Byssus Research Articles

The Widespread Prevalence and Functional Significance of Silk-Like Structural Proteins in Metazoan Biological Materials.

In nature, numerous mechanisms have evolved by which organisms fabricate biological structures with an impressive array of physical characteristics. Some examples of metazoan biological materials include the highly elastic byssal threads by which bivalves attach themselves to rocks, biomineralized structures that form the skeletons of various animals, and spider silks that are renowned for their exceptional strength and elasticity. The remarkable properties of silks, which are perhaps the best studied biological materials, are the result of the highly repetitive, modular, and biased amino acid composition of the proteins that compose them. Interestingly, similar levels of modularity/repetitiveness and similar bias in amino acid compositions have been reported in proteins that are components of structural materials in other organisms, however the exact nature and extent of this similarity, and its functional and evolutionary relevance, is unknown. Here, we investigate this similarity and use sequence features common to silks and other known structural proteins to develop a bioinformatics-based method to identify similar proteins from large-scale transcriptome and whole-genome datasets. We show that a large number of proteins identified using this method have roles in biological material formation throughout the animal kingdom. Despite the similarity in sequence characteristics, most of the silk-like structural proteins (SLSPs) identified in this study appear to have evolved independently and are restricted to a particular animal lineage. Although the exact function of many of these SLSPs is unknown, the apparent independent evolution of proteins with similar sequence characteristics in divergent lineages suggests that these features are important for the assembly of biological materials. The identification of these characteristics enable the generation of testable hypotheses regarding the mechanisms by which these proteins assemble and direct the construction of biological materials with diverse morphologies. The SilkSlider predictor software developed here is available at https://github.com/wwood/SilkSlider.

Sugary interfaces mitigate contact damage where stiff meets soft.

The byssal threads of the fan shell Atrina pectinata are non-living functional materials intimately associated with living tissue, which provide an intriguing paradigm of bionic interface for robust load-bearing device. An interfacial load-bearing protein (A. pectinata foot protein-1, apfp-1) with L-3,4-dihydroxyphenylalanine (DOPA)-containing and mannose-binding domains has been characterized from Atrina's foot. apfp-1 was localized at the interface between stiff byssus and the soft tissue by immunochemical staining and confocal Raman imaging, implying that apfp-1 is an interfacial linker between the byssus and soft tissue, that is, the DOPA-containing domain interacts with itself and other byssal proteins via Fe3+–DOPA complexes, and the mannose-binding domain interacts with the soft tissue and cell membranes. Both DOPA- and sugar-mediated bindings are reversible and robust under wet conditions. This work shows the combination of DOPA and sugar chemistry at asymmetric interfaces is unprecedented and highly relevant to bionic interface design for tissue engineering and bionic devices.

Trophic structure of the fouling community in Odessa Bay (Black Sea)

The trophic structure of the coastal fouling community of Odessa Bay (Black Sea), which was composed of 10 species of macrophytes, 57 invertebrate species and 4 species of fish, was determined. The basic trophic relationship between organisms composing the community is shown. A minimization of interspecific trophic competition within the community is noted. The main sources of food material entering the fouling community were determined. We show that a significant proportion of food in the form of detritus, dissolved organic matter and small planktonic organisms enters the community from the water column. Filtration and pumping activity of sestonophage-organisms, particularly mussels, helps to attract food material to the community. Primary producers of the community are macrophytes and microphytes, which develop on account of their photosynthetic activity and ensure the provision of food to herbivores. The trophic group of detritophages consumes different fractions of the detritus which accumulates in the byssus threads of bivalve molluscs. In this context, mussel druses act as sediment traps, collecting detritus. Numerous polyphages, which are essentially omnivores and do not usually lack food material, were noted in the community. A small group of carnivorous invertebrates, whose representatives actively attack small animals, was identified. The abundance of these species in the community was about 1%, and their biomass less than 0.6%. Fish living in macrophyte weeds are the consumers in the community. We determined that the highest relative abundance (over 36%) in the fouling community was reached by sestonophages and polyphages. We found that the undisputed leader in the relative biomass (over 97%) in the fouling community ofOdessaBaywas the sestonophages (mainly composed of mussels). We determined that the trophic structure index of the community was 0.94, which confirms the significant dominance in biomass of bivalves over other species in the fouling community.

