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ACh and 5-HT induced changes in the concentration of cytosolic inositol trisphosphate (InsP3) and inositol bisphosphate (InsP2) in the ABRM of Mytilus edulis L.

1. For determination of the phosphoinositides and inositol phosphates present in anterior byssus retractor muscle (ABRM) of Mytilus edulis fiber bundles of this muscle were incubated with [3H]-inositol. Close-to-equilibrium labelling was achieved after 14-17 hr of incubation. 2. The phosphoinositides formed during incubation were identified as phosphatidylinositolphosphates by thin layer chromatography and as glycerophosphoryl esters by anion-exchange chromatography after deacylation. Besides PtdIns, PtdInsP and PtdInsP2 two labelled products are formed, which could not be identified. 3. Inositol phosphates were separated by anion-exchange chromatography. InsP, InsP2 and InsP3 are present, while InsP4 seemed to be absent. 4. Incubation of pre-labelled fibers with ACh induces the accumulation of InsP3 and InsP2 immediately. While 5-Ht accomplishes the accumulation after a lag time of 25 sec. The concentration of cytosolic InsP does not change.

Acetylcholine↗

The FMRFamide-related decapeptide of Mytilus contains a D-amino acid residue.

1. An FMRFamide-related decapeptide isolated from the anterior byssus retractor muscle (ABRM) of the bivalve mollusc, Mytilus edulis, was shown to have D-Leu as the second amino acid residue. 2. The excitatory effects of the peptide (Mytilus-FFRFamide) on the ABRM were not changed appreciably by substituting an L-Leu residue for the D-Leu residue.

Animals↗

Crystalline style and tissue redistribution in Perna viridis as indicators of Cu and Pb bioavailabilities and contamination in coastal waters.

The concentrations of Cu, Pb, and Zn in the crystalline style (CS) and in the remaining soft tissues (ST) of the green-lipped mussel Perna viridis from 10 geographical sites along the coastal waters off peninsular Malaysia were determined. The CS, compared with the remaining ST, accumulated higher levels of Cu in both contaminated and uncontaminated samples, indicating that the style has a higher affinity for the essential Cu to bind with metallothioneins. The similar pattern of Cu accumulation in the different ST of mussels collected from clean and Cu-contaminated sites indicated that the detoxification capacity of the metallothioneins had not been overloaded. For Pb, higher levels of the metal in the CS than in the remaining ST were found only in mussels collected from a contaminated site at Kg. Pasir Puteh. This indicated a tissue redistribution of Pb due to its binding to metallothioneins for Pb detoxification and the potential of the CS as an indicator organ of Pb bioavailability and contamination. For Zn, the above two phenomena were not found since no obvious patterns were observed (lower levels of Zn in the CS than in the remaining ST) in contaminated and uncontaminated samples due to the mechanism of partial regulation. Generally, all the different STs studied (foot, mantle, gonad, CS, gill, muscle, and byssus) are good biomonitoring tissues for Cu and Pb bioavailabilities and contamination. Among these organs, the CS was found to be the best organ for biomonitoring Cu. The present data also suggest the use of the tissue redistribution of Pb in P. viridis as an indicator of Pb bioavailability and contamination in coastal waters.

Animals↗

Biological factors influencing tissue compartmentalization of trace metals in the deep-sea hydrothermal vent bivalve Bathymodiolus azoricus at geochemically distinct vent sites of the Mid-Atlantic Ridge.

In this study, we investigated on concentrations of trace metals (Al, Cd, Mn, Co, and Hg) in the hydrothermal bivalve Bathymodiolus azoricus, a dominant species at most vent sites along the Mid-Atlantic Ridge (MAR), and in its endosymbiont bacteria and commensal parasite Branchipolynoe seepensis. Comparison of our results with data from the literature on non-hydrothermal bivalves suggests lack of "extreme" uptake of trace metals by B. azoricus, except for Hg concentration which exceeded manyfold previously reported values. Mussels collected from three geochemically distinct vent sites, Menez Gwen, Lucky Strike, and Rainbow, along the MAR showed significant differences in tissue concentration of metals. Proportionality of metals in soft tissues of mussels reflected variation of water chemistry at different vents, which in turn conserved the order of trace metal prevalence in undiluted fluids. There were significant tissue-specific differences in trace metal compartmentalization for all metals investigated. Byssus thread contained the highest metal concentration among examined tissues, and thus it is suggested to be an important detoxification route. Size-dependent differences in metal concentrations were detected only for Hg, revealing a general trend of small mussels accumulating more metal than big mussels. Endosymbiont bacteria are shown to exclusively sequester Al from the host gill and contribute to removal of other toxic metals in mussels from Menez Gwen. The commensal parasite present in all mussels from Lucky Strike had higher tissue concentrations of Mn, Al, and Co than the host gill, unlike Cd and Hg which were considerably lower in the former, and thus its role in detoxification remains unclear. Bioaccumulation potential of vent bivalves and associated organisms are quantified as concentration factors and compared to make inferences on the putative role of the endosymbiont bacteria and the commensal parasite in detoxification of trace metals.

