Effect of fructose 2,6-bisphosphate and ammonium ions on AMP-mediated activation of phosphofructokinase from the posterior adductor muscle of the sea musselMytilus galloprovincialis

The effects of different modulators on the phosphofructokinase (PFK) activity from the posterior adductor muscle of the sea musselMytilus galloprovincialis Lmk. were studied in mussels collected from N.W. Spain in spring/summer, 1988. Adenosine monophosphate (AMP), fructose 2,6-bisphosphate (Fru 2,6-P₂) and ammonium ions individually activated PFK. AMP and Fru 2,6-P₂ reduced ATP-mediated inhibition and the value ofS _0.5 [concentration of fructose 6-phosphate (Fru 6-P) for half-maximum velocity], whereas ammonium ions increasedV _max (the rate at the saturating concentrations of Fru 6-P). Fru 2,6-P₂ and ammonium ions both enhanced the effect of AMP, but in different ways: Fru 2,6-P₂ reduced apparentK _m for AMP (concentration of AMP for half-maximum activation) without affectingK _m , whereas ammonium ions only altered the latter. The sharp increases in the values ofS _0.5 for Fru 6-P and of apparentK _m for AMP that are caused by a drop in pH were reduced by the presence of Fru 2,6-P₂, but not by ammonium ions. The results shows that Fru 2,6-P₂ may contribute to maintain the PFK activity during hypoxia periods, whereas ammonium ions (in the presence of high AMP concentrations) may activate the enzyme during the first few hours of post-hypoxia re-immersion.     

Biochemical biomarkers and overall health status of mussels Mytilus galloprovincialis exposed to nickel and chromium

This study examined the effects of nickel and chromium exposure on two biochemical biomarkers, glutathione peroxidase (GPX) and acetylcholinesterase (ACHE), in parallel with an indicator of overall health, Scope for Growth (SFG), in the mussel Mytilus galloprovincialis to evaluate their ecological relevance and to assess their potential use as biomarkers of Ni and Cr pollution. SFG integrates major physiological responses into an index that represents the energy status of the animals and is considered an ecologically relevant biomarker. Following exposure to a range of Ni and Cr concentrations effects of Ni and Cr were evident on GPX and SFG but not on ACHE. Accumulation in the mussels’ tissues increased according to exposure concentrations. Adverse effects on the energy status and induction of GPX were found at the higher exposure concentrations while possible hormetic effects on the energy status and moderate GPX suppression were associated with lower exposure concentrations. Nevertheless GPX was negatively correlated with SFG suggesting that GPX responses reflect the overall health of the organisms and thus show potential as an early warning signal of population level effects of Ni and Cr. On the other hand, our results indicate that ACHE is not a suitable biomarker for Ni and Cr pollution.     

Biomass measurements and weight-to-weight conversion factors: a comparison of methods applied to the mussel Mytilus galloprovincialis

Wet weight to ash-free dry weight conversion factors were calculated for the bivalve Mytilus galloprovincialis Lamarck; specimens were collected in November 1989 in the Magra Estuary (eastern Ligurian Sea, Italy). Ash-free dry weight was determined by four different methods: (A) mechanical separation of shell and body; (B) shell dissolution with HCl; (C) dissolution of organic matter with NaClO; (D) incineration of the whole organism. The results obtained by these four methods were significantly different, indicating that caution is necessary when comparing biomass data obtained by different methods. Incineration and decalcification with HCl seem to be the best procedures: they not only combine accuracy with speed, but are also the most universally applicable, being equally suitable for soft-bodied or hard-bodied organisms. Mechanical separation can lead to an underestimation of the biomass, whereas treatment with NaClO can lead to its overestimation. However, no method can be considered the best in absolute terms, the final choice depending on the particular weight measurement required (e.g. shell-free dry weight) and the nature of the organism concerned (e.g. an organism with a large carbonate content).     

Studies on cytochrome P450 in Mytilus galloprovincialis: induction by Na-phenobarbital and ability to biotransform xenobiotics

Mytilus galloprovincialis has a hepatopancreatic monooxygenase activity cytochrome P₄₅₀-dependent. The present paper studies the induction of this activity in the hepatopancreatic microsomes after treatment of M. galloprovincialis with Na-phenobarbital. Mussels were collected from the Gulf of La Spezia in 1985. We measured the increase in levels of cytochrome P₄₅₀, b₅, and P₄₂₀ and the 7-ethoxycoumarin deethylase activity. Furthermore, the fraction of mouse liver was replaced by the correspondent mussels' hepatopancreatic fraction in the yeast genotoxicity test to determine the ability of mussels to biotransform cyclophosphamide and styrene oxide. The results suggest that edible mussels are capable of detoxicating styrene oxide but not of bioactivating cyclophosphamide.     

Deep-sea caging of the mussel Mytilus galloprovincialis: Potential application in ecotoxicological studies

We experimented with caging the Mediterranean mussel (Mytilus galloprovincialis) at various depths for 69 d to measure basic physiological parameters, histological response and bio-accumulation of contaminants in a deep-sea contaminated area. In preliminary experiments, we demonstrated, under artificial pressure conditions, the ability of mussels Mytilus galloprovincialis to tolerate rapid immersion (at a speed of up to 120 m min^?1). In situ experiments were performed using submerged lines enabling mussels to be maintained at depths ranging of 40–1550 m with survival rates ranging from 80 to 38%, respectively. No significant differences in condition indexes were observed between treated and control specimens. However, histological observations demonstrated a clear reduction in thickness of the digestive epithelium with increasing depth exposure. By determining the contaminants in caged mussels, we found the following values for chromium accumulation: 27.4 μg g^?1 dry weight at 580 m depth and 9.8 μg g^?1 dry weight at 1550 m. Selected stations were located downstream of an industrial effluent at 420 m. The biological and environmental consequences of deep-sea contamination demonstrate the suitability of caged mussels for monitoring contaminant accumulation.     

Interactions of 233Pa with tissues of Mytilus galloprovincialis and Carcinus mediterraneus

Uptake of ²³³Pa from sea water by mussels Mytilus galloprovincialis L. and shore crabs Carcinus mediterraneus Csrn. was studied. Results indicate a high contamination factor in digestive tract, gills and skeleton of both investigated species and an increment of the contamination factor in mussel byssus. In spite of this, reproductive system, muscle and hemolymph were only negligibly contaminated. The presence of complexone EDTA in sea water has no effect on uptake of ²³³Pa in the animal tissues tested.     

A proteomic approach to the study of the marine mussels Mytilus edulis and M. galloprovincialis

In this study, a proteomic approach was applied for the generation of reference maps and subsequently to detect, quantify and compare the global protein expression between two related species of marine mussels, Mytilus edulis and Mytilus galloprovincialis, growing in their own geographical habitats. A comparative study of the protein profiles generated from analytical two-dimensional electrophoresis gels was performed, and changes in protein expression were analysed quantitatively by computer analysis. An average of 1,278 spots per gel was detected in 16 individuals (8 M. edulis and 8 M. galloprovincialis); however, not all spots were included in the study. Expression of 420 spots was compared, and significant differences in the intensity levels were detected in 37 protein spots (8.8%). Fifteen proteins showed higher expression in M. edulis, and 22 proteins, in M. galloprovincialis. The technique of peptide mass fingerprinting using MALDI-TOF (matrix-assisted laser desorption ionisation/time-of-flight) and/or nanoelectrospray double subfragmentation mass spectrometry enabled the unambiguous identification of 15 of these 37 differentially expressed proteins. Most of the identified proteins can be grouped basically into four broad functional classes: cytoskeletal and myofibrillar proteins, proteins associated with stress response, proteins associated with the storage or production of energy, and proteins related to rearrangement in the synthesis of native structures. These results expand our understanding of the molecular differentiation of the two mussel taxa and serve as a useful base for future ecological, physiological and genetic studies.     

Mytilus galloprovincialis and Parapenaeus longirostris as bioindicators of heavy metal and organochlorine pollution

The worth of the data obtained from Mytilus galloprovincialis and Parapenaeus longirostris for the purpose of assessing the extent to which the areas they come from are polluted by heavy metals (Fe, Zn, Cu, Pb, Mn, Ni, Cd, Cr, Co) and chlorinated hydrocarbons (PCBs, p,p'DDE, p,p' DDT, p,p'DDD, BHCs, heptachlor epoxide, dieldrin, endrin), was thoroughly investigated. The work embraced the accuracy of the determinations, the spot and area variability, the overall coefficient of variation, the effect of the season and the size, together with, in the case of P. longirostris, the differences observed in four sections of the Saronikos Gulf, in Greece, the degree of pollution, which was estimated independently by measuring the concentration in the water of nutrients (phosphate, ammonia, nitrite, nitrate, silicate) and dissolved oxygen. The study proved the feasibility of employing the organisms as bioindicators of pollution. It also established that the individual variability of response, the season and the size do not cause as much assessment error as the commonly huge temporal fluctuations and the frequent inadequacies of the analytical procedures. A scheme is described for the collection of a truly representative sample.     

Decomposition of faecal matter and somatic tissue of Mytilus galloprovincialis: changes in organic matter composition and microbial succession

Variations in the biochemical composition of pseudo-faeces and faeces egested by Mytilus galloprovincialis (Lamarck) and in detritus derived from the somatic tissue of mussels during the decomposition process were investigated by means of two intensive experiments. During the degradation process, the biochemical composition of pseudo-faeces and faeces showed a clear increase in protein content related to the microbial colonization. Changes also occurred in the biochemical composition of particulate organic matter (POM) in the surrounding water due to faecal matter decomposition, heterotrophic utilization and conversion of particulate carbohydrates and proteins to the dissolved pool. The study of production and heterotrophic utilization of the POM derived from the somatic tissue of M. galloprovincialis collected in the Gulf of Tigullio, Italy in 1990 indicates that this kind of material is rapidly decomposable and largely available for benthic consumers. Bacteria utilized selectively the different compounds, and proteins proved to be the most suitable substrate for bacterial growth. The input of organic detritus into the experimental system resulted in an enhancement of bacterial activity and consequently of the RNA/DNA ratio. Bacterial DNA accounted on average for 17% of particulate DNA. During decomposition processes, nutrient release was about ten times higher than the value calculated from individual excretion rates, indicating that mussel beds may be important sites for nutrient regeneration. Carbon conversion efficiency for bacteria growing on faecal matter was, on average, 17.2%. The potential importance of faecal output and bacterial production as a carbon resource for benthic communities near mussel culture areas is discussed.     

Synergistic effects of lead,salinity and temperature on embryonic development of the mussel Mytilus galloprovincialis

Combined effects of lead, salinity and temperature on the embryonic development of the mussel Mytilus galloprovincialis Lmk. were studied under laboratory conditions. The basic experimental design was a 4x6 factorial experiment using 4 lead concentrations (100, 250, 500 and 1000 ppb Pb²⁺) and 6 salinity levels (from 25 to 37.5 with 2.5 intervals). These factorial designs were carried out at three constant temperatures (150, 17.50 and 20°C). The statistical analysis indicated that salinity changes have more effect on the embryonic development than temperature. Optimal development was observed at 34.8 and 15.6°C, which is in accordance with observations in the field. The effect of lead was mininal in optimal salinity and temperature conditions. The deleterious effect of lead on the embryonic development was especially conspicuous at 20°C. Since in nature spawning occurs at temperatures inferior to 20°C, lead will probably not drastically decrease the potential recruitment of mussel spat in the littoral populations of the northern Adriatic Sea, where the salinity of the water is relatively stable. Under experimental conditions, lead caused a delay or inhibition of the embryonic development with the occurrence of a large number of abnormal larvae.     

