BULLETIN OF THE NAGOYA UNIVERSITY MUSEUM No. 19

ISSN 1346-8286 BULLETIN OF THE NAGOYA UNIVERSITY MUSEUM No. 19 THE NAGOYA UNIVERSITY MUSEUM Chikusa-ku, Nagoya 464-8601, Japan 2003

BULLETIN OF THE NAGOYA UNIVERSITY MUSEUM No. 19

EDITORIAL BOARD Bulletin of the Nagoya University Museum EDITOR-IN-CHIEF ADACHI Mamoru Geology EDITOR AKIYAMA Masanori History HIRUNAGI Kanjun Animal Morphology HOSHINO Mitsuo Petrology MIYAJI Akira Art History NIIMI Michiko Environmental Archaeology NISHIDA Sachiko Plant Taxonomy NISHIKAWA Teruaki Animal Taxonomy OKUYAMA Takashi Wood Physics OZAWA Tomowo Paleontology SUZUKI Kazuhiro Geochronology HAGA Syoji History TANAKA Tsuyoshi Geochemistry TSUKADA Kazuhiro Tectonics YOSHIDA Hidekazu Environmental Geology

Immunocytochemical and Ultrastructural Analysis of Opsin- and Vasoactive Intestinal Peptide (VIP)- like Immunoreactive Neurons in the Lateral Septum of the Pigeon HIRUNAGI Kanjun First discovery of Bugula stolonifera Ryland, 1960 (Phylum Bryozoa) in Japanese waters, as an alien species to the Port of Nagoya Joachim SCHOLZ, NAKAJIMA Kiyonori, NISHIKAWA Teruaki, KASELOWSKY Jürgen and MAWATARI F. Shunsuke

Bulletin of the Nagoya University Museum No. 19 Contents Immunocytochemical and Ultrastructural Analysis of Opsin- and Vasoactive Intestinal Peptide (VIP)- like Immunoreactive Neurons in the Lateral Septum of the Pigeon HIRUNAGI Kanjun... 001 First discovery of Bugula stolonifera Ryland, 1960 (Phylum Bryozoa) in Japanese waters, as an alien species to the Port of Nagoya Joachim SCHOLZ, NAKAJIMA Kiyonori, NISHIKAWA Teruaki, KASELOWSKY Jürgen and MAWATARI F. Shunsuke... 009 Salt-Making Pottery Found in the Ohara-Shell Mound of Iwaki City, Fukushima Prefecture... WATANABE Makoto and MORI Yasumichi... 021 Study of plug-shaped clay ear ornaments in Jomon period... YOSHIDA Yasuyuki... 029 A Study on Yak as Statues of Early Indian Art... NAGATA Kaoru... 055 The Inscriptions at AjaNTA Caves... FUKUYAMA Yasuko... 073 List of the medical moulages transferred from the School of Medicine to the Museum at Nagoya University... NISHIDA Sachiko, KOBAYASHI Miya, ADACHI Ayumi, ITO Yuji, ICHIMURA Takuya, OSAKA Chieko and KIM Kyonja... 087 A newly-established questionnaire database and its reference system TSUKADA Kazuhiro... 105 Latest arrival: Serial histological light-microscoopic sections of human embryonal and fetal specimens... NOZAKI Masumi... 121 Records of 5 th NUM Special Exhibition Afghanistan Nagoya University Expedition 1968... HIRUNAGI Kanjun... 125 Records of 6 th NUM Special Exhibition Lost Heritage Bamian in Afghanistan... NIIMI Michiko... 135 Record of the 2 nd NUM Special Display C. W. Hufeland and Japanese Medicine in Edo Period, with Supplementary Explanatory Notes... NISHIKAWA Teruaki... 149

Bull. Nagoya Univ. Museum No. 19, 1 8, 2003 Immunocytochemical and Ultrastructural Analysis of Opsin- and Vasoactive Intestinal Peptide (VIP)- like Immunoreactive Neurons in the Lateral Septum of the Pigeon HIRUNAGI Kanjun The Nagoya University Museum, Chikusa-ku Nagoya, 464-8601, Japan Summary CSF-contacting neurons of the lateral septum are considered as a putative deep brain photoreceptor in the avian brain. By means of immunocytochemistry using antibodies to a visual pigment (RET-P1, a monoclonal antibody against opsin, Barnstable, 1980) and vasoactive intestinal peptide (VIP), I demonstrated clusters of immunoreactive small neurons in the lateral septum of the pigeon. Opsin-like immunoreactive neurons and VIP-like immunoreactive neurons have similar morphological features. Their perikarya accumulate in the subependymal regions of the ventromedial walls of both lateral ventricles. The labelled neurons are multipolar or bipolar with pyriform or spindle-shaped cell bodies. Immunoreactive CSF-contacting neurons contact the CSF via a process that penetrates the ependyma and terminates in a single knob-like swelling. Immunoreactive fibers originating in the group of cell bodies seem to give rise to dense terminal-like structures in the septal area. Immuno-electron-microscopic investigations of these neurons revealed an accumulation of VIP- and opsin immunoreactive dense-core vesicles (100-150 nm in diameter) in ventricular terminals, perikarya and neuronal terminal-like structure with VIP- and RET-P1-immunolabelling respectively. Based on these evidence it seems clear that VIP-and opsin-like immunoreactive neurons of this study are the same as the neurons that express both opsin- and VIP-like immunoreactivity in the ateral septum of the ring dove (Silver et al., 1988). In this study double immunolabelling using VIP and RET-P1 antibodies shows the coexistence of VIP and opsin in the same dense vesicles. Introduction Immunohistochemical studies reveal small neurons with an accumulation of VIP-like immunoreactive (ir) in the lateral septum in avian brains (Hirunagi et al., 1995; Hof et al., 1991; Korf and Fahrenkrug, 1984; Kuenzel and Blähser, 1994; Silver et al., 1988; Yamada et al., 1982), some of which are cerebrospinal fluid (CSF)-contacting. Recently, this type of small neuron has been considered as a candidate for the deep encephalic photoreceptor of avian and reptilian brains. First, Silver et al. demonstrated opsin-like immunoreactivity in this type of neurons of the lateral septum of birds using an opsin antibody, RET-P1, raised against a membrane fraction of rat retina (Barnstable, 1980; Silver et al., 1988). In that study, they established that some VIP-like ir neurons of the lateral septum co-express RET-P1 immunoreactivity with an immuno-fluorescent double labelling method. More recently, opsin-like immunoreactivity has been reported in similar CSFcontacting neurons of the identical region of the lateral septum of the lizard Anolis carolinensis (Foster et al., 1993). Furthermore, VIP-like ir CSF-contacting neurons have been demonstrated in the same region of the lateral septum of several reptilian species (Hirunagi et al., 1993). With regard to the subcellular localization of VIP-like antigen, Hirunagi et al. showed that VIP-like ir CSF-contacting neurons have VIP-like ir dense vesicles (approximately 100-150 nm diameter) in 1

