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The byssus is a structure unique to bivalves. Byssal threads composed of many proteins extend like tendons from muscle cells, ending in adhesive pads that attach underwater. Crucial to settlement and metamorphosis, larvae of virtually all species are byssate. By contrast, in adults, the byssus is scattered throughout bivalves, where it has had profound effects on morphological evolution and been key to adaptive radiations of epifaunal species. I compare byssus structure and proteins in blue mussels ( Mytilus ), by far the best characterized, to zebra mussels ( Dreissena polymorpha ), in which several byssal proteins have been isolated and sequenced. By mapping the adult byssus onto a recent phylogenomic tree, I confirm its independent evolution in these and other lineages, likely parallelisms with common origins in development. While the byssus is superficially similar in Dreissena and Mytilus , in finer detail it is not, and byssal proteins are dramatically different. I used the chromosome-scale D. polymorpha genome we recently assembled to search for byssal genes and found 37 byssal loci on 10 of the 16 chromosomes. Most byssal genes are in small families, with several amino acid substitutions between paralogs. Byssal proteins of zebra mussels and related quagga mussels ( D. rostriformis ) are divergent, suggesting rapid evolution typical of proteins with repetitive low complexity domains. Opportunities abound for proteomic and genomic work to further our understanding of this textbook example of a marine natural material. A priority should be invasive bivalves, given the role of byssal attachment in the spread of, and ecological and economic damage caused by zebra mussels, quagga mussels and others. This article is part of the Theo Murphy meeting issue 'Molluscan genomics: broad insights and future directions for a neglected phylum'.

Background: The Mediterranean mussel Mytilus galloprovincialis is an ecologically and economically relevant edible marine bivalve, highly invasive and resilient to biotic and abiotic stressors causing recurrent massive mortalities in other bivalves. Although these traits have been recently linked with the maintenance of a high genetic variation within natural populations, the factors underlying the evolutionary success of this species remain unclear., Results: Here, after the assembly of a 1.28-Gb reference genome and the resequencing of 14 individuals from two independent populations, we reveal a complex pan-genomic architecture in M. galloprovincialis, with a core set of 45,000 genes plus a strikingly high number of dispensable genes (20,000) subject to presence-absence variation, which may be entirely missing in several individuals. We show that dispensable genes are associated with hemizygous genomic regions affected by structural variants, which overall account for nearly 580 Mb of DNA sequence not included in the reference genome assembly. As such, this is the first study to report the widespread occurrence of gene presence-absence variation at a whole-genome scale in the animal kingdom., Conclusions: Dispensable genes usually belong to young and recently expanded gene families enriched in survival functions, which might be the key to explain the resilience and invasiveness of this species. This unique pan-genome architecture is characterized by dispensable genes in accessory genomic regions that exceed by orders of magnitude those observed in other metazoans, including humans, and closely mirror the open pan-genomes found in prokaryotes and in a few non-metazoan eukaryotes.

An L-shaped shell deformity (LSSD) on the posterior shell edge is known exclusively in wild mytilid mussels infected with photosynthetic Coccomyxa-like algae. LSSD forms due to the appearance of extra shell material; it only occurs if the mussel is heavily infected with the alga. Traditionally, observation of high amount of the green spots (algal colonies) on a large area of host soft tissues (most of the mantle and in adductor muscle) has been used to indicate a high infection rate. We examined 300 Mytilus spp. (100 small, 20-30 mm; 200 large, 40-60 mm) with a high degree of LSSD (parameter "d" > 5 mm) from the Lower St. Lawrence Estuary (Québec, Canada). Green spots were absent in two large mussels, and were only present along the mantle posterior edge in 14 large mussels; other individuals had high infection levels. Our observations suggest that some individuals could be in a state of remission, or, even more optimistically - mussels may be able to resist the pathogen. LSSD is the stable through-time marker for detection of mytilid mussels that are or were infected with Coccomyxa algae, and, thus, may provide information for the study of mussel immunity and control of alga distribution/migration in coastal waters worldwide., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Changes in the cell type composition of the digestive gland epithelium constitute a common and recognized biological response to stress in mussels. Usually, these changes are identified as alterations in the relative proportion of basophilic cells, determined in tissue sections stained with hematoxylin-eosin (H&E) and measured in terms of volume density of basophilic cells (Vv BAS ) after stereological quantification. However, the identification and discrimination of basophilic cells may be a difficult issue, even for a trained operator, especially when, in circumstances of environmental stress, basophilic cells lose their basophilia and the perinuclear area of digestive cells gains basophilia. Thus, the present study was aimed at exploring the best available practices (BAPs) to identify and discriminate basophilic cells on tissue sections of mussel digestive gland. In a first step, a thorough screening of potentially suitable staining methods was carried out; the final selection included several trichrome staining methods and some of their variants, as well as toluidine-based stains. Next, the sample processing (fixation/dehydration steps) was optimized. Toluidine-eosin (T&E) staining after fixation in 4% formaldehyde at 4 °C for 24 h was considered the BAP to identify and discriminate basophilic cells in the digestive gland of mussels. Using the mussel Mytilus galloprovincialis as a target organism, this approach was successfully applied to quantify Vv BAS values after automated image analysis and compared with the conventional H&E staining in different field and laboratory tests. It is worth noting that Vv BAS values were always higher after T&E staining than after H&E staining, apparently because discrimination of basophilic cells was enhanced. Thus, until more data are available, any comparison with Vv BAS values obtained in previous studies using H&E staining must be done cautiously. Finally, the T&E staining was successfully used to discriminate basophilic cells in tissue sections of other marine molluscs of ecotoxicological interest, including Mytilus edulis, Mytilus trossulus, Crassostrea gigas and Littorina littorea., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Mytilus galloprovincialis female specimens were collected from two mussel farms located in two sites next to Castel dell'Ovo, a historical complex located in the Naples Bay. Such sites were named, respectively, A-area and B-area for the different microbiological parameters so that mussels from A-area can be sold without purification, whereas mussels from B-area must be purified before sale. The mussels were collected during the nonreproductive (summer 2009) and reproductive periods (autumn 2009). Gonadosomatic index, structural organization of the ovary, presence of apoptosis, estrogen receptors expression, as well as the bisphenol A (BPA) content in the ovaries, were evaluated. Ovaries from specimens collected in area B showed a different and significant distribution of the investigated biomarkers as well as of BPA content in respect to those measured in the A-area specimens, confirming that mussels are valid sentinel organisms to biomonitor in the Naples bay too., (© 2018 Wiley Periodicals, Inc.)