Extraordinary structure and properties of mussel byssus protein fibers

A naturally available single protein fiber that is stiff and strong at one end but at the same time highly flexible with moderate strength at the other end is quite exceptional. Such exceptional protein fibers called byssus threads are produced by mussels. A unique arrangement of collagen proteins along the length of the fibers and a specific amount and distribution of the β-sheet and α-helix regions provide extraordinary properties to byssus threads. Due to the unique configuration of the threads and a distinct adhesive plaque, mussels are able to adhere to substrates and withstand large amounts of external forces. However, significant variations in composition and tensile properties exist between the mussels threads obtained from different species and even along the length of a single byssal thread. Similarly, environmental conditions such as the presence of salt water and chemicals affect the properties of the fibers. Extensive studies have been done to understand the composition, the structure and the properties of the byssal threads. This review provides an insight into the unique structure and properties of the byssal threads and discusses the potential of developing biomimetic materials based on the mussel byssal threads.

Morphology and taxonomy of Isognomon spathulatus (Reeve, 1858), a cryptic bivalve from the mangroves of Thailand.

Isognomon spathulatus (Reeve, 1858) is redescribed based on type material and original collections from Kungkrabaen Bay, Thailand. The species agrees with previously described isognomonids in most conchological and anatomical features, but possesses a suite of diagnostic characters, including a comma-shaped outline of the nacreous border, an uncoiled ventral diverticulum of the stomach, and the thickened mantle lobes with granulated cells. This study is the most comprehensive morphological analysis to date for any species of Isognomonidae Woodring, 1925 (1828). It describes and illustrates a number of previously unrecognized or underutilized anatomical characters of potential phylogenetic significance: the morphology of the byssal threads (cross-sectional shape, plumate rootlets, and the shape of adhesive disks), the presence and extent of the interdemibranchial buttresses, the presence of secretory cells in the central zone of the mantle, the shape of the ventral diverticulum of the gastric chamber, the presence of the typhlosolar guard ridge, and the position of the renal pore. A comparison is made between I. spathulatus and morphologically similar Isognomon ephippium (Linnaeus, 1758) with which it has been previously synonymized. Pearls of both species are described and illustrated. Individuals of I. spathulatus inhabit mangroves, where they attach by byssus to prop roots, typically in parapatry with individuals of I. ephippium that occupy adjacent mudflats. The spacial distribution and diverging adaptive strategies (pertaining to physical stabilization and response to predation) displayed by the two isognomonid species are considered in the light of the ecological speciation theory.

Functional morphology ofCardiomya cleryana(d'Orbigny, 1842) (Bivalvia: Anomalodesmata: Cuspidariidae) from Brazilian waters: new insights into the lifestyle of carnivorous bivalves

Of the more than 800 Recent species of Anomalodesmata only 16 have been studied alive. The Septibranchia, comprising a number of carnivorous bivalve superfamilies, are no exception to this generalization and, until recently, no living member of this group has been studied since the 1980s. Collected from the shallow, shelf seabed off Brazil,Cardiomya cleryanais one of only a few species to have its morphology described in recent years and the first ever anomalodesmatan to be filmed alive. Important anatomical features such as a greatly extensible foot to secure itself in the sediment with a single byssal thread, exhalant siphon inter-tentacular projections and micro-papillae on the surface of the siphonal tentacles are described. Observations on the species’ behaviour have revealed a lifestyle hitherto not recorded for any septibranch, indeed any anomalodesmatan. The anchoring mechanism ofC. cleryanausing an extraordinarily long byssal thread is described for the first time. The life position ofC. cleryanais at 45° to the sediment-water interface whereas other cuspidariids have been considered to orient themselves vertically. An anatomical comparison betweenCardiomyaandBathyneaerahas revealed close affinities. This study thus provides new insights into the morphology and behaviour of the Cuspidariidae.

