Initial Shell Formation Research Articles

The Increased Expression of an Engrailed to Sustain Shell Formation in Response to Ocean Acidification.

Engrailed is a transcription factor required in numerous species for important developmental steps such as neurogenesis, segment formation, preblastoderm organization, and compartment formation. Recent study has proved that engrailed is also a key gene related to shell formation in marine bivalves. In the present study, the expression pattern of an engrailed gene (Cgengrailed-1) in Pacific oyster Crassostrea gigas under CO2-driven acidification was investigated to understand its possible role in the regulation of shell formation and adaptation to ocean acidification (OA). The open reading frame (ORF) of Cgengrailed-1 was obtained, which was of 690 bp encoding a polypeptide of 229 amino acids with a HOX domain. Phylogenetic analysis indicated that the deduced amino acid sequence of Cgengrailed-1 shared high homology with other engraileds from Drosophila melanogaster, Mizuhopecten yessoensi, and Crassostrea virginica. The mRNA transcripts of Cgengrailed-1 were constitutively expressed in various tissues with the highest expression levels detected in labial palp and mantle, which were 86.83-fold (p < 0.05) and 75.87-fold (p < 0.05) higher than that in hepatopancreas. The mRNA expression of Cgengrailed-1 in mantle decreased dramatically after moderate (pH 7.8) and severe (pH 7.4) acidification treatment (0.75- and 0.15-fold of that in control group, p < 0.05). The results of immunofluorescence assay demonstrated that the expression level of Cgengrailed-1 in the middle fold of mantle increased significantly upon moderate and severe acidification treatment. Moreover, after the oyster larvae received acidification treatment at trochophore stage, the mRNA expression levels of Cgengrailed-1 increased significantly in D-shape larvae stages, which was 3.11- (pH 7.8) and 4.39-fold (pH 7.4) of that in control group (p < 0.05). The whole-mount immunofluorescence assay showed that Cgengrailed-1 was mainly expressed on the margin of shell gland, and the periostracum in trochophore, early D-shape larvae and D-shape larvae in both control and acidification treatment groups, and the intensity of positive signals in early D-shape larvae and D-shape larvae increased dramatically under acidification treatment. These results collectively suggested that the expression of Cgengrailed-1 could be triggered by CO2-driven acidification treatment, which might contribute to induce the initial shell formation in oyster larvae and the formation of periostracum in adult oyster to adapt to the acidifying marine environment.

Ocean acidification inhibits initial shell formation of oyster larvae by suppressing the biosynthesis of serotonin and dopamine

Ocean acidification has severely affected the initial shell formation of marine bivalves during their larval stages. In the present study, it was found that dopamine (DA) content in early D-shape larvae was significantly higher than that in trochophore and D-shape larvae, while the serotonin (5-HT) content in early D-shape larvae and D-shape larvae was obviously higher than that in trochophore. Incubation of trochophore with 5-HT or DA could accelerate the formation of calcified shell, and the treatments with selective antagonists of receptors for 5-HT and DA (Cg5-HTR-1 and CgD1DR-1) obviously inhibited the formation of calcified shells. When oyster larvae were subjected to an experimental acidified treatment (pH 7.4), the biosynthesis of 5-HT and DA was inhibited, while the mRNA expression levels of the components in TGF-β pathway were significantly up-regulated in D-shape larvae. Moreover, the phosphorylation of TIR and the translocation of smad4 were hindered upon acidification treatments, and the expression patterns of chitinase and tyrosinase were completely reverted. These results collectively suggested that monoamine neurotransmitters 5-HT and DA could modulate the initial shell formation in oyster larvae through TGF-β smad pathway by regulating the expression of tyrosinase and chitinase to guarantee the chitin synthesis for shell formation. CO2-induced seawater acidification could suppress the biosynthesis of 5-HT and DA, as well as the activation of TGF-β smad pathway, which would subvert the expression patterns of chitinase and tyrosinase and cause the failure of initial shell formation in oyster early D-shape larvae.