Robust ecological forecasting requires accurate predictions of physiological responses to environmental drivers. Energy budget models facilitate this by mechanistically linking biology to abiotic drivers, but are usually ground-truthed under relatively stable physical conditions, omitting temporal/spatial environmental variability. Dynamic Energy Budget (DEB) theory is a powerful framework capable of linking individual fitness to environmental drivers and we tested its ability to accommodate variability by examining model predictions across the rocky shore, a steep ecotone characterized by wide fluctuations in temperature and food availability. We parameterized DEB models for co-existing mid/high-shore (Mytilus galloprovincialis) and mid/low-shore (Perna perna) mussels on the south coast of South Africa. First, we assumed permanently submerged conditions, and then incorporated metabolic depression under low tide conditions, using detailed data of tidal cycles, body temperature and variability in food over 12 months at three sites. Models provided good estimates of shell length for both species across the shore, but predictions of gonadosomatic index were consistently lower than observed. Model disagreement could reflect the effects of details of biology and/or difficulties in capturing environmental variability, emphasising the need to incorporate both. Our approach provides guidelines for incorporating environmental variability and long-term change into mechanistic models to improve ecological predictions.

Determining the magnitude and causes of intrinsic variability is a main issue in the analysis of bivalve growth. Inter-individual variability in bivalve growth has been attributed to differences in the physiological performance. This hypothesis has been commonly tested comparing the physiological rates of fast and slow growers after size differentiation has occurred. This experimental design may detect a link between growth and physiological performance, but we cannot interpret the posterior physiological performance as a driver for the prior growth variability. Considering these limitations, this work introduces a new methodological framework for the analysis of bivalve growth variability. We have conducted sequential measurements of size and physiological performance (feeding, digestion and metabolic rates) in even-sized mussels growing under homogeneous environmental conditions. This experimental design allows us to distinguish between changes over time within individuals, i.e. growth and trends in the physiological rates, from differences between individuals with respect to a baseline level. In addition, Functional Data Analysis provides powerful tools to summarize all the information obtained in the exhaustive sampling scheme and to test whether differences in the physiological performance enhance growth dispersion. Our results report an increasing dispersion in both size and physiological performance over time. Although mussels grew during the experiment, it is difficult to detect any increasing or decreasing temporal pattern in their feeding, digestion and metabolic rates due to the large inter-individual variability. Comparison between the growth and physiological patterns of mussels with final size above (fast growers) and below (slow growers) the median found that fast growers had larger feeding and digestion rates and lower metabolic expenditures during the experimental culture than mussels with slow growth, which agrees with the hypothesis of a physiological basis for bivalve growth variability., Competing Interests: The authors have declared that no competing interests exist.

In vivo confocal Raman microscopy (CRM), polarized light microscopy and Fourier transform infrared spectroscopy (FTIR) were used to determine if a significant amount of amorphous calcium carbonate (ACC) exists within larval shells of Baltic mytilid mussels ( Mytilus edulis -like) and whether the amount of ACC varies during larval development. No evidence for ACC was found from the onset of shell deposition at 21 h post-fertilization (hpf) until 48 hpf. Larval Mytilus shells were crystalline from 21 hpf onwards and exhibited CRM and FTIR peaks characteristic of aragonite. Prior to shell deposition at 21 hpf, no evidence for carbonates was observed through in vivo CRM. We further analysed the composition of larval shells in three other bivalve species, Mercenaria mercenaria , Crassostrea gigas and Crassostrea virginica and observed no evidence for ACC, which is in contrast to previous work on the same species. Our findings indicate that larval bivalve shells are composed of crystalline aragonite and we demonstrate that conflicting results are related to sub-optimal measurements and misinterpretation of CRM spectra. Our results demonstrate that the common perception that ACC generally occurs as a stable and abundant precursor during larval bivalve calcification needs to be critically reviewed., (© 2018 The Author(s).)

A α-carbonic anhydrase (CA, EC 4.2.1.1) has been purified and characterized biochemically from the mollusk Mytilus galloprovincialis. As in most mollusks, this α-CA is involved in the biomineralization processes leading to the precipitation of calcium carbonate in the mussel shell. The new enzyme had a molecular weight of 50 kDa, which is roughly two times higher than that of a monomeric α-class enzyme. Thus, Mytilus galloprovincialis α-CA is either a dimer, or similar to the Tridacna gigas CA described earlier, may have two different CA domains in its polypeptide chain. The Mytilus galloprovincialis α-CA sequence contained the three His residues acting as zinc ligands and the gate-keeper residues present in all α-CAs (Glu106-Thr199), but had a Lys in position 64 and not a His as proton shuttling residue, being thus similar to the human isoform hCA III. This probably explains the relatively low catalytic activity of Mytilus galloprovincialis α-CA, with the following kinetic parameters for the CO 2 hydration reaction: k cat = 4.1 × 10 5 s -1 and k cat /K m of 3.6 × 10 7 M -1 × s -1 . The enzyme activity was poorly inhibited by the sulfonamide acetazolamide, with a K I of 380 nM. This study is one of the few describing in detail the biochemical characterization of a molluskan CA and may be useful for understanding in detail the phylogeny of these enzymes, their role in biocalcification processes and their potential use in the biomimetic capture of the CO 2 .

