A novel ornithine-containing tripeptide isolated from the extract of the brackish-water bivalve Corbicula japonica

Previous studies have demonstrated that frozen preparations of the brackish-water bivalve Corbicula japonica significantly increase the content of free ornithine found in its extracts. Here we report a novel ornithine-containing tripeptide commonly found in C. japonica, which is believed to be the source of increased free ornithine. The new peptide, named acorbine, was isolated from extracts of this bivalve obtained using ultra-filtration and gel permeation chromatography. Acorbine is comprised of N(2)-[N(2)-(beta-alanyl)-L-ornithyl]-L-ornithine as determined by amino acid composition analysis, N- and C-terminal amino acid analyses, proton nuclear magnetic resonance spectrometry, and chirality analysis of the ornithine residue. The total amount of beta-alanine and ornithine in the extract remained constant regardless of the temperature at which the bivalve was processed. The amount of free beta-alanine and ornithine increased significantly when the bivalve was frozen, with a corresponding decrease in peptidic beta-alanine and ornithine. The results suggest that changing the growth conditions triggers tripeptide proteolysis within the bivalve, which ultimately manifests in increased free beta-alanine and ornithine.     

Activities of the three ammonia-forming enzymes in the tissues of the brackish-water bivalve Corbicula japonica

• 1.1. Activities of the three ammonia-forming enzymes, glutamate dehydrogenase, AMP deaminase and serine dehydrase (SerDH), were measured in tissues of gill, digestive diverticula, mantle and foot muscle of the brackish-water bivalve Corbicula japonica.
• 2.2. High levels of SerDH activity were detected in gill and digestive diverticula, while the activity levels of the other two enzymes were low.
• 3.3. The result suggests the significance of SerDH in amino acid degradation of this species.

     

Effect of metabolic inhibitors on hyperosmotically induced free amino acid accumulation in the isolated foot muscle of the brackish-water bivalve Corbicula japonica

• 1.1. Accumulation of free amino acids (FAA) in the isolated foot muscle of the brackish-water bivalve Corbicula japonica during the initial stage of hyperosmotic stress was quantitatively and qualitatively similar to that in the foot of the intact animal.
• 2.2. Aminooxyacetate (AOA), a transaminase inhibitor, markedly inhibited alanine accumulation and promoted ornithine accumulation in the isolated foot. Iodoacetate (IAA), a glycolytic inhibitor, caused no significant alteration in the alanine level and the TLC pattern of FAA. Both the inhibitors scarcely influenced the pool size of total ninhydrin positive substances (NPS).
• 3.3. A major part of the carbon of accumulated FAA during the initial stage of hyperosmotic stess did not seem to arise from glycolysis.
• 4.4. Free d-alanine as well as l-alanine accumulated in isolated foot muscle exposed to hyperosmotic stress.

     

Effects of biological behaviors of benthic bivalve (Corbicula japonica) on its passive transport

Hydrobiologia - The bivalve Corbicula japonica is a popular food in Japan. The abundance of the clam in a lake changes over time. Clam transport is assumed to be passive even after the clam’s...     

Purification and some properties of alanine racemase from a bivalve mollusc Corbicula japonica

A brackish-water mollusc, Corbicula japonica, uses large quantities of D- and L-alanine as intracellular osmotically active solutes, osmolytes, for regulation of intracellular osmolarity. We purified alanine racemase from the mantle of C. japonica to characterize its enzymological properties. The molecular masses of the enzyme were estimated to be 41 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 140 kDa by gel filtration on high-performance liquid chromatography, suggesting the trimeric or tetrameric nature of the enzyme. Neither dialysis nor chromatographic procedures in the absence of pyridoxal 5'-phosphate led to loss of enzyme activity, although carbonyl reagents, hydroxylamine and phenylhydrazine, inhibited the activity. These results suggest that alanine racemase of the animal may bind pyridoxal 5'-phosphate tightly as a cofactor. Kinetic experiments using the partially purified enzyme revealed that alanine was the sole substrate among 17 kinds of L-amino acids tested. The Lineweaver-Burk plot for L-alanine as substrate resulted in Km value of 22.6 mM, and the value for D-alanine was 9.2 mM. Together with the previous evidence that D- and L-alanine levels of this animal change with the external salinity maintaining the D-/L-alanine ratio at unity, the present results seem to indicate that the physiological role of alanine racemase in this animal is to supply D-alanine as a main intracellular osmolyte. J. Exp. Zool. 289:1-9, 2001.     