Metabolomic and transcriptomic profiling reveals the alteration of energy metabolism in oyster larvae during initial shell formation and under experimental ocean acidification

Marine bivalves secrete calcified shells to protect their soft bodies from predation and damages, which is of great importance for their survival, and for the safety of the coastal ecosystem. In recent years, larval shell formation of marine bivalves has been severely affected by ocean acidification (OA), and previous study indicated that OA might affect such process by disrupting endogenous energy metabolism. Developmental stages from trochophore to D-shape larvae are extremely important for initial shell formation in oyster since a calcified shell was formed to cover the chitin one. In the present study, metabolomic and transcriptomic approaches were employed to investigate the energy metabolism of oyster larvae during initial shell (prodissoconch I, PDI shell) formation and under experimental OA treatment. Totally 230 chemical compounds were identified from the present dataset, most of which were highly expressed in the “middle” stage (early D-shape larvae) which was critical for PDI shell formation since a calcified shell was formed to cover the chitin one. Several compounds such as glucose, glutarylcarnitine (C5), β-hydroxyisovaleroylcarnitine, 5-methylthioadenosine (MTA), myristoleate (14:1n5) and palmitoleate (16:1n7) were identified, which were involved in energy metabolic processes including amino acid oxidation, glycolysis, pentose phosphate pathway and fatty acid metabolism. In addition, mRNA expressions of genes related to protein metabolism, glycolysis, lipid degradation, calcium transport and organic matrix formation activities were significantly down-regulated upon experimental OA. These results collectively suggested that formation of the initial shell in oyster larvae required endogenous energy coming from amino acid oxidation, glycolysis, pentose phosphate pathway and fatty acid metabolism. These metabolic activities could be severely inhibited by experimental OA, which might alter the allocation of endogenous energy. Insufficient endogenous energy supply then suppressed the mobilization of calcium and resulted in a failure or delay in PDI shell formation.

Assessment of Shell Strength During Solidification in the Mold Cracking Simulator (MCS) Test

To properly model the cracking susceptibility during solidification under continuous casting conditions, it is essential to have accurate data. Such data for the mechanical properties of steel during solidification are scarce if not non-existent. An experimental tool called the Mold Cracking Simulator (MCS) has been used to simulate the initial shell formation under continuous casting conditions. As part of the test, the shell is mechanically subjected to deformation. A mathematical model has been developed to translate the force and elongation measured during the MCS trials into stress–strain components. To test the model and validate the assumptions, two steel grades were tested, a peritectic steel grade and a higher-alloyed grade. The results show that the reproducibility of the test is very good and the stress–strain curves are consistent with the steel composition. Moreover, the metallographic and fractographic analysis of the deformed MCS samples shows that the microstructure is comparable to that of a continuously cast product and the cracks generated are interdendritic, i.e., hot tears.

Gene expression correlated with delay in shell formation in larval Pacific oysters (Crassostrea gigas) exposed to experimental ocean acidification provides insights into shell formation mechanisms

BackgroundDespite recent work to characterize gene expression changes associated with larval development in oysters, the mechanism by which the larval shell is first formed is still largely unknown. In Crassostrea gigas, this shell forms within the first 24 h post fertilization, and it has been demonstrated that changes in water chemistry can cause delays in shell formation, shell deformations and higher mortality rates. In this study, we use the delay in shell formation associated with exposure to CO2-acidified seawater to identify genes correlated with initial shell deposition.ResultsBy fitting linear models to gene expression data in ambient and low aragonite saturation treatments, we are able to isolate 37 annotated genes correlated with initial larval shell formation, which can be categorized into 1) ion transporters, 2) shell matrix proteins and 3) protease inhibitors. Clustering of the gene expression data into co-expression networks further supports the result of the linear models, and also implies an important role of dynein motor proteins as transporters of cellular components during the initial shell formation process.ConclusionsUsing an RNA-Seq approach with high temporal resolution allows us to identify a conceptual model for how oyster larval calcification is initiated. This work provides a foundation for further studies on how genetic variation in these identified genes could affect fitness of oyster populations subjected to future environmental changes, such as ocean acidification.