Background: Mytilisepta virgata is a marine mussel commonly found along the coasts of Japan. Although this species has been the subject of occasional studies concerning its ecological role, growth and reproduction, it has been so far almost completely neglected from a genetic and molecular point of view. In the present study we present a high quality de novo assembled transcriptome of the Japanese purplish mussel, which represents the first publicly available collection of expressed sequences for this species., Results: The assembled transcriptome comprises almost 50,000 contigs, with a N50 statistics of ~1 kilobase and a high estimated completeness based on the rate of BUSCOs identified, standing as one of the most exhaustive sequence resources available for mytiloid bivalves to date. Overall this data, accompanied by gene expression profiles from gills, digestive gland, mantle rim, foot and posterior adductor muscle, presents an accurate snapshot of the great functional specialization of these five tissues in adult mussels., Conclusions: We highlight that one of the most striking features of the M. virgata transcriptome is the high abundance and diversification of lectin-like transcripts, which pertain to different gene families and appear to be expressed in particular in the digestive gland and in the gills. Therefore, these two tissues might be selected as preferential targets for the isolation of molecules with interesting carbohydrate-binding properties. In addition, by molecular phylogenomics, we provide solid evidence in support of the classification of M. virgata within the Brachidontinae subfamily. This result is in agreement with the previously proposed hypothesis that the morphological features traditionally used to group Mytilisepta spp. and Septifer spp. within the same clade are inappropriate due to homoplasy.

Investigations on asymmetries showed that deviations from perfect bilateral symmetry are interpreted as environmental changes inducing developmental instability. Since morphological abnormalities increase with pollution, deformations may be considered indicators of the organism exposition to pollution. Therefore, the onset of asymmetry in otherwise normally symmetrical traits has been used as a measure of some stresses as well. In this context, we studied how marine pollution affects the valve morphological alterations in the mussel Mytilus galloprovincialis. We used 180 specimens (30 per site) from the aquaculture area of Goro (River Po delta, northern Adriatic Sea), translocated, and released within 50 × 50 × 50 cm cages in five sites: two disturbed and one undisturbed near Naples (eastern Tyrrhenian Sea), and one disturbed and one undisturbed near Siracusa (western Ionian Sea). Disturbed sites were stressed by heavy industrialization and heavy tankers traffic of crude and refined oil, and were defined basing on sediment contamination. In particular, by the cone-beam computed tomography we obtained 3D virtual valve surfaces to be analyzed by the geometric morphometric techniques. Specifically, we focused the levels of the shell shape fluctuating asymmetry in relation to the degrees of marine pollution in different sites of the Tyrrhenian Sea. The Mahalanobis distances (interpreted as proxy of the individual shape asymmetry deviation from the mean asymmetry) significantly regressed with the sediment contamination gradient. Indeed, although the left-right differences were normally distributed in each studied site, the individual asymmetry scores (IAS) significantly varied amongst the investigated sites. IAS showed higher values in disturbed areas than those of undisturbed ones in both Tyrrhenian and Ionian Sea. Our results are consistent with past studies on molluscans and other taxa, demonstrating some detrimental effects of chemicals on organisms, although the investigated morphological marker did not discriminate the real disturbance source. Our findings indicate that the mussels act as a prognostic tool for sea pollution levels driving detrimental effects on benthic community.

Dense aggregations of foundation species often mitigate environmental stresses for organisms living among them. Considerable work documents such benefits by comparing conditions inside versus outside these biogenic habitats. However, environmental gradients commonly arise across the extent of even single patches of habitat-forming species, including cases where stresses diverge between habitat interiors and edges. We ask here whether such edge effects could alter how habitat-forming species influence residents, potentially changing the strength or direction of interactions (i.e., from stress amelioration to exacerbation). We take as a model system the classic marine foundation species, Mytilus californianus, the California mussel. Results demonstrate that mussel beds both increase and decrease thermal stresses. Over a distance of 6 to 10 cm from the bed interior to its upper surface, peak temperatures climb from as much as 20 °C below to 5 °C above those of adjacent bedrock. This directional shift in temperature modification affects interactions with juvenile mussels, such that thermal stresses and associated mortality risk are higher at the bed surface, but substantially reduced deeper within the adult matrix. These findings provide a case example of how stress gradients generated across biogenic habitats can markedly alter ecological interactions even within a single habitat patch.

