Tropomyosin isoforms present in the sea anemone,Anthopleura japonica (Anthozoa,Cnidaria)

Five isoforms of tropomyosin, designated as TMa, TMb, TMc, TMd, and TMe, were detected in the sea anemone, Anthopleura japonica. The apparent molecular weights of these isoforms were estimated to be approximately 30 kD to 37.5 kD, and their pI values were approximately 4.55 (TMa and TMb) and 4.65 (TMc, TMd, and TMe). Although sea anemone tropomyosin isoforms have the ability to bind to rabbit skeletal muscle actin, they preferably bind to actin at higher concentrations of Mg(2+) (10-20 mM) and slightly lower pH (6.2-7.2) than those used in conventional conditions. Antigenic properties of sea anemone tropomyosin seemed to be considerably specific to each isoform. Distribution of tropomyosin isoforms in the sea anemone body was somewhat portion-specific. TMa, TMb, and TMe were detected similarly in the extracts from tentacle, oral disc, column, mouth, and pedal disc. Although TMc and TMd were detected abundantly in the tentacle extract and moderately in the column and mouth extracts, these components were not contained in the pedal disc extract and detected only faintly in the oral disc extract.     

Ultrastructure of muscle cells in the adductor of the boring clam Tridacna crocea

The ultrastructure of the adductor muscle of the boring clam (Tridacna crocea) was investigated. The adductor was composed of opaque and translucent portions. The opaque portion contained smooth muscle cells; the translucent portion contained obliquely striated cells. Smooth muscle cells were classified, according to the statistically analyzed diameters of their thick myofilaments, into two types, S-1 and S-2. S-1 cells had thick myofilaments, 50–60 nm in diameter. S-2 cells had thick myofilaments of two sizes, about 55–65 nm and 85–100 nm in diameter, respectively. Obliquely striated muscle cells in the translucent portion were also classified into two types: O-1 cells, with thick myofilaments 30–35 nm in diameter, and O-2 cells, with myofilaments of 50–60 nm.     

Ultrastructural differences between the anterior and posterior adductors of the strawberry cockle, Fragum unedo

Adductors of Fragum unedo were observed ultrastructurally and their muscle cells were classified according to the statistically analyzed diameter of their thick myofilaments. Two types of smooth muscle cells were observed in the opaque portion of the anterior adductor: A-type cells containing thick myofilaments of about 46 nm in diameter and B-type cells having 62 nm thick myofilaments. The posterior adductor was also composed of two kinds of cells: the B-type cell, which had thick myofilaments of about 67 nm in diameter, and the C-type, containing thick myofilaments of 90 nm. Two types of oblique-striated cells were commonly recognized in the translucent portions of anterior and posterior adductors. Our observations thus indicate that the posterior adductor generally consists of cells which have thicker myofilaments than the ones of the anterior adductor.     

Extraction and Identification of the Pigment in the Adductor Muscle Scar of Pacific Oyster Crassostrea gigas

In this study, UV (ultraviolet) and IR (infrared radiation) spectral analysis were integrated to identify the pigment in the adductor muscle scar of the Pacific oyster Crassostrea gigas. The pigment was extracted from the adductor muscle scars of cleaned oyster shells that were pulverized, hydrolyzed in hot hydrochloric acid, purified with diethyl ether, and dissolved in 0.01 mL/L NaOH. The maximum absorption of the pigment in the UV absorption spectrum within the range of 190–500 nm was observed between 210–220 nm. The UV absorbance decreased with increasing wavelength which was consistent with the UV spectral absorption characteristics of melanin. In addition, Fourier transform infrared spectroscopy scanning revealed characteristic absorption peaks that emerged near 3440 cm^-1 and 1630 cm^-1, which was consistent with infrared scanning features of eumelanin (a type of melanin). This study has demonstrated for the first time that the pigment in the adductor muscle scar of the Pacific oyster is melanin, hinting that the adductor muscle could be another organ pigmenting the mollusc shell with melanin other than mantle.     