Animals↗

Antibacterial activities in various tissues of the horse mussel, Modiolus modiolus.

A search for antibacterial activity in different organs/tissues of the horse mussel, Modiolus modiolus, was conducted. Dried samples were extracted with 60% (v/v) acetonitrile, containing 0.1% (v/v) trifluoroacetic acid. Due to high salt content, two liquid phases were obtained; an acetonitrile-rich phase (ACN extract) and an aqueous phase. The aqueous phase was further subjected to solid phase extraction (SPE). Eluates from SPE and ACN extracts were tested for antibacterial, lysozyme, and toxic activity. Antibacterial activity was demonstrated in extracts from several tissues, including plasma, haemocytes, labial palps, byssus, mantle, and gills. Some of the extracts were sensitive to proteinase K treatment, indicating antibacterial peptides and/or proteins. Lysozyme-like activity and toxic activity against Artemia salina nauplii was detected in fractions from the gills, mantle, muscle, and haemocytes. Results from this study indicate that M. modiolus is a promising source for identifying novel drug lead compounds.

Animals↗

Upper temperature tolerances of exotic brackish-water mussel, Mytilopsis leucophaeata (Conrad): an experimental study.

The dark false mussel, Mytilopsis leucophaeata is an important mussel colonising the brackish-water systems of temperate and subtropical regions. Of late it has earned notoriety as a biofouling species in industrial cooling water systems. However, there are no published data on the temperature tolerance of this species. This paper presents data on the upper temperature tolerance of this mussel from the view point of biofouling control using thermal methods. In addition to mortality, response of physiological activities such as oxygen consumption, filtration rate, foot activity and byssus thread production were also studied at temperatures varying from 5 to 35 degrees C. Experiments were also carried out to understand the effect of mussel size, breeding condition, nutritional status and acclimation conditions (temperature and salinity) on the mortality pattern. The physiological activities were significantly reduced at temperatures beyond 27.5 degrees C and ceased at 35 degrees C. In 20 mm size group mussels exposed to 37 degrees C, 50% mortality was observed after 85 min and 100% mortality after 113 min. The effect of mussel size on mortality at different temperatures was significant, with the larger size group mussels showing greater resistance. M. leucophaeata collected during the non-breeding season (December-April) were more tolerant to temperature than those collected during the breeding season (June-October). Nutritional status of the mussel had no significant influence on the thermal tolerance of the mussel: fed and starved (non-fed) mussels succumbed to temperature at comparable rates. The effect of acclimation temperature and acclimation salinity on M. leucophaeata mortality at different temperatures was significant. Survival time increased with increasing acclimation temperature and decreased with increasing salinity. In comparison with other co-occurring species such as Mytilus edulis and Dreissena polymorpha, M. leucophaeata appears to be more tolerant to high temperature stress.

Adaptation, Physiological↗

Impact of low doses of tritium on the marine mussel, Mytilus edulis: genotoxic effects and tissue-specific bioconcentration.

