The blue mussel inside: 3D visualization and description of the vascular-related anatomy of Mytilus edulis to unravel hemolymph extraction.

The blue mussel Mytilus edulis is an intensely studied bivalve in biomonitoring programs worldwide. The lack of detailed descriptions of hemolymph-withdrawal protocols, particularly with regard to the place from where hemolymph could be perfused from, raises questions regarding the exact composition of aspirated hemolymph and does not exclude the possibility of contamination with other body-fluids. This study demonstrates the use of high resolution X-ray computed tomography and histology combined with 3D-reconstruction using AMIRA-software to visualize some important vascular-related anatomic structures of Mytilus edulis. Based on these images, different hemolymph extraction sites used in bivalve research were visualized and described, leading to new insights into hemolymph collection. Results show that hemolymph withdrawn from the posterior adductor muscle could be extracted from small spaces and fissures between the muscle fibers that are connected to at least one hemolymph supplying artery, more specifically the left posterior gastro-intestinal artery. Furthermore, 3D-reconstructions indicate that puncturing hemolymph from the pericard, anterior aorta, atria and ventricle in a non-invasive way should be possible. Hemolymph withdrawal from the heart is less straightforward and more prone to contamination from the pallial cavity. This study resulted simultaneously in a detailed description and visualization of the vascular-related anatomy of Mytilus edulis.

Hierarchically-structured metalloprotein composite coatings biofabricated from co-existing condensed liquid phases.

Complex hierarchical structure governs emergent properties in biopolymeric materials; yet, the material processing involved remains poorly understood. Here, we investigated the multi-scale structure and composition of the mussel byssus cuticle before, during and after formation to gain insight into the processing of this hard, yet extensible metal cross-linked protein composite. Our findings reveal that the granular substructure crucial to the cuticle's function as a wear-resistant coating of an extensible polymer fiber is pre-organized in condensed liquid phase secretory vesicles. These are phase-separated into DOPA-rich proto-granules enveloped in a sulfur-rich proto-matrix which fuses during secretion, forming the sub-structure of the cuticle. Metal ions are added subsequently in a site-specific way, with iron contained in the sulfur-rich matrix and vanadium coordinated by DOPA-catechol in the granule. We posit that this hierarchical structure self-organizes via phase separation of specific amphiphilic proteins within secretory vesicles, resulting in a meso-scale structuring that governs cuticle function.

The impact of ocean acidification on the byssal threads of the blue mussel (Mytilus edulis).

Blue mussel (Mytilus edulis) produce byssal threads to anchor themselves to the substrate. These threads are always exposed to the surrounding environmental conditions. Understanding how environmental pH affects these threads is crucial in understanding how climate change can affect mussels. This work examines three factors (load at failure, thread extensibility, and total thread counts) that indicate the performance of byssal threads as well as condition index to assess impacts on the physiological condition of mussels held in artificial seawater acidified by the addition of CO2. There was no significant variation between the control (~786 µatm CO2 / ~7.98 pH/ ~2805 µmol kg-1 total alkalinity) and acidified (~2555 µatm CO2 / ~7.47 pH/ ~2650 µmol kg-1 total alkalinity) treatment groups in any of these factors. The results of this study suggest that ocean acidification by CO2 addition has no significant effect on the quality and performance of threads produced by M. edulis.

Blue mussel shell shape plasticity and natural environments: a quantitative approach.

Shape variability represents an important direct response of organisms to selective environments. Here, we use a combination of geometric morphometrics and generalised additive mixed models (GAMMs) to identify spatial patterns of natural shell shape variation in the North Atlantic and Arctic blue mussels, Mytilus edulis and M. trossulus, with environmental gradients of temperature, salinity and food availability across 3980 km of coastlines. New statistical methods and multiple study systems at various geographical scales allowed the uncoupling of the developmental and genetic contributions to shell shape and made it possible to identify general relationships between blue mussel shape variation and environment that are independent of age and species influences. We find salinity had the strongest effect on the latitudinal patterns of Mytilus shape, producing shells that were more elongated, narrower and with more parallel dorsoventral margins at lower salinities. Temperature and food supply, however, were the main drivers of mussel shape heterogeneity. Our findings revealed similar shell shape responses in Mytilus to less favourable environmental conditions across the different geographical scales analysed. Our results show how shell shape plasticity represents a powerful indicator to understand the alterations of blue mussel communities in rapidly changing environments.

Does the membrane pacemaker theory of metabolism explain the size dependence of metabolic rate in marine mussels?

