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Carbohydrate-binding modules (CBMs) are non-catalytic protein domains that bind to carbohydrates, and have been well studied in microorganisms. Endogenous CBMs in aquatic invertebrates, however, have not yet been identified, and little is known about their ecological significance to wetland environments. Using an approach of characterizing a recombinant CBM (CjCel9A) from a brackish bivalve, Corbicula japonica, this work identified CjCel9A-CBM’s cellulose-binding activity. Scatchard plot analysis in the study of CjCel9A-CBM binding to α-cellulose showed a high corresponding partitioning coefficient (Kr) of 20.33, indicating CjCel9A-CBM’s high affinity for cellulose. In addition, this affinity tolerated a high ion concentration buffer system, consistent with C. japonica’s adaption to brackish wetland environments. Moreover, immuno-scanning electron microscopy (immuno-SEM) suggested that CjCel9A-CBM binds to α-cellulose unevenly, which was further determined to be caused by its higher affinity for crystalline cellulose (Cellulose I, mostly seen in plant leaves). Together, these findings suggest that CjCel9A-CBM is capable of immobilizing its associated catalytic domain on environmental crystalline cellulose (i.e., fallen leaves) in wetland sediments. Most importantly, they could provide a reasonable answer to a question recognized broadly in wetland ecologists, namely, why many wetland sediments have constant cellulase activities, although the sediments are being washed almost every day.

Seven species of the freshwater snail genus Semisulcospira, which are indigenous taxa of the largest lake in Japan, Lake Biwa, have been introduced into 17 localities, including five newly recorded localities. Among these species, S. dilatata Watanabe and Nishino, 1995, S. habei Davis, 1969, and S. rugosa Watanabe and Nishino, 1995 were firstly recorded as introduced species. The alien Semisulcospira snails could be identified based on the combination of three morphological characters: longitudinal ribs number of penultimate whorl of adult shell, spire angle of adult shell, and shell height of the largest embryonic shell. Accordingly, morphological identification of introduced Semisulcospira populations recorded in previously published studies was revisited. The introductions of Semisulcospira snails have been suggested to have occurred due to contamination of seedlings of other shellfish and fishes at most sites. Taking into account the potential impacts of accidental species introductions, seedlings should be carefully managed so as not to unintentionally introduce Semisulcospira snails.

Recently, numerous species of aquatic invertebrates inhabiting wetlands have been shown to possess endogenous cellulase, following the discovery that termites have cellulase genes encoded in their own genome rather than relying on symbiotic bacteria for decomposing cellulose. Wetlands have been empirically shown to play an important role in the decomposition of land‐originating hard‐to‐degrade polysaccharides such as cellulose. However, the mechanism that connects the cellulase producer and the wetlands remains unknown, which makes it very difficult to evaluate the ecological function of wetlands. Here we found that a macrobenthic bivalve, Corbicula japonica, secretes its cellulase to the wetland sediment. Secreted cellulases are immobilized in the components of the sediment. Moreover, adding cellulose or glucose to C. japonica could trigger its cellulase secretion level. These findings suggest a novel wetland cellulose decomposition mechanism. The decomposition ability of wetlands was previously ascribed only to microbes and/or invertebrates that contain cellulases. Our findings suggest that benthic animals supply wetlands with their enzymes as decomposition agents, while wetland sediments serve as immobilization scaffolds for the enzymes. This system, which was named by us an “environmental bioreactor system,” could provide a key function in wetlands.

Phenoloxidases (POs) play a crucial role in melanization of crustaceans. There are at least two types of POs characterized in crustaceans: the conventional type (POα here) that is expressed in hemocytes and POβ, a secreted protein synthesized in the hepatopancreas. We investigated the source of PO activity in the hemolymph of a lobster and determined the kinetic parameters of mono- and di-PO activities. In the lobster hemolymph, POα, which formed a hexamer similar to both POβ and hemocyanin, contributed to PO activity, whereas the amount of POβ was low. Kinetic analyses using purified prophenoloxidase of crustaceans showed that lobster POα has a higher rate constant, while shrimp POβ has higher specificity in both mono- and di-PO reactions, when tyramine and dopamine were employed as substrates. There should be at least two types of PO molecules in crustacean hemolymph, but the dominant PO molecule type varies among species.

Scyphozoa, Cubozoa and Hydrozoa are classes in the phylum Cnidaria that undergo metagenesis involving a dramatic morphological transition. In Scyphozoa and Cubozoa, when exposed to species- or strain-specific transition-inducing stimuli, asexually reproducing benthic polyps transform into sexually reproducing planktonic medusae. In Hydrozoa, exposure to species- or strainspecific transition-inducing stimuli causes formation of medusa buds in the polyp's body. In Aurelia aurita (Linnaeus, 1758) (Scyphozoa, Semaeostomeae), polyp-to-jellyfish transition is induced by some simple indole compounds. However, whether indole compounds can induce polyp-to-jellyfish transition in Cubozoa and Hydrozoa remains unknown. In the present study, we show that an indole compound, 5-methoxy-2-methylindole, induces polyp-to-jellyfish transition in Scyphozoa and Cubozoa. This inducing action suggests that the downstream steps of polyp-to-jellyfish transition are regulated by the same biochemical reactions in Scyphozoa and Cubozoa, irrespective of the type of transition-inducing environmental stimuli.

Hypoxia often occurs in summer and causes deleterious effects on marine benthic animals. A marine annelid, Capitella teleta, has tolerance to hypoxia, as shown by the fact that it inhabits organically polluted area, where severe hypoxia is often observed. To understand how this species adapts to the environment, we focused on its hypoxia sensor, and we showed that TRPAbasal was a possible contributor to hypoxia detection in C. teleta. To examine the involvement of TRPA1 in the response of C. teleta to hypoxia, we exposed C. teleta to hypoxic water with or without a TRPA1-specific inhibitor, A-967079. Hypoxic stimulation induced escape behavior of C. teleta from the sediment, and this behavior was suppressed by the inhibitor. The cloned TRPA gene from C. teleta was phylogenetically categorized into TRPAbasal, and contains an oxygen-dependent degradation domain, which is important for the detection of hypoxia. Whole-mount in situ hybridization analysis showed that the gene was transcribed in the prostomium, where sensing functions are localized. These results suggested that the worm has a hypoxia-sensing system possibly utilizing CtTRPAbasal, and this system contributes to expanding the organism's niches in hypoxic environments by detecting whether hypoxia exceeds a level that would imperil its survival.

