An ultrastructural study of byssus stem formation in Mytilus californianus

In Mytilus californianus, root lamellae of the byssus stem are formed by two morphologically distinct exocrine cell types. Type 1 cells contain large ellipsoid granules which are ultrastructurally identical to those of the collagen gland associated with byssus thread formation: these granules are secreted only at the base of the stem generator. Type 2 cells contain small cylindroid granules which are secreted only from the lateral surfaces of generator septa. The resultant matrix is biphasic because the two secretions are incompletely mixed. Lamellar sheets of matrix are propelled outward by the action of cilia and are molded into a cylinder at the neck region of the stem. However, the stem retains a lamellar pattern. Byssus threads are attached to the stem by flattened rings formed from thread material which is secreted into the cervical crevice surrounding the neck. The microanatomy of the stem forming region is described and a new term, “stem generator,” is proposed for this organ.     

Specialized cilia of the byssus attachment plaque forming region in Mytilus californianus

The distal depression of the ventral pedal groove of Mytilus californianus was investigated by scanning and transmission electron microscopy. This part of the byssus forming system is responsible for the formation of the attachment plaque of the byssus thread. The longitudinal pedal ducts open into this area and the floor of the distal depression is covered by specialized cilia which terminate as biconcave flattened discs or “paddles.” The disc is formed by a 360° curvature of the axoneme tip within the ciliary membrane. The diameter of the disc is about 1.33 μ while that of the shaft portion is 0.24 μ. There are about 11 cilia per square micron of surface area and the necks of the cilia are separated from each other by a web-like extension of apical cytoplasm extending from the epithelial cells. It is proposed that these specialized cilia function as microscopic spatulas for the application of the adhesive plaque material to substrate surfaces. The pattern of surface convection currents seen in vivo tends to support this hypothesis.     

Linking adhesive and structural proteins in the attachment plaque of Mytilus californianus

The byssal attachment of California mussels Mytilus californianus provides secure adhesion in the presence of moisture, a feat that still eludes most synthetic polymers. Matrix-assisted laser desorption ionization mass spectrometry was used to probe the footprints of byssal attachment plaques on glass cover slips for adhesive proteins. Besides the abundant mcfp-3 protein family (Zhao, H., Robertson, N. B., Jewhurst, S. A., and Waite, J. H. (2006) J. Biol. Chem. 281, 11090-11096), two new proteins, mcfp-5 and mcfp-6, with masses of 8.9 kDa and 11.6 kDa, respectively, were identified in footprints, partially characterized and completely sequenced from a cDNA library. mcfp-5 resembles mcfp-3 in its basic pI and abundant 3,4-dihydroxyphenyl-L-alanine (Dopa; 30 mol %), but is distinct in two respects: it is more homogeneous in primary sequence and is polyphosphorylated. mcfp-6 is basic and contains a small amount of Dopa (<5 mol %). In contrast to mcfp-3 and -5, tyrosine prevails at 20 mol %, and cysteine is present at 11 mol %, one-third of which remains thiolate. Given the oxidative instability of Dopa and cysteine at pH 8.2 (seawater), we tested the hypothesis that thiols serve to scavenge dopaquinones by adduct formation. Plaque footprints were hydrolyzed and screened for cysteine dopaquinone adducts using phenylboronate affinity chromatography. 5-S-Cysteinyldopa was detected at nearly 1 mol %. The results suggest that mcfp-6 may provide a cohesive link between the surface-coupling Dopa-rich proteins and the bulk of the plaque proteins.     

Identification and estimation of dopamine released from the anterior byssus retractor muscle of Mytilus californianus in response to nerve stimulation

• 1.1. Small quantities of sea water were recyclically perfused over the surface of paired anterior byssus retractor muscles of Mytilus californianus.
• 2.2. Dopamine was identified in the perfusate by thin-layer chromatography.
• 3.3. Stimulation of the pedal ganglion caused the dopamine content of the perfusate to increase.
• 4.4. A significant increment of release of dopamine was detected at stimulation frequencies above 3 Hz and increased progressively with increase in stimulation frequency.
• 5.5. The possibility of a role for dopamine as a relaxing or inhibitory neurotransmitter in Mytilus is considered in relation to the present and related evidence and to the actions of 5-HT, the probable relaxing transmitter.