Despite growing scientific, public and regulatory concern over the discharge of radioactive substances, no serious attempts have been made to develop a rationale to evaluate the impact of environmentally relevant radionuclides in the aquatic environment. In this study, we have evaluated the genotoxic effects and tissue-specific concentration of tritium (added as tritiated water, HTO) in the adult life stage of the edible mussel, Mytilus edulis. The genotoxic effects were quantified in terms of the induction of: (a) micronuclei (MN), and (b) DNA single-strand breaks/alkali-labile sites using alkaline single-cell gel electrophoresis (Comet assay) in the haemocytes of exposed animals. The assays were optimised and validated using a range of concentrations (18-56 mgl(-1)) of ethylmethane sulfonate (EMS), a direct-acting reference genotoxic agent, over different exposure periods. Mussels were exposed to a series of concentrations of HTO equivalent to a dose range from 12 to 485 muGyh(-1) for 96 h, and different tissues and organs were then extracted and analysed. The study revealed a dose-dependent increase in the response for both the MN test and the Comet assay and for both EMS and HTO. In addition, HTO delivering dose rates below 500 muGyh(-1) was shown to be capable of inducing genetic damage in the haemocytes of these bivalves. The study also showed that inorganic tritium accumulated differentially in mussel tissues in a dose-dependent manner, with the gut accumulating the highest amount of radioactivity, followed by the gill, mantle, muscle, foot and byssus thread. The faeces and pseudo-faeces accumulated least radioactivity over the exposure period. Differential accumulation of radionuclides has significant implications for biomonitoring programmes, for this and other aquatic organisms. The study also suggests that the generic dose limits recommended by the International Atomic Energy Agency for the protection of aquatic biota might not be applicable to all aquatic organisms.

Animals↗

Suppression of crossbridge motions of isolated thick myofilaments in ATP-free medium by thiourea.

Thiourea is known to suppress the contractile response of Mytilus anterior byssus retractor muscle and toad sartorious muscle following electrical or chemical stimulation without abolishing of the electrical responses. In addition, it blocks the shortening of glycerinated sartorious muscle induced by Ca2+. With dynamic laser light scattering method we found that thiourea suppresses the increase of the average linewidth of the photoelectron count autocorrelation function, gamma, of isolated thick filaments of Limulus striated muscle induced by depletion of ATP. The results obtained suggest that thiourea prevents the crossbridges from moving away from the thick myofilament backbones which will, in turn, prohibit the crossbridges from interacting with the thin myofilaments.

Adenosine Triphosphate↗

Structures and actions of Mytilus inhibitory peptides.

Two congeneric peptides that inhibit contraction of the anterior byssus retractor muscle of Mytilus edulis were isolated from the pedal ganglia of the mussel. Their structures were determined to be H-Gly-Ser-Pro-Met-Phe-Val-NH2 and H-Gly-Ala-Pro-Met-Phe-Val-NH2. These hexapeptides also showed inhibitory action on contractions in several other molluscan muscles, such as the cardiac muscle of Meretrix lusoria and the penis retractor muscle of Achatina fulica.

Amino Acid Sequence↗

The myosin cross-bridge cycle and its control by twitchin phosphorylation in catch muscle.

The anterior byssus retractor muscle of Mytilus edulis was used to characterize the myosin cross-bridge during catch, a state of tonic force maintenance with a very low rate of energy utilization. Addition of MgATP to permeabilized muscles in high force rigor at pCa > 8 results in a rapid loss of some force followed by a very slow rate of relaxation that is characteristic of catch. The fast component is slowed 3-4-fold in the presence of 1 mM MgADP, but the distribution between the fast and slow (catch) components is not dependent on [MgADP]. Phosphorylation of twitchin results in loss of the catch component. Fewer than 4% of the myosin heads have ADP bound in rigor, and the time course (0.2-10 s) of ADP formation following release of ATP from caged ATP is similar whether or not twitchin is phosphorylated. This suggests that MgATP binding to the cross-bridge and subsequent splitting are independent of twitchin phosphorylation, but detachment occurs only if twitchin is phosphorylated. A similar dependence of detachment on twitchin phosphorylation is seen with AMP-PNP and ATPgammaS. Single turnover experiments on bound ADP suggest an increase in the rate of release of ADP from the cross-bridge when catch is released by phosphorylation of twitchin. Low [Ca(2+)] and unphosphorylated twitchin appear to cause catch by 1) markedly slowing ADP release from attached cross-bridges and 2) preventing detachment following ATP binding to the rigor cross-bridge.

Adenosine Diphosphate↗

An x-ray diffraction study of contracting molluscan smooth muscle.