Innervation of bivalve larval catch muscles by serotonergic and FMRFamidergic neurons

Bivalve larvae use catch muscles for rapid shell closure and maintenance of the closed condition. We used specific antibodies against the muscle proteins together with phalloidin and neuronal markers, FMRFamide and serotonin (5-HT), to analyze mutual distribution of muscle and neuronal elements in larvae of the mussel, Mytilus trossulus, and the oyster, Crassostrea gigas. At trochophore and early veliger stages no anatomical connections between muscular and nervous system were detected. By the pediveliger stage the 5-HT innervation of the anterior adductor developed in oyster only, while rich FMRFa innervation of the adductor muscles developed in both species. Possible roles and mechanisms of FMRFamide and serotonin in the regulation of the catch state are discussed.     

Regulation of a truncated isoform of AMP-activated protein kinase α (AMPKα) in response to hypoxia in the muscle of Pacific oyster Crassostrea gigas

AMP-activated protein kinase α (AMPKα) is a key regulator of energy balance in many model species during hypoxia. In a marine bivalve, the Pacific oyster Crassostrea gigas, we analyzed the protein content of adductor muscle in response to hypoxia during 6 h. In both smooth and striated muscles, the amount of full-length AMP-activated protein kinase α (AMPKα) remained unchanged during hypoxia. However, hypoxia induced a rapid and muscle-specific response concerning truncated isoforms of AMPKα. In the smooth muscle, a truncated isoform of AMPKα was increased from 1 to 6 h of hypoxia, and was linked with accumulation of AKT kinase, a key enzyme of the insulin signaling pathway which controls intracellular glucose metabolism. In this muscle, aerobic metabolism was maintained over the 6 h of hypoxia, as mitochondrial citrate synthase activity remained constant. In contrast, in striated muscle, hypoxia did not induce any significant modification of neither truncated AMPKα nor AKT protein content, and citrate synthase activity was altered after 6 h of hypoxia. Together, our results demonstrate that hypoxia response is specific to muscle type in Pacific oyster, and that truncated AMPKα and AKT proteins might be involved in maintaining aerobic metabolism in smooth muscle. Such regulation might occur in vivo during tidal intervals that cause up to 6 h of hypoxia.     

Cloning of cDNAs and hybridization analysis of lysozymes from two oyster species, Crassostrea gigas and Ostrea edulis

In bivalve molluscs including oysters, lysozymes play an important role in the host defense mechanisms against invading microbes. However, it remains unclear in which sites/cells the lysozyme genes are expressed and which subsequently produced the enzyme. This study cloned lysozyme cDNAs from the digestive organs of Pacific oyster Crassostrea gigas and European flat oyster Ostrea edulis. Both complete sequences of two oysters' lysozymes were composed of 137 amino acids. Two translated proteins present a high content in cysteine residues. Phylogenetic analyses showed that these oysters' lysozymes clustered with the invertebrate-type lysozymes of other bivalve species. In the Pacific oyster, lysozyme mRNA was expressed in all tissues except for those of the adductor muscle. In situ hybridization analyses revealed that lysozyme mRNA was expressed strongly in basophil cells in the digestive gland tubule of C. gigas, but not in digestive cells. Results indicated that the basophil cells of the oyster digestive gland are the sites of lysozyme synthesis.     

