Smooth Adductor Research Articles

First record of blister pearls in the oyster Hyotissa hyotis (Linné, 1758) from Pliocene deposits at Sidi Brahim, Lower Chelif Basin (north-west Algeria)

Fossil pearls are rare but important palaeoecological indicators in proving the former presence of parasites. A single right valve of Hyotissa hyotis from the Pliocene of Sidi Brahim shows numerous blister pearls inside the adductor muscle imprint. At the same locality, numerous shells with smooth adductor scars and without any blisters, have been collected. The structures in the studied valve can be assigned to blister pearls with high confidence due to their similarity to other Cenozoic pearls from Austria. The blister pearls likely formed as a reaction to parasite infestation. It is possible that some parasites especially targeted areas associated with the adductor muscles in the oyster genus Hyotissa, because similar blister pearls have previously been described in a congeneric species, H. squarrosa, from the Miocene of Austria.

MicroRNA expression signature in the striated and smooth adductor muscles of Yesso scallop Patinopecten yessoensis

Increasing evidences point to the potential role of microRNAs (miRNAs) in muscle growth and development in animals. However, knowledge on the identity of miRNAs and their targets in molluscs remains largely unknown. Scallops have one large adductor muscle, composed of fast (striated) and slow (smooth) muscle types, which display great differences in muscle fibers, meat quality, cell types and molecular components. In the present study, we performed a comprehensive investigation of miRNA transcriptomes in fast and slow adductor muscles of Yesso scallop Patinopecten yessoensis. As a result, 47 differentially expressed miRNAs representing ten miRNA families were identified between the striated and smooth adductor muscles. The KEGG enrichment analysis of their target genes were mainly associated with amino acid metabolism, energy metabolism and glycan biosynthesis. The target genes of miR-133 and miR-71 were validated by the dual-luciferase reporter assays and miRNA antagomir treatment in vivo. The identification and functional validation of these different miRNAs in scallops will greatly help our understanding of miRNA regulatory mechanism that achieves the unique muscle phenotypes in scallops. The present findings provide the direct evidences for muscle-specific miRNAs involved in muscle growth and differentiation in molluscs.

First Immunodetection of Sensory and Nervous Systems of Parasitic Larvae (Glochidia) of Freshwater Bivalve Nodularia douglasiae.

Most freshwater mussels have an unusual life cycle that requires host fish species for larval (glochidia) development and dispersal. Glochidia have a unique morphological structure that adapts to parasitic lifestyles and survival. The morphology of the glochidial shells of most Unionoidea, a group of freshwater bivalve mollusks, has been studied in detail using light and scanning electron microscopy. This study summarizes our data on the glochidia shell morphology of the Asian mussel Nodularia douglasiae from two localities in the Primorsky Territory, the Russian Far East. In contrast to the shell morphology of glochidia, little is known about the neurodevelopment of the Unionoidea. Herein, we first demonstrate that the structures of the sensory, muscle, and nervous systems of the glochidia larvae of N. douglasiae differ dramatically from those of the comparable larval systems of marine bivalve species, as revealed through alpha-acetylated tubulin, serotonin (5-HT), and FMRFamide antibodies as well as phalloidin for detection of F-actin and whole-mount confocal microscopy. We found that the glochidia sensory system included four pairs of tubulin-lir multicilia hair cells. Non-ciliar tubulin-lir cells synthesize the neuropeptide FMRFamide and are identified as afferent neurons collecting information from peripheral tubulin-lir hair sensory cells to nervous regulators. The glochidia’s muscular system was represented by a smooth adductor, retractors, and minor muscle bundles associated with the shell and visceral organs. The 5-HT-lir larval system is arranged most simply and consists of two immunopositive neurons innervating the adductor. The FMRFamide-lir system is more complicated and consists of several neuronal centers comprising neuronal bodies and their neurites in different areas of the larva. The FMRFamide-lir neurons are closely associated with sensory hair cells, and others, together with 5-HT-lir neurons, may be involved in the anlagen of adult ganglia. Thus, the nervous system of N. douglasiae glochidia is drastically different from other mollusks and lophotrochozoans because of the absence of an apical organ and the location and composition of FMRFamide and 5-HT cells. Morphological, molecular, and behavioral investigations of Unionoidea taxa need to be further conducted to investigate the parasite-host relationship, nerve-dependent regulation of parasite behavior, and evolution of mollusks.