Ecological gradients and relative abundance of native (Mytilus trossulus) and invasive (Mytilus galloprovincialis) blue mussels in the California hybrid zone

Marine communities are experiencing unprecedented rates of species homogenization due to the increasing success of invasive species, but little is known about the mechanisms that allow a species to invade and persist in a new habitat. In central California, native (Mytilus trossulus Gould 1850) and invasive (Mytilus galloprovincialis Lamarck 1819) blue mussels and their hybrids co-exist, providing an opportunity to analyze the mechanisms that determine the distributions of these taxa. Spatial and temporal variation in temperature and salinity and the relative frequencies of these mussel taxa were examined between 2000 and 2004 at four sites in San Francisco Bay and four in Monterey Bay, which were chosen for their different positions along inferred estuarine/oceanic gradients in the hybrid zone. Mussels were genetically identified as the parent species or hybrids by amplifying regions of two species-specific loci: the adhesive byssal thread protein (Glu-5′) and the internal transcribed spacer region of ribosomal DNA (ITS 1). The proportion of M. trossulus at the eight hybrid zone sites correlated negatively with average salinity (R ²=0.60) and positively with maximal temperature (R ²≥0.72), a somewhat unexpected result given what is known about the phylogeography of this species. The proportion of M. galloprovincialis showed the opposite pattern. The proportion of hybrids was correlated neither with habitat temperature nor salinity. Genotypes of mussel populations at an additional 13 sites from Coos Bay, Oregon (latitude 43.35°N) to Long Beach, California (latitude 33.72°N), sampled at various intervals between 2000 and 2004, were also determined. This survey confirmed previous reports that the hybrid zone lies between Monterey and the Cape Mendocino region (latitudes 36.63°N–40.5°N). Within Monterey and San Francisco Bays, however, the temporal comparisons (1990s vs. 2000s) revealed abrupt changes in the proportions of the two parent species and their hybrids on annual and decadal time scales. These changes indicate that the blue mussel populations are in a highly dynamic state. The survey also showed that, regardless of habitat, M. trossulus is consistently of smaller average size than either M. galloprovincialis or hybrids.

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Mytilus galloprovincialis as bioindicator in embryotoxicity testing to evaluate sediment quality in the lagoon of Venice (Italy)

The possibility of using Mytilus galloprovincialis from natural populations of the lagoon of Venice in toxicity bioassays based on embryo development, according to international standard methods, was investigated. In order to valuate this method for the lagoon, iterative steps including evaluation of reproducibility, sensitivity and discriminatory capacity towards some pure substances and environmental samples were performed. Evaluation of sensitivity towards a reference toxicant (copper) evidenced good replicability and repeatability. Results showed that natural population is better than hatchery population for performing bioassays. Evaluation of applicability to elutriates revealed that the method is able to discriminate among sediments of the lagoon of Venice at different typologies and levels of contamination, showing an important effect for almost all study sites.     

Filtration of the enteric bacteria Escherichia coli by two filter-feeding bivalves,Venus verrucosa and Mytilus galloprovincialis

We investigated the filtration and utilization of the enteric bacteria Escherichia coli by two suspensionfeeding bivalves, Venus verrucosa (Linné), collected, during April 1990, in Port-Vendres harbour (France), and Mytilus galloprovincialis (Lamarck) collected from rearing units in the bay of Banyuls-sur-mer during June 1990. Because of a significant decrease in the concentration of culturable E. coli in filtered seawater, we used ¹⁴C glutamic acid to label the bacteria. Labelling efficiency was low (20%) compared to the 30% reported for the marine bacteria Lactobacillus sp. by Amouroux (1982). However, the labelling by this radioisotope was very stable, enabling its use to monitor filtration. Concentrations of culturable E. coli decreased more rapidly in the presence of M. galloprovincialis than in the presence of V. verrucosa. In both bivalve species, changes of radioactivity within the bivalve, particulate organic matter (POM), dissolved organic matter (DOM) and CO₂ compartments were similar and resulted from the interaction of several processes: filtration, biodeposition, and recycling. This interaction complicates the determination of the actual ingestion and assimilation rates, and necessitates the use of mathematical modelling.     

