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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.

There are limited studies on the mineralogy of tidal flats in Japan even though the composition would affect the functions of water purification and organic matter preservation. This study investig...

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.

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.

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

The environments around deep-sea hydrothermal vents are very harsh conditions for organisms due to the possibility of exposure to highly toxic compounds and extremely hot venting there. Despite such extreme environments, some indigenous species have thrived there. Alvinellid worms (Annelida) are among the organisms best adapted to high-temperature and oxidatively stressful venting regions. Although intensive studies of the adaptation of these worms to the environments of hydrothermal vents have been made, little is known about the worms' sensory adaptation to the severe chemical conditions there. To examine the sensitivity of the vent-endemic worm Paralvinella hessleri to low pH and oxidative stress, we determined the concentration of acetic acid and hydrogen peroxide that induced avoidance behavior of this worm, and compared these concentrations to those obtained for related species inhabiting intertidal zones, Thelepus sp. The concentrations of the chemicals that induced avoidance behavior of P. hessleri were 10-100 times lower than those for Thelepus sp. To identify the receptors for these chemicals, chemical avoidance tests were performed with the addition of ruthenium red, a blocker of transient receptor potential (TRP) channels. This treatment suppressed the chemical avoidance behavior of P. hessleri, which suggests that TRP channels are involved in the chemical avoidance behavior of this species. Our results revealed for the first time hypersensitive detection systems for acid and for oxidative stress in the vent-endemic worm P. hessleri, possibly mediated by TRP channels, suggesting that such sensory systems may have facilitated the adaptation of this organism to harsh vent environments.

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.

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.

六価クロムは強い毒性をもつことからその水質汚染が問題となっており，簡便な除去方法の開発が急務となっている。本研究では海藻を原料として作製した炭に注目し，その六価クロム除去能を調べた。36 種類の海藻について検討した結果，コンブ目チガイソ科の海藻を原料とした炭に強い六価クロムの除去効果が確認された。チガイソ科のワカメを原料とした炭を用いて調べた結果，六価クロムの除去効果はフコイダンによることが示唆された。ワカメ炭は各種市販セメント廃液中に含まれる六価クロム除去に有効であることが確認された。

養殖の副産物として大量に廃棄されているマガキ貝殻を原料とした凝集沈澱剤を開発した。焼成温度（200℃～600℃）が凝集沈殿効果に及ぼす効果を検討した結果，400℃ 焼成が最も効果的であった。比表面積及び粒度分布測定の結果，焼成温度が高いほど比表面積及び粒径は小さくなった。これらの結果から，400℃ 焼成の貝殻がもっとも高い凝集沈殿効果を示したのは，400℃ がフロック形成に有効な小さな粒径と，フロック強度に必要な大きな比表面積を貝殻に付与するのに最適な焼成温度であるためと推測された。

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.