Molecular Characterization and Expression Pattern of Paramyosin in Larvae and Adults of Yesso Scallop.

Simple SummaryParamyosin is an important myofibrillar protein in smooth muscle in molluscs that is not present in vertebrate muscles. This study characterized its sequence feature and expression patterns in Yesso scallop Patinopecten yessoensis and revealed the unique phosphorylation sites in scallops. The mRNA and protein expression of paramyosin was mainly found in foot and smooth adductor muscle. At late larval stages, strong paramyosin mRNA signals were detected in the symmetric positions of anterior and posterior adductor muscles. The present findings support that paramyosin may serve as the most important component of smooth muscle assembly during muscle development and catch regulation in scallops.Paramyosin is an important myofibrillar protein in molluscan smooth muscle. The full-length cDNA encoding paramyosin has been identified from Yesso scallop Patinopecten yessoensis. The length of paramyosin molecule has been found to be 3715 bp, which contains an open reading frame (ORF) of 2805 bp for 934 amino acid residues. Characterization of P. yessoensis paramyosin reveals the typical structural feature of coiled-coil protein, including six α-helix (α1-α6) and one coil (η) structures. Multiple phosphorylation sites have been predicted at the N-terminus of paramyosin, representing the unique phosphorylation sites in scallops. The highest levels of mRNA and protein expression of paramyosin have been found in foot and the smooth adductor muscle. According to whole-mount in situ hybridization (WISH), strong paramyosin mRNA signals were detected in the symmetric positions of anterior and posterior adductor muscles at late larval stages. These findings support that paramyosin may serve as the most important components for myogenesis and catch regulation in scallops. The present findings will not only help uncover the potential function of myofibrillar proteins in molluscs but also provide molecular evidence to infer evolutionary relationships among invertebrates.

Identification and characterization of phosphoproteins in the striated and smooth adductor muscles of Yesso scallop Patinopecten yessoensis

Many proteins are known to be phosphorylated, affecting important regulatory factors of muscle quality in the aquatic animals. The striated and smooth adductor muscles of Yesso scallop Patinopecten yessoensis were used to investigate muscle texture and identify phosphoproteins by histological methods and phosphoproteomic analysis. Our present study reveals that muscle fiber density is in relation to meat texture of the striated and smooth adductor muscles. The phosphoproteomic analysis has identified 764 down-regulated and 569 up-regulated phosphosites on 743 phosphoproteins in the smooth muscle compared to the striated part. The identification of unique phosphorylation sites in glycolytic enzymes may increase the activity of glycolytic enzymes and the rate of glycolysis in the striated adductor muscle. The present findings will provide new evidences on the role of muscle structure and protein phosphorylation in scallop muscle quality and thus help to develop strategies for improving meat quality of scallop products.

Characterization of paramyosin protein structure and gene expression during myogenesis in Pacific oyster (Crassostrea gigas).

Paramyosin is a key component of thick filaments in invertebrate muscles. In this study, we isolated the full length cDNA of paramyosin from Pacific oyster (Crassostrea gigas), and determined its pattern of expression during myogenesis. The full length paramyosin (CgPM) cDNA contains an open reading frame (ORF) of 2586bp encoding a 861-amino acid protein. Sequence analysis revealed an assembly competence domain (ACD) and a heptad repeat (d-e-f-g-a-b-c) with 28-residue repeat zones in the CgPM primary structure, a characteristic of coiled-coil protein. Quantitative analysis of CgPM expression revealed a sharp increase in trochophore stage, and peaked at the D-shaped stage. Strong CgPM expression was found in smooth adductor muscle, followed by striated adductor muscle and mantle tissue. Whole-mount in situ hybridization (WISH) showed a restricted pattern of CgPM expression in adductor muscle, larval velum retractor and foot muscles at the umbo and eyed larval stages. These data indicate that CgPM is strongly expressed during larval myogenesis in C. gigas, which provides the basis for further functional studies of paramyosin in oyster to better understand the molecular and cellular mechanisms of muscle formation in mollusks.