The role of shore crabs and mussel density in mussel losses at a commercial intertidal mussel plot after seeding

Mussel losses peak after relaying seed on culture plots. The present paper is an attempt to examine the role of shore crab predation and initial mussel density on mussel losses in mussel bottom culture using an intertidal culture plot as a case study. Because of their small size and loose attachment, mussels are particularly vulnerable to predation at this stage, as well as to handling stress and intraspecific competition. In the experimental field plots (1 × 1 m) in the intertidal Oosterschelde, three different densities (1, 5 and 10 kg m−2) of mussel seed are laid, with half of the experimental plots protected from predation by means of exclosures. Duration of the experiment was 5 weeks (August–September 2012) post-seeding. Protection was the major factor accounting for biomass production, followed by mussel density. Loss rates increased with mussel density, both in the exclosures and in the exposed plots. Losses in the exclosures with the lowest density were still 45 %. There are indications that handling stress prior to the start of the experiment played a major role in these losses. At the higher densities in the exclosures, losses increased to 72.1 % and were not significantly different between 5 and 10 kg m−2. About one-third of the total loss (32.6 %) was attributed to shore crab predation. The number of shore crabs observed on the plots did not differ between treatments. Byssal thread development during the experimental period was followed and was found to be a slow process that was insufficient to protect mussels from crab predation at the sheltered experimental location.

Spatial distribution of proteins in the quagga mussel adhesive apparatus

The invasive freshwater mollusc Dreissena bugensis (quagga mussel) sticks to underwater surfaces via a proteinacious ‘anchor’ (byssus), consisting of a series of threads linked to adhesive plaques. This adhesion results in the biofouling of crucial underwater industry infrastructure, yet little is known about the proteins responsible for the adhesion. Here the identification of byssal proteins extracted from freshly secreted byssal material is described. Several new byssal proteins were observed by gel electrophoresis. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to characterize proteins in different regions of the byssus, particularly those localized to the adhesive interface. Byssal plaques and threads contain in common a range of low molecular weight proteins, while several proteins with higher mass were observed only in the plaque. At the adhesive interface, a plaque-specific ~8.1 kDa protein had a relative increase in signal intensity compared to the bulk of the plaque, suggesting it may play a direct role in adhesion.

Development of collagen-byssus protein hydrolysate matrix to improve elasticity in scaffolds for vascular tissue engineering applications

Event Abstract Back to Event Development of collagen-byssus protein hydrolysate matrix to improve elasticity in scaffolds for vascular tissue engineering applications Erica Rosella1, Caroline Loy1, Lucie Levesque1, Bernard Drouin1, 2, Isabelle Marcotte3 and Diego Mantovani1 1 Laval University, Lab. for Biomaterials & Bioengineering (CRC-I), Dept. Min-Met-Materials Engineering & CHU de Quebec, Canada 2 Cegep Garneau, Department of Physics, Canada 3 Université de Québec à Montréal, Department of Chemistry, Canada Type-1 collagen from rat-tail tendon has been widely used as scaffold in the field of tissue engineering. In the form of hydrogel, collagen serves as a matrix that mechanically and biologically supports three-dimensional cell growth and tissue regeneration. Considering their application as scaffold for small caliber engineered blood vessels (d<6 mm), the ability to withstand blood cyclic stress and compliance are crucial requirements. To address the elasticity in the collagen scaffold, we have explored the addition in the collagen hydrogel of byssus protein hydrolysate (BPH)[1] extracted from mussel byssal threads, which show remarkable combination of strength and extensibility. In vitro self-assembly process leading to hydrogel gelation was optimized at pH 10 in 48-multiwell plates. The protocol of collagen gelation at pH 10 was adapted and modified from the work of Achilli et al[2]. Collagen buffer solutions with 101 mM of salt concentration (NaCl and NaOH) was used to solubilize the BPH, and mixed with the collagen solution to reach a final protein concentration of 2.8 mg/mL. The effect of temperature (4 and 22°C) on the gelation process was assessed by turbidimetric measurements. The resulting microstructure and matrix density were observed by scanning electron microscopy (SEM). The swelling ratio of the scaffold was also determined and compression-relaxation tests were carried to investigate the mechanical properties of the scaffold under compression. Figure 1 shows the results of the turbidimetry measurements where the gelation time (t1/2) is defined as the required time to reach half of the final absorbance. The gelation time of collagen-BPH gel is longer (3.4 and 4.5 days) compared to the collagen control which took 4 and 13 hours at respective temperatures of 22°C and 4°C. Comparison of SEM images of collagen and collagen-BPH mixture at 22°C shows a denser matrix of collagen-BPH at the microstructural level (Figure 2). Moreover, collagen-BPH has a lower (58±5%) water uptake ratio than collagen (71±8%), suggesting greater chemical interactions within the gel leading to a more compact matrix. Compression test shows that BPH incorporation increases scaffold relaxation time, which corresponds to the higher residual force. The compressive and tensile modulus also increases by 12±4% compared to collagen control at pH 10. In conclusion, the addition of BPH to collagen hydrogel increases the density and strength of the scaffold, and shows great potential to improve the collagen matrix elasticity. Further work is required to assess the biological properties of this new scaffold. Figure 1. Turbidity measurements for collagen-BPH gels prepared at pH 10 at 4 °C and 22 °C Figure 2. SEM images of collagen control (left) and collagen-BPH (right) samples prepared at pH 10 and 22°C This work was partially funded by NSERC-Canada, FRQ-NT-Quebec, CFI-Canada, and the Centre Québécois sur les Matériaux Fonctionnels (CMQF).