Carbon and nitrogen stable isotope ratios (δ13C and δ15N) of sympatric nereidid polychaetes including Hediste diadroma, H. atoka, Tylorrhynchus osawai, and Simplisetia erythraeensis were examined at six stations along the Japanese coast to reveal the trophic niches of nereidid polychaetes in estuarine benthic food webs. The species studied exhibited a large degree of spatial variation in δ13C values (− 25.3 to − 13.7‰) among stations, suggesting site-based differences in nereidid dietary components. The low δ13C values of nereidids suggest that terrestrial plant materials are important components of their diets in river-affected estuarine habitats, which were also shown by a δ13C-based stable isotope mixing model (SIAR). δ13C values of H. diadroma and H. atoka were 0.3–3.1‰ lower than those of the sympatric T. osawai and S. erythraeensis, while no significant difference was found between Hediste species. A biochemical assay revealed cellulase activity in H. diadroma, H. atoka, and T. osawai (S. erythraeensis was not examined), while the apparent molecular size of cellulase differed between the two genera. These observations suggest that some nereidids can directly assimilate terrestrial plant matters using cellulases, although the dietary importance of terrestrial matter varies among genera. Species with the ability to digest cellulose may be better able to colonize river-dominated estuarine habitats, where refractory terrestrial detritus is a major carbon source.

Shelk2, a novel shell matrix protein from the Pacific oyster, Crassostrea gigas, is reported to be involved in shell biosynthesis of the prismatic layer. Results of RNAi experiment on shelk2 showed that Shelk2 has a key role in shell regeneration. When dsRNA of shelk2 was injected into the adductor muscle of Pacific oyster, the prismatic layer did not grow normally during shell regeneration. Observation of regenerated shell using scanning electron microscopy (SEM) revealed that the size of each column in the prismatic layer was reduced, and the edge of the column top looked rounder. From these results, it was deduced that the columns were less tightly bound with each other than in normally regenerated shells. Furthermore, the surface of the column appeared to be rough. Unexpectedly, the expression level of shelk2 mRNA was not reduced but remarkably enhanced by the knockdown experiment. Further experiments including gene and protein expression will be necessary for a better understanding of its function and role in oyster shell regeneration.

We assessed cellulase activity in several crustaceans of different taxonomic groups inhabiting the mangrove areas of Malaysia to clarify whether these animals could digest cellulose derived from mangrove trees. We investigated four Copepod species, two Mysida species, and ten Decapoda species. Three copepod species demonstrated multiple cellulase active bands that differed among species in a zymographic assay, suggesting that these animals were equipped with distinct cellulases. Interestingly, the way the cellulase were expressed in the zymographic assay differed, even among members of the same species collected at different locations, suggesting that cellulase expression patterns of copepod species are regulated by environmental factors. Although no significant cellulase activity was detected in two of the Mysida species, widespread distribution of cellulases was also detected in decapod species. Multiple common active bands in the various organs of decapod species were detected by the zymographic assay, while remarkable activity was detected in the hepatopancreas in the reducing sugar assay. The above findings suggested that cellulases are synthesized in the hepatopancreas and then secreted into digestive tracts such as the stomach and intestine. The present study shows that various crustaceans comprising most of the biotic resources in mangrove areas may be there because of their ability to digest cellulose. Discipline: Aquaculture Additional key words: Cellulase, Copepod, Decapoda, Digestion, Mysida The present study was funded by the Japan International Research Center for Agricultural Sciences (JIRCAS) and partly by Grants-inAid for Scientific Research from the Japan Society for the Promotion of Science (nos. 22405030 and 25292113) * Corresponding author: e-mail toyohara@kais.kyoto-u.ac.jp Received 13 May 2014;accepted 3 September 2014. Introduction All zooplanktons are heterotrophic, although the individual species differ in how they obtain organic energy; classified as herbivores, carnivores, detritivores and omnivores (Lalli & Parson 1997a). In temperate areas, zooplankton mainly feed on phytoplankton and are classified as carnivores (Bouillon et al. 2000). In tropical mangrove areas, however, the amount of phytoplankton is assumed to decrease due to the low transparency of water caused by the suspended clay particles (Okamura et al. 2010, Robertson et al. 1992). Conversely, a remarkable number of zooplankton inhabit mangrove areas, including small crustaceans such as mysid crustaceans, despite the turbid water (Hanamura et al. 2008). Therefore, it seems likely that small crustaceans in the mangrove areas feed on alternative carbon sources and not solely on phytoplankton. Recently, our studies showed that various aquatic invertebrates in temperate zones could break down cellulose using cellulases (Niiyama & Toyohara 2011). In addition

Iron is one of the essential trace elements for humans. In this study, the iron contents in fresh, dried, and toasted nori (Pyropia yezoensis) were analyzed. The mean iron content of fresh, dried, and toasted nori were 19.0, 22.6, and 26.2 mg/100 g (dry weight), respectively. These values were superior to other food of plant origin. Furthermore, most of the iron in nori was maintained during processing, such as washing, drying, and toasting. Then, the form of iron in fresh, dried, and toasted nori was analyzed. As a result, an iron storage protein ferritin contributed to iron storage in raw and dried nori, although the precise rate of its contribution is yet to be determined, while ferritin protein cage was degraded in the toasted nori. It is the first report that verified the ferritin contribution to iron storage in such edible macroalgae with commercial importance.

Chlorophyll c2 in a Sargassum horneri extract suppressed the degranulation of rat basophilic leukaemia RBL-2H3 cells. In this contribution, the mode of action of S. horneri extract was revealed. The extract inhibited the elevation of intracellular Ca 2+ concentration induced by the antigen. Immunoblot analysis showed that S. horneri extract down-regulated the phosphorylation of both Syk and PI3K in the signalling pathways involved in degranulation triggered by antigen–antibody interaction. In addition, oral administration of S. horneri extract inhibited mice nasal rubbing behaviour sensitized with anti-DNP IgE. These findings suggest that S. horneri extract has anti-allergic activity via the suppression of degranulation of mast cells and basophils.