Unlabelled: Mussels attach to various submerged surfaces by using the byssus, which contains different proteins and is a promising source of water-resistant bio-adhesives for potential use in biotechnological and medical applications. The protein composition of the byssus has not yet been fully understood although at least eleven byssal proteins were characterized previously. In order to increase genomic resources and identify new byssal proteins from mussel Mytilus coruscus, high-throughput Illumina sequencing was undertaken on the foot, and 79,997,776 paired-ends reads were generated, yielding a library containing 88,825ft unigenes. The M. coruscus byssus was divided into three parts, the proximal thread, the distal thread, and the plaque. Byssal proteins from each part of the byssus were analyzed by shotgun-LTQ analysis. The MS/MS spectra were searched against the foot unigenes dataset and 48 byssal proteins were identified from the M. coruscus byssus. From the whole set, 17, 5, and 11 proteins were exclusive to the proximal thread, the distal thread, and the plaque, respectively. These data can be used as a resource for further studies on the roles of byssal proteins in the deposition of different byssus parts (thread vs. plaque) or in the different mechanical properties (tenacity vs. adhesion)., Biological Significance: Byssal proteins are the major component that controls different aspects of the byssal formation process and thus a source of bioactive molecules that would offer interesting perspectives in biomaterials and bio-adhesive fields. In this paper, we characterized the protein set from different partsof Mytilus coruscus byssus by a combination of transcriptome/proteome technical. A whole set of 48 byssal proteins were described here, including proteins of collagen-like, C1q domain-containing, protease inhibitor-like, tyrosinase-like, SOD, and others. Thread (the distal portion and the proximal portion) and plaque showed distinct protein composition. Of the whole byssal protein set, 11 are exclusive to the plaque, 17 are exclusive to the proximal thread, and 5 are exclusive to the distal thread. Only four proteins are shared by all the three parts of the byssus. The new byssal proteins reported here represent a significant expansion of the knowledge base of Mytilus byssal proteins, and are important for further exploring the mechanism of adhesion in mussel., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Two blue mussel lineages of Pliocene origin, Mytilus edulis (ME) and M. trossulus (MT), co-occur and hybridize in several regions on the shores of the North Atlantic. The two species were distinguished from each other by molecular methods in the 1980s, and a large amount of comparative data on them has been accumulated since that time. However, while ME and MT are now routinely distinguished by various genetic markers, they tend to be overlooked in ecological studies since morphological characters for taxonomic identification have been lacking, and no consistent habitat differences between lineages have been reported. Surveying a recently discovered area of ME and MT co-occurrence in the White Sea and employing a set of allozyme markers for identification, we address the issue whether ME and MT are true biological species with distinct ecological characteristics or just virtual genetic entities with no matching morphological and ecological identities. We find that: (1) in the White Sea, the occurrence of MT is largely concentrated in harbors, in line with observations from other subarctic regions of Europe; (2) mixed populations of ME and MT are always dominated by purebred individuals, animals classified as hybrids constituting only ca. 18%; (3) in terms of shell morphology, 80% of MT bear a distinct uninterrupted dark prismatic strip under the ligament while 97% of ME lack this character; (4) at sites of sympatry MT is more common on algal substrates while ME mostly lives directly on the bottom. This segregation by the substrate may contribute to maintaining reproductive isolation and decreasing competition between taxa. We conclude that while ME and MT are not fully reproductively isolated, they do represent clearly distinguishable biological, ecological and morphological entities in the White Sea. It remains to be documented whether the observed morphological and ecological differences are of a local character, or whether they have simply been overlooked in other contact zones.

Mytilus californianus is a foundation species of rocky shores of western North America. Its dominance depends on rapid growth to large sizes, which confers an advantage in size-dependent species interactions. Initial rates of growth and final (terminal) sizes of the mussels depend on environmental factors. Prior comparisons of growth made over large spatial scales (tens of meters to hundreds of kilometers) indicate that temperature, submergence time, and wave exposure affect growth. However, there are few studies quantifying variation in temperature, wave force, and mussel growth parameters at small scales within local populations-that is, meter-level increments. Such measures are necessary to better understand the consequences of the complex spatial mosaic of physical factors in the intertidal zone. We measured variation in temperature, wave force, size-specific shell growth, and terminal size at 3-4-m intervals along horizontal contours within two mussel beds separated by 15 ds of latitude. Both mussel beds showed the same general trends: growth rates attenuated along gradual clines from low and wave-exposed to high shore and sheltered. For example, young adults from low and wave-exposed microhabitats grew 9- and 6-fold higher than those from high-shore-wave-sheltered points. While higher flow may promote growth by enhancing feeding, it also appears to exert a positive effect by moderating energetically costly temperature stress. Consistent with the growth rate findings, cumulative degree-hours explained 83% and 69% of the variation of terminal sizes in regressions for the two locations., (© 2015 Marine Biological Laboratory.)

In this paper, we consider the problem of testing nonequivalence of several independent normal population means. It is a well-known problem to test the equality of several means using the analysis of variance (ANOVA). Instead of determining the equality, one may consider more flexible homogeneity, which allows a predetermined level of difference. This problem is known as testing nonequivalence of populations. We propose the plug-in statistics for two different measures of variability: the sum of the absolute deviations and the maximum of the absolute deviations. For each test, the least favorable configuration (LFC) to ensure the maximum rejection probability under the null hypothesis is investigated. Furthermore, we demonstrate the numerical studies based on both simulation and real data to evaluate the plug-in tests and compare these with the range test., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: The Mediterranean mussel Mytilus galloprovincialis is marine bivalve with a relevant commercial importance as well as a key sentinel organism for the biomonitoring of environmental pollution. Here we report the RNA sequencing of the mussel digestive gland, performed with the aim: a) to produce a high quality de novo transcriptome assembly, thus improving the genetic and molecular knowledge of this organism b) to provide an initial assessment of the response to paralytic shellfish poisoning (PSP) on a molecular level, in order to identify possible molecular markers of toxin accumulation., Results: The comprehensive de novo assembly and annotation of the transcriptome yielded a collection of 12,079 non-redundant consensus sequences with an average length of 958 bp, with a high percentage of full-length transcripts. The whole-transcriptome gene expression study indicated that the accumulation of paralytic toxins produced by the dinoflagellate Alexandrium minutum over a time span of 5 days scarcely affected gene expression, but the results need further validation with a greater number of biological samples and naturally contaminated specimens., Conclusion: The digestive gland reference transcriptome we produced significantly improves the data collected from previous sequencing efforts and provides a basic resource for expanding functional genomics investigations in M. galloprovincialis. Although not conclusive, the results of the RNA-seq gene expression analysis support the classification of mussels as bivalves refractory to paralytic shellfish poisoning and point out that the identification molecular biomarkers of PSP in the digestive gland of this organism is problematic.

