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.     

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.     

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.     

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.     

Isolation of collagen granules from the foot of the sea mussel,Mytilus californianus

• 1.1. Membrane bound granules were isolated from homogenates of the foot of Mytilus californianus and shown to correspond to the ellipsoid collagen granules described by Tamarin & Keller (1972).
• 2.2. Protein was extracted from the purified granules by standard procedures for preparation of collagen and its amino acid composition was compared with that of vertebrate collagen and different parts of the byssus secreted by glands in the mussel's foot.
• 3.3. Hydrolysates of collagen extracted from granules contained approximately 8 mole % hydroxyproline and 33 mole % glycine and were similar in composition to byssus threads.
• 4.4. Analyses of other parts of the byssus indicated that they contained collagen in addition to other protein components.

     

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.     

A single heat-stress bout induces rapid and prolonged heat acclimation in the California mussel,Mytilus californianus

Climate change is not only causing steady increases in average global temperatures but also increasing the frequency with which extreme heating events occur. These extreme events may be pivotal in determining the ability of organisms to persist in their current habitats. Thus, it is important to understand how quickly an organism''s heat tolerance can be gained and lost relative to the frequency with which extreme heating events occur in the field. We show that the California mussel, Mytilus californianus—a sessile intertidal species that experiences extreme temperature fluctuations and cannot behaviourally thermoregulate—can quickly (in 24–48 h) acquire improved heat tolerance after exposure to a single sublethal heat-stress bout (2 h at 30 or 35°C) and then maintain this improved tolerance for up to three weeks without further exposure to elevated temperatures. This adaptive response improved survival rates by approximately 75% under extreme heat-stress bouts (2 h at 40°C). To interpret these laboratory findings in an ecological context, we evaluated 4 years of mussel body temperatures recorded in the field. The majority (approx. 64%) of consecutive heat-stress bouts were separated by 24–48 h, but several consecutive heat bouts were separated by as much as 22 days. Thus, the ability of M. californianus to maintain improved heat tolerance for up to three weeks after a single sublethal heat-stress bout significantly improves their probability of survival, as approximately 33% of consecutive heat events are separated by 3–22 days. As a sessile animal, mussels likely evolved the capability to rapidly gain and slowly lose heat tolerance to survive the intermittent, and often unpredictable, heat events in the intertidal zone. This adaptive strategy will likely prove beneficial under the extreme heat events predicted with climate change.     

Molecular population genetics of the male and female mitochondrial DNA molecules of the California sea mussel, Mytilus californianus

The presence of two gender-associated mitochondrial genomes in marine mussels provides a unique opportunity to investigate the dynamics of mtDNA evolution without complications inherent in interspecific comparisons. Here, we assess the relative importance of selection, mutation, and differential constraint in shaping the patterns of polymorphism within and divergence between the male (M) and female (F) mitochondrial genomes of the California sea mussel, Mytilus californianus. Partial sequences were obtained from homologous regions of four genes (nad2, cox1, atp6, and nad5) totaling 2307 bp in length. The M and F mtDNA molecules of M. californianus exhibited extensive levels of nucleotide polymorphism and were more highly diverged than observed in other mytilids (overall Tamura-Nei distances >40%). Consistent with previous studies, the M molecule had significantly higher levels of silent and replacement polymorphism relative to F. Both genomes possessed large numbers of singleton and low-frequency mutations that gave rise to significantly negative Tajima's D values. Mutation-rate scalars estimated for silent and replacement mutations were elevated in the M genome but were not sufficient to account for its higher level of polymorphism. McDonald-Kreitman tests were highly significant at all loci due to excess numbers of fixed replacement mutations between molecules. Strong purifying selection was evident in both genomes in keeping the majority of replacement mutations at low population frequencies but appeared to be slightly relaxed in M. Our results suggest that a reduction in selective constraint acting on the M genome remains the best explanation for its greater levels of polymorphism and faster rate of evolution.     

Nitric oxide generation by hemocytes of the mussel Mytilus galloprovincialis.