Numerical analysis of the influence of carbon content on the initial shell of continuous casting slab

ABSTRACTA numerical model for calculation of temperature and thermal stress in initial steel shell in plate and grooved slab continuous casting moulds was established to predict the influence of steel carbon content on the temperature and stress fields. The calculated results show that the temperature non-uniformity and surface stress of the initial shell increase as the steel temperature decreases. Among the steel grades with carbon contents of 0.06, 0.17, 0.45 and 0.80 wt-%, the initial shell surface stress of steel with 0.17 wt-% (peritectic steel) is the highest. In the grooved mould the temperature non-uniformity and surface stress of the initial shell are significantly lower than those in the plate mould. At 1.7 s after initial shell formation, the non-uniformity of shell surface temperature and stress in the grooved mould are four to nine times and three to nine times, respectively, lower than those in the plate mould.

Dynamics of nuclear receptor gene expression during Pacific oyster development.

BackgroundNuclear receptors are a highly conserved set of ligand binding transcription factors, with essential roles regulating aspects of vertebrate and invertebrate biology alike. Current understanding of nuclear receptor regulated gene expression in invertebrates remains sparse, limiting our ability to elucidate gene function and the conservation of developmental processes across phyla. Here, we studied nuclear receptor expression in the early life stages of the Pacific oyster, Crassostrea gigas, to identify at which specific key stages nuclear receptors are expressedResultsWe used quantitative RT-PCR to determine the expression profiles of 34 nuclear receptors, revealing three developmental key stages, during which nuclear receptor expression is dynamically regulated: embryogenesis, mid development from gastrulation to trochophore larva, and late larval development prior to metamorphosis. Clustering of nuclear receptor expression patterns demonstrated that transcriptional regulation was not directly related to gene phylogeny, suggesting closely related genes may have distinct functions. Expression of gene homologs of vertebrate retinoid receptors suggests participation in organogenesis and shell-formation, as they are highly expressed at the gastrulation and trochophore larval initial shell formation stages. The ecdysone receptor homolog showed high expression just before larval settlement, suggesting a potential role in metamorphosis.ConclusionThroughout early oyster development nuclear receptors exhibited highly dynamic expression profiles, which were not confined by gene phylogeny. These results provide fundamental information on the presence of nuclear receptors during key developmental stages, which aids elucidation of their function in the developmental process. This understanding is essential as ligand sensing nuclear receptors can be disrupted by xenobiotics, a mode of action through which anthropogenic environmental pollutants have been found to mediate effects.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-016-0129-6) contains supplementary material, which is available to authorized users.

The Carbonate Chemistry of the “Fattening Line,” Willapa Bay, 2011–2014

Willapa Bay has received a great deal of attention in the context of rising atmospheric CO2 and the concomitant effects of changes in bay carbonate chemistry, referred to as ocean acidification, and the potential effects on the bay’s naturalized Pacific oyster (Crassostrea gigas) population and iconic oyster farming industry. Competing environmental stressors, historical variability in the oyster settlement record, and the absence of adequate historical observations of bay-water carbonate chemistry all conspire to cast confusion regarding ocean acidification as the culprit for recent failures in oyster larval settlement. We present the first measurements of the aqueous CO2 partial pressure (PCO2) and the total dissolved carbonic acid (TCO2) at the “fattening line,” a location in the bay that has been previously identified as optimal for both larval oyster retention and growth, and collocated with a long historical time series of larval settlement. Samples were collected from early 2011 through late 2014. These measurements allow the first rigorous characterization of Willapa Bay aragonite mineral saturation state (Ωar), which has been shown to be of leading importance in determining the initial shell formation and growth of larval Crassostrea gigas. Observations show that the bay is usually below Ωar levels that have been associated with poor oyster hatchery production and with chronic effects noted in experimental work. Bay water only briefly rises to favorable Ωar levels and does so out of phase with optimal thermal conditions for spawning. Thermal and carbonate conditions are thus coincidentally favorable for early larval development for only a few weeks at a time each year. The limited concurrent exceedance of thermal and Ωar thresholds suggests the likelihood of high variability in settlement success, as seen in the historical record; however, estimates of the impact of elevated atmospheric CO2 suggest that pre-industrial Ωar conditions were more persistently favorable for larval development and more broadly coincident with thermal optima.