The mechanics of tessellations - bioinspired strategies for fracture resistance.

Faced with a comparatively limited palette of minerals and organic polymers as building materials, evolution has arrived repeatedly on structural solutions that rely on clever geometric arrangements to avoid mechanical trade-offs in stiffness, strength and flexibility. In this tutorial review, we highlight the concept of tessellation, a structural motif that involves periodic soft and hard elements arranged in series and that appears in a vast array of invertebrate and vertebrate animal biomaterials. We start from basic mechanics principles on the effects of material heterogeneities in hypothetical structures, to derive common concepts from a diversity of natural examples of one-, two- and three-dimensional tilings/layerings. We show that the tessellation of a hard, continuous surface - its atomization into discrete elements connected by a softer phase - can theoretically result in maximization of material toughness, with little expense to stiffness or strength. Moreover, the arrangement of soft/flexible and hard/stiff elements into particular geometries can permit surprising functions, such as signal filtering or 'stretch and catch' responses, where the constrained flexibility of systems allows a built-in safety mechanism for ensuring that both compressive and tensile loads are managed well. Our analysis unites examples ranging from exoskeletal materials (fish scales, arthropod cuticle, turtle shell) to endoskeletal materials (bone, shark cartilage, sponge spicules) to attachment devices (mussel byssal threads), from both invertebrate and vertebrate animals, while spotlighting success and potential for bio-inspired manmade applications.

Functional morphology, biology and sexual strategy of the circumboreal, adventitious crypt-building,Crenella decussata(Bivalvia: Mytiloidea: Crenellidae)

The anatomy ofCrenella decussata(Mytiloidea) is described. Individuals of this circumboreal species occupy granular crypts composed of sand grains held in place by mucus. The swollen basal region of the tubule is occupied by an individual, which connects to the sediment surface by two posterior tubes accommodating the inhalant and exhalant streams. There is reduction in musculature and, most importantly, anterior foreshortening of the outer ctenidial demibranchs and loss of the labial palps. This creates an anterior space in the mantle for the initial brooding of fertilized ova by females to the prodissoconch stage. Subsequently, these larvae are transferred to the exhalant tube of the crypt wherein they attach by a single fine byssal thread and are further brooded until the crawl-away juvenile stage is attained. Experimental studies of larval behaviour suggest that parental pheromones sustain the female/offspring bond. Newly hatched individuals responded to parental exhalant water by actively attaching themselves using a byssal thread. This response persisted for 28 days, but not after 55 days when, we suggest, the pheromonal response ceases and offspring are developed sufficiently to take up life in their own nests. Offspring retrieved from parental crypts and fed continuously reached an average shell length of 500 μm after 7.5 months. Brooded offspring thus appear to rely on embryonal energy resources until post-metamorphosis, after which suspension feeding becomes essential for further growth and development before the parental crypt is vacated.

Attachment features of mytilids in ecosystems with mobile substrate:Brachidontes rodrigueziiin San Antonio Bay (Patagonia, Argentina)

The force required to dislodge mussels from the substrate is known as attachment strength. This feature has been mostly studied in mytilids inhabiting the intertidal of consolidated rocky substrates, whereas it has been less studied in sedimentary substrates. The aim of this study was to evaluate the attachment strength and the number of byssal threads ofBrachidontes rodrigueziiin two sites with mobile substrates in San Antonio Bay, Argentina [Punta Verde (PV) and Punta Delgado (PD)]. PV has relatively higher current velocities and coarser grain size than PD. Along coastal line transects at two different levels of the mid-intertidal of each site, the attachment strength was measured. The number of byssal threads in collected mytilids and the weight of the sediment adhered to them were recorded in the lab. The attachment strength, the number of byssal threads and the sediment adhered to the byssus differed significantly between levels and sites. Mytilids from the mid-level of both sites where the density was relatively lower, had a significantly greater number of byssal threads and higher weight of sediment adhered. This study reveals that a high amount of coarse sediment adhered to the byssus affects the attachment strength ofB. rodrigueziiin ecosystems relative to unstable substrates. We underline the importance of mobile substrates in understanding attachment features (attachment strength and byssal threads) of mytilids in this unstable ecosystem.

Pollution of the coastal waters in the Possyet Bay (Peter the Great Bay, Japan Sea) in conditions of current economic activity

Impact of economic activity on environment quality is assessed for the coastal waters of the Possyet Bay (Peter the Great Bay, Japan Sea). Total volume of the sewage water discharged from coastal sources to the bay and total amount of principal pollutants wasting this water body are calculated. Specific index of sewage for the Possyet Bay is estimated as 0.081 g/m 3 that is rather high value. Distribution of petroleum hydrocarbons in the coastal waters is considered: their concentration ranges from 0.025 to 0.065 mg/L. Following to biotesting results, the worst water quality is in the Reid Pallada Bay where the highest inhibitory effect on the byssus thread formation of mussel Crenomytilus grayanus is observed.

Mechanical homeostasis of a DOPA-enriched biological coating from mussels in response to metal variation.

Protein-metal coordination interactions were recently found to function as crucial mechanical cross-links in certain biological materials. Mussels, for example, use Fe ions from the local environment coordinated to DOPA-rich proteins to stiffen the protective cuticle of their anchoring byssal attachment threads. Bioavailability of metal ions in ocean habitats varies significantly owing to natural and anthropogenic inputs on both short and geological spatio-temporal scales leading to large variations in byssal thread metal composition; however, it is not clear how or if this affects thread performance. Here, we demonstrate that in natural environments mussels can opportunistically replace Fe ions in the DOPA coordination complex with V and Al. In vitro removal of the native DOPA-metal complexes with ethylenediaminetetraacetic acid and replacement with either Fe or V does not lead to statistically significant changes in cuticle performance, indicating that each metal ion is equally sufficient as a DOPA cross-linking agent, able to account for nearly 85% of the stiffness and hardness of the material. Notably, replacement with Al ions also leads to full recovery of stiffness, but only 82% recovery of hardness. These findings have important implications for the adaptability of this biological material in a dynamically changing and unpredictable habitat.

Role of Sacrificial Protein-Metal Bond Exchange in Mussel Byssal Thread Self-Healing.

Marine mussels tether to seashore surfaces with byssal threads, proteinaceous fibers that effectively dissipate energy from crashing waves. Protein-metal coordination bonds have been proposed to contribute to the characteristic mechanical and self-healing properties of byssal threads; however, very little is understood about how these cross-links function at the molecular level. In the present study, combined Raman and X-ray absorption spectroscopy (XAS) measurements were employed to confirm the presence of protein-Zn(2+) coordination bonds in the mussel byssus and to monitor transitions in the coordination structure during thread deformation and self-healing. Results indicate that Zn(2+) coordination bonds, primarily mediated via histidine, are ruptured during thread yield and reformed immediately following thread relaxation. Mechanical healing, on the other hand, is correlated with the transition toward shorter coordination bond lengths. Calculation of the healing activation energy suggests that protein-Zn bond exchange provides a primary rate-limiting step during healing.

Byssogenesis in the juvenile pink heelsplitter mussel, Potamilus alatus (Bivalvia: Unionidae).

The North American pink heelsplitter (Potamilus alatus) differs from most freshwater mussels in China by the ability to secrete an ephemeral byssus during its juvenile stage. In the present study, light microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) were used to investigate this ephemeral byssal structure, and amino acid composition was analyzed and compared with that of other species. The results revealed that the byssus consists of a long byssal thread and a few adhesive plaques which are randomly set up along the thread and assembled by petioles. There is a thin but distinctive cuticle with a continuous homogeneous matrix surrounding the byssal thread. Structural variation occurred when the byssal thread was differentially stretched. Four-stranded helical primary fasciculi, which form a stable rope-like structure, become evident after removal of the cuticle. The primary fasciculi consist of bundles of hundreds of parallel secondary fasciculi, each measuring about 5 μm in diameter. All evidence indicates that the byssus of the pink heelsplitter has a significantly different macrostructure and microstructure than the permanent byssus of the marine mussel Mytilus. Byssogenesis ceases when juveniles exceed 30 mm in length, although it varies greatly even among juveniles of similar size. Byssus formation is influenced by substrate type. The unique characteristics of the byssus have important advantages for survival, transition, and aggregation during the early life history. This study not only provides first insight into the structure of the ephemeral byssus and its relationship to freshwater mussel development and growth, but also suggests possibilities for the synthesis of novel biopolymer materials particularly useful in freshwater ecosystems.

Water-worn shell and pebbles in shell middens as proxies of palaeoenvironmental reconstruction, shellfish procurement and their transport: A case study from the West Coast of South Africa

Abstract Geoarchaeological studies today are a vital component of archaeological research. The sedimentary environments of coastal settings in particular are highly dynamic and governed by a variety of factors among which changes in sea level play an important role. The object of such studies often involves the study of sediments deposited by natural factors in overlapping geological and anthropogenic contexts. Archaeomalacological studies conducted on shell middens along the West Coast of South Africa in the last two decades have also identified the presence of additional natural components that became incorporated into archaeological sites through active but inadvertent human agency. These sediments are relatively large particles (2–20 mm) of water-worn shells and water-worn pebbles (WWSP) that became entangled among the byssus threads that rocky shore mussels use for attaching themselves to hard substrate. Prehistoric shellfish foraging and subsequent transport of rocky shore mussels along with their byssus contents to campsites ensured the inclusion of WWSP into archaeological middens. This study shows that WWSP abundances and the proportion of their organic fraction (water worn shell) can be used as proxies for coastal palaeoenvironmental reconstruction and as a complement to archaeomalacological studies for inferring technologies involved in shellfish collection and transport. Conclusions presented here probably apply to other similarly configured shorelines and shell assemblages of comparable age from elsewhere in southern Africa and beyond. Follow-up studies from this sub-region and beyond might confirm and/or qualify the use of WWSP observations in other coastal settings.