Filamin isoforms in molluscan smooth muscle

The role of filamin in molluscan catch muscles is unknown. In this work three proteins isolated from the posterior adductor muscle of the sea mussel Mytilus galloprovincialis were identified by MALDI-TOF/TOF MS as homologous to mammalian filamin. They were named FLN-270, FLN-230 and FLN-105, according to their apparent molecular weight determined by SDS-PAGE: 270kDa, 230kDa and 105kDa, respectively. Both FLN-270 and FLN-230 contain the C-terminal dimerization domain and the N-terminal actin-binding domain typical of filamins. These findings, together with the data from peptide mass fingerprints, indicate that FLN-270 and FLN-230 are different isoforms of mussel filamin, with FLN-230 being the predominant isoform in the mussel catch muscle. De novo sequencing data revealed structural differences between both filamin isoforms at the rod 2 segment, the one responsible for the interaction of filamin with the most of its binding partners. FLN270 but not FLN230 was phosphorylated in vitro by cAMP-dependent protein kinase. As for the FLN-105, it would be an N-terminal proteolytic fragment generated from the FLN-270 isoform or a C-terminally truncated variant of filamin. On the other hand, a 45-kDa protein that copurifies with mussel catch muscle filamins was identified as the mussel calponin-like protein. The fact that this protein coelutes with the FLN-270 isoform from a gel filtration chromatography suggests a specific interaction between both proteins.     

The food chain dynamics of the oyster,clam, and mussel in an aquaculture food chain

The food chain dynamics of the edible mussel Mytilus edulis L., the American oyster Crassostrea virginica (Gmelin) and the hard clam Mercenaria mercenaria (L.) were investigated in large experimental tanks with flowing, filtered sea water and controlled addition of phytoplankton. The feeding rate of the mussel (5.36 μg carbon removed/l/g C animal was higher than that of the oyster (3.92) and clam (3.03) but the ecological efficiencies (net production/ingested food) × 100 of the clam (23.69 %) and the oyster (18.38 %) were higher than that of the mussel (10.01 %).The food chain efficiencies (net production/available food) were lower than the ecological efficiencies, suggesting under-exploitation of the available food. The clam, although having a lower feeding rate, was more efficient in utilizing the food it filtered and so showed the highest net production.The rates (μg-at/l/g C animal) of regeneration of nutrients, especially total inorganic nitrogen (mussel, 2.1723 × 10^?3; oyster, 7.4270 × 10^?3; and clam, 8.1750 × 10^?3) along with reported high biodeposition rates of bivalves suggest that multi-species aquaculture systems would be more efficient and productive than one-species systems.     

Tissue specificity of tropomyosin from the crayfish, Cambarus clarki

The molecular heterogeneity and tissue specificity of crustacean tropomyosin were investigated, using muscle and nonmuscle tissues from the crayfish, Cambarus clarki. In muscle, three types of tropomyosin isoforms were found on two-dimensional gel electrophoresis. One of them was specific to cardiac muscle, and the other two were shared by skeletal and visceral muscles. In nonmuscle tissues, four types of isoforms were found on two-dimensional gel electrophoresis and in immunoreplica tests using an antiserum against crayfish skeletal muscle tropomyosin. Two of them were common to the muscle isoforms, but the other two were not detected in muscles. Furthermore, nonmuscle tissues contained several peculiar isoforms, the electrophoretic mobilities of which were considerably higher than those of the other isoforms mentioned above. When tropomyosin was purified from the mid-gut gland, these isoforms with high mobilities were found in the crude tropomyosin preparation. These results showed that the crayfish tropomyosin was heterogeneous and that the isoforms were distributed in a tissue-specific manner, like vertebrate tropomyosin. However, the results did not coincide with those of our previous study on horseshoe crab tropomyosin, which showed molecular heterogeneity but no tissue specificity. In view of the difference in the isoform distributions between the two major groups (Crustacea and Merostomata) of Arthropoda, the significance of the tissue specificity of tropomyosin isoforms was discussed.     

Factors influencing in vitro killing of bacteria by hemocytes of the eastern oyster (Crassostrea virginica).