According to the membrane pacemaker theory of metabolism (MPT), allometric scaling of metabolic rate in animals is determined by the composition of cellular and mitochondrial membranes, which changes with body size in a predictable manner. MPT has been elaborated from interspecific comparisons in mammals. It projects that the degree of unsaturation of membrane phospholipids decreases in larger organisms, thereby lowering ion permeability of the membranes and making cellular, and thus whole-animal metabolism more efficient. Here, we tested the applicability of the MPT to a marine ectotherm, the mussel Mytilus edulis at the intraspecific level. We determined effects of body mass on whole-organism, tissue and cellular oxygen consumption rates, on heart rate, metabolic enzyme activities and on the lipid composition of membranes. In line with allometric patterns, the organismal functions and processes such as heart rate, whole-animal respiration rate and phospholipid contents showed a mass-dependent decline. However, the allometry of tissue and cellular respiration and activity of metabolic enzymes was poor; fatty acid unsaturation of membrane phospholipids of gill tissue was independent of animal size. It is thus conceivable that most of the metabolic allometry observed at the organismal level is determined by systemic functions. These whole-organism patterns may be supported by energy savings associated with growing cell size but not by structural changes in membranes. Overall, the set of processes contributing to metabolic allometry in ectotherms may differ from that operative in mammals and birds, with a reduced involvement of the mechanisms proposed by the MPT.

Intra-population variability of ocean acidification impacts on the physiology of Baltic blue mussels (Mytilus edulis): integrating tissue and organism response.

Increased maintenance costs at cellular, and consequently organism level, are thought to be involved in shaping the sensitivity of marine calcifiers to ocean acidification (OA). Yet, knowledge of the capacity of marine calcifiers to undergo metabolic adaptation is sparse. In Kiel Fjord, blue mussels thrive despite periodically high seawater PCO2, making this population interesting for studying metabolic adaptation under OA. Consequently, we conducted a multi-generation experiment and compared physiological responses of F1 mussels from 'tolerant' and 'sensitive' families exposed to OA for 1 year. Family classifications were based on larval survival; tolerant families settled at all PCO2 levels (700, 1120, 2400 µatm) while sensitive families did not settle at the highest PCO2 (≥99.8% mortality). We found similar filtration rates between family types at the control and intermediate PCO2 level. However, at 2400 µatm, filtration and metabolic scope of gill tissue decreased in tolerant families, indicating functional limitations at the tissue level. Routine metabolic rates (RMR) and summed tissue respiration (gill and outer mantle tissue) of tolerant families were increased at intermediate PCO2, indicating elevated cellular homeostatic costs in various tissues. By contrast, OA did not affect tissue and routine metabolism of sensitive families. However, tolerant mussels were characterised by lower RMR at control PCO2 than sensitive families, which had variable RMR. This might provide the energetic scope to cover increased energetic demands under OA, highlighting the importance of analysing intra-population variability. The mechanisms shaping such difference in RMR and scope, and thus species' adaptation potential, remain to be identified.

Experimental treatment of a refinery waste air stream, for BTEX removal, by water scrubbing and biotrickling on a bed of Mitilus edulis shells.

The paper presents the results of a two-stage pilot plant for the removal of benzene, toluene, ethylbenzene and xylene (BTEX) from a waste air stream of a refinery wastewater treatment plant (WWTP). The pilot plant consisted of a water scrubber followed by a biotrickling filter (BTF). The exhausted air was drawn from the main works of the WWTP in order to prevent the free migration to the atmosphere of these volatile hazardous contaminants. Concentrations were detected at average values of 12.4 mg Nm(-3) for benzene, 11.1 mg Nm(-3) for toluene, 2.7 mg Nm(-3) for ethylbenzene and 9.5 mg Nm(-3) for xylene, with considerable fluctuation mainly for benzene and toluene (peak concentrations of 56.8 and 55.0 mg Nm(-3), respectively). The two treatment stages proved to play an effective complementary task: the water scrubber demonstrated the ability to remove the concentration peaks, whereas the BTF was effective as a polishing stage. The overall average removal efficiency achieved was 94.8% while the scrubber and BTF elimination capacity were 37.8 and 15.6 g BTEX d(-1) m(-3), respectively. This result has led to outlet average concentrations of 1.02, 0.25, 0.32 and 0.26 mg Nm(-3) for benzene, toluene, ethylbenzene and xylene, respectively. The paper also compares these final concentrations with toxic and odour threshold concentrations.