The peptidyl prolyl cis/trans isomerase Pin1 enhances the uptake of triglycerides and the differentiation of fibroblasts into adipose cells in response to insulin stimulation. Pin1 downregulation could be a potential approach to prevent and treat obesity-related disorders. In order to identify an inhibitor of Pin1 that exhibited minimal cytotoxicity, we established a high-throughput screen for Pin1 inhibitors and used this method to identify an inhibitor from 1,056 crude fractions of two natural product libraries. The candidate, a phlorotannin called 974-B, was isolated from the seaweed, Ecklonia kurome. 974-B inhibited the differentiation of mouse embryonic fibroblasts and 3T3-L1 cells into adipose cells without inducing cytotoxicity. We discovered the Pin1 inhibitor, 974-B, from the seaweed, E. kurome, and showed that it blocks the differentiation of fibroblasts into adipose cells, suggesting that 974-B could be a lead drug candidate for obesity-related disorders.

We investigated the relationship between digestive function and ecological distribution for four related species of Gastropoda (Cerithidea rhizophorarum, Cerithidea cingulata, Batillaria multiformis, and Batillaria attra- mentaria) inhabiting the Tanaka River estuary (Mie Prefecture, Japan). We compared the digestive enzyme activities for four hard degradable carbohydrates, namely, cellulose, mannan, xylan, and laminarin. Among the investigated four species, Cerithidea rhizophorarum showed the highest cellulase, mannanase, xylanase, and laminarinase activi- ties, and was also dominantly distributed in the estuary reed bed. The results of CN stable isotopic analysis suggested that this species assimilated organic matter derived from reeds. Hard degradable carbohydrates, including plant com- ponents derived from the reed bed or from dry land, accumulate in the sediment of estuaries. Estuarine benthic ani- mals are assumed to consume these accumulated hard degradable carbohydrates, as well as microphytobenthos and particulate organic matter (including phytoplankton). Our present findings suggest that Cerithidea rhizophorarum is dominant in reed beds because it can more efficiently digest plant-derived carbohydrates than can Cerithidea cingu- lata, B. multiformis, and B. attramentaria. To the best of our knowledge, ours is the first study to demonstrate that the specific ecological distribution of related animal species with similar ecological traits can be explained by the effi- ciency of their digestive enzyme activity.

For the purpose of investigating the cellulose degradation mechanism in mangrove estuaries, cellulase activities of three Acetes shrimps (Acetes sibogae, A. japonicus and A. indicus) were examined in the Matang Mangrove Forest Reserve (MMFR). As a result of cellulose plate assay measurement, we detected the activities from all three species. SDS-PAGE zymographic analysis revealed that all Acetes shrimps demonstrated active bands at approximately 25 kDa and higher than 100 kDa. In addition, by quantifi cation of reducing sugar production, hepatopancreas samples of Acetes showed signifi cantly higher cellulase activity compared to whole body samples suggesting their endogenous origin. Our fi ndings that cellulase activities are widely distributed among the Acetes shrimps living in the MMFR, suggests that mangroves are indirectly utilized by higher trophic consumers.(Keywords: cellulase, mangrove, Acetes, hepatopancreas)

Cellulose is utilized as a nutritional source by various organisms. For a long time it was believed that only protozoa, bacteria, and fungi, in addition to plants and photosynthetic bacteria, are able to synthesize cellulases encoded by their own genes. However, the widespread distribution of cellulases throughout the animal kingdom has recently been recognized. Conventionally, animals digest cellulose utilizing cellulases derived from symbiotic bacteria in the digestive organs. However, recent molecular biological studies have shown that some cellulase genes are actually encoded on animal chromosomes. In addition, the homologous primary structure of cellulases obtained from various invertebrate phyla indicates the possible vertical transfer of the cellulase gene from ancient organisms that are now extinct. The results of studies on cellulases with unique enzymatic properties are expected to be applied to bioethanol production and aquaculture. In the present review, we describe cellulases, focusing primarily on aquatic invertebrates in which both endogenous and exogenous cellulases are involved in the breakdown of cellulose in the digestive organs.

The anti-allergic effect of alcohol extracts from edible seaweeds was evaluated by the degranulation of a rat basophilic leukemia cell line (RBL-2H3). Extracts prepared from the brown algae Sargassum horneri, Ecklonia cava and Ecklonia kurome, inhibited the degranulation of RBL-2H3 cells, whereas those from green algae and red algae did not. The active substance in S. horneri was purified and identified as chlorophyll c2, a major pigment in brown algae. The purified chlorophyll c2 preparation also suppressed the degranulation of RBL-2H3 cells. In contrast, chlorophyll a and b did not affect cell degranulation. Therefore, chlorophyll c2 from brown algae might be useful for the prevention or mitigation of allergic symptoms.

Iron is an essential element for virtually all kingdoms of life, and especially for primary producers in ocean ecosystems. To date, the molecular mechanism of iron utilization by macroalgae remains largely unknown. To elucidate the strategy of iron acquisition and storage in macroalgae, we focused on the function of the iron storage protein ferritin in the sea lettuce, Ulva pertusa, which has abundant iron content. Judging from the primary structure, U. pertusa ferritin (UpFer) can be classified as a land-plant-type ferritin, which is usually found in plastids. The gene of UpFer was expressed in the peripheral, central and rhizoid parts. Western blot analysis showed that UpFER was present and functioned in processed 26- and 22-kDa forms. Furthermore, recombinant UpFER had iron incorporation activity comparable to other ferritins. These results suggest that ferritin also functions as an iron storage protein as in unicellular algae and land plants.

During shell formation, little is known about the functions of organic matrices, especially about the biomineralization of shell prismatic layer. We identified a novel gene, shelk2, from the Pacific oyster presumed to be involved in the shell biosynthesis. The Pacific oyster has multiple copies of shelk2. Shelk2 mRNA is specifically expressed on the mantle edge and is induced during shell regeneration, thereby suggesting that Shelk2 is involved in shell biosynthesis. To our surprise, the database search revealed that it encodes a spider silk-like alanine-rich protein. Interestingly, most of the Shelk2 primary structure is composed of two kinds of poly-alanine motifs-GXNA(n)(S) and GSA(n)(S)-where X denotes Gln, Arg or no amino acid. Occurrence of common motifs of Shelk2 and spider silk led us to the assumption that shell and silk are constructed under similar strategies despite of their living environments.