the perikarya with dendritic processes and axon terminals of the duck (Hirunagi et al., 1995). This result suggests that VIP-like ir neurons contain VIP-like neuropeptide in these electron-dense vesicles. However, until now, the ultrastructural localization of opsin-like antigen has remained elusive. In the present study we demonstrate the ultrastructural localization of opsin- and VIPlike antigens in the neurons of the lateral septum of the pigeon. I used single labelling immunocytochemistry for RET-P1 and VIP and a dual-labelled method using both ABC labelling and immunogold-silver labelling at the electron microscopic level. Material and Methods Male and female adult pigeons were perfused transcardially with 0.75% NaCl followed by a mixture of 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1M phosphate buffer at ph 7.4. Brains were rapidly removed and a coronal block containing the lateral septum area post-fixed for overnight. Vibratome sections (50 µm thick) were cut and treated for the demonstration of neurons with RET-P1 and VIP immunoreactivities as described below. (1) Single label immunostaining for VIP; Sections were incubated in polyclonal porcine VIP antibody (1:1000, Cambridge Research Biochemicals, UK) for 72 hours at 4 C. For visualizing of immunoreaction, a PAP method was used. The details of immunostaining for VIP and the specifity of VIP antibody was described previously (Hirunagi et al., 1993; Hirunagi et al., 1995). (2) Single label immunostaining for RET- P1; Sections were incubated in monoclonal opsin antibody (RET-P1) (1: 20000) diluted in 0.1MPBS containing 1% BSA and 0.3% Triton X-100 for 72 hours at 4 C. The sites of the antibody-antigen binding were visualized with an avidin-biotin-peroxidase (ABC) procedure (Elite ABC kit, Vector Labs, Burlingame, CA) according to the protocol. Other sections were incubated in monoclonal opsin antibody (RET-P1) (1: 20000) for 72 hours at 4 C. Sections were then incubated in goat antimouse IgG bound to 1nm colloidal gold (AuroProbe One, Amersham, UK) diluted 1:50 in 0.1% gelatin PBS, BSA. Sections were fixed in 2% glutaraldehyde for 10 minutes. Silver intensification of gold was processed by the light-insensitive IntenSE-M Kit (Amersham, UK) following the manufacture s protocol. (3) Dual label immunostaining for VIP and RET-P1; Following incubation with RET-P1 as the first primary antisera, immunoreactivity was visualized with an ABC procedure as shown in (2). Sections were incubated in polyclonal porcine VIP antibody (1:1000, Cambridge Research Biochemicals, UK) as second primary antisera. Sections were then incubated in goat anti-rabbit IgG bound to 1 nm colloidal gold (AuroProbe One, Amersham, UK) diluted 1:50 in 0.1% gelatin PBS, BSA. Sections were fixed in 2% glutaraldehyde for 10 minutes. Silver intensification of gold was processed by the light-insensitive IntenSE-M kit (Amersham, UK), as above. For electron microscopy immunostained sections were osmicated and flat-embedded in Araldite. The details of these procedures have been described previously. Following contrast staining with uranyl acetate and lead citrate, ultra-thin sections were examined with a JEOL JEM 1210 electron microscope. Results Using polyclonal antibody against VIP and monoclonal antibody, RET-P1, against rat opsin, an accumulation of immunorective neurons is found in a highly circumscribed region of the lateral septum /nucleus accumbens with each label. Light- and electron-microscopic morphological features of VIP-like and opsin-like ir neurons are identical in the lateral septum of the pigeon. We show results of opsin-like and VIP-like ir neurons and double labelling with RET-P1 and VIP in Fig. 1, 2 respectively. Opsin-like ir perikarya accumulates in the subependymal regions of the ventromedial walls of both lateral ventricles at the level of the pars medialis of the lateral septal 2