A novel ornithine-containing tripeptide isolated from the extract of the brackish-water bivalve Corbicula japonica

Previous studies have demonstrated that frozen preparations of the brackish-water bivalve Corbicula japonica significantly increase the content of free ornithine found in its extracts. Here we report a novel ornithine-containing tripeptide commonly found in C. japonica, which is believed to be the source of increased free ornithine. The new peptide, named acorbine, was isolated from extracts of this bivalve obtained using ultra-filtration and gel permeation chromatography. Acorbine is comprised of N²-[N²-(β-alanyl)-l-ornithyl]-l-ornithine as determined by amino acid composition analysis, N- and C-terminal amino acid analyses, proton nuclear magnetic resonance spectrometry, and chirality analysis of the ornithine residue. The total amount of β-alanine and ornithine in the extract remained constant regardless of the temperature at which the bivalve was processed. The amount of free β-alanine and ornithine increased significantly when the bivalve was frozen, with a corresponding decrease in peptidic β-alanine and ornithine. The results suggest that changing the growth conditions triggers tripeptide proteolysis within the bivalve, which ultimately manifests in increased free β-alanine and ornithine.     

Seasonal characteristics of gametogenesis in the Corbicula japonica

A morphological study of gonads in Corbicula japonica made it possible to distinguish between five stages of gonad maturity. It has been established that stages of active gametogenesis, prespawning and of spawning go almost in parallel and in the shortest time. In July, with the beginning of spawning, gametogenesis does not stop, and oogenesis proceeds up to the end of August. Dynamics of changes of some chemical substances in oocytes was followed. High concentrations of, respectively, RNA, albumens, lipids, glycoproteins and proteoglycans were revealed in oocyte nuclei and nucleoli both at the beginning of the development, and at the stage of active gametogenesis. This testifies in favor of a synthetic activity of these cells.     

Immunohistochemical, in situ hybridization and biochemical studies on endogenous cellulase of Corbicula japonica

We previously reported on the endogenous cellulase gene of Corbicula japonica, CjCel9A. In this study, the tissue localization of the mRNA and translated products of CjCel9A was investigated in order to understand how this gene is physiologically involved in cellulose decomposition by C. japonica. Antiserum against recombinant CjCel9A protein was prepared. Multiple bands were observed mainly on western blot analysis of the crystalline style, and the band sizes partially corresponded to the active bands detected using zymographic analysis. In situ hybridization and immunohistochemical analyses clarified the exclusive production and secretion of this cellulase by the secretory cells localized in the epithelium of the digestive tubules in the digestive gland. These data strongly support our previous assumption that the endogenous cellulase of C. japonica is produced in the digestive gland and transported to the crystalline style to act as a component of its cellulolytic activity.     

The gametogenesis and sexual cycle of the bivalve Corbicula japonica Prime (1864) in the mouth of the Kievka River (Sea of Japan)

Oogenesis, spermatogenesis, and the sexual cycle of the bivalve Corbicula japonica in the estuary of the Kievka River (Primorye, Sea of Japan) were studied. The stages of development of germ cells and the role of auxiliary cells in gonads during vitellogenesis were described. The features of sperm morphology were recorded. It was shown that the reproductive cycle in C. japonica is determined by the water temperature, that gametogenesis starts at the boundary between April and May, that spawning occurs from mid-July to mid-September, and that gonads are in a stage of relative sexual inactivity in the autumn and winter.     

The impact of an introduced bivalve (Corbicula fluminea) on the benthos of a sandy stream

1. The effect of an exotic, burrowing bivalve ( Corbicula fluminea ) on the benthic fauna of a sandy-bottomed stream was assessed by field and laboratory experiments. Corbicula differs from other freshwater, non-native bivalves in that it both filter- and pedal-feeds and thus has the potential to influence the streambed community in different ways.
2. In the field, cages were used to vary the abundance of Corbicula in the streambed. Increasing abundance of Corbicula was negatively associated with the abundance of benthic bacteria and flagellates but had no apparent effect on other protists or meiofauna.
3. In the laboratory, we compared the effect of Corbicula on the benthic community when the bivalves were able both to filter- and pedal-feed with that when they were able only to filter-feed. Bivalves restricted to filter-feeding were placed in sediment lacking benthic fauna and organic matter, then the benthos in nearby natural sediment was compared with the community present when Corbicula was able to move freely through the sediment and both to filter- and pedal-feed. Corbicula able to pedal-feed were again associated with a decreased abundance of benthic flagellates and bacteria, as well as diatoms.     