We have been assessing the activity level of cellulase in wetland sediments to clarify the significance of cellulase for the turnover of plant cellulose in wetlands. In this study, we investigated the cellulose degrading function of sediment in wetlands to clarify the biochemical breakdown mechanism of cellulose. Specifically, we measured cellulase activities of sediments collected from wetlands of the Chikugo River (Fukuoka Prefecture), Midori River (Kumamoto Prefecture), Hamado River (Kumamoto Prefecture), Yodo River (Osaka Prefecture), and Tanaka River (Mie Prefecture). The results revealed that the activity levels differed significantly among rivers. Additionally, the cellulase activities of the sediment were not completely suppressed in the presence of chloramphenicol. These findings suggested that a portion of the cellulase activities were derived from cellulases binding to the components of sediments. Actually, sediments also showed the ability to bind fungal cellulase. Comparison of the binding ability of clay and plant residues, the main components of sediments, revealed that plant residues had significantly higher abilities to bind cellulase. This finding was supported by the fact that there was a strong correlation between the organic matter content in the sediment and the cellulase-binding ability (R = 0.66). Results of this study show that sediment complexes harboring cellulases might function as a bioreactor to degrade cellulose in wetlands.

Plant ferritins have some unique structural and functional features. Most of these features can be related to the plant-specific “extension peptide” (EP), which exists in the N-terminus of the mature region of a plant ferritin. Recent crystallographic analysis of a plant ferritin revealed the structure of the EP, however, two points remain unclear: (i) whether the structures of well-conserved EP of plant ferritins are common in all plants, and (ii) whether the EP truly contributes to the shell stability of the plant ferritin oligomer. To clarify these matters, we have cloned a green-plant-type ferritin cDNA from a green alga, Ulva pertusa, and investigated its crystal structure. Ulva pertusa ferritin (UpFER) has a plant-ferritin-specific extension peptide composed of 28 amino acid residues. In the crystal structure of UpFER, the EP lay on and interacted with the neighboring threefold symmetry-related subunit. The amino acid residues involved in the interaction were very highly conserved among plant ferritins. The EPs masked the hydrophobic pockets on the ferritin shell surface by lying on them, and this made the ferritin oligomer more hydrophilic. Furthermore, differential scanning calorimetric analysis of the native and its EP-deletion mutant suggested that the EP contributed to the thermal stability of the plant ferritin shell. Thus, the shell stability and surface hydrophobicity of plant ferritin were controlled by the presence or absence of the plant-ferritin-specific EP. This regulation can account for those processes such as shell stability, degradation, and association of plant ferritin, which are significantly related to iron utilization in plants.

To validate the mechanism of cellulose breakdown in cold climate wetlands, we investigated cellulase activity in sediments collected from 17 wetland sites in Hokkaido, the northern area of Japan. We evaluated cellulase activity by quantitative analysis of glucose released from carboxymethyl cellulose and found that sediments from peat fens demonstrated high activity, followed by sediments from lagoons and estuaries. Sediments from peat fens also contained greater amounts of organic matter, followed by lagoons and estuaries, thereby suggesting a strong positive correlation between organic matter content and cellulase activity. Evaluation of cellulase activity by qualitative cellulose zymographic analysis showed that various cellulases with different molecular sizes were implicated in cellulose breakdown in wetlands. Among them, cellulose breakdown in Meguma Pond (peat fen), Notsuke Gulf (peat fen), and Lake Utonai (lagoon) was potentially due to microorganism cellulase, while that in Lake Chobushi (lagoon) was ascribed to meiobenthos (Oligochaeta species) cellulase. The findings presented herein suggest that the origin and activity level of cellulase vary depending on the type of cold climate wetland.

Cellulase and hemicellulases (mannanase, xylanase, xyloglucanase, and licheninase) activities were measured in the extracts of 18 species of macrobenthos (an oligochaete, two polychaetes, two decapods, one amphipod, three bivalves, and nine gastropods) to check the relevance of macrobenthos to the breakdown process of hard-degradable carbohydrates derived from terrestrial plants. As a result, these enzyme activities were detected in all of the extracts of macrobenthos except for Tubificidae sp. High cellulase activities were detected in the extracts of the decapods (Neocaridina denticulate denticulate, Caridina multidentata) and the gastropods (Semisulcospira libertina, S. reiniana, Physa acuta, Terebralia palustris). However, only weak activities were detected in the polychaetes (Perinereis nuntia, P. aibuhitensis) and the bivalve (Ruditapes philippinarum). High mannanase and licheninase activities were detected in two decapod species (N. denticulate, C. multidentata), while high xylanase activities were seen in the gastropods (S. libertina, S. reiniana). High xyloglucanase activities were noted in the gastropod (T. palustris). These facts suggest that the activities of different types of hemicellulase were distributed rather unevenly among the macrobenthos. The present paper is the first to report the widespread distribution of cellulase and hemicellulase activities among ecologically important aquatic invertebrates, and the possible relevance of these species in the breakdown process of plant-derived hard-degradable carbohydrates.

Bivalves are often the dominant macrobenthos species in exposed sandy beach environments. However, our understanding of their recruitment processes before post-settlement stages on sandy beaches with highly energetic environments is incomplete. To clarify the characteristics of the free-swimming planktonic stage that affects recruitment efficiency in sandy shore ecosystems, we investigated the temporal (weekly–biweekly) variation of bivalve planktonic larval concentration coupled with oceanographic conditions on an exposed sandy shore on the sea of Kashima-nada, Japan, from summer 2003 to autumn 2005. Larvae were observed throughout the year, but the surge of larval concentration composed of sandy beach and sessile bivalves occurred most prominently in summer, from August to September. The peak concentration of larvae during this season was more than 1000 times higher than in other seasons. The larval concentration was positively correlated with water temperature and northward wind velocity and negatively correlated with each of the nutrient concentrations. On the other hand, chlorophyll a concentration and salinity seemed to have little effect on the larval concentration. Based on this fundamental knowledge, further investigations about planktonic larvae in sandy beaches are needed.