Fig 1. a. Accumulation of opsin-ir neurons in the lateral septum Arrows: Immunoreactive neurons Arrowheads: Basal processes V: Lateral ventricle Bar: 100 µm b. Opsin ir CSF-contacting neurons in the lateral septum Arrows: ventricular processes of opsin ir CSF-contacting neurons B: Basal process P: Ventricular process S: cell body V: Lateral ventricle Bar: 20 µm c. Opsin ir dense vesicles in immunoreactive neuron Arrows: immunoreactive vesicles E: Endoplasmic reticulum N: Nucleus Bar: 1 µm d. Opsin ir vesicles identified by 1 nm immunogold-silver enhanced method Arrowheads: Immunoreactive dense vesicles M: Mitochondria Bar: 1 µm e. Opsin ir ventricular process of the CSF-contacting neuron in the lateral septum C: Cilium in lateral ventricle Bar: 1 µm f. Opsin ir neuronal terminal near small neuron in the lateral septum M: Mitochondria N: nucleus of immunonegative neuron T: Opsin ir neuronal terminal Bar: 1 µm 3

Fig. 2. a. VIP ir neuron in the lateral septum B: Basal process EP: Ependymal cell N: Nucleus of VIP ir neuron Bar: 2 µm b. VIP ir ventricular process of the CSF-contacting neuron of the lateral septum EP: Ependymal cell M: Mitochondria in process V: Lateral ventricle Bar: 1 µm c. Double labelling of opsin and VIP antibodies Arrow indicates opsin ir dense vesicle. Arrowheads indicate both opsin (dense vesicles) and VIP (silver-enhanced gold particles) immunoreactivity on granules. ER: Endoplasmic reticulum Bar: 1 µm organ (Kuenzel and Blähser, 1994) (Fig. 1a). The labelled neurons are multipolar or bipolar with pyriform or spindle-shaped cell bodies. Some of these are CSF-contacting neurons, with a process that penetrates the ependyma and terminates in a single knob-like swelling (Fig. 1b). In addition to the cluster of cell bodies, ir fibers originating in the group of cell bodies course into deeper layers of the septal area. Some of them appear to be beaded axons (Fig. 1a). Using pre-embedding methods of ABC and 1 nm immunogold, electron- microscopic studies reveal that opsin-like neurons have an accumulations of dense-core vesicles with diameter of 100-150 nm in their perikarya and ventricular terminal. An ABC labelling demonstrates RET-P1-labelled dense vesicles near the endoplasmic reticulum in the cytoplasm (Fig. 1c) and in the ventricular terminal (Fig. 1e). With a 1nm immunogold silver labelling, silver-enhanced gold particles, which indicate the site of RET-P1 antigen, are frequently associated with dense vesicles (Fig. 1d). In the lateral septum, terminal formations with VIP or RET-P1 immunoreactivity are distributed. VIP-and RET-P1-labelled terminals lie in the same regions. Most prominent immunoreactive terminal formations are observed in the area of the nucleus septi lateralis. In this area opsin-like ir terminals form dense networks. They appeared as intensely immunoreactive spots at light-microscopic observations as VIP immunoreactive terminals that were reported our previous paper (Hirunagi et al., 1994). Electron-microscopic observations show that opsin-like ir terminals are observed near immunonegative small neurons in the lateral septum (Fig. 1f). VIP immunocytochemistry shows an accumulation of VIP ir neurons in the subependymal region of the lateral septum. This type of neurons project VIP-like ir 4

basal processes (Fig. 2a) and ventricular processes in the lateral ventricle (Fig. 2b). In ventricular swellings, dense vesicles and mitochondria are frequently observed (Fig. 2 b). A double-labelling survey, combined ABC method for RET-P1 antibody and immunogold-silver method for VIP antibody, reveals that RET-P1-labelled dense-core vesicles appear to coexpress VIP-like immunoreactivity. Silver-enhanced gold particles which indicate the localization of VIP antigen are preferentially associated with RET-P1-immunolabelled dense core-vesicles near the endoplasmic reticulum in the cytoplasm (Fig, 2c). Control sections in which second primary antibody (VIP antibody) was omitted from the protocol showed ABC immunolabelling corresponding to the first primary antibody alone. Discussion There is a lot of experimental evidence for the existence of a deep encephalic photoreceptor within avian brain (Kuenzel, 1993; Oksche, 1991; Perera and Follett, 1992). However, to date there is no morphological evidence to support the existence of photoreceptor-like structures such as the outer segment of photoreceptor of retina in the avian forebrain (Oksche, 1991). The sites of deep brain photoreceptors of birds remain unidentified. According to recent opsin-immunohistochemical studies, the lateral septum is a candidate for a photoreception site in birds (Silver et al., 1988). The present immunocytochemical observations clearly indicated that opsin-like and VIP-like ir CSFcontacting neurons are important components of the lateral septum region in the pigeon. Electron microscopic observations revealed that opsin- and VIP-like ir CSF-contacting neurons have similar ultrastructural features. This type of small neurons have a ventricular bulbous terminal that contains numerous mitochondira and large dense vesicles. Similar electron microscopic features have been reported in a recent study of rudimentary photoreceptor cells in the pineal organ of a marsupial, Didelphis albiventris (del C. González and Affanni, 1995). In reptilian and avian brains, VIP- and opsin-like ir CSF-contacting neurons are reported in the identical regions of several species. A CSF-contacting neuron of the lateral septum is a putative deep encephalic photoreceptor of the avian and reptilian species, mainly because of its expression of opsin-like immunoreactivity using immunohistochemical method in reptilian and avian species. This study demonstrated that a CSF-contacting neuron located in the lateral septum has opsinlike immunoreactivity in the pigeon. However, immunohistochemical results regarding the existence of opsin-like immunoreactive neurons in the avian lateral septum are controversial. Using a monoclonal anti-opsin antibody RET-P1, Silver et al. (1988) described neurons with opsin-like immunoreactiveity in the septal and infundibular hypothalamic region of doves, ducks, quail, sparrows and junco (Silver et al., 1988). Furthermore, it appears that the same cells in doves were stained with Cos-1 (a monoclonal anti-opsin antibody raised against a chicken cone visual pigment) (Silver, 1990). On the other hand, Garcia-Fernández and Foster (1994) have failed to identify any opsin-immunopositive cells in the identical regions of the quail or chick brain, although they could show opsin-like ir hypothalamic CSF-contacting neurons in the the larval lamprey using the antibodies (García-Fernández and Foster, 1994). Furthermore, no specific staining of opsin and retinal S-antigen was detected in the lateral septal cells and hypothalamic cells in birds (Foster and Korf, 1987). Recently, rhodopsin cdna was cloned from the pigeon lateral septum fraction. An antibody (RhoN) which recognizes the amino-terminal region of pigeon rhodopsin was prepared for use in an immunohistochemical study to identify photoreceptor cells in the lateral septum of the pigeon. Immunoreactive CSF-contacting neurons were detected in the lateral septum of the pigeon (Wada et al., 1998). It is difficult to say whether the disagreement between these result depends on some species differences or different types of opsin antibodies used. 5