Slow shell building, a possible trait for resistance to the effects of acute ocean acidification

Increasing anthropogenic carbon dioxide is altering marine carbonate chemistry through a process called ocean acidification. Many calcium carbonate forming organisms are sensitive to changes in marine carbonate chemistry, especially mollusk bivalve larvae at the initial shell building stage. Rapid calcification, limited energy reserves, and more exposed calcification surfaces, are traits at this stage that increase vulnerability to ocean acidification through our previously argued kinetic-energetic hypothesis. These developmental traits are common to broadcast spawning bivalve species that are the focus of most ocean acidification studies to date. Some oyster species brood their young, which results in slower development of the embryos through the initial shell formation stage. We examined the responses of the brooding Olympia oyster, Ostrea lurida, during their initial shell building stage. We extracted fertilized eggs from, O. lurida, prior to shell development, then exposed developing embryos to a wide range of marine carbonate chemistry conditions. Surprisingly, O. lurida showed no acute negative response to any ocean acidification treatments. Compared to the broadcast spawning Pacific oyster, Crassostrea gigas, calcification rate and standardized endogenous energy lipid consumption rate were nearly 10 and 50 times slower, respectively. Our results suggest that slow shell building may lessen the energetic burden of acidification at this stage and provides additional support for our kinetic-energetic hypothesis. Furthermore, these results may represent an example of exaptation; fitness conveyed by a coopted trait that evolved for another purpose, a concept largely lacking in the current perspective of adaptation and global climate change.

Analysis of the Particle Formation Process of Structured Microparticles.

The particle formation process for microparticles of cellulose acetate butyrate dried from an acetone solution was investigated experimentally and theoretically. A monodisperse droplet chain was used to produce solution microdroplets in a size range of 55-70 μm with solution concentrations of 0.37 and 10 mg/mL. As the droplets dried in a laminar air flow with a temperature of 30, 40, or 55 °C, the particle formation process was recorded by two independent optical methods. Dried particles in a size range of 10-30 μm were collected for morphology analysis, showing hollow, elongated particles whose structure was dependent on the drying gas temperature and initial solution concentration. The setup allowed comprehensive measurements of the particle formation process to be made, including the period after initial shell formation. The early particle formation process for this system was controlled by the diffusion of cellulose acetate butyrate in the liquid phase, whereas later stages of the process were dominated by shell buckling and folding.

Effects of Co-spray Drying of Surfactants with High Solids Milk on Milk Powder Wettability

Dairy industries often spray lecithin, a food-grade surfactant, to spray-dried whole milk powders while fluidisation to produce instant powders. Though adding surfactant to milk feed was often reported to improve the wettability of dried powders, this approach was not favourably used. The present study investigated the effects of surfactant addition into high solids milk feed before drying on the wettability of whole milk particles. Adding either 0.1 wt.% Tween 80 or 0.1 wt.% lecithin to un-concentrated whole milk led to a significant wettability improvement of spray-dried powders. At higher feed solids levels of 23 and 33 wt.%, the wetting process of pure milk powders was comparatively rapid, but the surfactant-added powders showed similar wettability to the pure milk powders. The development of powder wettability as drying progressed was investigated using single droplet-drying technique for 32 and 43 wt.% whole milk in the presence and absence with surfactants. The technique captured an advanced shell formation during drying of higher solids milk. The wettability of surface shells formed by surfactant-added milk was similar to those formed by pure milk throughout drying, from initial shell formation to final drying stage. By contrast, coating surfactants on the outer layer of particles being dried could substantially improve wettability. The rapid shell formation and the slow material diffusion owing to the high medium viscosity were considered the main factors limiting the migration of surfactant molecules towards droplet surface during drying.

A developmental and energetic basis linking larval oyster shell formation to acidification sensitivity

AbstractAcidified waters are impacting commercial oyster production in the U.S. Pacific Northwest, and favorable carbonate chemistry conditions are predicted to become less frequent. Within 48 h of fertilization, unshelled Pacific oyster (Crassostrea gigas) larvae precipitate roughly 90% of their body weight as calcium carbonate. We measured stable carbon isotopes in larval shell and tissue and in algal food and seawater dissolved inorganic carbon in a longitudinal study of larval development and growth. Using these data and measured biochemical composition of larvae, we show that sensitivity of initial shell formation to ocean acidification results from diminished ability to isolate calcifying fluid from surrounding seawater, a limited energy budget and a strong kinetic demand for calcium carbonate precipitation. Our results highlight an important link between organism physiology and mineral kinetics in larval bivalves and suggest the consideration of mineral kinetics may improve understanding winners and losers in a high CO2 world.