We examined the feeding niche of four species of molluscs by analysis of their stable isotope signatures and cellulase activities to determine if they could utilize terrestrial organic matter. The molluscs and potential food sources were collected from the upper, middle and lower estuary of the Yura River from spring 2007 to winter 2008. All species showed positive cellulase activity which highlighted their potential to digest terrestrial organic matter. Consumption and assimilation of terrestrial organic matter by estuarine molluscs however varied spatially and temporally, reflecting species-specific differences in feeding niche and in response to variations in food availability in the estuary. Thus, terrestrial primary production in the catchment area supports secondary production of molluscs in the Yura River estuary.

SUMMARY Corbicula japonica is a typical brackish water bivalve species belonging to the order Veneroida, and it is the most important inland fishery resource in Japan. Corbicula japonica has been suggested to assimilate organic matter from terrestrial plants, unlike Ruditapes philippinarum and Mactra veneriformis, which selectively assimilate organic matter of marine origin. This led us to hypothesize that C. japonica, despite being a suspension feeder, could assimilate cellulosic materials derived from terrestrial plants. In the present study, we measured cellulase and hemicellulase activities in the crystalline styles of C. japonica and other commercially important Veneroida bivalve species in Japan: Ruditapes philippinarum, Meretrix lamarckii and Meretrix lusoria. Corbicula japonica demonstrated notably higher cellulase, xylanase and β-mannanase activities than the other marine bivalves, suggesting that this species possesses a far greater biochemical capacity to break down the structural polysaccharides of plant cell walls than the other species. In contrast, the β-1,3-glucanase and pectinase activities of C. japonica were similar to or even lower than those of the others. This is possibly due to the presence of these polysaccharides in the cell walls of diatoms, a principal food of most marine bivalves. Although direct evidence is lacking, the high cellulase, xylanase and β-mannanase activities of C. japonica may result from adaptation to an upstream estuarine environment where phytoplankton and diatoms are scarce, but plant-derived substances are abundant.

Studies on the cellulose utilization by animals have been conducted in keeping with the recent developments in molecular biology. In mollusks, endogenous cellulases have been reported from blue mussel, abalone, and freshwater snail. We previously reported the possibility of cellulose assimilation by Corbicula japonica , a representative bivalve dominant in brackish water environments in Japan, and the cloning of its endogenous cellulase (β-1,4-glucanase) gene (Sakamoto, K., Touhata, K., Yamashita, M., Kasai, A. and Toyohara, H., 2007. Cellulose digestion by common Japanese freshwater clam Corbicula japonica . Fish. Sci. 73, 675–683). However, the gene of β-glucosidase, another enzyme essential for the complete cellulose decomposition to glucose units, has not yet been isolated from the mollusk. Therefore, we attempted the molecular cloning of endogenous β-glucosidase from C. japonica and succeeded in the isolation of a cDNA with a 2832-bp open reading frame (ORF) encoding 943 amino acid residues ( CjCel1A ). CjCEL1A has 2 repeated GHF-1(Glycosyl Hydrolase family 1)-like domains and showed high similarity with known insect β-glucosidases and mammalian lactase-phlorizin-hydrolases. Reverse transcription (RT)-PCR analysis and in situ hybridization revealed that CjCEL1A is likely to be produced in the secretory cells in the digestive gland, suggesting that CjCEL1A is a digestive β-glucosidase of C. japonica and is not derived from symbionts.

We previously reported endogenous Glycoside Hydrolase Family (GHF) 9 β-1,4-glucanase gene, CjCel9A, from common Japanese freshwater clam Corbicula japonica. Here we identified another endogenous β-1,4-glucanase genes which belong to GHF45 (CjCel45A, CjCel45B). Both genes encode ORF of 627 bp corresponding to 208 amino acids. CjCel45A and CjCel45B are different in 5′ and 3′-untranslated regions and six nucleotides in the ORF. CjCEL45 has only one GHF45 catalytic domain without any carbohydrate binding modules as is the case with other molluskan GHF45 enzymes. Phylogenetic analysis and genomic structure of CjCel45 gene implies that this gene is likely to be acquired from fungi by common ancestor of mollusks. Reverse transcription (RT)-PCR analysis and in situ hybridization revealed that CjCel45A is likely to be expressed in the secretory cells in the digestive gland, suggesting that this cellulase is produced in the same site as CjCEL9A. CjCEL45A was successfully expressed in E. coli cells and zymographic analysis of the recombinant CjCEL45A showed that CjCEL45A is a functional β-1,4-glucanase. The finding of multiple cellulase genes in C. japonica strongly supports our hypothesis that this species function as a cellulose decomposer in estuarine environments.

The relative contributions of larval recruitment and post-settlement mortality to the establishment of the zonation of Acropora communities, a dominant group in reef corals, remain unknown. Our field research revealed that the relative abundances of juveniles between two morphological categories of acroporid corals were different on shallow and deep slopes: tabular species dominated in shallow waters (1.5-2.5m), whereas arborescent species dominated in deep waters (5.5-11m). These abundances were consistent with the distributional patterns of the adults, suggesting that the zonation structure is formed during the initial life stages of settlement and early post-settlement periods. To determine to what extent Acropora zonation is determined by depth-related differences in initial mortality among species, a transplant experiment using artificially settled corals (10 days after settlement) of four dominant species (the shallow-dominants Acropora digitifera and A. selago and the deep-dominants A. muricata and A. tenuis) was performed at four depths (1.5, 2.5, 5.5, and 11m). Survivorship of all four species was lower at 5.5m than the other depths and that the survival rate of A. muricata was much higher than that of other species at all depths. However, there was no species-specific survival related to adaptation to a particular habitat (i.e., depth), implying that Acropora spats do not possess species-specific tolerance for the environmental stresses of their own particular habitat, at least during the first 6 months after settlement. Post-settlement survival rate during this period is therefore not a major factor in forming the vertical zonation pattern of these Acropora species in subtropical reefs around Japan.