RET-P1 ir cells of the lateral septum coexpress VIP-like immunoreactivity in the brains of ring dove, Japanese quail and duck (Silver et al., 1988). In the present study, we confirmed the colocalization of RET-P1 and VIP in neurons of the lateral septum in the pigeon at the electron microscopic level using a dual labeling method. Our electron microscopic results suggested that RET-P1 and VIP antigens colocalize in a dense core vesicle which is found in the perikarya and terminals. With regard to the subcelluar localization of VIP-like specific antigen, electron microscopic analysis has shown that VIP-like ir neurons of the lateral septum have an accumulation of immunostained large dense core vesicles (approximately 150 nm in diameter) in their cytoplasm (Hirunagi et al., 1995). Similar VIP immunoreactive core vesicles were reported in the terminal formations of the lateral septum of duck (Hirunagi et al., 1995) and pigeon (Hirunagi et al., 1994). About the subcellular localization of RET-P1 antigen, electron microscopy revealed the distribution of RET-P1 antigen in the rat retina (Fekete and Barnstable, 1983). Immunoreactivity of RET- P1 antibody can be seen in the plasma membranes of both outer segments and inner segments of rod photoreceptors. And a more diffuse reaction product was reported within the peripheral cytoplasm of the inner segments. In this study, RET-P1 immunoreactivity has been observed in the entire neuronal elements including axon and dendritic processes of CSF-contacting neurons. Ventricular bulbus terminals of CSF-contacting neurons have RET-P1 immunoreactivity. However, no lamellar membrane structures, similar to those found in the outer segments of retinal photoreceptors, have been identified electron-microscopically in the CSF-contacting neurons of lateral septum of birds. Confirmation of these CSF-contacting neurons as photoreceptors awaits physiological evidence. Acknowledgements I would like to thank Drs. S. Ebihara and A. Adachi (Nagoya Univ.) and Dr. C. Blakemore for the gift of the antibody. I would also like to thank Dr. R. Silver (Columbia Univ.) for her kind comments in this paper. References Barnstable, C.J. (1980) Monoclonal antibodies which recognize different cell types in the rat retina, Nature, 286, 231-235. del C. González, M.M. and Affanni, J.M. (1995) Cells of photoreceptor line in the pineal organ of the adult marsupial, Didelphis albiventris, Cell Tissue Res., 282, 363-366. Fekete, D.M. and Barnstable, C.J. (1983) The subcellular localization of rat photoreceptor-specific antigens, J. Neurocytol., 12, 785-803. Foster, R.G., García-Fernández, J.M., Provencio, I. and DeGrip, W.J. (1993) Opsin localization and chromophore retinoids identified within the basal brain of the lizard Anolis carolinensis, J. Comp. Physiol. A, 172, 33-45. Foster, R.G. and Korf, H.-W. (1987) Immunocytochemical markers revealing retinal and pineal but not hypothalamic photoreceptor system in the Japanese quail, Cell Tissue Res., 248, 161-167. García-Fernández, J.M. and Foster, R.G. (1994) Immunocytochemical identification of photoreceptor proteins in hypothalamic cerebrospinal fluid-contacting neurons of the larval lamprey (Petromyzon marinaus), Cell Tissue Res., 275, 319-326. Hirunagi, K., Kiyoshi, K., Adachi, A., Hasegawa, M., Ebihara, S. and Korf, H.-W. (1994) Electron-microscopic investigations of vasoactive intestinal peptide (VIP)-like immunoreactive terminal formations in the lateral septum of the pigeon, Cell Tissue Res., 278, 415-418. Hirunagi, K., Rommel, E. and Korf, H.-W. (1995) Ultrastructure of cerebrospinal fluid-contacting neurons immunoreactive to vasoactive intestinal peptide and properties of the blood-brain barrier in the lateral septal organ of the duck, Cell Tissue Res., 279, 123-133. 6

