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MicroRNAs (miRNAs) regulate post-transcription gene expression by targeting genes and play crucial roles in diverse biological processes involving body color formation. However, miRNAs and miRNA-targets underlying shell color polymorphism remain largely unknown in mollusca. Using four shell colors full-sib families of the Pacific oyster Crassostrea gigas, we systematically identified miRNAs and miRNA-targets in the mantles, which organ could produce white, golden, black or partially pigmented shell. RNA sequencing and analysis identified a total of 53 known miRNA and 91 novel miRNAs, 47 of which were detected to differentially express among six pairwise groups. By integrating miRNA and mRNA expression profiles, a total of 870 genes were predicted as targets of differentially expressed miRNAs, mainly involving in biomineralization and pigmentation through functional enrichment. Furthermore, a total of four miRNAs and their target mRNAs were predicted to involve in synthesis of melanin, carotenoid or tetrapyrrole. Of them, lgi-miR-317 and its targets peroxidase and lncRNA TCONS_00951105 are implicated in acting as the competing endogenous RNA to regulate melanogenesis. Our studies revealed the systematic characterization of miRNAs profiles expressed in oyster mantle, which might facilitate understanding the intricate molecular regulation of shell color polymorphism and provide new insights into breeding research in oyster.

The colour of oyster shells is a very diverse characteristic morphotype, forming intriguing vivid patterns both on the inside and outside of the shell. In the present study, we have identified for the first time, the presence of several porphyrins as constituents of the shell pigmentation of the Crassostrea gigas oyster consumed worldwide. The precise molecular structures of halochromic, fluorescent and acid-soluble porphyrins, such as uroporphyrin and turacin, are unambiguously determined by reverse phase liquid chromatography combined with high resolution mass spectrometry. Their presence account for the purple colouration of shells but also for the dark colouration of adductor muscle scars. We have also defined the endogenous origin of these porphyrins, specifically secreted or accumulated by the shell forming tissue. These findings are pioneering analytical proofs of the existence of the haem pathway in the edible oyster Crassostrea gigas, evidenced by the chemical identification of haem side-products and supported by the recent publication of the corresponding oyster genome.

The eastern oyster plays a vital role in estuarine habitats, acting as an ecosystem engineer and improving water quality. Populations of Chesapeake Bay oysters have declined precipitously in recent decades. The fossil record, which preserves 500 000 years of once-thriving reefs, provides a unique opportunity to study pristine reefs to establish a possible baseline for mitigation. For this study, over 900 fossil oysters were examined from three Pleistocene localities in the Chesapeake region. Data on oyster shell lengths, lifespans and population density were assessed. Comparisons to modern Crassostrea virginica , sampled from monitoring surveys of similar environments, reveal that fossil oysters were significantly larger, longer-lived and more abundant than modern oysters from polyhaline salinity zones. This pattern results from the preferential harvesting of larger, reproductively more active females from the modern population. These fossil data, combined with modern estimates of age-based fecundity and mortality, make it possible to estimate ecosystem services in these long-dead reefs, including filtering capacity, which was an order of magnitude greater in the past than today. Conservation palaeobiology can provide us with a picture of not just what the Chesapeake Bay looked like, but how it functioned, before humans. This article is part of a discussion meeting issue 'The past is a foreign country: how much can the fossil record actually inform conservation?'

The function of Late Archaic period (5000-3000 B.P.) shell rings has been a focus of debate among archaeologists for decades. These rings have been variously interpreted as a product of seasonal feasting/ceremonial gatherings, quotidian food refuse generated by permanent dwellers, or a combination of seasonal and perennial activities. Seasonality of shell rings can be assessed by reconstructing the harvest time of oysters (Crassostrea virginica), the primary faunal component of shell rings. We estimated the timing of oyster harvest at St. Catherines Shell Ring (Georgia, USA) by statistical modeling of size frequency distributions of the impressed odostome (Boonea impressa), a parasitic snail inadvertently gathered by Archaic peoples with its oyster host. The odostome samples from three archaeological excavation units were evaluated against resampling models based on monthly demographic data obtained for present-day populations of Boonea impressa. For all samples, the harvest was unlikely to start earlier than late fall and end later than late spring, indicating that shell deposits at St. Catherines Shell Ring formed seasonally with substantial harvesting activities restricted to non-summer months. For all samples, the resampling models indicated that harvesting activities likely occurred during multiple months. However, these analytical outcomes would also be expected in the case of extensively time-averaged records of short-term, non-summer harvest events. Regardless of the exact harvest duration, the results point to seasonal harvesting and suggest that Archaic populations may have opted out of consuming summer oysters to focus on other resources, avoid unpalatable food, decrease pathogen risks, or ensure sustainable harvesting., Competing Interests: The authors have declared that no competing interests exist.

Restoration efforts with native eastern oyster, Crassostrea virginica, in Chesapeake Bay and elsewhere have been limited by shell availability, necessitating the use of alternative structures as subtidal reefs, yet these have rarely been evaluated quantitatively. We quantified population structure, density, abundance and biomass of eastern oyster and hooked mussel, Ischadium recurvum, on a concrete modular reef (75 m2 surface area over 5 m2 of river bottom) deployed subtidally at 7 m depth in the Rappahannock River, Virginia during October, 2000. After nearly 5 y (May 2005), we took 120 stratified random samples over the reef. The reef was heavily colonized by 28-168 oysters and 14-2177 mussels m-2 surface area. These densities translate to 1085 oysters and 8617 mussels m-2 river bottom, which are the highest recorded for artificial oyster reefs. Size structure of oysters reflected four year classes, with over half of oysters more than 1 y old and of reproductive age. Oyster biomass (1663 g dry mass m-2 river bottom) and condition index were equally high, whereas parasite prevalence and intensity were low. Oyster density correlated positively in a sigmoid fashion with mussel density up to high densities, then declined. This modular reef is one of the most successful artificial reefs for eastern oyster and hooked mussel restoration, and details features that are conducive for successful settlement, growth and survival in subtidal habitats., Competing Interests: The authors have declared that no competing interests exist.

Golden shell color and mineral content are important economic traits of Pacific oyster (Crassostrea gigas). In this study, we mapped a series of quantitative trait loci (QTLs) that control zinc (Zn) and magnesium (Mg) content, shell color and growth performance to two sex-averaged linkage maps from the FAM-A and FAM-B families. In total, ten QTLs were identified in seven linkage groups (LGs) in the FAM-B family, and seven QTLs were identified in four linkage groups in the FAM-A family. Two QTLs affecting the trait of golden shell color were identified in LG8 of the FAM-A and LG10 of the FAM-B families, which could explain 20.2 and 10.5% of the phenotypic variations, respectively. Two QTLs for Zn content were identified that could contribute to 17.9 and 34.44% of the phenotypic variations in FAM-A. Six QTLs for Zn and Mg contents were identified in four LGs (LG1, LG2, LG5, and LG9) in FAM-B, which explained 13.5-26.7% of the phenotypic variations. In addition, seven QTLs related to oyster growth were recognized in both FAM-A and FAM-B families accounting for 14.6-36.7% of the phenotypic variations. All of the DNA markers in QTL regions were blasted and 14 genes associated with above traits were identified. The mRNA expression of these genes was determined by quantitative RT-PCR. These QTLs and candidate genes could be used as potential targets for marker-assisted selection in C. gigas breeding.

Many prey react to predation risk by altering their phenotype to reduce their chances of being consumed but incur reductions in growth and fecundity when reacting to predators. To determine when to produce defenses, prey collect information and evaluate the costs and benefits of defense induction. Resource availability can affect prey ability and willingness to incur defense costs. When resources are scarce, defenses may suffer disproportionate decreases in energy allocation if defenses would further reduce prey access to resources or if resources are needed to maintain metabolic functions. We tested the effects of predation risk and resource availability on plastic defenses in eastern oysters Crassostrea virginica and present novel findings that oysters continued to produce defended shells in response to predators when resources were limited, even though they grew smaller, lighter shells when deprived of food in control conditions. Predation risk affected all three tested shell metrics (area, weight, and strength), but food availability did not. Although low food levels often limit expression of predator defenses, predator cues caused oysters to build shells that were larger and heavier, with a similar trend for shell strength, in treatments with both low and high food levels, suggesting that predation is an important pressure in this system. The differences between predator and control treatments were greater under conditions of low food availability, and thus, resource availability may influence interpretations of plastic responses to predators.

Estuaries around the world are in a state of decline following decades or more of overfishing, pollution, and climate change. