Preparation and properties of mitochondria from tissues of the sea mussel Mytilus edulis L

• 1.1. A method is described for the preparation of coupled mitochondria from hepatopancreas, mantle and adductor muscle of the sea mussle, Mytilus edulis.
• 2.2. NADH is a non-penetrant, whereas succinate, glutamate and malate plus pyruvate have a clear stimulatory effect on respiration. Proline does not stimulate respiration of sea mussel mitochondria.
• 3.3. No P/0 ratios could be calculated as after the addition of ADP the mitochondria remain in the active state (state III), even after enough oxygen is consumed for complete phosphorylation. The reason for this observation is discussed.
• 4.4. Mitochondrial densities based on cytochrome b levels of two tissues of the mussel are compared to rat liver. Within the mussel the mantle contains twice the amount of cytochrome of adductor muscle.

     

Prolyl 4-hydroxylase in the foot of the marine mussel Mytilus edulis L.: purification and characterization

The mussel foot secretes a variety of unusual hydroxyproline-containing collagenous and noncollagenous proteins. Prolyl 4-hydroxylase acting on one or more of the secreted proteins was isolated from the foot by using conventional gel filtration and ion exchange chromatography. Mr of the intact enzyme was 230,000 (alpha 2 beta 2) composed of two subunits with Mr of 60,000 (alpha) and 57,000 (beta) as estimated by HPLC gel filtration and SDS-PAGE. The enzyme utilized (Pro-Pro-Gly)10 as a substrate with an apparent Km value of 0.17 mM. Cofactors and inhibitors were very similar to animal, plant, and microbial prolyl hydroxylases previously described. The enzyme had a relatively sharp pH optimum in the range of 7.8-8.3 and the hydroxyproline formed increased in proportion to the rise in the temperature between 5 and 20 degrees C. No detectable hydroxylation occurred with poly-L-proline or the unhydroxylated decapeptide analog (Ala-Lys-Pro-Ser-Tyr-Pro-Pro-Thr-Tyr-Lys) of the polyphenolic protein. Kinetic studies, however, revealed that the mussel prolyl 4-hydroxylase was competitively inhibited by poly-L-proline and uncompetitively inhibited by the decapeptide. These results suggest that the decapeptide binds the enzyme-substrate i.e. (Pro-Pro-Gly)10 complex. It is not yet clear whether this enzyme acts exclusively on collagenous substrates or whether its catalytic purview extends as well to the polyphenolic protein.     

Seasonal variations in the anaerobic metabolism of the mussel Mytilus edulis (L.)

• 1.1. The incorporation of atmospheric ¹⁴CO₂ into the ethanol soluble metabolites of the tissues of the mussel, Mytilus edulis, exposed to the atmosphere, was measured monthly between July 1976 and June 1977.
• 2.2. The total incorporation of ¹⁴CO₂ was found to be greater in the summer than in the winter and these seasonal effects were not temperature-dependent.
• 3.3. The incorporation of ¹⁴CO₂ into the individual metabolites is discussed in relation to possible seasonal variations in the operation of the anaerobic succinate pathway and seasonal variations of tissue glycogen concentrations.

     

Nuclear and cytosolic distribution of metallothionein in the blue mussel Mytilus edulis L

Four tissues from the blue mussel, Mytilus edulis L., were examined for the presence of nuclear metallothionein (MT), and the nuclear:cytosolic (N:C) MT ratios and nuclear MT:DNA ratios investigated. Gill, digestive gland, gonad and posterior adductor muscle tissues were dissected, homogenized and subjected to differential centrifugation in order to isolate the nuclear and cytosolic fractions, which were then analyzed for MT and DNA. MT was present in all samples of the nuclear fractions from all four tissues. The nuclear MT concentration was either lower or the same as the cytosolic MT concentration from the same tissue. The mean N:C MT ratio of the digestive gland was significantly lower than that of the gill. The mean nuclear MT:DNA ratio of the digestive gland was significantly higher than that of the gill and posterior adductor muscle. In addition to being the first report of nuclear MT in bivalves, we showed that N:C MT ratios and nuclear MT:DNA ratios differ among tissues of the same organism. This raises important questions concerning the regulation of nuclear MT concentrations and the role of nuclear MT in metal regulation and DNA protection.     