Investigating the spatio-temporal patterns of planktonic larvae is fundamental to studies regarding stock assessment and larval dispersal of commercial and non-commercial, i.e. invasive or rare marine invertebrates. Because of the difficulty involved in morphological identification of marine invertebrate larvae, various molecular methods based on PCR have been used to enhance taxonomic resolution. In previous studies, different methods for the preservation or pretreatment of larvae were applied in each case. However, no comparative studies have been conducted to determine the optimal method for PCR testing for bivalve larvae, and no information is available regarding the selection of an appropriate method.This study compared the PCR success rate of 6 pretreatment methods for larvae of the Mediterranean blue mussel, which was preserved using different fixatives (70% ethanol, 100% ethanol, 70% acetone and 10% formalin). The results revealed that the success rate of PCR was different for each pretreatment; moreover, the use of ammonium sulphate and Tween 20 buffer with proteinase K digestion was found to be the most effective method. Some pretreatments showed lower success rates for long-fixed larvae than for short-fixed larvae for formalin-fixed larvae; however, the success rate of PCR amplification for ethanol-fixed larvae pretreated by this method did not decrease through 1-year fixation. In addition, this pretreatment showed a high success rate for different fixation periods. These findings suggest that the selection of the pretreatment method is critically important for successfully amplifying larval DNA and that the pretreatment involving the use of ammonium sulphate prior to PCR amplification enables the use of fixatives for preserving bivalve larvae. This method will be utilized in various field studies and molecular genetic studies.

Cellulose digestion by Corbicula japonica was investigated according to the hypothesis that without any symbiotic aid, this organism can utilize cellulose as a carbon source. Enzymatic studies revealed the complete cellulase activity of this species, and molecular cloning resulted in the isolation of cDNA with an ORF encoding a 596-amino-acid protein that shares significant homology with abalone and termite cellulases with an amino acid identity of 52.2% and 50.5%, respectively. The isolated cellulase had a carbohydrate-binding module at the N-terminal region that was also reportedly present in abalone cellulase, and its mRNA were specifically expressed in the digestive gland. These findings strongly support the assumption that C. japonica has an endogenous cellulose, as well as abalones and termites. It is further believed that C. japonica plays an important roll in decomposing cellulose, and consequently contributes to the carbon-cycle in the aquatic environment, as termites do in terrestrial forests.

Carbon and nitrogen stable isotope ratios in tissue of the bivalve corbicula Corbicula japonica and particulate organic matter (POM) were measured in Lake Jusan, Lake Ogawara and Lake Shinji, which are the foremost fishing grounds for the corbicula in Japan, to determine their food sources. The bivalves in Lake Ogawara and Lake Shinji showed enriched isotope composition, while those in Lake Jusan were depleted. In addition, the difference in the isotope ratios between the sampling sites was remarkable in Lake Jusan. Chlorophyll concentrations were significantly higher in Lake Ogawara and Lake Shinji than those in the inflow rivers, although that in Lake Jusan was equivalent to that in the river. Residence time of river water was estimated at 1 day, 455 days and 88 days in Lake Ogawara and Lake Shinji assimilate autochthonous phytoplankton, while those in Lake Jusan assimilate terrestrial matter in the upper reaches and marine phytoplankton in the lower reaches because of low production in the lake.

The post-mortem changes of type I and V collagens in Japanese flounder muscle during chilled storage were examined. The muscle softened significantly within 12 h under chilled storage. Both type I and V collagens isolated by ion-exchange chromatographies showed no remarkable changes in band patterns even after 24 h of storage, suggesting that degraded collagen molecules may be removed from the collagen fraction by a conventional preparation method. In contrast, type I and V collagen molecules were detected by Western blotting with each specific antibody in a saline extract and gradually increased during chilled storage. These results suggest that both type I and V collagens may participate in the post-mortem softening of fish muscle.

To elucidate the involvement of amino acid transporter in osmotic adaptation of oysters, the expression of the amino acid transporter gene in response to environmental osmotic changes was investigated. As expected, the expression of the amino acid transporter gene was increased by hyper-osmotic stress, probably to elevate intracellular osmolality. Unexpectedly, the expression was also increased by hypo-osmotic stress, and the level of expression was higher than that induced by hyper-osmotic stress. To identify the region regulating the expression of the oyster amino acid transporter gene in response to changes in environmental osmolality, the 5′-flanking region of approximately 2.3 kb upstream from the translation start site was cloned. Expression vectors with luciferase as a reporter gene driven by 5′-flanking regions with different lengths were constructed and their promoter activities were compared. As a result, the osmo-responsive regulatory region responding to osmolality by both hyper- and hypo-osmolality was found within 132 bp from the transcription start site.

Expression of taurine transporter in response to osmotic stress was investigated at the protein level in the mantle of the Mediterranean blue mussel by using the specific antibody raised against the carboxy-terminal region of the deduced amino acid sequence of mussel taurine transporter. Immunohistochemical observation revealed that taurine transporter was expressed in the mantle and the expression was up-regulated in response to hypo-osmotic stress, while down-regulated in response to hyper-osmotic stress. Western blot analysis revealed major protein bands corresponding to 62 kDa and 65 kDa. In response to hypo-osmotic stress, the 62 kDa band became more intense, while it became less intense when the ambient osmolality was elevated. These results suggested that the 62 kDa taurine transporter would be implicated in hypo-osmotic adaptation.

Expression of HSP70 in response to heat-shock was investigated at the protein and mRNA levels in Mediterranean blue mussel. Western and Northern blot analyses revealed that HSP70 was expressed following heat-shock in the mantle at both protein and mRNA levels, suggesting that gene expression of HSP70 is implicated in the cellular response to heat-shock stress in mussel. It was then attempted to clone HSP70 cDNA in order to determine the primary structure of mussel HSP70. As a result, two full-length cDNA encoding HSP70 were isolated from a cDNA library prepared from the heat-shocked mantle. The isolated cDNA consist of single open reading frames of 2067 bp and 1911 bp which encode proteins of 689 amino acids and 637 amino acids, respectively. Both HSP70 cDNA encode an ATPase do main, and a substrate-binding do main in addition to a Glu-Glu-Val-Asp (EEVD) peptide motif that is specific for cytosolic HSP70. These findings suggest that the cDNA clones obtained in the present study encode cytosolic HSP70.