Domoic acid uptake and elimination kinetics in oysters and mussels in relation to body size and anatomical distribution of toxin.

Toxin accumulation by suspension-feeding qualifier depends on a balance between processes regulating toxin uptake (i.e. ingestion and absorption of toxic cells) and elimination (i.e. egestion, exchange among tissues, excretion, degradation and/or biotransformation) during exposure to toxic blooms. This laboratory study compares the size-specific uptake and elimination kinetics of domoic acid (DA) from Pseudo-nitzschia multiseries in two co-occurring bivalves, the oyster Crassostrea virginica and the mussel Mytilus edulis. Domoic acid concentrations were measured in visceral and non-visceral tissues of different-sized oysters and mussels during simultaneous long-term exposure to toxic P. multiseries cells in the laboratory, followed by depuration on a non-toxic algal diet. Mussels attained 7-17-fold higher DA concentrations than oysters, depending on the body size and exposure time, and also detoxified DA at higher rates (1.4-1.6 d(-1)) than oysters (0.25-0.88 d(-1)) of a comparable size. Small oysters attained markedly higher weight-specific DA concentrations (maximum=78.6 µg g(-1)) than large, market-sized individuals (≤ 13 µg g(-1)), but no clear relationship was found between body size and DA concentration in mussels (maximum=460 µg g(-1)). Therefore, differential DA accumulation by the two species was, on average, approximately 3-fold more pronounced for large bivalves. An inverse relationship between DA elimination rate and body size was established for oysters but not mussels. Elimination of DA was faster in viscera than in other tissues of both bivalves; DA exchange rate from the former to the latter was higher in oysters. The contribution of viscera to the total DA burden of mussels was consistently greater than that of other tissues during both uptake (>80%) and depuration (>65%) phases, whereas it rapidly decreased from 70-80% to 30-40% in oysters, and this occurred faster in smaller individuals. Residual DA concentrations (≤ 0.25 µg g(-1)) were detected at later depuration stages (up to 14 d), mainly in viscera of oysters and non-visceral tissues of mussels, suggesting that a second, slower-detoxifying toxin compartment exists in both species. However, a simple exponential decay model was found to adequately describe DA elimination kinetics in these bivalves. The lower capacity for DA accumulation in oysters compared to mussels can thus only be explained by the former's comparatively low toxin intake rather than faster toxin elimination.

Relevance of mytilid shell microtopographies for fouling defence--a global comparison.

Prevention of epibiosis is of vital importance for most aquatic organisms, which can have consequences for their ability to invade new areas. Surface microtopography of the shell periostracum has been shown to have antifouling properties for mytilid mussels, and the topography shows regional differences. This article examines whether an optimal shell design exists and evaluates the degree to which shell microstructure is matched with the properties of the local fouling community. Biomimics of four mytilid species from different regional provenances were exposed at eight different sites in both northern and southern hemispheres. Tendencies of the microtopography to both inhibit and facilitate fouling were detected after 3 and 6 weeks of immersion. However, on a global scale, all microtopographies failed to prevent fouling in a consistent manner when exposed to various fouling communities and when decoupled from other shell properties. It is therefore suggested that the recently discovered chemical anti-microfouling properties of the periostracum complement the anti-macrofouling defence offered by shell microtopography.

Aging reduces reproductive success in mussels Mytilus edulis.

The present study was aimed to determine whether reproductive success constantly increases with age in a relatively short-lived invertebrate with continuous growth - the bivalve mollusc Mytilus edulis or there is an age-related decline such as observed in species with finite growth (mammals, insects, nematodes, etc.). We studied the reproductive output and viability of the offspring during early embryogenesis in females of different sizes and ages, and used allometric approaches to correct for the effects of the body size and to discern pure age-specific effects on these reproductive traits. We have also determined contributions of females of different age and size classes to the total larval pool of a population. Both gonadosomatic index and individual fecundity significantly decreased in the course of aging if the size of the animals was accounted for. The proportion of normally developing embryos declined from almost 100% to 60% in females of 2-10-year-old. We suggest that animals with infinite growth and "slow aging", such as molluscs, undergo senescence, the physiological manifestations of which can be masked by a more pronounced effect of continuously increasing size.

Comment on "Divergent induced responses to an invasive predator in marine mussel populations".