     

Respiratory exchange of Mytilus californianus

Summary The oxygen uptake of Mytilus californianus between 12 and 24° C. varies greatly with different samples, from approximately 0.3 to 2.0 cc. per 10 gms. of wet weight.. Similar values are found for the carbon dioxide production. Respiratory quotients as high as 2.2 were found; they are explained by the buffering action of the shell. Toxic mussels do not differ from normal ones in their gas metabolism.Aided by Grants from the James Moffitt Fund.     

Composition and ultrastructure of the byssus of Mytilus edulis

Three regions of the byssus of the marine mussel Mytilus edulis L. are distinct in structural organization at the macroscopic and microscopic level and in amino acid composition. The threads that emanate from the stem at the base of the foot are divided into two regions. The proximal, elastic region has a crimped, densely staining cortex enclosing an interior matrix of spiral fibers, and its amino acid composition reflects protein heterogeneity. The more distal, rigid region has a straight, tubular cortex surrounding an inner matrix of linearly arranged bundles of fibrils and has a composition approximating pure collagen. The plaque, or disc-shaped portion, which mediates attachment to various substrates, is distinguished by a surface matrix of collagen-like fibers similar to those of the thread region and anchored on an inner spongy matrix. Compositional evidence exists for a collagenous component, a catechol-rich protein, and at least one other accessory protein in the plaque.     

The digestive system of Mytilus californianus Conrad

Conclusions No evidence of a definitely pathological nature was found during the course of these studies, although certain findings indicate that there are factors which may be considered as characteristic of mussels, and other similar bivalves, which are highly toxic. The quantities of poison present in the digestive diverticula of a single mussel were so small that its demonstration by histo-chemical methods was impossible. The observations have shown, also, that the mussels are not affected physiologically by the ingestion of this poisonous substance which is so highly toxic to warm blooded animals. However, these findings are taken from observations which have been made in a restricted area, and may be considerably in error if applied to another. Therefore, further studies must be carried on elsewhere and the results compared with these before conclusions may be drawn with any degree of certainty.A direct relationship between the quantity and nature of the stored food material in the digestive diverticula and the seasonal variations in the plankton has been definitely established.Aided by Grants from the James Moffitt Fund.     

The physiological ecology of Mytilus californianus Conrad

Summary The rates of oxygen consumption, filtration and ammonia excretion by Mytilus californianus have been related to body size and to ration. The rate of oxygen consumption (VO₂) by individuals while immersed, measured on the shore, resembled rates recorded for mussels starved in the laboratory. VO₂ by M. californianus was relatively independent of change in temperature, with a Q ₁₀ (13–22° C) of 1.20. In contrast, the frequency of heart beat was more completely temperature dependent [Q ₁₀ (13–22° C)=2.10]. Filtration rate showed intermediate dependence on temperature change [Q ₁₀ (13–22° C)=1.49] up to 22° C, but declined at 26° C. Both VO₂ and filtration rate declined during starvation. The utilisation efficiency for oxygen was low (approx. 4%) between 13 and 22° C, but increased to 10% at 26° C. Three components of the routine rate of oxygen consumption are recognised and estimated; viz. a basal rate (0.136 ml O₂ h^-1 for a mussel of 1 g dry flesh weight), a physiological cost of feeding (which represented about 6% of the calories in the ingested ration), and a mechanical cost of feeding which was three times higher than the physiological cost. The ratio oxygen consumed to ammonia-nitrogen excreted was low, and it declined during starvation. These data are compared with previously published measurements on Mytilus edulis, and the two species of mussel are shown to be similar in some of their physiological characteristics, though possibly differing in their capacities to compensate for change in temperature. For M. californianus, the scope for growth was highest at 17–22° C and declined at 26° C; it is suggested that exposure to temperatures in excess of 22° C, as for example during low tides in the summer, might result in a cumulative stress on these populations of mussels by imposing a metabolic deficit which must be recovered at each subsequent high tide. The high mechanical cost of feeding imposes a more general constraint on the scope for activity of the species.     

Algal-induced spawning in the marine mussel Mytilus californianus 4

The characteristics of algal-induced spawning of the marine mussel Mytilus californianus were studied. Exposure of mature individuals to culture suspensions of the unicellular alga Pseudoisochrysis paradoxa elicited copious and synchronous release of gametes. Alkaline conditions were necessary to make the animals responsive to the spawning stimulus provided by the algae. The filtered, cell-free fraction of the algal suspensions also stimulated spawning, suggesting that an active principle is secreted into the culture media by the algae.