We investigated the friction and wear behavior as well as the mechanical properties of the periostracum of Mytilus sp. Tribological properties were determined with a reciprocal sliding microtribometer, while mechanical characterization was performed using a nanoindenter. Measurements were performed in dry and wet conditions. On the dry periostracum we found a low friction coefficient of 0.078±0.007 on the young parts and a higher one of 0.63±0.02 on the old parts of the shell. Under wet, saline, conditions we only observed one average coefficient of friction of 0.37±0.01. Microscopic ex situ analysis indicated that dry periostracum wore rather rapidly by plowing and fatigue, while it exhibited a high wear resistance when immersed in salt water. The Young's modulus and hardness of the periostracum were also investigated in both dry and wet conditions. Under dry conditions the Young's modulus of the periostracum was 8±3GPa, while under wet conditions it was 0.21±0.05GPa. The hardness of dry periostracum samples was 353±127MPa, whereas the hardness of wet samples was 5±2MPa. It was found that, in the wet state, viscous behavior plays a significant role in the mechanical response of the periostracum. Our results strongly indicate that the periostracum can provide an important contribution to the overall wear resistance of Mytilus sp. shell., (Copyright © 2014. Published by Elsevier Ltd.)

The structure, organic matrix, and mineral structure of the scar (the interface between the adductor muscle and the shell) in Mytilus galloprovincialis were investigated. The scar was found to be a hierarchically multilayered structure composed of organic matrix and structurally different minerals. Different from the aragonite structure of the nacre, we have identified the top layer of the scar to contain structurally organized columnar calcite. This is the first report on calcite-containing scar. Study of the organic matrix showed that there was at least one protein that seemed to be preferentially localized in this columnar layer. Since the scar is the most important stress distribution site in the mussel, the function of the columnar structure and the matrix protein was discussed in relation to a similar structure at the tendon-bone connection site.

Coping with environmental stress may involve combinations of behavioral and physiological responses. We examined potential interactions between adult mussels' simple behavioral repertoire - opening/closing of the shell valves - and thermal stress physiology in common-gardened individuals of three Mytilus congeners found on the West Coast of North America: two native species (M. californianus and M. trossulus) and one invasive species from the Mediterranean (M. galloprovincialis). We first continuously monitored valve behavior over three consecutive days on which body temperatures were gradually increased, either in air or in seawater. A temperature threshold effect was evident between 25 and 33°C in several behavioral measures. Mussels tended to spend much less time with the valves in a sealed position following exposure to 33°C body temperature, especially when exposed in air. This behavior could not be explained by decreases in adductor muscle glycogen (stores of this metabolic fuel actually increased in some scenarios), impacts of forced valve sealing on long-term survival (none observed in a second experiment), or loss of contractile function in the adductor muscles (individuals exhibited as many or more valve adduction movements following elevated body temperature compared with controls). We hypothesize that this reduced propensity to seal the valves following thermal extremes represents avoidance of hypoxia-reoxygenation cycles and concomitant oxidative stress. We further conjecture that prolonged valve gaping following episodes of elevated body temperature may have important ecological consequences by affecting species interactions. We then examined survival over a 90 day period following exposure to elevated body temperature and/or emersion, observing ongoing mortality throughout this monitoring period. Survival varied significantly among species (M. trossulus had the lowest survival) and among experimental contexts (survival was lowest after experiencing elevated body temperature in seawater). Surprisingly, we observed no cumulative impact on survival of 3 days relative to 1 day of exposure to elevated body temperature. The delayed mortality and context-specific outcomes we observed have important implications for the design of future experiments and for interpretation of field distribution patterns of these species. Ultimately, variation in the catalog of physiological and behavioral capacities among closely related or sympatric species is likely to complicate prediction of the ecological consequences of global change and species invasions.

Hexavalent chromium Cr(VI) is an important contaminant released from both domestic and industrial effluents, and represents the predominant chemical form of the metal in aquatic ecosystems. In the marine bivalve Mytilus galloprovincialis exposure to non-toxic, environmentally relevant concentrations of Cr(VI) was shown to modulate functional parameters and gene expression in both the digestive gland and hemocytes. In this work, the effects of exposure to Cr(VI) (0.1-1-10 μg L(-1) animal(-1) for 96 h) in mussel gills were investigated. Gill morphology and immunolocalization of GSH-transferase (GST), of components involved in cholinergic (AChE and ChAT), adrenergic (TH) and serotoninergic (5-HT(3) receptor) systems, regulating gill motility, were evaluated. Total glutathione content, activities of GSH-related enzymes (glutathione reductase - GSR, GST), of catalase, and of key glycolytic enzymes (phosphofructokinase - PFK and pyruvate kinase - PK) were determined. Moreover, mRNA expression of selected Mytilus genes (GST-π, metallothionein isoforms MT10 and MT20, HSP70 and 5-HT receptor) was assessed by RT-q-PCR. Cr(VI) exposure induced progressive changes in gill morphology and in immunoreactivity to components involved in neurotransmission that were particularly evident at the highest concentration tested, and associated with large metal accumulation. Cr(VI) increased the activities of GST and GSR, and total glutathione content to a different extent at different metal concentrations, this suggesting Cr(VI) detoxication/reduction at the site of metal entry. Cr(VI) exposure also increased the activity of glycolytic enzymes, indicating modulation of carbohydrate metabolism. Significant changes in transcription of different genes were observed. In particular, the mRNA level for the 5-HTR was increased, whereas both decreases and increases were observed for GST-π, MT10, MT20 and HSP70 mRNAs, showing sex- and concentration-related differences. The results demonstrate that Cr(VI) significantly affected functional and molecular parameters in mussel gills, and indicate that this tissue represents the major target of exposure to environmentally relevant concentrations of the metal., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The present study investigates the histochemical localization of N-acetyl-β-hexozaminidase (Hex), acid phosphatase (AcP) and β-glucuronidase (β-Gus) in the digestive gland of mussels Mytilus galloprovincialis, as well as the clarification of suitable enzyme for biomarkers' application dealing with lysosomes. The results show more intense and homogenous localization of Hex, in relation to AcP and β-Gus and, thus, Hex histochemistry is supported as more suitable procedure for the evaluation of "lysosomal membrane stability" and "morphometrical alterations of lysosomes". The affection of lipofuscin granules on lysosomal enzymes' activity is also discussed. Additionally, the present study examines the response of small- and large-sized mussels M. galloprovincialis by assessing the "lysosomal membrane stability", "morphometrical alterations of lysosomes", "lysosomal response index (LRI)" and "structural epithelial changes in digestive tubules". The results indicate appreciable alterations of the above parameters in large-sized mussels, supporting their greater influence by the environmental factors, in relation to small-sized ones., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