Molecular characterization and expression profiles of myosin essential light chain gene in the Pacific oyster Crassostrea gigas.

In molluscs, muscle contraction is triggered by a direct binding of Ca2+ to myosin. The myosin essential light chain (MELC) provides the Ca2+ binding site and is of importance for motor function and regulation. In this study, the complete cDNA sequence of MELC gene of the Pacific oyster, Crassostrea gigas, was obtained, and the expression profiles were performed in different tissues and different embryo-larval development stages. The results showed that the full length of C. gigas MELC (CgMELC) cDNA was 659bp, containing a 5'-untranslated region of 73 bp, a 3'-untranslated region of 112bp. The open reading frame encoded a 157 amino acid peptide. The protein sequence of CgMELC contained a conserved EF-hand calcium binding motif, and showed a high sequence identity (68.4-100%) with other bivalves. Quantitative analysis of CgMELC mRNA in tissues indicated that CgMELC was expressed at the highest level in the striated adductor muscle, followed by the smooth adductor muscle and mantle. During the development of embryos and larvae, quantitative analysis and whole-mount in situ hybridization revealed that CgMELC was expressed starting from the blastula stage and abundantly expressed in trochophore and D-shaped larvae, indicating that CgMELC might also be involved in regulation of larval muscle system development. Our data provided valuable information for further researches on the function of MELC and regulation of muscle contraction in oysters.

Thermostability of striated and smooth adductor muscle tropomyosins from Yesso scallop Mizuhopecten yessoensis

The striated and smooth adductor muscle tropomyosins (TMs) of Yesso scallop Mizuhopecten yessoensis have been known to express different isoforms, but have not been characterized in detail to date. In the present study, TMs from both muscles of Yesso scallop were purified and their stabilities were compared by circular dichroism (CD) spectrometry and differential scanning calorimetry (DSC). From the CD data, the apparent melting temperature and the apparent free energy of folding at 20 degrees C were calculated to be 30.5 degrees C and -13.4 kJ/mol, and 36.0 degrees C and -31.9 kJ/mol for the striated and smooth muscle TMs, respectively. From the DSC data, DeltaH values were calculated to be 1.87 x 10(3) and 2.19 x 10(3) kJ/mol for striated and smooth muscle TMs. These results suggest that smooth muscle TM has higher thermostability than striated muscle TM. The amino acid residues responsible for such stability difference were considered to be the six amino acid substitutions in the middle region of the TM molecules.

Mitochondrial plasticity in brachiopod (Liothyrella spp.) smooth adductor muscle as a result of season and latitude

Habitat temperature and mitochondrial volume density (Vv(mt,mf)) are negatively correlated in fishes, while seasonal acclimatization may increase Vv(mt,mf) or the surface density of the mitochondrial cristae (Sv(im,mt)). The effect of temperature on invertebrate mitochondria is essentially unknown. A comparison of two articulate brachiopod species, Liothyrella uva collected from Rothera Station, Antarctica in summer 2007, and Liothyrella neozelanica collected from Fiordland, New Zealand in winter 2007 and summer 2008, revealed a higher Vv(mt,mf) in the Antarctic brachiopod. The Sv(im,mt) was, however, significantly lower, indicating the Antarctic brachiopods have more, less reactive mitochondria. L. uva, from the colder environment, had larger adductor muscles in both absolute and relative terms than the temperate L. neozelanica. Furthermore, a seasonal comparison (winter vs. summer) in L. neozelanica showed that the absolute and relative size of the adductor increased in winter, Vv(mt,mf) was unchanged, and Sv(im,mt) was significantly increased. Thus, seasonal acclimatization to the cold resulted in the same number of more reactive mitochondria. L. neozelanica was clearly able to adapt to seasonal changes using a different mechanism, i.e. primarily through regulation of cristae surface area as opposed to mitochondrial volume density. Furthermore, given the evolutionary age of these living fossils (i.e. approximately 550 million years), this suggests that mitochondrial plasticity has roots extending far back into evolutionary history.