Hirunagi, K., Rommel, E., Oksche, A. and Korf, H.-W. (1993) Vasoactive intestinal peptide-immunoreactive cerebrospinal fluid-contacting neurons in the reptilian lateral septum/nucleus accumbens, Cell Tissue Res., 274, 79-90. Hof, P.R., Dietl, M.M., Charnay, Y., Martin, J.-L., Bouras, C., Palacio, J.M. and Magistretti, P.J. (1991) Vasoactive intestinal peptide binding sites and fibers in the brain of the pigeon Columba livia: an autoradiographic and immunohistochemical study, J. Comp. Neurol., 305, 393-411. Korf, H.-W. and Fahrenkrug, J. (1984) Ependymal and neuronal specializations in the lateral ventricle of the Pekin duck, Anas platyrhynchos, Cell Tissue Res., 236, 217-227. Kuenzel, W.J. (1993) The search for deep encephalic photoreceptors within the avian brain, using gonadal development as a primary indicator, Poult. Sci., 72, 959-967. Kuenzel, W.J. and Blähser, S. (1994) Vasoactive intestinal polypeptide (VIP)-containing neurons: distribution throughout the brain of the chick (Gallus domesticus) with focus upon the lateral septal organ, Cell Tissue Res., 275, 91-107. Oksche, A. (1991) The development of the concept of photoneuroendocrine system: historical perspective. In D.C. Klein, R.Y. Moore and S.M. Reppert (Eds.), Suprachiasmatic Nucleus, The Mind s Clock, Oxford University Press, New York, pp. 5-11. Perera, A.D. and Follett, B.K. (1992) Photoperiodic induction in vitro: the dynamics of gonadotropin-releasing hormone release from hypothalamic explants of Japanese quail, Endocrinology, 131, 2898-2908. Silver, R. (1990) Avian behavioral endocrinology: status and prospects. In W.M., S. Ishii and C.G. Scanes (Eds.), Endocrinology of Birds: Molecular to Behavioral, Japan Sci. Soc. (Springer -Verlag), Tokyo, Berlin, pp. 261-272. Silver, R., Witkovsky, P., Horvath, P., Alones, V., Barnstable, C.J. and Lehman, M.N. (1988) Coexpression of opsin- and VIP-like immunoreactivity in CSF-contacting neurons of the avian brain, Cell Tissue Res., 253, 189-198. Wada, Y., Okano, T., Adachi, A., Ebihara, S. and Fukada, Y. (1998) Identification of rhodopsin in the pigeon deep brain. FEBS lett., 424, 53-56. Yamada, S., Mikami, S. and Yanaihara, N. (1982) Immunohistochemical localization of vasoactive intestinal polypeptide (VIP)-containing neurons in the hypothalamus of the Japanese quail, Coturnix coturnix, Cell Tissue Res., 226, 13-26. 7

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Bull. Nagoya Univ. Museum No. 19, 9 19, 2003 First discovery of Bugula stolonifera Ryland, 1960 (Phylum Bryozoa) in Japanese waters, as an alien species to the Port of Nagoya Joachim SCHOLZ 1), NAKAJIMA Kiyonori 2), NISHIKAWA Teruaki 3), KASELOWSKY Jürgen 4) and MAWATARI F. Shunsuke 5) 1,4) Forschungsinstitut und Naturmuseum Senckenberg, Section Marine Evertebrates III (Bryozoology), Senckenberganlage 25, D-60325 Frankfurt am Main, Germany 1,2) Port of Nagoya Public Aquarium, 1-3, Minatomachi, Minato-ku, Nagoya 455-0033, Japan 1,3) The Nagoya University Museum, Chikusa-ku, Nagoya 464-8601, Japan 1,5) Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan Abstract Samples taken in 1997 from the Port of Nagoya, Japan, revealed the first record of the cheilostome bryozoan, Bugula stolonifera Ryland in Japanese waters. The species, which has been reported within the last years from several localities over the world, has probably been imported by ships coming to the Port of Nagoya. The species record illustrates the changes to which the Japanese bryozoan communities are subjected due to neozoan immigration. 1. Introduction Bryozoans represent a phylum of colonial, suspension feeding animals. They are abundantly found in both cool waters and tropical reefs, silicoclastic shelves, polar regions, and even in the deep sea realm. The phylum is the only major group of exclusively clonal animals. Colonies are formed by repeated budding of genetically (but not always morphologically) identical, physically connected, intercommunicating member zooids (McKinney and Jackson, 1989; Cook, 1988). The modular construction of their fixosessile, mostly calcifying colonies makes it possible to reconstruct life histories and interactive relationships on substratum surfaces. Skeletal growth modifications of both single units (zooids) and colonies (zoaria) preserve the influence of the outside environment at the time of growth. The large percentage of surface occupied by laminar bryozoans despite the small biomass makes them important in any biological interactions on hard substrata (Riedl, 1966). Bryozoans are frequently the dominant biota covering the surfaces of dead coral reef colonies. This suggests that bryozoans serve as a major frame-binding agent for maintaining the integrity of the reefs, in opposition to bioeroding biota (Soule and Soule, 1972). Fouling bryozoa are an economic problem since for example in boats and ships, a 1 mm thick layer of slime can cause a 15% loss in ship speed compared to values obtained for a clean hull (Lewthaite et al., 1985). Bryozoans are frequently overgrowing and suffocating marine oyster beds, and fresh-water bryozoans may foul water system distribution pipes (Cuffey, 1970). Today, the arrival of fouling bryozoans settling e.g. on ship hulls is rapidly altering the species composition of bryozoans on a world wide scale, occasionally with dramatical impacts (see Gordon 9