This study aimed to estimate the heritabilities of three economically important traits (total weight, shell shape and meat yield) in Australian blue mussels. The estimates were derived using a pedigree reconstructed from a suite of both published and newly developed microsatellite markers. A total of 135 microsatellite loci were tested, of which 10 loci produced consistent PCR amplification and reliable results across all samples (74 full-sibling families including 74 pairs of parents and 2536 offspring). Lack of polymorphism at the non-repetitive region of the adhesive protein gene confirmed that the broodstock were derived from a single species. A total of 1538 progenies (62.5%) could be assigned to single parent pairs, and the remainder were assigned to two families or more, so were discarded from further analysis. Heritabilities for total weight, shell shape and meat yield were low (0.051 ± 0.027, 0.085 ± 0.038 and 0.049 ± 0.028, respectively) but reflected large environmental variation rather than limited genetic variation, suggesting a family-based breeding programme could improve these traits. The genetic correlation between weight and meat yield, expressed as percentage of total mussel which was not shell, was negative, while the genetic correlation between meat yield and shell shape was weakly positive., (© 2011 Blackwell Verlag GmbH.)

Mytilus coruscus is one of the most important cultured species of marine shellfish in China. Using an expressed sequence tag-library and two microsatellite-enriched genomic libraries of M. coruscus, we isolated and characterized 12 polymorphic microsatellites in a test population; the number of alleles ranged from three to seven, and the observed and expected heterozygosities ranged from 0.0333 to 0.8571 and from 0.3452 to 0.8267, respectively. Four loci deviated from Hardy-Weinberg equilibrium. These polymorphic microsatellite loci can be employed in genetic diversity analysis and molecular marker-assisted breeding of M. coruscus.

Background: Mytilus species are important in marine ecology and in environmental quality assessment, yet their molecular biology is poorly understood. Molecular aspects of their reproduction, hybridisation between species, mitochondrial inheritance, skewed sex ratios of offspring and adaptation to climatic and pollution factors are priority areas., Methodology/principal Findings: To start to address this situation, expressed genetic transcripts from M. galloprovincialis were pyrosequenced. Transcripts were isolated from the digestive gland, foot, gill and mantle of both male and female mussels. In total, 175,547 sequences were obtained and for foot and mantle, 90% of the sequences could be assembled into contiguous fragments but this reduced to 75% for the digestive gland and gill. Transcripts relating to protein metabolism and respiration dominated including ribosomal proteins, cytochrome oxidases and NADH dehydrogenase subunits. Tissue specific variation was identified in transcripts associated with mitochondrial energy metabolism, with the digestive gland and gill having the greatest transcript abundance. Using fragment recruitment it was also possible to identify sites of potential small RNAs involved in mitochondrial transcriptional regulation. Sex ratios based on Vitelline Envelop Receptor for Lysin and Vitelline Coat Lysin transcript abundances, indicated that an equal sex distribution was maintained. Taxonomic profiling of the M. galloprovincialis tissues highlighted an abundant microbial flora associated with the digestive gland. Profiling of the tissues for genes involved in intermediary metabolism demonstrated that the gill and digestive gland were more similar to each other than to the other two tissues, and specifically the foot transcriptome was most dissimilar., Conclusions: Pyrosequencing has provided extensive genomic information for M. galloprovincialis and generated novel observations on expression of different tissues, mitochondria and associated microorganisms. It will also facilitate the much needed production of an oligonucleotide microarray for the organism.

The effect of spawning events of the mussel Mytilus galloprovincialis on both quantitative and qualitative values of byssus secretion and its associated attachment force was investigated. Byssogenesis rates and absorption efficiency values were significantly reduced after spawning of individuals. However, the maintenance of individuals under sub-optimal conditions (lack of microalgae in the diet) for a week caused no effect on thread's number. Surprisingly, the attachment force varied within a narrow range of values (1.7-1.9 N) with the exception of a significant drop in the experimental group spawned and kept unfed (1.0 N; P < 0.001), most likely due to a similar pattern of the thread's thickness variability. Qualitative analysis concerned to the amino acid composition of the byssus highlighted a higher presence of the basic residues histidine and lysine in threads secreted by spawned individuals. The presence of both histidine and lysine residues in the byssal collagen is associated to the formation of cross-links and specifically histidine has a functionality with a pronounced effect on metal chelation to stabilise the integrity of the byssus. Results reported here evidence the necessity to integrate all components that eventually determines the attachment strength of the mussels to get more insight to the plasticity of such secretion. Morphology of the byssus (thickness) secreted under different endogenous conditions of mussels was the major parameter to explain variability in the attachment force. Moreover, aminoacidic composition as quality term of the byssus secreted may also contribute to understand the plasticity of this secretion and needs to be extended in further surveys.