Multiple invertebrate lysozymes in blue mussel (Mytilus edulis)

Initial analyses of lysozyme activities in individual blue mussels Mytilus edulis indicated variations in features of activity from the crystalline style to the remaining body parts (the soft body). Two separate larger scale lysozyme isolations were performed employing extracts from 1000 styles and 50 soft bodies, respectively. The soft body origin contained one, or one major, lysozyme that was purified to homogeneity. This 13 kDa protein, designated bm-lysozyme, was sequence-analysed and found to represent the product of a recently published invertebrate-type lysozyme gene from M. edulis. Three additional lysozymes were isolated from the style extract and one of them was fully purified. All four lysozymes showed different profiles of enzymatic features such as responses to pH, ionic strengths and divalent cations. From the results and the profound differences demonstrated we believe that the observed multiple forms of lysozyme activities in blue mussel reflect multiple genes instead of individual lysozyme variants and that the lysozymes serve different functions in the blue mussel.     

Biodeposit production by the mussel Mytilus edulis L. on rocky shores

Seasonal changes in the amount of biodeposit (faeces and pseudofaeces) produced by the mussel Mytilus edulis L., which is one of the representative suspension-feeders in the rocky intertidal and shallow subtidal regions of Mutsu Bay, were studied in the laboratory. The effects of water temperature, light, food concentration, flow rate, body size, age, and spawning on biodeposit production were investigated. More biodeposit was produced in summer than in other seasons. Throughout the year, the amount of biodeposit was positively correlated with body size. Relatively more biodeposit was produced by smaller than by larger individuals. A M. edulis population living in one square meter was estimated to produce 9.20 kg of faeces and 2.71 kg of pseudofaeces per year (dry wt). More biodeposit was produced at water temperatures of 17.6–20.2° C than at 4.5–7.6° C and 25.2–26.0° C. The optimum temperature for biodeposit production was found to be ≈ 20.0 °C. When kept in the dark, M. edulis produced more biodeposit than in the light. When food concentration is increased, more psuedofaeces are produced; the amount of faeces, however, remains constant. With increasing flow rate, the amount o f biodeposit per h increased but the biodeposition rate decreased. Larger amounts of faeces and smaller amounts of pseudofaeces were produced by younger mussels than by older ones of a similar size. Spawning also affected biodeposit production.     

Pharmacological induction of larval settlement and metamorphosis in the blue mussel Mytilus edulis L

The blue mussel Mytilus edulis L. is an important aquaculture and fouling species in northern seas. Although the general role of chemical cues for settlement of larvae of the blue mussel has been proposed, few studies have focused on induction of settlement and metamorphosis by pharmacological agents. In this study, the induction of larval settlement of the blue mussel by pharmacological compounds was investigated through a series of laboratory experiments with an aim of identifying artificial cues for laboratory bioassay systems in fouling and antifouling research. Gamma-aminobutiric acid (GABA), dihydroxyphenyl L-alanine (DOPA), isobutyl methylxanthine (IBMX) and acetylcholine chloride (ACH) at 10(-7)-10(-2) M as well as KCl at 10-40 mM K+ in excess of the level in normal seawater were tested for their inductive effect on larval settlement. In filtered seawater (FSW) < 9% of the larvae settled after 48 h. Elevated K+ and GABA levels had no effect on larval settlement and metamorphosis. DOPA at 10(-5) M and IBMX at 10(-6)-10(-4) M induced 41-83% larval settlement and ACH at 10(-7)-10(-5) M induced < 40% larval settlement. While the highest settlement rates were observed after 48 h exposure to the chemical, most of the larvae settled within 24 h. Compounds at concentrations of 10(-3)-10(-2) M were either toxic to larvae or retarded the growth of the post-larvae shell. Juveniles resulting from induction by lower concentrations of chemicals had a very high survival rate, completed metamorphosis and grew as well as the juveniles that metamorphosed spontaneously. IBMX at 10(-6)-10(-4) M and L-DOPA at 10(-5) M are effective agents for induction of settlement and metamorphosis for future studies using juvenile M. edulis.     