One of the biggest and long-standing difficulties in investigation of larval ecology in the field is species-level identification. In the present study, we developed polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) analysis based on the large subunit (LSU) rRNA gene (rDNA) D1/D2/D3 region for identification of multiple species of bivalve larvae using 14 species of bivalve collected from Maizuru Bay. The LSU rDNA D1/D2/D3 region of all analyzed species could be amplified by PCR using bvD1f/bvD3r primers, and RFLP analysis by HaeIII digestion on the PCR products showed species-specific fragment patterns. Furthermore, this analysis applied to single bivalve larvae in Maizuru Bay revealed efficient amplification of the target region and the species-specific pattern from 80% of the larvae, 75% of which showed a pattern that matched a certain pattern of the adult bivalves. In addition, the analysis of inter- and intraspecies variation of the LSU rDNA D1/D2/D3 region using the sequence data of the genus Crassostrea from the DDBJ database showed high applicability of this RFLP analysis on closely related species. Because of the wide applicability and technical simplicity, this method can become the standard for the identification of bivalve larvae species once the sequence data of the LSU rDNA D1/D2/D3 region of many bivalve species have been accumulated.

We have cloned a cDNA encoding the matrix metalloproteinase (MMP) from oyster Crassostrea gigas. The clone contains a 1797 bp open reading frame encoding a protein of 599 amino acids. Oyster MMP (Cg-MMP) has a transmembrane domain at its C-terminal and a furin/prohormone convertase cleavage site at the end of a propeptide domain, which are commonly observed in membrane-type MMPs (MT-MMPs). This suggests that Cg-MMP is an MT-MMP. The deduced amino acid sequence of oyster MMP shares approximately 30% identity with human MT4-MMP and MMPs from fruit fly and hydra. Cg-MMP mRNA was detected in the gill, mantle and adductor muscle, and more intense signals in the northern blot analysis were recognized in the gill and adductor muscle. Similar tissue distribution was observed for tissue inhibitor of metalloproteinase (Cg-TIMP) in oyster. In response to hypoxic stress, the abundance of Cg-MMP mRNA was elevated in the gill, while that of Cg-TIMP mRNA remained almost constant. These findings suggest that promotion of collagen metabolism may be implicated in the hypoxic adaptation in oyster.

Post-mortem muscle tenderization during chilled storage is thought to result from loss of physical strength due to the disintegration of intramuscular connective tissue. Although metalloproteinases may be involved in the disintegration, details of the proteolytic processes occurring during chilled storage remain to be elucidated. To determine the involvement of matrix metalloproteinase-9 (MMP-9) in the post-mortem disintegration of intramuscular connective tissue, we produced recombinant Japanese flounder MMP-9 (jfMMP-9) in COS-7 cells. The recombinant jfMMP-9 showed gelatinolytic activity in the zymographic analysis at chilled temperature, but no apparent proteolytic activity against the triple helical domain of types I and type V collagens. In contrast, recombinant jfMMP-9 solubilized type I collagen from a crude connective tissue preparation at chilled temperature. This finding, together with the fact that a protein corresponding to MMP-9 was immunologically detected in the muscle extract, indicates that MMP-9 may be involved in the disintegration of the intramuscular connective tissue that induces the post-mortem tenderization of fish muscle during chilled storage.

We have cloned for the first time two cDNAs encoding distinct types of tissue inhibitor of metalloproteinases-2 (TIMP-2) from teleost fish, Japanese flounder, and designated these types as jfTIMP-2a and jfTIMP-2b. The open reading frames of the jfTIMP-2a and jfTIMP-2b cDNAs are composed of 663 and 657 nucleotides and 221 and 219 amino acids, respectively. Both jfTIMP-2s contain 12 cysteine residues, which might form six disulfide bonds as in other animals' TIMP-2s. The predicted full-length amino acid sequence of jfTIMP-2a has lower identity to jfTIMP-2b (63%) than to those of human (74%) and chicken (73%) TIMP-2s, but higher than to those of other human TIMPs (TIMP-1: 39%, TIMP-3: 43%, TIMP-4: 45%), indicating that jfTIMP-2a is a common TIMP-2, while jfTIMP-2b is unique to Japanese flounder. However, the C-terminal region including the last three disulfide bonds of jfTIMP-2b has higher amino acid identity to those of other animal TIMP-2s than to that of jfTIMP-2a. Reverse-transcribed polymerase chain reaction (RT-PCR) analysis showed the mRNAs of jfTIMP-2a and jfTIMP-2b to be ubiquitously expressed in all tissues examined, but with different expression patterns. These findings suggest that the two distinct jfTIMP-2s might perform different functions in teleost tissues.

In order to identify free amino acids (FAA) that are importantas intracellular osmolytes in Crassostrea gigas, we investigatedthe change in FAA content in the mantle exposed to an abrupt decreaseor increase in salinity. In hypo-osmotic adaptation, most FAA showedremarkable and synchronous decreases from 2 to 8 h, suggestingthat the non-selective efflux of FAA was mainly responsible forthe decrease in FAA. Taurine that accounted for approximately 80% oftotal FAA content contributed most significantly to the hypo-osmoticadaptation. In hyper-osmotic adaptation, significant increases inglycine, alanine, β-alanine, proline, arginine and taurinewere observed. Of these, alanine showed an immediate increase thatis important to short-term adaptation to hyper-osmolality, whiletaurine showed a slower and substantial increase that contributesto a long-term adaptation to hyper-osmolality.

We previously cloned cDNA of type V/XI collagen α1 chain (ColVa1) gene from cultured cells derived from red sea bream embryo. We raised an antibody against the deduced C-telopeptide of ColVa1 in order to detect the translation products of this cDNA and their degradation products in red sea bream muscle. To improve its specificity, the antibody was purified from rabbit antiserum by use of an affinity column cross-linked with recombinant C-terminal peptide of ColVa1 produced by Epicurian coli. The purified antibody recognized a band corresponding to the α chain of type V/XI collagen in western blot analysis of the extract of cultured cells. The antibody also recognized two bands in acid-soluble and pepsin-solubilized collagens, indicating that the translation products of the ColVa1 gene are present in muscle and that bands correspond to α and β chains of type V/XI collagen. A band corresponding to a molecular weight of approximately 65 k was detected in the NaOH extracts of muscle, suggesting that type V/XI collagen α1 chain is restrictedly digested in red sea bream muscle.