The phagocytic activity of Mytilus galloprovincialis hemocytes is thought to be associated with NADPH-oxidase activity of the plasma membrane, thus producing superoxide anions. Few studies, however, have been devoted to nitric oxide release by these haemocytes. We investigated NO generation in M. galloprovincialis in order to understand its role in the defensive mechanisms of these organisms. The presence of NO-synthase-like enzymatic activity in protein homogenates from M. galloprovincialis hemocytes was revealed by the conversion of radiolabelled L-arginine to L-citrulline. We observed partial inhibition of the luminol-dependent chemiluminescence of stimulated M. galloprovincialis hemocytes by both NO-synthase inhibitors and superoxide dismutase, indicating that peroxynitrite (which results from the reaction between nitric oxide and superoxide anions) partially mediated this chemiluminescence. Furthermore, we confirmed the production of nitric oxide by M. galloprovincialis by highlighting the nitric oxide-synthase-dependence of the nitrate and nitrite production of stimulated hemocytes.     

Transport of the aromatic amino acids into isolated rat liver cells. Properties of uptake by two distinct systems.

The transport of the aromatic amino acids into isolated rat liver cells was studied. There was a rapid and substantial binding of the aromatic amino acids, L-alanine and L-leucine to the plasma membrane. This has important consequences for the determination of rates of transport and intracellular concentrations of the amino acids. Inhibition studies with a variety of substrates of various transport systems gave results consistent with aromatic amino acid transport being catalysed by two systems: a 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH)-insensitive aromatic D- and L-amino acid-specific system, and the L-type system (BCH-sensitive). The BCH-insensitive component of transport was Na+-independent and facilitated non-concentrative transport of the aromatic amino acids; it was unaffected by culture of liver cells for 24 h, by 48 h starvation, dexamethasone phosphate or glucagon. Kinetic properties of the BCH-inhibitable component were similar to those previously reported for the L2-system in liver cells. The BCH-insensitive component was a comparatively low-Km low-Vmax. transport system that we suggest is similar to the T-transport system previously seen only in human red blood cells. The results are discussed with reference to the importance of the T- and L-systems in the control of aromatic L-amino acid degradation in the liver.     

Probing the adhesive footprints of Mytilus californianus byssus

California mussels Mytilus californianus owe their tenacity to a holdfast known as the byssus, a fibrous extracellular structure that ends distally in flattened adhesive plaques. The "footprints" of freshly secreted plaques deposited onto glass coverslips were shown by matrix-assisted laser desorption ionization time of flight mass spectrometry to consist chiefly of proteins ranging in mass from 5200 to 6700 Da. These proteins, variants of a family known as mcfp3 (M. californianus foot protein 3), were purified from acetic acid/urea extracts of plaques and foot tissue. Mcfp3 appears to sort into fast and slow electrophoretic variants. Both are rich in Gly and Asn and exhibit post-translational hydroxylation of Tyr and Arg to Dopa and 4-hydroxyarginine, respectively, with the fast variant containing more than twice as much Lys + Arg. Both the slow and fast variants were partially sequenced from the N terminus, and the complete sequences of 12 variants were deduced from cDNA using degenerate oligonucleotides, PCR, and rapid amplification of cDNA ends. Mcfp3s are highly polar molecules and contain up to 28 mol % Dopa, which remains intact and may be crucial for adhesion to metal and mineral surfaces.     

Uptake of arsenic-betaines by the mussel Mytilus edulis

Mussels (Mytilus edulis) were exposed to trimethyl(carboxymethyl)arsonium bromide (arsenobetaine, C-1 betaine), trimethyl(2-carboxyethyl)arsonium bromide (C-2 betaine), or trimethyl(3-carboxypropyl)arsonium bromide (C-3 betaine). Arsenic was accumulated by the mussels in all cases but the efficiency of uptake decreased with the number of methylene units in the carboxyalkyl group. Arsenobetaine (C-1 betaine) was the most readily accumulated, followed by the C-2 betaine (70% as efficient as arsenobetaine) and the C-3 betaine (∼7%). Chromatographic analysis (HPLC-ICPMS) of extracts of the mussels demonstrated that the arsenic compounds were accumulated uncahanged. A 46-day depuration period which followed exposure did not significantly reduce the arsenic concentration in any of the three groups. Comparison with previous data on accumulation of arsenic compounds by M. edulis indicates that uptake may be influenced by the presence of a quaternary arsonium group and the zwitterionic nature of the arsenic-betaines.     

