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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

The role of the nervous system in algae-induced gamete release by Mytilus californianus

Algae-induced spawning in M. californianus was associated with an increase in dopamine in the visceral ganglia and a decrease in serotonin in the pedal ganglia. Lesioning studies indicate that nervous communication between the pedal and visceral ganglia plays a major role in gamete release.     

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).     

Transport of amino acids by isolated gills of the mussel Mytilus californianus Conrad.

The unidirectional influx of cycloleucine into in vitro preparations of gill tissue of the mussel, Mytilus californianus, was determined. Influx was found to be linear for at least an hour, and the kinetics of cycloleucine influx conformed to Michaelis-Menten type kinetics. The transport mechanism(s) for cycloleucine is relatively specific for the L-enantiomorph of neutral amino acids, and is capable of accumulating cycloleucine to intracellular concentrations much higher than those of the surrounding medium. Evedence is presented that the transport of amino acids by gill tissue plays a significant role in whole animal nutrition.     

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.

     

Heterologous expression of Mytilus californianus foot protein three (Mcfp-3) in Kluyveromyces lactis

Mytilus californianus foot protein three (Mcfp-3) was successfully expressed in the yeast, Kluyveromyces lactis. The first nine amino acids (YPYDVPDYA) from the human-influenza-virus hemagglutinin (HA) protein were fused to the amino terminus of Mcfp-3 (HA-Mcfp-3) to facilitate identification and purification. HA-Mcfp-3 was purified to a concentration of 1mg/L using HA affinity chromatography. The recovered polypeptide was resolved by SDS-PAGE and migrated primarily at 36 kDa, an increase of approximately 29 kDa over the calculated molecular weight of a HA-Mcfp-3 monomer. Significantly, release of Mcfp-3 by enterokinase treatment coincided with the formation of high molecular weight complexes. It is noteworthy that the complexes mimicked the previously reported insolubility of Mcfps found in vivo to denaturing and reducing conditions. These data demonstrate the successful expression of Mcfp-3 in K. lactis and show an intrinsic ability of Mcfp-3 to self-assemble into stable, higher molecular weight forms.     

Evaluation of methods for improved detection of Cryptosporidium spp. in mussels (Mytilus californianus)

Bivalve molluscs concentrate Cryptosporidium oocysts from fecal-contaminated aquatic environments and are therefore useful in monitoring water quality. A real-time TaqMan polymerase chain reaction (PCR) system was developed to allow for large scale quantitative detection of Cryptosporidium spp. in mussels (Mytilus californianus). The TaqMan sensitivity and specificity were compared to conventional PCR and direct immunofluorescent antibody (DFA) assays, with and without immunomagnetic separation (IMS), to identify the best method for parasite detection in mussel hemolymph, gill washings and digestive glands. TaqMan PCR and two conventional PCR systems all detected 1 or more oocysts spiked into 1 ml hemolymph samples. The minimum oocyst detection limit in spiked 5 ml gill wash and 1 g digestive gland samples tested by TaqMan PCR and DFA was 100 oocysts, with a 1 log(10) improvement when samples were first processed by IMS. For tank exposed mussels, TaqMan and conventional PCR methods detected C. parvum in <5% of hemolymph samples. No gill washings from these same mussels tested positive by TaqMan PCR or DFA analysis even with IMS concentration. All methods detected the highest prevalence of C. parvum-positive samples in digestive gland tissues of exposed mussels. In conclusion, the most sensitive method for the detection of C. parvum in oocyst-exposed mussels was IMS concentration with DFA detection: 80% of individual and 100% of pooled digestive gland samples tested positive. TaqMan PCR was comparable to conventional PCR for detection of C. parvum oocysts in mussels and additionally allowed for automated testing, high throughput, and semi-quantitative results.     

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

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

Molecular Epidemiology of Cryptosporidium spp. and Giardia spp. in Mussels (Mytilus californianus) and California Sea Lions (Zalophus californianus) from Central California

Cryptosporidium and Giardia are of public health importance, with recognized transmission through recreational waters. Therefore, both can contaminate marine waters and shellfish, with potential to infect marine mammals in nearshore ecosystems. A 2-year study was conducted to evaluate the presence of Cryptosporidium and Giardia in mussels located at two distinct coastal areas in California, namely, (i) land runoff plume sites and (ii) locations near sea lion haul-out sites, as well as in feces of California sea lions (CSL) (Zalophus californianus) by the use of direct fluorescent antibody (DFA) detection methods and PCR with sequence analysis. In this study, 961 individual mussel hemolymph samples, 54 aliquots of pooled mussel tissue, and 303 CSL fecal samples were screened. Giardia duodenalis assemblages B and D were detected in hemolymph from mussels collected near two land runoff plume sites (Santa Rosa Creek and Carmel River), and assemblages C and D were detected in hemolymph from mussels collected near a sea lion haul-out site (White Rock). These results suggest that mussels are being contaminated by protozoa carried in terrestrial runoff and/or shed in the feces of CSL. Furthermore, low numbers of oocysts and cysts morphologically similar to Cryptosporidium and Giardia, respectively, were detected in CSL fecal samples, suggesting that CSL could be a source and a host of protozoan parasites in coastal environments. The results of this study showed that Cryptosporidium and Giardia spp. from the feces of terrestrial animals and CSL can contaminate mussels and coastal environments.     

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.