We quantified imposex in file dogwinkles (Nucella lima) and tributyltin (TBT) contamination in bay mussels (Mytilus trossulus) from a number of sites in Auke Bay, Alaska, previously studied in the late 1980s. Imposex occurrence and TBT contamination have generally declined in spatial extent and severity over time. However, high levels of TBT contamination (0.069 microg TBT/g wet tissue wt.) and imposex (100% of females affected) are still present near a large boat docking facility in the bay and deserve continued attention because of the importance of this bay to the local economy and fisheries.

For lasting holdfast attachment, the mussel Mytilus californianus coats its byssal threads with a protective cuticle 2-5 microm thick that is 4-6 times stiffer than the underlying collagen fibers. Although cuticle hardness (0.1 GPa) and stiffness (2 GPa) resemble those observed in related mussels, a more effective dispersion of microdamage enables M. californianus byssal threads to sustain strains to almost 120% before cuticle rupture occurs. Underlying factors for the superior damage tolerance of the byssal cuticle were explored in its microarchitecture and in the cuticular protein, mcfp-1. Cuticle microstructure was distinctly granular, with granule diameters (approximately 200 nm) only a quarter of those in M. galloprovincialis cuticle, for example. Compared with homologous proteins in related mussel species, mcfp-1 from M. californianus had a similar mass (approximately 92 kDa) and number of tandemly repeated decapeptides, and contained the same post-translational modifications, namely, trans-4-hydroxyproline, trans-2,3-cis-3,4-dihydroxyproline, and 3,4-dihydroxyphenylalanine (Dopa). The prominence of isoleucine in mcfp-1, however, distinguished it from homologues in other species. The complete protein sequence deduced from cDNAs for two related variants revealed a highly conserved consensus decapeptide PKISYPPTYK that is repeated 64 times and differs slightly from the consensus peptide (AKPSYPPTYK) of both M. galloprovincialis and M. edulis proteins.

The mussel byssus is a unique holdfast structure employed by marine mussels to colonize diverse substrates. The byssus consists of extracellular threads with mainly proteinaceous components. Individual threads reveal high tensile strength at their distal end and high elasticity in their proximal portion. Our studies show that proteins of the distal part are oriented along the thread axis and are well-ordered with a high beta-structural content. In contrast, proteins of the proximal part are less ordered and are not as well-oriented with primarily alpha-helical structure. The detected differences in the structural features of the proteins along a byssus thread are likely an important basis for its gradual mechanical properties.

The level of the acid DNase activity in the hemocytes and digestive gland of the mussel Mytilus galloprovincialis after exposure to model marine pollutants, a detergent, gasoline and a copper salt, as well as to unknown environmental mixture at selected sampling sites, was investigated. The specific enzyme activity in unexposed mussels from mariculture area was higher in hemocytes than in digestive gland. Concentration and time effect patterns of DNase activity revealed tissue- and pollutant-specific responses to model marine pollutants. Since in some cases the pollutant effect could not be detected by measurement of acid DNase in single tissue only, digestive gland/hemocyte (Hep/Hem) ratio was introduced. The Hep/Hem ratio enabled the detection of pollutant effect at the significance level. Field investigations indicated that the digestive gland is a suitable tissue for discrimination of polluted areas from maricultured area. Additionally, the Hep/Hem ratio enabled differentiation within a group of polluted sampling sites that differ in the type of pollutants and/or environmental conditions.

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.

The marine mussel Mytilus californianus Conrad inhabits the most wave-exposed regions of the rocky intertidal by dint of its extraordinary tenacity. Tenacity is mediated in large part by the byssus, a fibrous holdfast structure. M. californianus byssal threads, which are mechanically superior to the byssal threads of other mytilids, are composed almost entirely of a consortium of three modular proteins known as the preCols. In this study, the complete primary sequence of preCols from M. californianus was deduced and compared to that of two related species with mechanically inferior byssal threads, M. edulis Linnaeus and M. galloprovincialis Lamarck in order to explore structure-function relationships. The preCols from M. californianus are more divergent from the other two species than they are from one another. However, the degree of divergence is not uniform among the various domains of the preCols, allowing us to speculate on their mechanical role. For instance, the extra spider silk-like runs of alanine-rich sequence in the flanking domains of M. californianus may increase crystalline order, enhancing strength and stiffness. Histidine-rich domains at the termini, in contrast, are highly conserved between species, suggesting a mechanical role common to all three. Mechanical testing of pH-treated and chemically derivatized distal threads strongly suggests that histidine side chains are ligands in reversible, metal-mediated cross-links in situ. By combining the mechanical and sequence data, yield and self-healing in the distal region of threads have been modeled to emphasize the intricate interplay of enthalpic and entropic effects during tensile load and recovery.

Background: Chitin self-assembly provides a dynamic extracellular biomineralization interface. The insoluble matrix of larval shells of the marine bivalve mollusc Mytilus galloprovincialis consists of chitinous material that is distributed and structured in relation to characteristic shell features. Mollusc shell chitin is synthesized via a complex transmembrane chitin synthase with an intracellular myosin motor domain., Results: Enzymatic mollusc chitin synthesis was investigated in vivo by using the small-molecule drug NikkomycinZ, a structural analogue to the sugar donor substrate UDP-N-acetyl-D-glucosamine (UDP-GlcNAc). The impact on mollusc shell formation was analyzed by binocular microscopy, polarized light video microscopy in vivo, and scanning electron microscopy data obtained from shell material formed in the presence of NikkomycinZ. The partial inhibition of chitin synthesis in vivo during larval development by NikkomycinZ (5 microM - 10 microM) dramatically alters the structure and thus the functionality of the larval shell at various growth fronts, such as the bivalve hinge and the shell's edges., Conclusion: Provided that NikkomycinZ mainly affects chitin synthesis in molluscs, the presented data suggest that the mollusc chitin synthase fulfils an important enzymatic role in the coordinated formation of larval bivalve shells. It can be speculated that chitin synthesis bears the potential to contribute via signal transduction pathways to the implementation of hierarchical patterns into chitin mineral-composites such as prismatic, nacre, and crossed-lamellar shell types.