A tetrazolium dye reduction assay was used to study factors governing the killing of bacteria by oyster hemocytes. In vitro tests were performed on bacterial strains by using hemocytes from oysters collected from the same location in winter and summer. Vibrio parahaemolyticus strains, altered in motility or colonial morphology (opaque and translucent), and Listeria monocytogenes mutants lacking catalase, superoxide dismutase, hemolysin, and phospholipase activities were examined in winter and summer. Vibrio vulnificus strains, opaque and translucent (with and without capsules), were examined only in summer. Among V. parahaemolyticus and L. monocytogenes, significantly (P < 0.05) higher levels of killing by hemocytes were observed in summer than in winter. L. monocytogenes was more resistant than V. parahaemolyticus or V. vulnificus to the bactericidal activity of hemocytes. In winter, both translucent strains of V. parahaemolyticus showed significantly (P < 0.05) higher susceptibility to killing by hemocytes than did the wild-type opaque strain. In summer, only one of the V. parahaemolyticus translucent strains showed significantly (P < 0.05) higher susceptibility to killing by hemocytes than did the wild-type opaque strain. No significant differences (P > 0.05) in killing by hemocytes were observed between opaque (encapsulated) and translucent (nonencapsulated) pairs of V. vulnificus. Activities of 19 hydrolytic enzymes were measured in oyster hemolymph collected in winter and summer. Only one enzyme, esterase (C4), showed a seasonal difference in activity (higher in winter than in summer). These results suggest that differences existed between bacterial genera in their ability to evade killing by oyster hemocytes, that a trait(s) associated with the opaque phenotype may have enabled V. parahaemolyticus to evade killing by the oyster's cellular defense, and that bactericidal activity of hemocytes was greater in summer than in winter.     

High molecular weight calcium binding protein in the microsome of scallop striated muscle

In the microsome of scallop adductor striated muscle, 30K, 55K, 90K, and 360K proteins were detected as calcium binding proteins by 45Ca autoradiography on the transferred nitrocellulose membrane after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). The 360K protein was directly extracted with Triton X-100 from the whole homogenate of striated portion of scallop adductor muscle and purified through DEAE cellulose and hydroxyapatite column chromatography. This purified scallop high molecular weight calcium binding protein (SHCBP) showed a faster mobility in SDS PAGE in the presence of Ca2+ than in its absence. The decrease of tryptophan fluorescence had a half maximum near pCa 7 and was slightly co-operative with Mg2+. UV absorbance was slightly increased with Ca2+. The CD spectrum also changed with Mg2+ and Ca2+. These results reflect that this SHCBP binds calcium ions under near physiological conditions. SHCBP-like high molecular weight calcium binding proteins were also detected in the smooth muscle portion of adductor muscle and branchiae of scallop by 45Ca autoradiography, but not in liver. The adductor muscle of clam had a high molecular weight calcium binding protein whose molecular weight was a little smaller than that of SHCBP. The foot of turban shell had the same molecular weight calcium binding protein as SHCBP. Stains-all, a cationic carbocyanine dye, which has been reported to stain calcium binding proteins blue, stained SHCBP blue. The spectrum of SHCBP stained with Stains-all was very similar to that of calsequestrin. Although the function of SHCBP is still unknown, it might be expected to correspond to calsequestrin of vertebrate skeletal muscle, a calcium sequestering protein, in the sarcoplasmic reticulum.     

Biphasic expression of slow myosin light chains and slow tropomyosin isoforms during the development of the human quadriceps muscle

Using a two-dimensional electrophoresis technique coupled with sensitive silver staining, we have investigated the chronology of appearance of the myosin light chain and tropomyosin isoforms during early stages of human quadriceps development. Our results show that slow myosin light chains and the slow tropomyosin isoform are not detected at 6 weeks of gestation. These isoforms transiently appear between 12.5 weeks and 15 weeks of gestation and then disappear. The slow myosin light chains are re-expressed at 31 weeks of gestation and the slow tropomyosin isoform later at 36 weeks of gestation, and normally remained expressed into the adulthood. Our study thus reveals a biphasic expression of the slow myosin light chains and the slow tropomyosin isoform in developing human quadriceps muscle.     