A key requirement for the development of an efficient and economically viable molluscan mariculture program is the capability to induce copious, synchronous spawning of gravid individuals. This procedure should be easily managed and have no deleterious effects on the gametes, fertilization, or the ontological development of the animals. Miyazaki [1] induced spawning in male oysters by exposing them to an extract of green algae. Recently it was reported that the marine mussels, M. californianus, could be induced to spawn by exposing them to cultures of the marine algae, P. paradoxa [2], This paper summarizes our efforts to characterize this spawning response of mussels to high concentrations of cultured marine algae.     

Screening of ESTs from Mytilus for the detection of SSR markers in Mytilus californianus

A set of expressed sequence tag‐simple sequence repeat (EST‐SSR) markers for the genus Mytilus was developed through bioinformatic mining of the GenBank public database. A total of 33 782 EST sequences from GenBank were downloaded and screened for di‐, tri‐ and tetranucleotide, with 1274 EST containing SSR markers. Nine microsatellite markers were characterized in Mytilus californianus with a number of alleles per locus ranging from two to six, and total observed and expected heterozygosities ranging from 0.490 to 0.730 and from 0.510 to 0.860 respectively. Cross‐species amplification was achieved in several other species, confirming the usefulness of these markers in Mytilus genetics.     

The structure and formation of the byssus attachment plaque in Mytilus

The byssus attachment plaque and the tissues responsible for its formation were studied in M. califomianus by light microscopy and by transmission and scanning electron microscopy. It was shown that the plaque consists of at least three phases which ultrastructurally resemble three secretions considered to be collagen, mucoid material and polyphenol. The mucoid and polyphenol appear to mix as a colloidal suspension in which the latter is the continuous phase and forms the definitive bonding surface. Plaque collagen represents an extension of thread material into the cementing substance. Stimulated secretion within the ducts and distal depression of the mussel's foot shows a continuum of increasing heterogeneity from the inner toward the outer regions. This reflects the distribution of exocrine cell apices wherein exocytosis of polyphenol granules predominate deeply, mucous granules superficially and collagen granules in between. It is proposed that the morphology of the plaque conforms to theoretical physical-chemical requirements for adhesion under water.     

High-resolution analysis of metabolic cycles in the intertidal mussel Mytilus californianus

Inhabitants of the marine rocky intertidal live in an environment that alternates between aquatic and terrestrial due to the rise and fall of the tide. The tide creates a cyclical availability of oxygen with animals having access to oxygenated water during episodes of submergence, while access to oxygen is restricted during aerial emergence. Here we performed liquid chromatography and gas chromatography-mass spectrometry enabled metabolomic profiling of gill samples isolated from the California ribbed mussel, Mytilus californianus, to investigate how metabolism is orchestrated in this variable environment. We created a simulated intertidal environment in which mussels were acclimated to alternating high and low tides of 6 h duration, and samples were taken every 2 h for 72 h to capture reproducible changes in metabolite levels over six high and six low tides. We quantified 169 named metabolites of which 24 metabolites cycled significantly with a 12-h period that was linked to the tidal cycle. These data confirmed the presence of alternating phases of fermentation and aerobic metabolism and highlight a role for carnitine-conjugated metabolites during the anaerobic phase of this cycle. Mussels at low tide accumulated eight carnitine-conjugated metabolites, arising from the degradation of fatty acids, branched-chain amino acids, and mitochondrial β-oxidation end products. The data also implicate sphingosine as a potential signaling molecule during aerial emergence. These findings identify new levels of metabolic control whose role in intertidal adaptation remains to be elucidated.     

Potassium contracture of anterior byssus retractor muscle of Mytilus edulis

• 1.1. The potassium contracture in the anterior byssus retractor muscle of Mytilus edulis relaxed spontaneously, and the relaxation was accelerated by 5-HT (10⁻⁶ M), but the contractile activation and inactivation was not affected significantly.
• 2.2. By reducing [Ca]₀, the “activation curve” was shifted downward at higher [K]₀, and the “inactivation curve” was shifted toward lower [K]₀ and the rate of inactivation was increased.
• 3.3. The steady-state inactivation was maintained for at least 2 hr without complete inactivation.
• 4.4. The half-inactivation time was dependent on [K]₀, while the half-relaxation time in the contracture induced by conditioning K solutions was not.