Rapid evolution of sessility in an endemic species flock of the freshwater bivalve Corbicula from ancient lakes on Sulawesi, Indonesia

The fauna of ancient lakes frequently contains taxa with highly derived morphologies that resulted from in situ radiation of lacustrine lineages with high antiquity. We employed a molecular mtDNA phylogeny to investigate this claim for corbiculid freshwater bivalves in two ancient lake systems on the Indonesian island Sulawesi. Among the otherwise mobile corbiculid species flock, only one taxon, Posostrea anomioides, in the ancient Lake Poso exhibits a unique habit, i.e. cementing one valve to the substrate. Our data show that Corbicula on Sulawesi is polyphyletic, with the endemic riverine taxa in terminal position, and the lacustrine species flock being paraphyletic. Surprisingly, Posostrea is not confirmed as a genus distinct from Corbicula and genetic distances suggest a rather recent origin from the only other corbiculid species endemic to Lake Poso, the non-cementing Corbicula possoensis. While the cementing anomioides, despite its unique behavioural and morphological characteristics, clusters together with non-sessile Corbicula species, the latter exhibit strong genetic distances in the absence of morphological disparity and fall into several genetically rather distinct clades. These findings suggest that developmental plasticity of animals in ancient lakes rather than the antiquity of lineages might account for the unique morphology of some species.     

Spatio-temporal and intra-specific variations in the physiological and biochemical condition of the invasive bivalve Corbicula fluminea

Hydrobiologia - Corbicula fluminea is a known aquatic invasive species responsible for several ecological and economic impacts. In Portugal, it is found in all major watersheds, although to varying...     

Purification, molecular cloning, and application of a novel sphingomyelin-binding protein (clamlysin) from the brackishwater clam, Corbicula japonica

A novel sphingomyelin-binding protein (clamlysin) was purified from the foot muscle of a brackishwater clam, Corbicula japonica. The purified 24.8-kDa protein lysed sheep, horse and rabbit erythrocytes and the hemolytic activity was inhibited by sphingomyelin, but not other phospholipids or glycosphingolipids. The open reading frame of the clamlysin gene encoded a putative 26.9-kDa protein (clamlysin B) which showed high sequence similarity with the actinoporin family. A surface plasmon resonance assay confirmed that clamlysin B specifically bound to sphingomyelin. Furthermore, two cDNA variants of clamlysin, encoding putative 31.4 kDa (clamlysin A) and 11 kDa (clamlysin C) proteins, were isolated. Only the 31.4-kDa variant was found to exhibit sphingomyelin-binding activity. Clamlysin A and B, but not C, shared a sequence (domain II) conserved in all known sphingomyelin-binding proteins. Domain II fused with a glutathione S-transferase bound to sphingomyelin. Horse erythrocytes, mouse melanoma B16 and GM95 cells, and Chinese hamster ovary CHO-K1 cells, but not the same cells treated with bacterial sphingomyelinase, were immunostained with clamlysin B. These results indicate that clamlysin B binds to the sphingomyelin of living cells and thus would be useful as a molecular probe to detect sphingomyelin.     

Ammonia-nitrogen production by the bivalve molluscTapes japonica and its recovery by the red seaweedHypnea musciformis in a tropical mariculture system

Production and recovery of ammonia-N was studied in the second and third trophic levels of a mariculture system on St. Croix, US Virgin Islands. The diatomChaetoceros curvisetus, grown on nutrients in artificially upwelled deep water from 870 m depth, was the food source forTapes japonica. Consumption of the phytoplankton byT. japonica increased throughout the day and decreased at night, and was related to corresponding changes in algal culture density. Feeding efficiency was highest at night. Ammonia-N production by the clams fluctuated over a typical 24 h period; dropping during the day and increasing at night. The ammonia-N concentration in the shellfish tank effluent was inversely related to the quantity of phytoplankton consumed by the clams. At all ration levels the small clams produced ammonia-N at a greater rate than large clams.H. musciformis fragmented and washed out of some tanks; the fragmentation was related to high ammonia-N concentrations in the inflowing water. High light intensity and temperature alone do not appear to cause fragmentation, but may have induced a trace nutrient deficiency inH. musciformis grown in the ammonia-rich seawater. Where fragmentation was not obvious, ammonia-N uptake per gH. musciformis was highly correlated with the average ammonia-N concentration of the inflowing seawater both day and night. Percent uptake of ammonia-N increased with increasing concentration of the nutrient in the inflowing seawater, reaching a plateau of about 70% uptake of the available ammonia-N at concentrations above 4 g-at/l.     