Gene expression analyses of essential catch factors in the smooth and striated adductor muscles of larval, juvenile and adult great scallop (Pecten maximus)

The scallop adductor muscle consists of striated fibres responsible for the fast closure of the shells, and smooth fibres able to maintain tension in a prolonged state of contraction called catch. Formation of the force-bearing catch linkages has been demonstrated to be initiated by dephosphorylation of the key catch-regulating factor twitchin by a calcineurin-like phosphatase, while the involvement of other thick filament proteins is uncertain. Here we report on the development of catchability of the adductor smooth muscle in the great scallop (Pecten maximus) by analysing the spatio-temporal gene expression patterns of the myosin regulatory light chain (MLCr), twitchin, myorod and calcineurin using whole mount in situ hybridization and real-time quantitative PCR. The MLCr signal was identified in the retractor and adductor muscles of the pediveliger larvae, and the juvenile and adult scallop displayed abundant mRNA levels of MLCr in the smooth and striated adductor muscles. Twitchin was mainly expressed in the smooth adductor muscle during metamorphosis, whereas the adult striated adductor muscle contained seven-folds higher twitchin mRNA levels compared to the smooth portion. Calcineurin expression predominated in the gonads and in the smooth adductor, and five-folds higher mRNA levels were measured in the smooth than in the striated fibres at the adult stage. In contrast to the other genes examined, the expression of myorod was confined to the smooth adductor muscle suggesting that myorod plays a permissive role in the molluscan catch muscles, which are first required at the vulnerable settlement stage as a component of the predator defence system.

Thermally induced gelation of paramyosin from scallop adductor muscle

Thermally induced gelation of paramyosin from scallop smooth adductor muscle was investigated by dynamic rheological measurements under various conditions. The paramyosin thermal gel was produced at pH 6.5 and 7.2 at temperatures above 30°C through a two-step increase in storage (G′) and loss (G″) moduli; these values were higher than in gels produced from actomyosin at a high temperature. The thermal gel properties were very firm and brittle. In contrast, one main peak of G′ was observed during gelation at pH 8.0. The gel produced at pH 8.0 was more transparent and less soluble in a 6 M urea-0.5 M NaCl solution than those formed either at pH 6.5 or 7.2. These differences in the thermal gel properties are presumed to derive from the pH dependence of the gel matrix-forming process, such as oxidative cross-linking between cysteine residues, rather than from the thermal unfolding of the paramyosin molecules. The thermal gelation profile of chymotrypsin-digested paramyosin showed marked depression of G′ at high temperature.

The genomic structure of the scallop, Patinopecten yessoensis, troponin C gene: a hypothesis for the evolution of troponin C

Two cDNAs encoding troponin C (TnC) isoforms are isolated from the scallop, Patinopecten yessoensis, striated adductor muscle. The sequential differences between these isoforms, named TnC(long) and TnC(short), are restricted in several residues of the C-terminal region. TnC(long) is commonly expressed in both the striated and the smooth adductor muscle; however, TnC(short) is only in the striated adductor muscle. The TnC gene is a single copy gene in the scallop, thus they are expressed through the alternative splicing from the same gene. The scallop TnC gene is constructed from five exons and four introns, and positions of introns are identical with chordate TnC genes, although the scallop TnC possesses no corresponding intron to the fourth intron of chordates. The loss of this intron is also observed in Drosophila TnC; these may be remnants of their ancestor, namely the early metazoan TnC gene might be a five exons-four introns structure. In addition, the absence of the corresponding intron is also observed among protostomian calmodulins (CaMs), a molecule closely related to TnC. This suggests that the common ancestor gene of the TnC superfamily might also be a five exons-four introns structure. Assuming this to be true, the discordance of the fourth intron positions observed among members of the family is well explained by the evolutionary independent gain of the intron on each member's lineage.