Ultrastructure and cytochemistry of the integument of Modiolicola gracilis,parasitic copepod in mussel gills (Mytilus galloprovincialis and Mytilus edulis)

Of mussels taken from the Ebro Delta River (E. Spain), 3% have a nonmodified copepod, Modiolicola gracilis, in the gill tissues. The cuticle of different segments of the body has an epicuticle with two layers, which show external microvilli-like projections. Weakly positive reactivity to the PTA technique has been detected in the external region. The procuticle has the helicoidal architecture of the chitinous tegument in arthropods, whereas the cuticle shows discontinuities in the regions of ducts in tegumental glands. The integument is comprised of three types of cells. Epidermal cells are flat with numerous mitochondria. Muscle cells show well-developed mitochondria with several longitudinally distributed cristae. A third and secretory cell shows a well-developed rough endoplasmic reticulum and Golgi complex in the basal zone. Its apical portion is full of secretory granules. Through the cuticle, these integumental glands open directly to the cuticular surface via a short duct coated by epicuticle. The composition and specializations of this complex cuticular architecture differ markedly from those shown by an endoparasitic copepod detected in the digestive gland of the mussel. It does not appear that the specializations detected in the cuticle of M. gracilis lead to any histopathological alteration in host tissues. © 1994 Wiley-Liss, Inc.     

3H]spiroperidol binding in crude membrane suspensions of gill tissue from the marine mussel, Mytilus californianus

Saturable, high-affinity binding sites for [3H]spiroperidol can be demonstrated in crude suspensions of mussel gill tissue. This binding shows stereospecificity toward the d- and l-isomers of butaclamol. Competition studies show that both dopamine and serotonin can displace [3H]spiroperidol from binding sites at nanomolar concentrations. Evidence is presented that suggests that the [3H]spiroperidol-binding sites can be divided into two distinct groups: those with high affinity for dopamine and those with high affinity for serotonin.     

Transport of neutral amino acids by human erythrocytes

The transport of several neutral amino acids by human erythrocytes in vitro was studied. The measurements made included steady-state distributions, kinetics of initial rates of uptake, effects of monovalent cations and anions, general mutual inhibitory interactions, kinetics of inhibitions, effluxes, ability to produce accelerative exchange diffusion, and the inhibitory action of the thiol reagent N-ethylmaleimide. The results are interpreted as showing that the human erythrocyte membrane possesses several distinct transport systems for these amino acids, including one Na⁺-dependent system and one dependent on both Na⁺ and a suitable anion, that are qualitatively similar to those systems previously described in pigeon erythrocytes and mammalian reticulocytes. Quantitatively, however, the systems differ among the different kinds of red cell and a major difference lies in their abilities to produce accelerative exchange diffusion.     

Transport of sugars or amino acids increases potassium efflux from isolated enterocytes

A technique to isolate epithelial cells from rabbit jejunum using hyaluronidase is described. The cells obtained retained their abilities to accumulate sugars and potassium (86Rb) against concentration gradients. Potassium efflux was monitored using cells preloaded with 86Rb and the rate constant of efflux was seen to increase when actively transported sugars or amino acids are added to the bathing medium. The increase is related to the transport of the non-electrolyte, but not to volume regulatory events.     

Interactions of organic pollutants with gills of the bivalve molluscs Anodonta californiensis and Mytilus californianus: uptake and effect on membrane fluxes. II

1. The uptakes of 2,4,5-T, glyphosate, parathion, paranitrophenol, naphthalene, glycine, and inulin by gills of the bivalve molluscs Anodonta californiensis (freshwater) and Mytilus californianus (marine) show non-polar compounds are taken up to a greater extent than polar compounds except where active transport occurs. 2. The uptake of glycine by M. californianus is reduced by pollutants containing complexing functional groups but not by non-polar compounds. 3. The uptake of parathion alters the polyphosphate-inorganic phosphate balance in M. californianus. 4. The uptakes of pollutants parallel their toxicities toward rats.