Enhanced understanding of physicochemical constraints on Corbicula japonica habitat in Lake Shinji assisted by machine learning

The catch of Corbicula japonica is one of the top three in Japan's inland water fisheries. Most of the Japanese fishing stock of this bivalve comes from Lake Shinji. Since the 1980's the catch of this species declined drastically with strong inter-annual variation. New recruitment and poor growth of the clams were thought to be the main reasons for this decline. This bivalve has a poor transportation ability after the settlement. Therefore, it is also important to know their suitable habit that is affected by multi interacting parameters of water qualities and sediments. In order to get a better understanding of their habitat, we used machine learning models to test abiotic limiting factors. The database that we used in our study included 337 sampling stations with 7 physicochemical variables and the Corbicula japonica counting which was divided into two size categories: small (shell length < 4 mm) and large (shell length ≥ 4 mm). Due to their low self-transportation ability, their survival is primarily influenced by the site environment, such as water quality and sediment conditions. To extract physicochemical thresholds that directly impact these clam populations, we applied a coupled methodology based on a random forest model and partial dependence plots.We highlighted that the use of three different populations groups for each size category, allowed us to significantly increase the accuracy of our model to predict groups. Moreover, our results show that the three most influential predictors were, in order of importance, the water depth, ignition loss of the bottom sediments and silt clay (diameter ≤ 0.063 mm) content of the bottom sediments; with, respectively, the following physicochemical limitations for the preferable habitat: 4 m, 10% and 45%. These limitations are based on the thresholds that we extracted from the coupling between a Random Forest model and the partial dependence plots. Our findings emphasize that this coupled methodology has the advantage of being able to manage the zero values of population and give graphical interpretation of the limiting environmental factors. Consequently, the results that we presented here clearly demonstrate a further step towards comprehension of the inter-annual variations in the fishery resources of Corbicula japonica after accounting for the impact of new recruitment. In addition, this kind of numerical tool could help the local government to manage the clam's population by restoring and conserving the water quality and sediment conditions of the lake.     

Kinetic properties and structural characteristics of an unusual two-domain arginine kinase of the clam Corbicula japonica

Arginine kinase (AK) from the clam Corbicula japonica is a unique enzyme in that it has an unusual two-domain structure with molecular mass of 80 kDa. It lacks two functionally important amino acid residues, Asp-62 and Arg-193, which are conserved in other 40 kDa AKs and are assumed to be key residues for stabilizing the substrate-bound structure. K m arg and Vmax values for the recombinant two-domain AK were determined. These values were close to those of usual 40 kDa AKs, although Corbicula AK lacks the functionally important Asp-62 and Arg-193. Domain 2 of Corbicula AK was separated from the two-domain enzyme and was expressed in Escherichia coli. Domain 2 still exhibited activity. However, kinetic parameters for domain 2 appeared to be slightly, but significantly, different from those of two-domain AK. Thus, it is likely that the formation of the contiguous dimer alters the kinetic properties of its constituent domains significantly. Comparison of K d arg and K m arg for two-domain AK and its domain 2 showed that the affinity of the enzyme for arginine is greater in the presence of substrate ATP than in its absence. Presumably this difference is correlated with the large structural differences in the enzyme in the presence or absence of substrate, namely open and closed structures. We expressed three mutants of Corbicula AK domain 2 (His-60 to Gly or Arg, Asp-197 to Gly), and determined their K m arg and Vmax values. The affinity for the substrate arginine in mutant enzymes was reduced considerably, accompanied by a decrease in Vmax. These results suggest that His-60 and Asp-197 affect the substrate binding system, and are consistent with the hypothesis that a hydrogen bond is formed between His-60 and Asp-197 in Corbicula AK as a substitute for the Asp-62 and Arg-193 bond in normal AKs.     

Influence of aqueous leaf extract of common lambsquarters and NaCl salinity on the germination, growth, and nutrient content of wheat

Aqueous leaf extract of common lambsquarters (Chenopodium album L.) was evaluated alone or in combination with NaCl salinity for its influence on germination, seedling growth and contents of Na, K, and Ca by shoots and roots of wheat. The leaf extract and NaCl alone or in combination did not have any significant effect on germination, but the shoot and root lengths of seedlings and their dry weight decreased significantly by the treatments. Root growth was affected more than the shoot. The combination of leaf extract and NaCl drastically reduced the growth more than the separate effects of these stress treatments. The incorporation of leaf extract in the growth media decreased the content of Na by shoot, whereas the contents of K and Ca increased. NaCl treatments in combination with leaf extract increased the content of Na. Similar increases were observed for K and Ca in shoot, while these nutrients were increased in roots compared to control.