and Mawatari, 1992). Bryozoans may even be distributed by aeroplanes, such as probably happened during the Second World War, when European Conopeum seurati were introduced to the remote Chatham island lagoon by allied Sunderland flying boats (Scholz, 2001). As early as 1971, J. Ryland cited the number of 54 species of bryozoans known to colonize ship s hulls, and 139 fouling bryozoans form ships, buoys, wrecks, and harbour installations. For fouling bryozoans it is typical that they do not have a competitive advantage over less-generalist species, but may thrive in the particular conditions of ports and harbours since the mature colonies are more tolerant of the wider ranges of the prevailing temperature, salinity, turbidity, and pollution (literature reviewed by Gordon and Mawatari, 1992). For the Japanese islands, little is known on the magnitude of this event, although it is readily evident that for example harbour sites or floating piers are heavily colonized by dense clusters of fouling species of Watersipora subovoidea, Bugula neritina and some other species (Nandakumar et al., 1993). Aside from this comparatively recent contribution, we have to rely on data that record the state of the art as it has been several decades ago (A. Grischenko, Sapporo, pers. comm.), such as the articles of Yanagi and Okada (1918) and S. Mawatari (1953), and a consideration of the neozoan impact must be part of any new taxonomic inventory of the Japanese seas. Every species counts, and in the following, we report on the arrival of a new fouling bryozoan to Japan. Furthermore, a state of the art of biodiversity research of Japanese bryozoans is given. Sea of Japan Nagoya Osaka Tokyo Pacific Ocean Sampling site Port of Nagoya Port of Nagoya Public Aquarium 100m Garden pier Ise Bay 2000m Fig. 1: Map showing the sampling site in the Port of Nagoya, Middle Japan. 10

2. Material and Methods The specimen, registered as NUM-Az 0363 in The Nagoya University Museum, was collected on15 Nov. 1997 from a vinyl-chloride panel set in a launch harbour in front of the Port of Nagoya Public Aquarium, northern end of Ise Bay on the Pacific coast of Middle Japan (Fig. 1). The Port of Nagoya is one of the biggest harbours in Japan, with arrivals of 9,231 (in 2001) and 9,132 (in 2002) foreign vessels (data from www.port-of-nagoya.jp). The panel had been set subtidally from 5 July 1997, just below the mean low water of spring tide. Water temperature and salinity at the sampling site were 21.8 C on an average (ranging from 11.3 to 31.6 C), and 31 (from 20 to 37 ), based on the daily data throughout 2000 and 2002, respectively. The specimen was fixed and preserved in 10% formalin. The sample was transferred to ethanol in a dehydration series, critical-point-dried and sputtered for SEM observation. 3. On the genus Bugula Oken, 1815 Species of Bugula have intrigued biologists for more than 100 years, due to their abundance and to the presence of spectacular, strange bird head avicularia (Fig. 2). For example, in the first 1969 edition of Ricketts famous account on the intertidal fauna of California, he states : The avicularia ( bird beaks ) of Bugula, thought to be defensive in function, are classic objects of interest to the invertebrate zoologist. It is a pity that these can be seen only with a microscope. If the movable beaks of avicularia were a foot or so long, instead of a fraction of a millimeter, newspaper photographers and reporters would flock to see them. The snapping process would be observed excitedly, some enterprising cub would certainly have one of his fingers snipped off, and the crowds would amuse themselves by feeding stray puppies into the pincers. Bird head avicularia are one of most useful features to identify Bugula species. This is important since the genus is diverse, and the ERMS (European Register of Marine Species) report (Hayward, 2001) cites 20 species of Bugula for European waters alone. The function of bird head avicularia, and avicularia in general is still obscure. The function was discussed in Darwin s Origin of Species, but Darwin and his successors provided inconclusive evidence on the evolutionary significance of avicularia. Defense against predators, cleaning, locomotion, nutrient storage, respiration, chemical defense, current flow modifications, or larval inoculation are among the functions possible for the diverse type of bryozoan avicularia (literature reviewed in Winston, 1984). For bird head avicularia, a very early observation may be the most probable explanation, pointing out a peculiar way of agriculture in invertebrates: On one occasion I saw one with the mandibles closed, grasping a tuft of confervoid-like substance, just like a bird with a wisp of hay in its beak. This is retained for some days, while the peculiar waving motion was still kept up. The only explanation seemed to be that the decaying conferva would attract minute infusoria, which would thus be brought within easy reach of the tentacles of the Polyzoon (Goldstein, 1880). Aside from the presence of bird head avicularia in most but not all species, the following description can be given for bryozoans of the genus Bugula: Colony are erect and branched, growing from an upright ancestrula (first zooid). The colony may be attached by rhizoids which issue from the autozooidal surfaces. Zooids arranged unilaminar in two or more series, alternating, the proximal end being forked. The basal and lateral walls are lightly calcified, and the membrane occupies most of the frontal surface. Spines are usually present. The ovicells (brood champers) are hyperstomial and globular (summarised from Ryland and Haward, 1977: p.150). 11

4. Results Bugula stolonifera Ryland, 1960 (Fig. 2) Bugula stolonifera Ryland, 1960, p.78; Ryland, 1962, p.50; Gordon and Mawatari, 1992, p.3. Colonies are greyish-buff. in a compact and non-spiral tuft 3-4 cm high. Branches with zooids in two series, with long and narrow zooids. Frontal membranes occupying 2/3 to 3/4 of the length of the zooid. the length of the avicularia not exceeding the length of the zooid, and avicularian beak is shorter than the head. Spines are rather slender (see Ryland, 1960; Ryland and Hayward, 1977; Zabala and Mauquer, 1988). The slender spines are important to distinguish the species from Bugula Fig. 2: Bugula stolonifera Ryland from the Port of Nagoya, Japan. A: Frontal and lateral surfaces of the erect colony. SEM Image, Critical-point-dried sample. B: Biserial row of zooids, bifurcating; surfaces are nearly free of microbial fouling, thus indicating the presence of secondary metabolites. C: Lateral view of autozooids and sections of the parent and daughter zooid, showing tentacles and bird head avicularia. D: Autozooid and part of neighboring zooids, showing orificial spines, frontal membranes, lateral walls, ovicells (brood chambers), and a bird head avicularium with mandible opened. E: Tentacle sheet, showing multiciliate cells and phytoplancton (diatoms), which is a food source of bryozoans. F: Single bird head avicularium with closed rounded mandible. 12