Preparation of aqueous core/silica shell microcapsules

Water core/silica shell microcapsules are prepared via the hydrolysis and subsequent polycondensation of tetraethoxysilane in a surfactant stabilised water-in-oil emulsion. The relationship between preparative conditions, including pH and silane concentration, has been related to final particle structure. Furthermore, the nature of the catalyst has been found to affect the mechanism by which the shells are formed, with an interfacial polymerisation proposed for ammonium hydroxide catalysed synthesis in agreement with previous reports and a new colloidosome assembly process for sodium hydroxide catalysis. In both cases shell aging processes are observed to continue beyond initial shell formation suggesting that trans-shell diffusion of reactants may be feasible, or that rapid hydrolysis is required in order to load high concentrations of the reactants into the internal phase before significant shell formation.

Microtexture of larval shell of oyster, Crassostrea nippona: A FIB-TEM study

The initial formation and subsequent development of larval shells in marine bivalve, Crassostrea nippona were investigated using the FIB-TEM technique. Fourteen hours after fertilization (the trochophore stage), larvae form an incipient shell of 100-150nm thick with a columnar contrast. Selected-area electron diffraction analysis showed a single-crystal aragonite pattern with the c-axis perpendicular to the shell surface. Plan-view TEM analysis suggested that the shell contains high density of {110} twins, which are the origin of the columnar contrast in the cross-sectional images. 72h after fertilization (the veliger stage), the shell grows up to 1.2-1.4mum thick accompanying an additional granular layer between the preexisting layer and embryo to form a distinctive two-layer structure. The granular layer is also composed of aragonite crystals sharing their c-axes perpendicular to the shell surface, but the crystals are arranged with a flexible rotation around the c-axes and not restricted solely to the {110} twin relation. No evidence to suggest the existence of amorphous calcium carbonate (ACC) was found through the observation. The well-regulated crystallographic properties found in the present sample imply initial shell formation probably via a direct deposition of crystalline aragonite.

Mass Production Methods for IFE Targets

We are performing research and development to increase production quantity and yield for Inertial Fusion Energy targets for laser fusion. A key component of the laser fusion target is an approximately 4 mm diameter foam shell. To facilitate large-scale production, research into optimization of foam shell gelation and hardening times to reduce non-concentricity of the foam shell is underway. Additionally, we are examining methods to modify the current laboratory bench scale process for initial foam shell formation, various fluid exchanges, and sealcoat chemistry into a continuous process in collaboration with Schafer Corporation. The proposed process utilizes porous tubing sections to perform fluid exchanges in a long (200 m-1 km) continuous path of tubing extending from the triple orifice generator currently used to encapsulate and form the foam shell.Real-time process control has been applied to the triple orifice generator to control the diameter of the foam shell. The system makes use of a pair of photodiode sensors in a closed loop feedback control system incorporating a variable speed process pump. Empirical results indicate the process control loop is capable of identifying wet shell diameters to an approximate standard deviation of 80 to 90 μm, on par with characterization results indicating true shell diameter standard deviations of 30-80 μm.

Solidification control in continuous casting of steel

An integrated understanding of heat transfer during solidification, friction/lubrication at solid-liquid interface, high temperature properties of the solidifying shell etc. is necessary to control the continuous casting process. The present paper elaborates upon the knowledge developed in the areas of initial shell formation, mode of mould oscillation, and lubrication mechanism. The effect of these issues on the caster productivity and the quality of the product has been discussed. The influence of steel chemistry on solidification dynamics, particularly with respect to mode of solidification and its consequence on strength and ductility of the solidifying shell, has been dealt with in detail. The application of these basic principles for casting of stainless steel slabs and processing to obtain good quality products have been covered.