Short-term impact of blue mussel dredging (Mytilus edulis L.) on a benthic community

The short-term effect of mussel dredging in a brackish Danish sound was studied. A commercial dredging track was identified and an analysis of the species composition inside the track and at an adjacent control area showed that dredging changed the community structure by reducing the density of polychaetes. In order to investigate the extent and the duration of the dredging impact experimental dredging was conducted. The experimental dredging removed 50% of the mussels in two dredged areas. Immediately after dredging, a significantly lower number of species was measured inside the mussel beds in dredged areas compared to control and boundary areas. This effect lasted for at least 40 days. The analysis of the species composition showed that the dredged area had a significantly lower density, particularly of polychaetes compared to the boundary area. An increased number of species was recorded outside the mussel beds just after dredging, but this effect lasted for less than 7 days. After dredging, brown shrimps, C. crangon invaded the dredged areas. This species is an important predator of smaller invertebrates, and it is suspected that it was feeding on small vulnerable polychaetes exposed at the sediment surface after dredging. The dredging process was observed to form 2–5-cm deep furrows in the seabed, but the sediment texture and the organic content of the sediment was not affected. The biomass accumulation of individual blue mussels was significantly lower in the dredged area compared to the boundary area. This indicates that the disturbance of the mussel bed structure reduced growth and that the lowering of intraspecific food competition caused by a reduced density of mussels did not increase the accumulation of biomass in the mussels which remained in the dredged area.     

Effects of ageing on nuclear DNA integrity and metabolism in mussel cells (Mytilus edulis L.)

As the age of a cell increases, so does the potential for DNA damage. Recent theories on ageing suggest accumulative DNA damage is the primary cause of cellular senescence, possibly due to the decreased ability of DNA to act as a template for gene expression.In this paper we investigate the effects of ageing on the level of nuclear DNA damage in tissues of wild mussels of three different age groups; 2–4 years (group I), 6–8 years (group II) and 10 years (group III). In the digestive gland and haemolymph cells, a significant age-dependent increase of DNA damage was observed, as evaluated by the fluorimetric alkaline DNA unwinding technique, which is able to detect both direct single strand DNA breaks as well as alkali-labile apurinic sites.In addition, the rate of DNA polymerase activity was studied in order to determine whether DNA damage was dependent on DNA alteration, or because of a reduced rate of DNA repair. Unscheduled DNA repair synthesis in isolated nuclei of digestive gland cells in older mussels, was significantly decreased in comparison to younger mussels (−42% in group II and −37% in group III, p<0.01). In the digestive gland, salt extraction gives a slight, but significant, decrease of aphidicolin-sensitive DNA polymerase activity in age group III of −25%, p<0.05.Finally, we looked at the age variation in relation to oxidative stress. This was evaluated by measuring malondialdehyde accumulation in mussel cells. Digestive gland cells of group III, showed a significant age-related increase in malondialdehyde content of 170%, p<0.01, indicative of enhanced peroxidative processes.Taken together, these data suggest that the accumulation of DNA damage in group II is mainly dependent on the impairment of DNA repair systems. This is contrary to group III DNA damage, where a possible relationship between oxidative stress and alteration of nuclear DNA metabolism is found, probably deriving from an antioxidant defence decline.     

Byssus thread strength in the mussel, Mytilus edulis

Mytilus edulis attaches to the substratum by means of a proteinaceous byssus complex. This consists of three portions: a root, embedded in the pedal tissues, a stem, continuous with the root but external to the body and a number of byssus threads attached proximally to the stem and distally to the substratum via adhesive discs. Byssus strength varies seasonally on the shore, in response to changes in wave action (Price, in press). As a decline in byssal attachment strength implies a decline in strength of the constituent threads, a study was undertaken to establish the extent to which byssus thread strength is determined by age. The ultimate tensile stress, ultimate tensile strain and Young's Modulus were measured in threads of known age and length and a stepped regression performed on the results. It was found that age and length correlate significantly with tensile stress and Young's Modulus. Length is a less important influence than age on tensile stress but has a greater effect than age on Young's Modulus. Tensile strain is independent of both length and age.     

