): 28S rDNA partial sequence of Amphicorina mobilis (Annelida: Sabellidae) by Ayako Tanaka.

Syllidae sp.: Observation of an unidentified species of the polychaete family Syllidae (Annelida: Syllidae) by Kentaro Miyashita.

Phylum Sipuncula

Phascolosoma sp.: Observation of Phascolosoma sp. (Sipuncula) by Yukiko Mizuo.

Phascolosoma scolops (Selenka & de Man, 1883): Cytochrome c oxidase subunit I partial sequence of Phascolosoma scolops (Sipuncula: Phascolosomatidae) by Yuuki Maruyama.


Phylum Mollusca

Acanthochitona achates (Gould, 1859): Cytochrome c oxidase subunit I partial sequence of Acanthochitona achates (Mollusca: Polyplacophora) by Masumi Abe.

Niveotectura pallida (Gould, 1859): 28S rDNA partial sequence of Niveotectura pallida (Mollusca: Gastropoda: Lottioidea) by Yuichi Matsumoto.


Phylum Arthropoda

Achelia japonica (Ortmann, 1890): 28S rDNA partial sequence of Achelia japonica (Arthropoda: Pycnogonida) by Yukino Miyoshi.

Ammothea hilgendrfi (Böhm, 1879): 28S ribosomal RNA gene partial sequences of Ammothea hilgendorfi (Pycnogonida: Pantopoda: Ammotheidae) by Naomi Takahashi.

Gammaroidea sp.: Cytochrome c oxidase subunit I partial sequence of an unidentified species of Gammaroidea (Crustacea: Amphipoda) by Takuya Akiyama.

Gammaroidea sp.: A partial sequence of 28S rDNA from an unidentified species of gammaridean (Crustacea: Amphipoda: Gammaridea) by Haruka Araki.

Gammaroidea sp.: 28S rDNA sequence of an unidentified species of gammaroid (Crustacea: Amphipoda: Gammaroidea) by Ryohei Niizawa.

Gammaroidea sp.: Observation of Gammaridea sp. (Arthropoda: Crustacea: Amphipoda) by Maho Ikoma.

Caprellidea sp.: Observation of an unidentified species of Caprellidea (Crustacea: Amphipoda) by Makoto Someya.

Anthuridea sp.: Observation of Anthuridea sp. (Arthoropoda: Crustacea: Isopoda) by Nobu Nagai.

Anthuridea sp.: Observation of an unidentified species of anthridean (Crustacea: Isopoda) by Yumi Yamagishi.

Anthuridea sp.: 28S rRNA partial sequence of an unidentified species of anthuridean (Crustacea: Isopoda) by Ruriko Mitani.

Dynoides dentisinus Shen, 1929: Observation of Dynoides dentisinus Shen, 1929 (Crustacea: Isopoda: Sphaeromatidae) by Tetsuro Oriyama.

Gaetice depressus (De Haan, 1833): 28S rDNA partial sequence of the crab Gaetice depressus (De Haan, 1833) (Crustacea: Decapoda: Bracyura: Varunidae) by Yasuyuki Nikami.


Phylum Echinodermata

Amphipholis kochii Lütken, 1872: 28S rDNA partial sequence of Amphipholis kochii (Echinodermata: Ophiuroidea) by Tadayuki Uramoto.

Stichopus japonicus (Selenka, 1867): Observation of Stichopus japonicus (Holothuroidea: Aspidochirotida: Stichopodidae) by Seiji Masuda.


Phylum Chordata

Botrylloides violaceus Oka, 1927: Cytochrome c oxidase subunit I partial sequence of the colonial ascidian Botrylloides violaceus Oka, 1927 (Ascidiacea: Styellidae) by Shoki Aoyama.

Ascidiacea sp.: Observation of an unidentified species of ascidian (Chordata: Ascidiacea) by Shohei Yamauchi.

Pisces sp.: Observation of an egg mass of an unidentified fish species (Chordata: Vertebrata: Pisces) by Nosaka Megumi .




References

Folmer, O., Black, M., Hoeh, W., Lutz, R. and Vrijenhoek, R. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3: 294–299.

Littlewood, D. T. 1994. Molecular phylogenetics ofcupped oysters based on partial 28S rRNA gene sequences. Molecular Phylogenetics and Evolution 3: 221–229.

Motoda, S. 1971. Oshoro Marine Biological Station, Hokkaido University, Japan. Bulletin of Plankton Society of Japan 18(1): 32–94.

Okada, S., Igarashi, T. and Kobayashi, K. 1971. Invertebrates and fishes of Oshoro Bay and neighbouring area. Bulletin of Plankton Society of Japan 18(1): 59–72.