Freeman and Byers (Reports, 11 August 2006, p. 831) presented evidence for the rapid evolution of antipredator defenses in the mussel Mytilus edulis. However, their analysis is confounded by three issues. Samples from some sites are likely to have included a second species, M. trossulus; their manipulation of chemical cues does not preclude other interpretations; and they failed to establish an adaptive significance to shell thickening.

Lysosomal responses and metallothionein induction in the blue mussel Mytilus edulis from the south-west coast of Iceland.

It has recently been emphasized that high levels of inorganic and organic micropollutants (particularly organometals, POPs and PAHs) may be present in coastal waters at high latitudes, stressing the need to evaluate the effects of contaminants on marine organisms from sub-arctic zones. With this aim, specimens of the blue mussel Mytilus edulis were sampled in polluted and reference areas along the south-west coast of Iceland in July 2004. Samples were collected from the intertidal zone at three sites in Reykjavik harbour which are differently exposed to contaminants, and at three reference coastal sites, two located along the Reykjanes Peninsula and the third one on the northern part of Hvalfjordur fiord. Lipofuscin content, neutral lipid accumulation and lysosomal enlargement were evaluated in digestive cells from cryostat sections of the mussel hepatopancreas, and quantified by automated image analysis. Metallothionein induction was also determined in the same tissue. Results indicate that mussels from the inner part of Reykjavik harbour, which is the most sheltered and most influenced by extensive shipping traffic, were the worst affected, with the highest values in neutral lipids, lipofuscin and lysosomal swelling. At the other two harbour sites, mussels exhibited lower values, similar to those observed in organisms collected in Hvalfjordur fiord and in bay of Osar. Mussels from Kuagerdi had the lowest values.

Divergent induced responses to an invasive predator in marine mussel populations.

Invasive species may precipitate evolutionary change in invaded communities. In southern New England (USA) the invasive Asian shore crab, Hemigrapsus sanguineus, preys on mussels (Mytlius edulis), but the crab has not yet invaded northern New England. We show that southern New England mussels express inducible shell thickening when exposed to waterborne cues from Hemigrapsus, whereas naïve northern mussel populations do not respond. Yet, both populations thicken their shells in response to a long-established crab, Carcinus maenas. Our findings are consistent with the rapid evolution of an inducible morphological response to Hemigrapsus within 15 years of its introduction.

[Comparative analysis of growth of edible mussel Mytilus edulis from different White Sea regions].

Growth properties were studied in edible mussel Mytilus edulis from different biotopes of the Kandalaksha Bay in the White Sea. Growth curves of the mussel shell were approximated by the von Bertalanffy equation. The highest growth rate, primarily dependent on hydrological conditions, was observed in mussels from the Chupa Bay (Levin Navolok fishery), while the lowest rate was observed in mussels from the Umba region (Turn Cape, Murmansk Region). Allometric relationships of the mussel shell were determined. Analysis of the relationship between the maximum width/convexity and their length demonstrated that this relationship is described by a single allometric equation for mussels from the Umba region, which indicates the homogeneity of this population. For mussels from the Chupa Bay, this relationship cannot be described by a single equation, which points to the population heterogeneity. For 90% mussels from this region, the shell width/convexity ratio ranged from 0.5 to 0.9; while for the remaining 10%, it ranged from 0.9 to 1.15.

Biomarkers of exposure and reproduction-related effects in mussels exposed to endocrine disruptors.

Biomarkers are useful tools to study the health of estuarine and marine ecosystems. Biomarkers can be measured in different organisms, but mussels have acquired a global importance as sentinels in marine pollution-monitoring programs. In the present work, we aimed to determine the effects of different endocrine disruptors in mussels by using peroxisome proliferation as a biomarker of exposure to organic pollutants and the levels of vitellogenin (Vtg)-like proteins as biomarker of endocrine disruption. In experiment 1, mussels Mytilus edulis were exposed for 3 weeks to North Sea crude oil (NSO 0.5 ppm) and a mixture of 0.5 ppm NSO, 0.1 ppm alkylphenol mix, and 0.1 ppm extra polycyclic aromatic hydrocarbons (PAHs) (MIX). In experiment 2, mussels were exposed for 3 weeks to diallylphthalate (DAP 50 ppb), bisphenol-A (BPA 50 ppb), and tetrabromodiphenylether (TBDE 5 ppb). Peroxisome proliferation was assessed by measuring acyl-CoA oxidase (AOX) activity and peroxisomal volume density (VVp) in digestive gland. Vtg-like protein levels were measured in gonads by the alkali-labile phosphate (ALP) method. Gonad was also analyzed histologically, and the gonad index (GI) calculated. Mussels exposed to NSO and MIX showed significantly increased AOX activities and VVP compared with control animals. Significantly higher VVP was also found in DAP- and TBDE-exposed mussels. Effects on ALP and GI depended significantly on sex and time of year. In female mussels, ALP levels and GI were lower in the NSO group. In male mussels, ALP levels were significantly increased in the MIX group. The volume density of athretic oocytes was higher in the NSO and MIX exposure groups than in controls, and gonad resorption was observed in the BPA exposure group. Our results confirm the usefulness of peroxisome proliferation as a biomarker of exposure to organic contaminants in mussels and indicate that changes in Vtg-like proteins could be used as potential indicator of pollutant effects on mussel reproduction.