Single and combined effects of heavy metals and hormones on lysosomes of haemolymph cells from the mussel Mytilus galloprovincialis

Effects of heavy metals on lysosomes were studied in living cells from the mussel (Mytilus galloprovincialis Lam.). Haemolymph cells were obtained from the mussel adductor muscle, stained with neutral red (NR), and analysed by digital imaging to evaluate NR retention times within lysosomes. Exposure to Hg²⁺, Cd²⁺ and Cu²⁺ induced a reduction of NR retention time, indicating lysosomal membrane destabilisation. The intensity of these effects was correlated with the metal affinity for sulfhydryls. In contrast, Zn²⁺ showed no effect on lysosomes. Moreover, 200 μM Zn²⁺ protected lysosomes against the effects of Cd²⁺ and Cu²⁺, but not against Hg²⁺. Cell loading with the fluorescent pH probe Lyso Sensor followed by digital imaging showed a rise of lysosomal pH induced by Cd²⁺ and Hg²⁺, while Zn²⁺ prevented the effect of Cd²⁺ and also partially that of Hg²⁺. The different protective effect of Zn²⁺ against Hg²⁺ suggests a dual action of Hg²⁺ on lysosomes, possibly involving both membrane destabilisation and proton pump inhibition. Cell exposure to 17 β-estradiol also caused a reduction of NR retention time, which was synergistic to that of Hg²⁺. This suggests a common pathway between metals and hormone, possibly involving Ca²⁺ signaling. Received: 17 November 1999 / Accepted: 29 June 2000     

Presence of hydrocarbon-degrading bacteria in the gills of mussel Mytilus galloprovincialis in a contaminated environment: a mesoscale simulation study

A number of previous studies have shown that the relationships of symbiosis existing between mussels and microorganisms are directly dependent on the environmental conditions. However, little is known about existing relationships between mussels and bacteria in hydrocarbon-impacted marine environments. The aim of this preliminary study is to investigate the presence of oil-degrading bacteria in the mussel Mytilus galloprovincialis during growth in polluted ecosystems. All the experiments were carried out in a mesocosm system designed to simulate chronic pollution and to enable direct exposure of mussels to chemicals. Quantitative (4′,6-diamidino-2-phenylindole, colony-forming units, Most Probable Number) analyses and screening (presence/absence) of metabolic functional genes were performed to analyse bacterial populations inside the gills of mussels exposed and not exposed to hydrocarbons. The data obtained show that the presence of hydrocarbons affected the abundance of bacteria inside the gills of specimens and determines selection for specific (hydrocarbon-degrading) bacteria (i.e. Alcanivorax sp. and Marinobacter sp.). However, is not yet clear whether the presence of such genera of bacteria inside the mussel is due to symbiosis or as a result of filtration.     

Lysosomal,tissue and cellular alterations in the gills,palps and intestine of the mussels Mytilus galloprovincialis,in relation to pollution

Lysosomal, tissue and cellular alterations of the gill, the palp and the intestine epithelium of the mussel Mytilus galloprovincialis (L.) collected from six stations along a closed estuarine system (Thermaikos Gulf, northern Greece) were monitored in May, August and December. Semi-quantitative evaluation of histopathological alterations was performed under the light microscope, while certain of the latter alterations were confirmed electron microscopically. In addition, morphometrical evaluation of the volume density of the lysosomes under the electron microscope was conducted. The observed alterations, as the detachment of gill epithelial cells from the basement membranes and the dilated extracellular spaces formed either between the lateral plasma membranes or between the infoldings of the basal plasma membrane in the palps and the intestine are possibly related to the degree of pollution at the examined stations. The particularly expanded extracellular spaces formed by basal plasma membrane infoldings in the intestine in spring may be attributable to red tide algal infection. On the other hand, differences in morphometric parameters of lysosomal structures indicated no direct relationships with pollution levels.Communicated by O. Kinne, Oldendorf/Luhe     