Glycolytic Enzymes in the Tissues of Three Species of Scallop (Bivalvia: Pectinidae)

The maximal activities of 15 enzymes were compared in the striated and smooth adductor muscle, mantle muscle, foot, digestive gland, testis, and ovary of three species of scallops Chlamys nobilis, Pecten albicans, and Patinopecten yessoensis. The patterns of glycolytic enzyme activities in these three species of scallop were similar. However, the maximal activities of phosphofructokinase and malate dehydrogenase in the striated adductor muscle and octopine dehydrogenase in the striated and smooth adductor muscle differ significantly between species. All enzymes except for hexokinase were detected at the highest activity in the adductor muscle. In the striated muscle octopine dehydrogenase activity was highest among pyruvate reductases, followed by other opine dehydrogenases, D-lactate dehydrogenase. and L-lactate dehydrogenase. In these scallops phosphoenolpyruvate carboxykinase activity was very low compared with pyruvate kinase. Gonads and digestive gland showed fairly high activities of malate dehvdrosenase. asoartate aminotransferase. and alanine aminotransferase.

Ultrastructural Studies of Calcium Location during the “Catch” Contraction of Clam Smooth Adductor Muscle Cells

Contraction of molluscan adductors has been classified into three states; 1) resting state, 2) contracted state, 3) prolonged “catch” state. Among these, the “catch” state is considered a peculiar state, which requires little expenditure of energy, but in which contraction can be maintained for long periods. It is not yet known whether “catch” muscle contraction is regulated by Ca, or where Ca translocates during resting state through “catch” state, if the muscle contraction is regulated by Ca. We attempted to observe Ca translocation in muscle cells during contraction by the K-pyroantimonate method. We fixed “catch” muscle cells for electron microscopy with fixative including K-pyroantimonate, and observed where electron-dense precipitates, in which Ca is concentrated, were located in the muscle cells in the three states of contraction. At the resting state, precipitate was located at cell peripheries, in positions such as at the inner surface of cell membranes and in sarcoplasmic reticular systems (SRs). In the contracted state, they were located within the cytoplasm. At the “catch” state, they were found in both the cytoplasm and at peripheries, although the number of precipitates in peripheral areas was small. Thus, we show that calcium translocates in the cells during resting-contraction-catch cycles of “catch” muscle contraction.

Troponin from smooth adductor muscle of Ezo-giant scallop.

Troponin which can confer Ca2+-sensitivity upon rabbit actomyosin Mg-ATPase activity has been prepared from the smooth adductor muscle of Ezo-giant scallop (Patinopecten yessoensis). The troponin comprises 40-, 20-, and 19-kDa components. In order to characterize the components, they were separated from each other by CM-Toyopearl column chromatography in the presence of 6 M urea. Consequently, the 20-kDa component was identified as troponin C, based on the Ca2+-binding ability. The amount of Ca2+ bound to the troponin C was estimated to be 0.75 mol/mol at 10(-4) M Ca2+ by the equilibrium dialysis method. The 19-kDa component was identified as troponin I on the basis of not only its inhibitory effect on rabbit actomyosin Mg-ATPase activity along with the smooth adductor tropomyosin, but also the releasing effect of the smooth adductor troponin C on the inhibition. On the other hand, the 40-kDa component was regarded as troponin T on the basis that it bound to F-actin-tropomyosin filament and was indispensable for conferring Ca2+-sensitivity upon rabbit actomyosin Mg-ATPase activity, along with troponin C and troponin I. The above assignments were confirmed by both amino acid analysis and immunoblotting using rabbit antisera raised against counterparts of scallop striated muscle troponin.

Tonic force maintenance after decay of active state in brachiopod smooth adductor muscle

1. Contraction of smooth adductor muscles (SmA) in the brachiopod,Laqueus californicus, is followed by very slow relaxation (R1/2=3 min). The period during which the cross-bridges are in the active state extends from the time of activation to a short time after peak tension is reached. The delayed relaxation phase represents tension maintenance in the absence of the active state. 2. The contraction characteristics of these muscles were compared to those during the catch contractions of mollusc adductor muscles. The immediate elasticity of brachiopod SmA, defined as the change in tension in response to quick length changes, is the same during the contraction and relaxation phases. The immediate elasticity of mollusc catch muscles is greater during the tonic relaxation (catch) than the active contraction phase (Pfitzer and Ruegg 1982). 3. When exposed to CO2, the SmA, whether relaxed or during contraction, enter a reversible rigor-like state of stretch resistance. The immediate elastic modulus during these conditions was the same as during normal contractions. 4. The results indicate that the same physical linkages, probably actomyosin cross-bridges, are responsible for all phases of the contraction/relaxation cycle and also during CO2 induced rigor-like stiffness.