Tyrosine and tyramine increase endogenous ganglionic morphine and dopamine levels in vitro and in vivo: cyp2d6 and tyrosine hydroxylase modulation demonstrates a dopamine coupling.

BACKGROUND: The ability of animals to make morphine has been in question for the last 30 years. Studies have demonstrated that animals do contain morphine precursors and metabolites, as well as the ability to use some morphine precursors to make morphine. MATERIAL/METHODS: The present study uses excised ganglia from the marine invertebrate Mytilus edulis as well as whole animals. Morphine and dopamine levels were determined by high performance liquid chromatography coupled to electrochemical detection and radioimmunoassay. Tissues and whole animals were also exposed to morphine precursors and exposed to the CYP2D6 inhibitor quinidine and the tyrosine hydroxylase inhibitor alpha-methyl-para-tyrosine (AMPT). Additionally, via RT-PCR, a cDNA fragment of the CYP2D6 enzyme in the ganglia of M. edulis was identified. RESULTS: Pedal ganglia incubated with either tyramine or tyrosine, or whole animals receiving injections, exhibited a statistically significant concentration- and time-dependent increase in their endogenous morphine and dopamine levels (2.51 +/- 0.76 ng/g for tyrosine and 2.39 +/- 0.64 ng/g for tyramine compared to approximately 1.0 ng/g morphine wet weight). Incubation with quinidine and/or AMPT diminished ganglionic morphine and dopamine synthesis at various steps in the synthesis process. We also demonstrated that CYP2D6 mediates the tyramine to dopamine step in this process, as did tyrosine hydroxylase in the step from tyrosine to L-DOPA. Furthermore, via RT-PCR, we identified a cDNA fragment of the CYP2D6 enzyme in the ganglia, which exhibits 94% sequence identity with its human counterpart. Evidence that tyrosine and tyramine were, in part, being converted to dopamine then morphine, and that this process can be inhibited by altering either or both CYP2D6 or tyrosine hydroxylase, is also provided. CONCLUSIONS: It appears that animals have the ability to make morphine. This process also appears to be dynamic in that the inhibition of one pathway allows the other to continue with morphine synthesis. Moreover, dopamine and morphine synthesis were coupled.

Structural characterization of the major extrapallial fluid protein of the mollusc Mytilus edulis: implications for function.

The major protein component of the extrapallial fluid of the mollusc Mytilus edulis has been previously isolated and partially characterized. It was postulated to play a role in shell mineralization because of its intriguing property of Ca(2+)-binding-induced self-assembling. However, it also binds other divalent ions, including Cd(2+), Cu(2+), Mn(2+), and Mg(2+). Herein is the initial report on the characterization of the primary structure of the extrapallial (EP) protein by RT-PCR and cDNA sequencing methods and by de novo peptide sequencing with mass spectrometry. The EP protein is comprised of 213 amino acids postcleavage of a signal peptide of 23 amino acids. The protein is rich in His, Glu, and Asp residues. The site of N-glycosylation, "NHTE", at amino acid positions 54-57 and the intramolecular disulfide bond between Cys 139 and Cys 171 of the protein have been characterized also. Sequence comparisons reveal that the EP protein possesses little homology to any presently known matrix proteins previously isolated from mollusc shells but rather it highly resembles a heavy metal binding protein and a histidine-rich glycoprotein, both from the hemolymph of M. edulis. The predicted domain profile and amino acid composition suggest that its N-terminus may be involved in calcium binding. The abundance of histidine residues of the protein may account for its heavy metal binding properties. Thus, the EP protein perhaps has multiple functions, serving as a Ca(2+)-transport protein, a shell matrix protein, and a heavy metal detoxification protein.