Variation in strength of attachment to the substrate explains differential mortality in hybrid mussel (Mytilus galloprovincialis and M. edulis) populations

Mussel samples were collected from a hybrid mussel (Mytilus galloprovincialis and M. edulis) Population at Croyde, southwest England, in January, March and May 1990. The strength of attachment of each mussel to the substrate was measured with a spring balance. A number of diagnostic characters were also recorded. These are shell lengh, width and height, mantle colour and genotype at two allozyme loci, esterase-D and octopine dehydrogenase. Multiple-regression analysis was used to assess the effect of the diagnostic characters on strength of attachment as dependent variable. Mussels possessing the relatively high shells and darker mantle colouration characteristic of M. galloprovincialis had higher values, on average, for strength of attachment than mussels resembling M. edulis. Phenotypically intermediate mussels had intermediate values for strength of attachment. The results suggest an adaptive difference which can account for reports of differential mortality acting in favour of M. galloprovincialiis.     

Hydrodynamic stress and habitat partitioning between indigenous (Perna perna) and invasive (Mytilus galloprovincialis) mussels: constraints of an evolutionary strategy

The ability of a mussel to withstand wave-generated hydrodynamic stress depends mainly on its byssal attachment strength. This study investigated causes and consequences of different attachment strengths of the two dominant mussels species on the South African south coast, the invasive Mytilus galloprovincialis and the indigenous Perna perna, which dominate the upper and the lower areas of the lower balanoid zone, respectively and co-exist in the middle area. Attachment strength of P. perna was significantly higher than that of M. galloprovincialis. Likewise solitary mussels were more strongly attached than mussels living within mussel beds (bed mussels), and in both cases this can be explained by more and thicker byssal threads. Having a wider shell, M. galloprovincialis is also subjected to higher hydrodynamic loads than P. perna. Attachment strength of both species increased from higher to lower shore, in response to a gradient of stronger wave action. The morphological features of the invasive species and its higher mortality rates during winter storms help to explain the exclusion of M. galloprovincialis from the low shore. The results are discussed in the context of the evolutionary strategy of the alien mussel, which directs most of its energy to fast growth and high reproductive output, apparently at the cost of reduced attachment strength. This raises the prediction that its invasive impact will be more pronounced at sites subject to strong but not extreme wave action.     

Hybridization between the blue mussels Mytilus galloprovincialis and M. trossulus along the Pacific coast of North America: evidence for limited introgression

Two species of blue mussel, Mytilus galloprovincialis and M. trossulus, co-occur and hybridize along the Pacific coast of North America. Using a set of polymerase chain-reaction (PCR)-based genetic markers which diagnostically identify these species, we show that they are sympatric from the Cape Mendocino region to the Monterey Peninsula in northern and central California, USA. Mussels with hybrid genotypes were detected in all populations sampled in the region of sympatry, and the frequency of hybrid genotypes in individual hybrid populations ranged from 13 to 44%. Significant frequencies of first-generation backcross genotypes were detected in two individual hybrid zone populations (Berkeley and Monterey Marina) and in the hybrid zone as a whole, indicating that the potential exists for introgression between M. galloprovincialis and M. trossulus. Despite this potential, we found no evidence of advanced introgression beyond first-generation backcrosses, suggesting that gene flow between M. galloprovincialis and M. trossulus has been quite limited. The frequency of mussels with M. trossulus and hybrid genotypes declined abruptly south of Monterey Peninsula, while the frequency of mussels with M. galloprovincialis and hybrid genotypes declined precipitously north of Cape Mendocino. These abrupt genetic discontinuities indicate that this blue mussel hybrid zone is presently positioned between two prominent coastal features and there is little, if any, export of alleles from the hybrid zone into bordering parental populations. Received: 20 August 1997 / Accepted: 26 October 1998