The living anterior byssus retractor muscle of Mytilus (ABRM), a smooth, "catch" muscle, has been studied by X-ray diffraction while relaxed and while tonically contracted. X-ray reflections were observed from the actin and paramyosin filaments and from the alpha-helical substructure of the paramyosin filaments. No differences in spacings or relative intensities were observed when the relaxed and contracting muscle patterns were compared. This result is consistent with a sliding filament mechanism involving an interaction between actin and paramyosin filaments.

Actins↗

X-ray structure analysis of thin filaments of a molluscan smooth muscle in the living relaxed state.

In the small-angle x-ray diffraction pattern of the living relaxed anterior byssus retractor muscle of Mytilus edulis, the thin filaments showed the following features. The 59.8-A reflection was much stronger and a little farther from the meridian than the 51.9-A reflection, although they are both contributions of the first-order Bessel function and are comparable with each other in the height from the equator. The 381-A reflection, given by the second-order Bessel function, was weaker than the 59.8-A reflection by more than the difference between the peak values of the first- and second-order Bessel functions, and was not so distant radially from the latter as estimated from the amount of peak shift brought about by the alteration of the Bessel order. A model of the thin filament was made on the basis of inverse Fourier transformation of the scattering amplitude, and the above features were explained by the characteristic shape of actin shown in this model. The actin subunits are elongated along the genetic left-hand helix with a pitch of 59.8 A, and are bonded together along the genetic helix in the inner part of the filament.

Animals↗

Elastic properties of isolated thick filaments measured by nanofabricated cantilevers.

Using newly developed nanofabricated cantilever force transducers, we have measured the mechanical properties of isolated thick filaments from the anterior byssus retractor muscle of the blue mussel Mytilus edulis and the telson levator muscle of the horseshoe crab Limulus polyphemus. The single thick filament specimen was suspended between the tip of a flexible cantilever and the tip of a stiff reference beam. Axial stress was placed on the filament, which bent the flexible cantilever. Cantilever tips were microscopically imaged onto a photodiode array to extract tip positions, which could be converted into force by using the cantilever stiffness value. Length changes up to 23% initial length (Mytilus) and 66% initial length (Limulus) were fully reversible and took place within the physiological force range. When stretch exceeded two to three times initial length (Mytilus) or five to six times initial length (Limulus), at forces approximately 18 nN and approximately 7 nN, respectively, the filaments broke. Appreciable and reversible strain within the physiological force range implies that thick-filament length changes could play a significant physiological role, at least in invertebrate muscles.

Actin Cytoskeleton↗

Regulation of catch muscle by twitchin phosphorylation: effects on force, ATPase, and shortening.

Recent experiments on permeabilized anterior byssus retractor muscle (ABRM) of Mytilus edulis have shown that phosphorylation of twitchin releases catch force at pCa > 8 and decreases force at suprabasal but submaximum [Ca2+]. Twitchin phosphorylation decreases force with no detectable change in ATPase activity, and thus increases the energy cost of force maintenance at subsaturating [Ca2+]. Similarly, twitchin phosphorylation causes no change in unloaded shortening velocity (Vo) at any [Ca2+], but when compared at equal submaximum forces, there is a higher Vo when twitchin is phosphorylated. During calcium activation, the force-maintaining structure controlled by twitchin phosphorylation adjusts to a 30% Lo release to maintain force at the shorter length. The data suggest that during both catch and calcium-mediated submaximum contractions, twitchin phosphorylation removes a structure that maintains force with a very low ATPase, but which can slowly cycle during submaximum calcium activation. A quantitative cross-bridge model of catch is presented that is based on modifications of the Hai and Murphy (1988. Am. J. Physiol. 254:C99-C106) latch bridge model for regulation of mammalian smooth muscle.

Adenosine Triphosphatases↗

Responses of the green-lipped mussel Perna viridis (L.) to suspended solids.