     

Environmental post-processing increases the adhesion strength of mussel byssus adhesive

Marine mussels (Mytilus trossulus) attach to a wide variety of surfaces underwater using a protein adhesive that is cured by the surrounding seawater environment. In this study, the influence of environmental post-processing on adhesion strength was investigated by aging adhesive plaques in a range of seawater pH conditions. Plaques took 8–12 days to achieve full strength at pH 8, nearly doubling in adhesion strength (+94%) and increasing the work required to dislodge (+59%). Holding plaques in low pH conditions prevented strengthening, causing the material to tear more frequently under tension. The timescale of strengthening is consistent with the conversion of DOPA to DOPA-quinone, a pH dependent process that promotes cross-linking between adhesive proteins. The precise arrangement of DOPA containing proteins away from the adhesive-substratum interface emphasizes the role that structural organization can have on function, an insight that could lead to the design of better synthetic adhesives and metal-coordinating hydrogels.     

Sequence and characterization of the sperm-specific protein phi 3 from Mytilus californianus

We have shown that the sperm-specific protein phi 3 from Mytilus californianus (Conrad) exhibits compositional microheterogeneity. For the first time, we have isolated and characterized the three major components of this protein. These fractions display different electrophoretic mobilities on Triton/urea/acetic acid polycrylamide gels. However, they have a very similar molecular mass of 5 +/- 0.1 kDa as measured by sedimentation equilibrium in the analytical ultracentrifuge. All of them show a marked trend toward aggregation. We have also established the sequence for each of these three fractions. The sequencing data suggest an even greater extent of microheterogeneity for this protein. The predicted secondary structure from the sequences, as well as infrared analyzes carried out on the native protein, suggest a structure organization into an alpha helix.     

Neurotransmitters and neuromodulators controlling the anterior byssus retractor muscle of Mytilus edulis.

1. The anterior byssus retractor muscle (ABRM) of Mytilus edulis is innervated by at least two kinds of nerves, excitatory and relaxing nerves. The principal neurotransmitters released from these nerves have been shown to be acetylcholine and serotonin, respectively. 2. Some other monoamines, such as dopamine and octopamine, and various peptides, such as FMRFamide-related peptides, Mytilus inhibitory peptides, SCP-related peptides and a catch-relaxing peptide, may also be involved in the regulation of the muscle as neurotransmitters or neuromodulators. 3. The ABRM seems to be typical of invertebrate muscles controlled by multiple neurotransmitters and neuromodulators.     

Histochemical and ultrastructural study on the innervation of the byssus glands of Mytilus galloprovincialis

The aluminium-formaldehyde (ALFA) histofluorescence method reveals an extensive plexus of brilliant greenish monoaminergic elements in the glandular zones of the Mytilus foot, while only scanty nerve fibres are acetylcholinesterase-positive. By electron microscopy, bundles of nerve fibres can be seen i) in close connection with the intrinsic musculature located in the connective septa among the glands, and ii) near the cell bodies and necks of all the byssus glands. The nerve fibres show varicosities containing three types of vesicles: small clear (50-60 nm), small granular (80-90 nm), and large granular (160-200 nm). The regions of close apposition between nerve terminals and muscle or gland cells generally do not show typical pre- or postsynaptic specializations. Along the pedal groove, mainly in the proximal two thirds of the foot, peripheral bipolar neurons can be detected, both by fluorescence and electron microscopy.     

The role of disturbance in the evolution of life history strategies in the intertidal mussels Mytilus edulis and Mytilus californianus

Summary The intertidal mussels Mytilus edulis and M. californianus compete for space on the west coast of North America. An analysis of differences in size, growth rate, age at first reproduction, life span, mode of reproduction, niche and habitats between these two species demonstrates that their life history strategies are different in several important ways. M. californianus is a larger, sturdier, slower growing mussel which has effective predator-deterring mechanisms and is an overall superior competitor for space in the intertidal. Its reproductive strategy is based on continually spawning at a very low level throughout a yearly cycle. M. edulis is a classic fugitive species which rarely attains a large size but matures early and is characterized by a single massive reproductive output each year. The differences noted in their life history strategies are very likely the result of either a) a pre-adaptation for coexistence, or b) a competitively-induced regional evolutionary divergence.In Washington a band of small M. edulis (removed from most predation and competition) exists in a high intertidal spatial refuge, and contributes relatively little to the gene pool. Larger individuals exist in disturbed areas lower in the intertidal zone. These lower zones represent a relatively high risk, and usually only temporary, habitat for M. edulis because of mortality due to competition and predation. Individuals, however, colonizing these two intertidal patches contribute 43 times as much as their high intertidal counterparts in terms of reproductive fitness (calculated on a populationwide basis).