Simulation of Shell Strength Properties by the SSCT Test.

Initial shell formation and surface quality in continuous casting are affected by thermal and mechanical stresses which may lead to crack formation during solidification. Thus, detailed knowledge of high temperature strength properties as function of steel composition is required under given conditions. To simulate shell straining by tensile force perpendicular to the main dendrite growth axis, the Submerged Split Chill Tensile (SSCT) test was (and still is) further developed. The present report details updated results for ferritic and austenitic iron, and compares different chill materials. The determined effect of P-content on shell strength is quantified. Primary dendrite arm spacing without and with load appears to indicate that tensile elongation is not uniformly distributed over the test length.

SHELL INORGANIC COMPOSITION AND ONSET OF SHELL MINERALIZATION DURING BIVALVE AND GASTROPOD EMBRYOGENESIS

Embryos of Spisula solidissima (Bivalvia); Crepidula fornicata, C. convexa, Ilyanassa obsoleta (Gastropoda: Prosobranchia); and Dendronotus frondosus, Aeolidia papillosa, and Hermissenda crassicornis (Gastropoda: Opisthobranchia) were reared in the laboratory and examined periodically to determine when shell mineralization began, as detected by birefringence under polarized light. Shell birefringence was detected at preveliger stages in all species. At onset of shell birefringence, samples were prepared for transmission electron microscopy to determine ifthe transitory shell field invagination (SFI) was still present. Contrary to previous reports for other molluscs, the present ultrastructural evidence indicates that at least for three species studied (S. solidissima, A. papillosa, and H. crassicornis), the SFI is still present during initial shell mineralization; thus for these three species the cells of the SFI may be involved in initial shell calcification. Electron cytochemical staining with pyroantimonate followed by electron probe microanalysis of thin sections indicated the presence of calcium within the lumen of the SFI in one species (H. crassicornis) fixed at onset of shell birefringence. These data suggest that the SFI may play an active role in initial molluscan shell formation. Analysis of veliger shell composition with scanning electron microprobe analysis indicated the variable presence of minor and trace amounts of Na, Mg, Al, P, S, Cl, and K in addition to large amounts of calcium. In two species (C. fornicata and A. papillosa) shell calcium carbonate was determined by X-ray diffraction analysis to be aragonitic.

Origin, morphology and fate of the nonmineralized shell of Coryphella salmonacea, an opisthobranch gastropod

Little is known about initial shell formation in molluscs or about the possible functions of the embryonic shell field invagination (SFI). The present paper describes formation and loss of an unusual shell in embryos of the nudibranch Coryphella salmonacea (Couthouy, 1838), collected in February–March, 1981–1984. The first organic shell material was attached to shell field cells just outside the SFI at two weeks after fertilization (5°C). As viewed with transmission electron microscopy, the organic shell in section consisted of two electron dense layers having a total thickness of 16 nm. The shell continued to grow through at least the fifth week of encapsulated development. In the eighth week, the shell still covered the visceral mass; there was no evidence that mantle tissue reflexed and covered the shell or that shell materials were resorbed by the mantle. The shell and sometimes the operculum were discarded within the embryonic capsule prior to hatching at the end of Week 8. The cup-like shell is unusual in that it was shed as a wrinkled organic sheet which apparently lacked mineralization. Fully-developed shells were non-birefringent and did not produce calcium peaks when examined by electron microprobe. Both the location of the first organic shell material outside the SFI and the apparent absence of shell mineralization in the presence of an SFI are consistent with the hypothesis that the SFI is nonsecretory. C. salmonacea is probably in the evolutionary process of losing its shell, a structure of little or no function during encapsulated, nonplanktonic development.

Morphology of aerosol particles generated from the evaporation of solution drops

Morphology of particles formed from the evaporation of solution droplets is examined. It is concluded that evaporation of solution droplets leads to initial shell formation. Final particle size, shape and structure are affected by rate of evaporation of the solution droplet and mass of solute present. Conditions conducive to solid particle formation include a nuclei-free environment, a high solubility solute and low drying rates. The frequent formation of non-solid particles necessitates the use of electron microscopy in particle size, shape and density verification.