The distribution of urocortin (UCN)-like material is investigated in the bivalve mollusc Mytilus galloprovincialis. Immunocytochemical data demonstrate that UCN-like molecules are present in ganglionic neurons, microglial cells and immunocytes. Moreover, a co-localization of UCN- and corticotrophin-releasing hormone (CRH)-like molecules is found in microglial cells and in immunocytes, but not in neurons. Following high salinity-stress experiments, immunoreactivity for UCN and CRH increased in ganglionic neurons and immunocytes. Our findings extend the number of molecules potentially used by molluscan immunocytes to confront stress situations and strengthen the idea of functional conservation of stress-related molecules during evolution.

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.

Maternal investment is a fundamentally important parameter in life-history theory and models, yet the scales at which it varies (among individuals vs. among populations) is rarely reported. In this study, variability in attributes of eggs and early larvae of Mytilus californianus was examined from four sites spanning Point Conception, California, in June and September 2001. The effects of female, site, and month were examined for the following variables: egg volume (microl), egg energy content (microg carbon per egg), and initial larval size (microm). The only significant effect on both egg traits was that of female. Females differed by up to 57% in mean egg volume and 116% in mean egg energetic content. Although there were significant effects of rearing environment, female, site, and month on initial larval size, variability in larval length was small compared to the egg traits. Mean larval length was maximally 11% different among females. Neither female body weight nor length was correlated to mean offspring traits, and there were also no significant relationships between egg traits and initial larval size. The primary source of variation in maternal investment in this system appears to be among individual females rather than over space or time.

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.

Variability of taurine (2-aminoethane sulfonic acid) was studied as a function of size in the mussel Mytilus galloprovincialis and tissue specificity. Isometric and/or allometric relationships were established with regard to total soft mass of the mussels between 20 and 60 mm shell length. Relative amounts of taurine dropped significantly with increasing mass of whole soft tissues with an allometric coefficient value of -0.15. The inverse relationship between taurine and increasing size of mussels was confirmed for gill epithelium and labial palp (allometric coefficient values of -0.16 and -0.10, respectively), tissues that, in turn, represented isometric functions with regard to total soft mass. Although relative amounts of taurine were significantly different in digestive gland, mantle and foot, relationships with increasing size of mussels remained unchanged in these tissues. Gill area of mussels was related to soft mass with an allometric coefficient of 0.70 by 2D Image Analysis, but increased to 0.85 when introducing a third dimension, i.e. gill thickness. Results are discussed according to gill structure analysis and taurine functionality.

During the EU project BEEP a battery of biomarkers was applied in flounder (Platichthys flesus) and the blue mussel (Mytilus edulis) collected at three locations off the Lithuanian coast (Baltic Sea) in June and September 2001 and 2002. The elevated biomarker responses in specimens sampled in September 2001 were apparently related to the extensive dredging activities in the Klaipeda port area and subsequent dumping of contaminated sediments. High concentrations of organic pollutants (organochlorines and PBDEs) were also measured in the tissues of both indicator species. In addition, response levels of genotoxicity, cytotoxicity, immunotoxicity as well as concentrations of PAH metabolites in the bile of flounder showed elevations in 2002 after an oil spill in the Būtinge oil terminal in November 2001. In flounder, biomarker measurements 10 months after the spill indicated recovery processes but in mussels a high level of genotoxicity could still be observed 22 months later. The present study illustrates the usefulness of the multi-biomarker approach in the detection of biological effects of pollution in this region of the Baltic Sea.

In the framework of the EU funded BEEP project a set of biomarkers, gross morphometric indices and tissue concentrations of selected organic pollutants were measured in flounder (Platichthys flesus) and mussels (Mytilus trossulus) collected twice a year (April and October) from three sites in the inner Gulf of Gdańsk between autumn 2001 and spring 2003. In flounder, seasonal differences in most biomarkers were observed, but no correlations with tissue pollutant levels could be found. In mussels, highly variable levels in biomarker responses were seen, but no clear seasonal or spatial trends, directly related to tissue concentrations, could be established. The observed biomarkers distribution the study sites are probably mostly caused by interannual, seasonal and individual variability and, in case of flounder, possibly by exchange of stocks between the sampling sites.

The byssal thread of a mussel is an extraorganismic connective tissue that exhibits a striking end-to-end gradient in mechanical properties and thus provides a unique opportunity for studying how gradients are made. Mfp-1 (Mytilus foot protein-1) is a conspicuous component of the protective outer cuticle of byssal threads given its high 3,4-dihydroxyphenylalanine (Dopa) content at 10-15 mol %. Amino acid analysis of mfp-1 extracted from successive foot sections of Mytilus galloprovincialis reveals a post-translationally mediated gradient with highest Dopa levels present in mfp-1 from the accessory gland near the tip of the foot decreasing gradually toward the base. The Dopa content of successive segments of byssal threads decreases from the distal to the proximal end and thus reflects the trend of mfp-1 in the foot. Inductively coupled plasma analysis indicates that certain metal ions including iron follow the trend in Dopa along the thread. Energy-dispersive x-ray spectrometry showed that iron, when present, was concentrated in the cuticle of the threads but sparse in the core. The axial iron gradient appears most closely correlated with the Dopa gradient. The direct incubation of mussels and byssal threads in Fe(3+) supplemented seawater showed that byssal threads are unable to sequester iron from the seawater. Instead, particulate/soluble iron is actively taken up by mussels during filter feeding and incorporated into byssal threads during their secretion. Our results suggest that mussels may exploit the interplay between Dopa and metals to tailor the different parts of threads for specific mechanical properties.