アカザラガイ横紋筋および平滑筋筋原線維の脱感作と再感作

By using the myofibrils prepared from akazara scallop striated and smooth adductor muscles, dissociation of myosin regulatory light chains and the relation of the regulatory light chain con-tent to Mg-ATPase activity were investigated. The regulatory light chain dissociation was found to occur more readily from the striated myofibrils than from the smooth ones. Moreover, it was confirmed that the complete removal of regulatory light chains was required for the complete loss of Ca-sensitivity of Mg-ATPase; and that the Mg-ATPase activity both in the presence and absence of Ca2+ changed in proportion to the regulatory light chain content. These results are very similar to those obtained with akazara myosin.

Isolation of troponins from striated and smooth adductor muscles of Akazara scallop.

Troponins which confer Ca-sensitivity to skeletal actomyosin ATPase were successfully isolated from striated and smooth adductor muscles of "Akazara" scallop (Chlamys nipponensis akazara). SDS-gel electrophoresis showed that striated and smooth adductor troponins were composed of three components having molecular weights of about 52K (52,000), 40K, and 20K, and about 40K, 21K, and 20K, respectively. The Mg-ATPase activity of actomyosin reconstituted from rabbit actin and either Akazara striated adductor myosin or smooth adductor myosin, along with the respective tropomyosin and troponin, indicated that the Ca2+ concentration required for the activation of actomyosin ATPase appeared to be favorable to myosin-linked regulation.

二枚貝平滑筋および横紋筋ミオシンの調節軽鎖の解離性

The temperature dependence of myosin Mg-ATPase activity and of the heat-dissociation rate of myosin regulatory light chains were studied by using the myosins from striated and smooth adductor muscles of akazara scallop, and from surf-clam foot muscle. 1. Mg-ATPase activity of akazara striated adductor myosin showed a significantly decreased Ca-sensitivity above 20°C, whereas those of akazara smooth adductor and surf-clam foot myosins were to a much less extent affected. 2. The regulatory light chains of akazara striated adductor myosin were readily dissociated by heat-treatment at 30°C, even in the presence of 2mM MgCl2, unlike those of surf-clam foot myosin 3. The Ca2+ concentration for 50% dissociation of regulatory light chains by heat (30°C)- treatment was estimated to be 4μM for akazara striated myosin, 0.8μM for akazara smooth myosin, 0.2μM for surf-clam foot mysoin. The Mg2+ concentration for the 50% dissociation was 240, 20, and 16μM, respectively.

Molecular organization of paramyosin in the core of molluscan thick filaments

An arrangement of paramyosin molecules in the polar part of molluscan thick filaments is proposed which accounts for the X-ray diffraction pattern of the smooth adductor muscle (other than the part ascribed to actin) and for the appearance of separated filaments in the electron microscope. The proposed structure is based on the PI arrangement of Cohen et al. (1971), and contains sets of parallel, equidistant molecules with successive molecules displaced along the molecular axis by 72 nm, which we call PI sheets. Every molecule belongs to two PI sheets which are nearly perpendicular. This array is not propagated throughout the filament, but is sheared periodically in the direction of the molecular (filament) axis by 2 5 × 72 nm . The shear occurs along parallel equidistant planes which are inclined to the PI sheets. The analysis of the X-ray data has been made possible by concentrating on those patterns from filaments in which the two sets of PI sheets appear to be mutually perpendicular, a condition brought about by bathing the muscle in aqueous acetone. In one set, there are four intermoleeular spaces between shear planes (this appears to be true at least for the smooth adductors of Ostrea edulis, Crassostrea angulata and Mercenaria mercenaria). In the other set, the number varies with species and probably lies between eight and ten in the first two and appears to be six in the last named species. The known paracrystalline nature of paramyosin filaments suggests that this number, though dominant in one species. is not exactly constant.