californica Roberston (1905) which was reported from Japan already, but judging from Soule et al. (1995), has more stout finger-like spines. Like many species of Bugula, B. stolonifera is a widely distributed neozoan. The type locality is Swansea, Wales. In the 1960 s and 70 s, it arrived in Australia and New Zealand. The report of Gordon and Mawatari (1992) does not mention any occurence in Japan. The species is not mentioned in the PhD thesis of S. Mawatari (1958) that focussed e.g. on the genus Bugula in Japan. SEM-images (Fig. 2) show that the colony surface is very clean. This phenomenon has been observed for many fouling bryozoans, which may probably be attributable to some anti-fouling chemicals produced by the colony. 5. Discussion Bugula stolonifera Ryland has been reported from the docks in the Southwest of the British Isles (Ryland, 1960). It settles between early summer to mid-autumn and is well known as a fouling species. It has been introduced to other countries, for example to New Zealand (Harger 1964, Gordon & Mawatari 1992), by ships. It has been recorded also from the Mediterranean sea, Ireland, Ghana, Massachusetts to Florida, Brazil and South Australia (Winston, 1982; Cook, 1985; Ryland and Hayward, 1991). Studies on bryozoans and bryozoan diversity have implications in very diverse fields of basic and applied research, four of them being listed below: 1) Bryozoans have been well established as biological indicators of anthropogenic influences in coastal waters (e.g. Soule and Soule, 1981; Scholz, 1991; Hillmer and Scholz, 1991; Winston, 1995). Thus, the contribution of bryozoans and other supension feeding benthos to the management equation cannot be ignored. 2) The development of molecular methods has led to the recognition of high marine microbial diversity, which can be used as a resource for the detection of novel marine natural products. Since the 1980 s, Bryozoans and their cultivatable bacterial associates have become a focal point for marine natural product research (e.g. Newman, 1996; Shellenberger and Ross, 1998; Pukall et al., 2001, 2003). 3) Bryozoans are excellent tools for paleoecological studies (e. g. Voigt, 1930; Hageman et al., 1998) since they commonly die in such a way that their relations to one another are preserved as they were in life (McKinney and Jackson, 1989). Presence or absence, zooid size, colonial form and integration of bryozoans contain conclusions about substrate, sedimentation, environmental stability and conditions, food supply and temperature (Smith, 1995). 4) Accordingly, bryozoans have become an example for the study of functional morphology (Schäfer, 1991), evolution (McKinney and Jackson, 1989, with review of literature) and zoogeographical aspects of speciation (Hayward, 1983; Soule and Soule, 1985a; Moyano, 1996). Data on bryozoans have significantly contributed the punctuated equilibrium theory (Gould, 2002 with review of literature), and biological individuality (Beklemishev, 1958-1960; Scholz and Levit, 2003). Yet, the use of bryozoan growth forms in the interpretation of palecology without adequate taxonomic classification is probably meaningless (Kelly and Horowitz, 1987), and non- or ill-classified organisms cannot be as a subject used in other fields of biology (Mawatari and Kajihara, 2003). Likewise, the utilisation of bryozoan data depends on continuous geographical coverage in collections. However, even in a comparatively well studied region such as the European coastal waters, monitoring activities have been subject to great variation through the last decades (Hayward, 2001). The aspect of fragmentary documentation is ever the more true for the modern Indo- 13

Pacific province were bryozoan communities constitute a considerable part of the sessile, benthic biomass, and thus achieve conspicuous presence over a huge ocean area. Especially the Western Pacific is considered to be the hot spot of bryozoan biodiversity (Gordon, 1984). With such a large number of regional bryozoan occurrences, and small numbers of systematists it would not be surprising if information about living specimens is rather insufficient (Winston, 1988), and what appears to be a simple request for the name of a bryozoan might be difficult or impossible to provide. Most of the taxonomical bryozoan monographs are several decades or a century old, and the oldfashioned style of descriptions and illustrations (for example in Ortmann 1890, Waters, 1909, or Canu and Bassler, 1929) precludes their reliable use in species identification. Therefore, the Grantin-aid projects for Monbusho International Scientific Research Programms have already supported re-descriptions of Japanese Bryozoa and other Japanese taxa (see Mawatari and Suwa, 1998). What has successfully been started for the Japanese fauna should now be applied on the whole Indo-Pacific. How biotas are affected by geographical isolation and environmental change is a question fundamental to ecology, historical biogeography, and phylogeography. A re-evaluation of types from Indo-Pacific collections (e.g. Waters, 1909, Red Sea; Ortmann, 1890, Japan; Harmer, 1915-1957, Indonesia; Canu and Bassler, 1929, Philippines), and modern state-of-the-art re-illustration of type material has now become necessary to have a new data base about the comparative morphology of Indopacific bryozoans. We should carefully consider both re-illustration of morphology, and geographical range. Specimens may not always be assigned unequivocally to a known species based on the holotype only. There is a need to understand morphological plasticity of species throughout their range of distribution. One of the first important achievements outlining regionalism and a high degree of speciation in tropical bryozoans is represented by the monograph of the Smittinidae of Hawaii (Soule and Soule, 1973), a family of ascophorine bryozoans that accordingly became known as marine Darwin Finches. During the past years, molecular biology has become a powerful tool in contribution to bryozoan taxonomy, on the grounds of combination with methods of traditional morphology (Levington et al., 2001). Meanwhile, studies on genetic relatedness have revealed metapopulations (Okamura, 2000), identified the presence of cryptic species in alleged cosmopolitan bryozoans (Hoare et al., 2001), and started to contribute to revised classifications of gymnolaemate bryozoans ( Dick et al., 2000, 2003). It has been found out that morphological differences are often, but not always consistent with taxonomical differences (Mackie et al., 2002). Accordingly, the statement made by McKinney and Jackson in 1989 that îmost descriptions of living bryozoans with calcified skeletons do not differ substantially from descriptions of fossil species in the same classî is only partly true today. Despite of the increasing demand for research in molecular biology and taxonomy of bryozoans, traditional morphology still has its values. Important regions of the Indo-Pacific like, for example, the Arabian (Persian) Gulf is still a terra incognita in terms of bryozoology. Aside from a brief account of Soule and Soule (1985b) on epiphytic bryozoans, nothing is known on the bryozoan fauna off the northern coast of Saudi Arabia. Recently, Azis et al. (2001) observed that bryozoans belong to the most important fouling organisms in the Persian Gulf region, but they did not provide a list of species that occur. The very diverse bryozoan fauna of Socotra (Yemen) is virtually unknown aside from a very preliminary account by Scholz et al. (2001), and a more extensive report is under way. The eastern coast of Africa is poorly studied, and aside from the report of Brood (1976) on certain taxa, and the Mauritius report of Hayward (1988), we still wait for an overview on the regional biodiversity of the phylum. Fortunately, for the Japanese bryozoans, the situation is not 14

so bad and there are numerous reports prepared by one of the authors (S. F. M.) and by his father Shizuo Mawatari. Some bryozoan studies have been conducted within the scope of the comparative survey Natural History Researches of northern Hokkaido of the National Science Museum, Tokyo, that outlined zoogeographical connections of the Hokkaido fauna to Russia (Mawatari et al., 1991), and triggered new research on comparative views of bryozoans from Northern Japan, Okhotsk, and the Kuril Arc (Grischenko et al., 2002). Previously, an inventorization of Recent marine Bryozoa living along Hokkaido Island was carried out by the efforts of Mawatari, S. (1957, 1973a, b, 1974), Mawatari, S.F. (1971, 1972), and by their collaborative researches (Mawatari and Mawatari, 1973, 1974; 1979, 1980, 1981a, b) with 22 cyclostome, 4 ctenostome and more than 130 cheilostome species reported, giving about 170 species in total. However, this is very preliminary number and more work needs to be done to receive a reliable data on bryozoan diversity of this region. Henceforth, a bryozoology PhD study (A. Grischenko, Hokkaido University, Sapporo) has been launched, and one of the goals of this study is to list all species of the bryozoans, that occur off the Oshoro Marine Biology Station, and other coastal areas of Hokkaido. Aside from the links to Russia that are evident in the bryozoans or the northern Japanese latitudes, there are surprising links of Recent Japanese bryozoans to the Southern Ocean (Gordon et al., 2002) that are also indicated by the fossil record (Brown, 1952: p.128). Compared with the bryozoans in Northern Japan, the warm water bryozoans of Japan that are influenced by the Kuroshio current are not so well known. The Bryozoa fauna of the cool temperate northern latitudes of Japan is strikingly different in both taxonomy, and growth morphologies. Therefore, for the bryozoans dwelling in the coastal seas off Honshu and Kyushu, we still have to rely on the historical accounts such as prepared by Ortmann (1890), Okada (1923), Saito (1931), and Sakakura (1935). Acknowledgements It is a pleasure to acknowledge valuable suggestions made by Mr. Andrei Grischenko, Hokkaido University, Sapporo, and the support of the staff of Port of Nagoya Public Aquarium for providing us data on the collection site. Furthermore, we would like to thank the Nagoya Port Authority for helpful support in field work. References Azis, P.K.A., Al-Tisan, I. and Sasikumar, N. (2001) Biofouling potential and environmental factors of seawater at a desalination plant intake. Desalination, 135, 69-83. Beklemishev, W. N. (1958-1960) Grundlagen der vergleichenden Anatomie der Wirbellosen. VEB Deutscher Verlag der Wissenschaften, Berlin, 441pp. (in 1958) and 403pp. (in 1960). Brood, K. (1976) Cyclostomatous Bryozoa from the coastal waters of East Africa. Zoologica Scripta, 5, 277-300. Brown, D.A. (1952) The Tertiary cheilostomatous Polyzoa of New Zealand. Trustees of the British Museum, London, 405 p. Canu, F. and Bassler, R.S. (1929) Bryozoa of the Philippine Region. Bulletin of the U.S. National Museum, 100, 1-685. Cook, P.L. (1985) Bryozoa from Ghana. A preliminary survey. Annales Musée de l Afrique centrale, Sciences zoologiques, Tervuren, 238, 1-315. Cook, P.L. (1988) Bryozoa. In Higgins, R.P. & Thiel, H. (eds.) Introduction to the Study of Meiofauna, pp. 438-442, Smithsonian Institution, Washington D.C. Cuffey, R.J. (1970) Bryozoan-Environment Interrelationships An Overview of Bryozoan Paleoecology and Ecology. Earth and Mineral Sciences, 39(6), 41-48. Dick, M.H., Freeland, J.R., Williams, L.P. and Coggeshall-Burr, M. (2000) Use of 16S mitochondrial ribosomal DNA sequences to investigate sister-group relationships among gymnolaemate bryozoans. Proceedings of 15

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