Local adaptation and species segregation in two mussel (Mytilus edulis x Mytilus trossulus) hybrid zones

Few marine hybrid zones have been studied extensively, the major exception being the hybrid zone between the mussels Mytilus edulis and Mytilus galloprovincialis in southwestern Europe. Here, we focus on two less studied hybrid zones that also involve Mytilus spp.; Mytilus edulis and Mytilus trossulus are sympatric and hybridize on both western and eastern coasts of the Atlantic Ocean. We review the dynamics of hybridization in these two hybrid zones and evaluate the role of local adaptation for maintaining species boundaries. In Scandinavia, hybridization and gene introgression is so extensive that no individuals with pure M. trossulus genotypes have been found. However, M. trossulus alleles are maintained at high frequencies in the extremely low salinity Baltic Sea for some allozyme genes. A synthesis of reciprocal transplantation experiments between different salinity regimes shows that unlinked Gpi and Pgm alleles change frequency following transplantation, such that post-transplantation allelic composition resembles native populations found in the same salinity. These experiments provide strong evidence for salinity adaptation at Gpi and Pgm (or genes linked to them). In the Canadian Maritimes, pure M. edulis and M. trossulus individuals are abundant, and limited data suggest that M. edulis predominates in low salinity and sheltered conditions, whereas M. trossulus are more abundant on the wave-exposed open coasts. We suggest that these conflicting patterns of species segregation are, in part, caused by local adaptation of Scandinavian M. trossulus to the extremely low salinity Baltic Sea environment.     

Organ specific changes in energy metabolism due to anaerobiosis in the sea mussel Mytilus edulis (L.)

• 1.1. Anaerobic energy metabolism was investigated in different organs of Mytilus edulis and the whole animal.
• 2.2. Succinate accumulates to high levels in most organs but remains low in the hemolymph.
• 3.3. After 16 hours propionate accumulation is observed in all organs. Experimental evidence is not sufficient yet to point out organs that produce more propionate than others.
• 4.4. Acetate is a minor end product.
• 5.5. Acetate and propionate are found in the hemolymph in amounts equal to those in the organs.
• 6.6. Animals incubated in oxygen-free seawater accumulate more end products than animals exposed to air, in the form of volatile fatty acids that are excreted into the incubation water.
• 7.7. Alanine and glutamine increase in the posterior adductor muscle. Aspartate decreases in the total animal, posterior adductor muscle and gills, while in the hemolymph decrease in alanine, asparagine, serine, threonine and proline are observed.

     

Picoeucaryot alga infecting blue mussel Mytilus edulis in southern Norway

During summer 2001, blue mussels Mytilus edulis with abnormal shell growth were collected near Krager?, southern Norway. The mussels had green spots in their mantle tissues, mainly posteriorly and ventrally, and in the adductor muscle. Mussels from 4 sites had a prevalence of green spots varying from 2 to 71% that correlated well with shell deformities. Histological examination revealed the presence of round or ovoid algae, 0.9 to 1.5 x 1.2 to 2.4 microm, free within haemocytes and in the lesions, characterised by an inflammatory response and the presence of cellular debris. The alga contain a relatively large nucleus, 1 chloroplast and 1 mitochondrion. Size and morphology suggest that the alga might be a picoeucaryot green alga. Infection of mussel tissues appears to start in the posterior mantle edge, near the siphons, and spread anterior-ventrally in the mantle connective and storage tissues-occasionally spots were also found in the gonad follicles. Large infected areas were also observed in sinuses within the adductor muscle. Only mussels that were 3 yr old or more were infected. Deformations apparently resulted from years of continuous shell formation by a contracted, partly deformed mantle. Most deformed mussels had eroded shells, allowing some light penetration through the exposed, thin nacre. Young, thin-shelled mussels were not infected. The present work suggests that the alga has, at least partially, a parasitic relationship with the mussels, and is associated with pathological alterations in mussel tissues.