Laboratory experiments were conducted to investigate the lethal and sublethal effects of suspended solids on the survival and physiological, behavourial and morphological changes of the green-lipped mussel Perna viridis collected from Tolo Harbour, Hong Kong. Results showed that P. viridis survived in all test conditions of suspended solids from 0 to 1,200 mg/l over a period of 96 h. Physiological responses of the green-lipped mussel under 14-d exposure of suspended solids from 0 to 600 mg/l, followed by 14-d recovery in natural seawater, revealed no significant changes (p > 0.05) in oxygen consumption and dry gonosomatic index for treatments in different concentrations of suspended solids and exposure time. Changes in clearance rate were only found to be significant (p < 0.001) with exposure time. Responses in behavourial and morphological changes of the green-lipped mussel were also studied under similar experimental treatments and exposure time. Byssus production was significantly (p < 0.001) related to exposure time. Gill damage, however, was significantly greater in treatments (p < 0.001). Present findings suggested that P. viridis could tolerate a high level of suspended solids in the laboratory. There were dose-dependent effects of suspended solids on morphology of gill filaments. Implications of survival and responses of the green-lipped mussel to suspended solids in the marine environment are discussed.

Adaptation, Physiological↗

Ultrastructural studies on paramyosin core filaments from native thick filaments in catch muscles.

Catch muscles of molluscs usually have thick filaments of about 100 nm in diameter. The filament is constructed of a paramyosin core filament and an outer layer of myosin molecules. Myosin molecules are situated on the paramyosin core filament towards both ends, and they consequently have polarity towards both ends. According to our observations on the paramyosin core filaments from the native thick filaments, they bear regular cross-striations on their surfaces of about 14.5 nm periodicity when incubated in a KCl solution for a short time. The periodic pattern is supposed to be representative of peculiar arrangements of paramyosin molecules in the core, but the periodic pattern disappeared during incubation in a solution of high concentration KCl for a prolonged time, and a 'Bear-Selby net' pattern appeared substitutionally. These 'Bear-Selby net' patterns were conveniently divided into 3 types among 4 paramyosin core filaments from 4 'catch' muscles; the adductors of a pecten, an oyster and a clam, and the byssus retractor of a mussel. The 'Bear-Selby net' of an oyster resembled that of a pecten. Purified paramyosin crystals from the 4 muscles showed a common periodicity of about 72.5 nm. Electrophoresis with SDS of the 4 paramyosins on 6% polyacrylamide gels revealed molecular weights at 104 kD from a pecten, 105 kD from an oyster, 103 kD from a clam and 105 kD from a mussel.

Actin Cytoskeleton↗

Uptake and depuration of 63Ni by Mytilus edulis.

The uptake and depuration of Ni (as radioisotope 63Ni) by Mytilus edulis (Mollusca: Bivalvia) has been investigated in a laboratory mesocosm. Nickel was reversibly held on low energy surface sites of estuarine suspended particulate matter (SPM), with a partition coefficient of the order 10(3) ml g-1, resulting in approximately 80% of radioisotopic Ni being in the dissolved phase. Mussels were fed a diet of radio-labelled estuarine SPM for 30 h in a flow-through exposure period and for a further 24 h in a static exposure period. Following exposure, the mussels were allowed to depurate in particle-free seawater. Pseudofaeces and faeces were collected throughout the experiment and tissues were dissected after 48 h of depuration. The 63Ni activity in the faeces was approximately 50% of that in the feedstock SPM, while the activity in the tissues was 2-29% of SPM activity. Tissue-specific accumulation occurred in the order viscera > byssus > mantle > adductor muscle > gill > foot. The results are discussed in the context of the bioavailability of Ni to mussels.

Animals↗

Histochemical detection of monoamine oxidase activity in smooth muscle and epithelial tissues of Mytilus edulis L. and Mytilus galloprovincialis Lmk.

Monoamine oxidase (MAO) activity was detected in the glio-interstitial tissue of the anterior byssus retractor muscle (ABRM) of Mytilus by the use of either dopamine or 5-HT as substrate and nitro blue tetrazolium salt as an electron receptor. Both substrates gave superimposable patterns. However, dopamine reactivity was very weak. Discrete but clear MAO activity was detected in the periphery of the smooth muscle cell, i.e., at the level of the peripherally localized mitochondria. The presence of MAO in the epithelium of the buccal palps was also demonstrated. These histochemical observations can be correlated with certain ultrastructural aspects, in particular the presence electron dense organelles profiles in glio-interstitial cells. This MAO activity is discussed in relationship with the high 5-HT content of ABRM and the control of its relaxation. Pigment formation and/or collagen tanning are discussed in terms of a possible relationship with inactivation of biogenic amines. These results complement previous data concerning the glio-interstitial tissue found in close association with muscle and nerve structures.

Animals↗