Sensitivity of veliger larvae of Mytilus edulis and mussel of various sizes to chlorination

The blue mussel Mytilus edulis is one of the dominant fouling organisms in cooling water systems. In this work, how veliger larvae and different size groups of the mussels responded against chlorine dosage was examined. Veliger larvae mortality was studied at different residual chlorine concentrations (0.05–0.5 mg L−1), and it was found that a chlorine dose of 0.5 mg L−1 is 4 times as effective as 0.05 mg L−1 and twice as effective as 0.1 mg L−1. Mortality of 100% for three size groups (1.4, 14, and 25 mm) and relative physiological activities of two size groups (14 and 25 mm) were observed. The exposure duration for 100% mortality of mussels decreased with the increasing residual chlorine concentration (0.1–4.0 mg L−1). Mussel size was also found to be an important factor, considering that the continuation times for mussel mortality were 28 h for the 1.4 mm and 410 h for the 25 mm size groups. All size groups showed progressive reduction in physiological activities, such as oxygen consumption, foot activity, and byssus thread production with increasing chlorine dose (0.05–1.0 mg L−1); the two data-sets were strongly correlated with each other. The results of this study should be of significance for optimizing the chlorine content, and minimize the environmental threat to industries where mussels are the dominant fouling organisms.

Embryonic and larval development of the invasive biofoulerMytilopsis sallei(Récluz, 1849) (Bivalvia: Dreissenidae)

The marine dreissenid bivalve Mytilopsis sallei is a fouling organism that has invaded habitats outside its original range. Understanding its early development will be useful for early detection in the environment, for species identification in ballast water and for development of control strategies targeted at early life stages, which will help us better manage this important invader. The processes of embryogenesis, shell formation and larval development of M. sallei are described here for the first time by using light and scanning electron microscopy. Released oocytes are 64 mm in diameter. Fertilized eggs were incubated at 27+ 1 8C. The trochophore, with an apical tuft and a prototroch, developed by 6.0+ 2.3 h postfertilization (hpf). At 16.5+ 4.2 hpf, D-shaped veligers with shell length (SL, mean+ SD) of 87.3+ 8.2 mm appeared, each possessing a velum and a calcified shell. At 2–3 d postfertilization (dpf ), the D-shaped veligers developed into umbonate larvae (SL ¼ 111.9+ 10.7 mm), the last obligate freeswimming veliger stage. Pediveligers (SL ¼ 232.8+ 37.1 mm) observed at 6–8 dpf could either swim using their velum or crawl with their foot. Pediveligers settled by secreting byssal threads and metamorphosed to plantigrades (SL ¼ 298.7+ 45.2 mm) 8–10 dpf. It is noteworthy that the larvae of this invasive bivalve are capable of settlement within 10 d. This is the first detailed study of early shell formation of a species of the family Dreissenidae. Shell field invagination appeared during gastrulation, secreting shell material by expanding over both sides in a saddle-shape during the trochophore stage.

Anti-predatory responses of the thick shell mussel Mytilus coruscus exposed to seawater acidification and hypoxia

Ocean acidification and hypoxia, both caused by anthropogenic activities, have showed deleterious impacts on marine animals. However, their combined effect on the mussel's defence to its predator has been poorly understood, which hinders us to understand the prey–predator interaction in marine environment. The thick shell mussel Mytilus coruscus and its predator, the Asian paddle crab Charybdis japonica were exposed to three pH levels (7.3, 7.7, 8.1) at two concentrations of dissolved oxygen (2.0 mg L−1, 6.0 mg L−1) seawater. The anti-predatory responses of mussels, in terms of byssus thread production were analyzed after 72 h exposure. During the experiment, frequency of shedding stalks (mussels shed their byssal stalks to release themselves from attachment and allow locomotion) and number of byssus threads increased with time, were significantly reduced by hypoxia and low pH levels, and some interactions among time, predator, DO and pH were observed. As expected, the presence of the crab induced an anti-predator response in M. coruscus (significant increases in most tested parameters except the byssus thread length). Acidification and hypoxia significantly reduced byssus thread diamter at the end of the experiment, but not the byssus thread length. Cumulative byssus thread length and volume were significantly impaired by hypoxia and acidification. Our results highlight the significance of anti-predatory responses for adult mussel M. coruscus even under a stressful environment in which stress occurs through ocean acidification and hypoxia. By decreasing the strength of byssus attachment, the chance of being dislodged and consumed by crabs is likely increased. Our data suggest that there are changes in byssus production induced by hypoxia and acidification, which may affect predation rates on M. coruscus in the field.