Ultrastructural investigations on the anterior adductor muscle of a brachiopoda, Lingula unguis

The brachiopoda, Lingula unguis, has a pair of anterior adductors located in the center of the shell. Each muscle consists of an opaque and a translucent portion which is constructed of smooth and obliquely-striated muscle respectively. According to our ultrastructural observations, the opaque portion seems to have two types of cells. They differ only in the diameters of their thick myofilaments. The fine structure of their cell organelles resembles each other. We measured the diameters of the thick myofilaments in each type of cell to distinguish between the two cell types. About 500 measurements of myofilament diameters were made for each type of cell and statistically analyzed. For one type of cell, the distribution of diameters of the thick myofilaments fit a normal distribution curve with a peak at 37-60 nm. The distribution of diameters of the thick myofilaments for the other type fit a curve in which two normal distribution curves having peaks at 37-60 and 75-97 nm respectively partially overlapped. According to these results, we suggest that the opaque portion contains two types of cells, each having a different distribution of thick myofilament sizes.     

Introduced Pacific oysters (Crassostrea gigas) in the northern Wadden Sea: invasion accelerated by warm summers?

Among the increasing number of species introduced to coastal regions by man, only a few are able to establish themselves and spread in their new environments. We will show that the Pacific oyster (Crassostrea gigas) took 17 years before a large population of several million oysters became established on natural mussel beds in the vicinity of an oyster farm near the island of Sylt (northern Wadden Sea, eastern North Sea). The first oyster, which had dispersed as a larva and settled on a mussel bed, was discovered 5 years after oyster farming had commenced. Data on abundance and size-frequency distribution of oysters on intertidal mussel beds around the island indicate that recruitment was patchy and occurred only in 6 out of 18 years. Significant proportions of these cohorts survived for at least 5 years. The population slowly expanded its range from intertidal to subtidal locations as well as from Sylt north- and southwards along the coastline. Abundances of more than 300 oysters m^–2 on mussel beds were observed in 2003, only after two consecutive spatfalls in 2001 and 2002. Analyses of mean monthly water temperatures indicate that recruitment coincided with above-average temperatures in July and August when spawning and planktonic dispersal occurs. We conclude that the further invasion of C. gigas in the northern Wadden Sea will depend on high late-summer water temperatures.Communicated by H.D. Franke     

Dynamic measurements of black oystercatcher (Haematopus bachmani) predation on mussels (Mytilus californianus)

Intertidal zone mussels can face threats from a variety of predatory species during high and low tides, and they must balance the threat of predation against other needs such as feeding and aerobic respiration. Black oystercatchers (Haematopus bachmani) on the Pacific coast of North America can depend on the mussel Mytilus californianus for a substantial portion of their diet. Observations suggest that oystercatchers tend to focus on mussels beginning to gape their valves during rising tides, following periods of aerial emersion. We present detailed, autonomous field measurements of the dynamics of three such predation events in the rocky intertidal zone. We measured accelerations of up to 4 g imposed on mussels, with handling times of 115–290 s required to open the shell and remove the majority of tissue. In each case a single oystercatcher attacked a mussel that had gaped the shell valves slightly wider than its neighbors as the rising tide began to splash the mussel bed, but no other obvious characteristic of the mussels, such as body temperature or orientation, could be linked to the oystercatcher's individual prey choice.     

A novel striated tropomyosin incorporated into organized myofibrils of cardiomyocytes in cell and organ culture

Striated muscle tropomyosin is classically described as consisting of 10 exons, 1a, 2b, 3, 4, 5, 6b, 7, 8, and 9a/b, in both skeletal and cardiac muscle. A novel isoform found in embryonic axolotl heart maintains exon 9a/b of striated muscle but also has a smooth muscle exon 2a instead of exon 2b. Translation and subsequent incorporation into organized myofibrils, with both isoforms, was demonstrated with green fluorescent protein fusion protein construct. Mutant axolotl hearts lack sufficient tropomyosin in the ventricle and this smooth/straited chimeric tropomyosin was sufficient to replace the missing tropomyosin and form organized myofibrils.