We have cloned a cDNA encoding the MMP-9 from a carp epidermal cell (EPC) cDNA library. The clone contains a 2025-base pair (bp) open reading frame encoding a protein of 674 amino acids. The deduced amino acid sequence shares 68% and 69% identity with medaka and Japanese flounder MMP-9. The hinge domain of the carp MMP-9, like those of the other non-mammalian species, lacks a type V collagen-like region that is typical of mammalian MMP-9. Gelatin zymography and immunoblot analysis of conditioned media of EPC cells and cDNA-transfected COS-7 cells detected a 76-kDa gelatinase. The apparent molecular mass of the carp zymogen is much smaller than those of its mammalian counterparts while almost identical with that of chicken 75-kDa gelatinase B-like enzyme. Although hypo-osmotic stress induced the elevation of MMP-9 mRNA level in EPC cells, no significant change in the protein in conditioned medium was detected during hypo-osmotic stress. Northern blot analysis detected a large amount of MMP-9 mRNA in carp kidney and spleen, suggesting the high expression of MMP-9 in blood cells, neutrophils, and macrophages. The smaller amount of MMP-9 mRNA was detected in gill, heart, fin, and eye, whereas none of the mRNA was detected in the hepatopancreas, intestine, brain, muscle, and skin.

SUMMARY: Toughness is one of the most important elements that define the commercial value of the raw meat of fish. Degradation of the extracellular matrix is thought to be a cause of postmortem tenderization of fish meat. A previous study has suggested that this tenderization is caused mainly by metalloproteinases. The present study seeks to identify the proteinase(s) involved in tenderization; hence, cloned cDNA of two gelatinases from Japanese flounder, which showed high homology with mammalian matrix metalloproteinase (MMP)-2 and MMP-9, were designated as jfMMP-2 and jfMMP-9, respectively. Northern blot analysis revealed that jfMMP-2 mRNA was expressed almost ubiquitously in adult tissues including the brain, muscle, gill, heart, gut, kidney, spleen, testis, and ovary. In contrast, the expression of jfMMP-9 mRNA was observed in those tissues which were abundant in blood cells, such as kidney, spleen, heart, and gill. Both recombinant proteins (jfMMP-2 and jfMMP-9) produced with the COS-7 cell system exhibited gelatin-degrading activity that was sensitive to 1,10-phenanthroline, a typical metalloproteinase inhibitor.

It is suspected that the proteolytic breakdown of extracellular matrix proteins is responsible for the postmortem tenderization of fish muscle during chilled storage. In order to identify the type(s) of proteinases involved in this phenomenon, the effect of proteinase inhibitors, EDTA (ethylenediamine tetraacetic acid), 1,10-phenanthroline, ρ-APMSF [(ρ-amidinophenyl) methanesulfonyl fluoride hydrochloride] and E-64 [L-trans-epoxysuccinyl-leucylamido-(4-guanidinobutane)] on tenderization was investigated by using Japanese flounder. Proteinase inhibitor solution was injected into a blood vessel in a caudal portion of live flounder and the firmness of muscle was then evaluated as a shear force value at 0 h and 6 h after death. Metalloproteinase inhibitors, EDTA and 1,10-phenanthroline, significantly suppressed postmortem tenderization. These findings suggest that metalloproteinases are candidates for proteinases involved in the postmortem tenderization of fish muscles. Although not significantly, p-APMSF, a serine proteinase inhibitor, partially suppressed muscle tenderization, which suggests that serine proteinases are also implicated in postmortem tenderization. A cysteine proteinase inhibitor, E-64, showed no effect, suggesting that cysteine proteinases are not involved.

SUMMARY: The distribution of surface immunoglobulin-positive (sIg+) cells in the spleen, the head-kidney, and the trunk-kidney of adult Japanese flounder Paralichthys olivaceus was investigated by a flow cytometric analysis using a monoclonal antibody FB17 against flounder immunoglobulin M. Although the percentage of sIg+ cells varied among organs, a good correlation was revealed between the percentage in the head-kidney and that in the trunk-kidney (r = 0.996, P < 0.001). In contrast, no correlation was obtained between the percentage of sIg+ cells in the spleen and the head-kidney or trunk-kidney. The head-kidney and the trunk-kidney contained cells bearing fewer sIg that were stained dimly by FB17 in addition to brightly stained sIg+ cells regarded as mature B cells, while the spleen contained only sIg+ cells stained brightly. Moreover, the immunocytochemical analyses showed that the head-kidney contains plasma cells with strongly stained cytoplasm in addition to B cells with stained cell surface, which suggests that the cells with fewer sIg detected by flow cytometric analysis are presumed to be at a stage during the processes of differentiation to plasma cells. These results suggest that there are some differences between the spleen and kidney in the role of the immune system and it is considered that differentiation of B cells to plasma cells occurs in the kidney.

Up-regulated genes of leucocytes expressing immunoglobulin (Ig+ leucocytes) of hirame rhabdovirus (HRV)-infected Japanese flounder were identified by differential hybridisation, using subtracted and un-subtracted cDNA probes. Ig+ leucocytes were separated from apparently healthy and HRV-infected Japanese flounder by the magnetic beads antibody method using mouse anti-Japanese flounder Ig monoclonal antibody (mab). A cDNA library was constructed from HRV-infected Japanese flounder leucocytes, and was screened with subtracted cDNA probes enriched in genes up-regulated by HRV infection. Fifty cDNAs were isolated for further analysis. These included cDNAs coding for homologues of interferon-inducible 56K protein (IFI56), Stat3, CEF-10, RGS5, inducible poly(A) binding protein, prolylcarboxylpeptidase, basigin III (Ig superfamily), MUC-18 (Ig superfamily), proteasome-nexin 1 (SERPIN), herpes virus entry mediator (TNFR family), collagenase III, gelatinase-b, megakaryocyte stimulating factor, Rab8-interacting protein, IgM, IgD and 20 unknown cDNA clones. The majority of these identified genes are reported for the first time in fish. From leucocytes mRNA for homologues of IFI56, CEF-10, Stat3, SERPIN and inducible poly (A) binding protein expression was shown to increase following HRV infection.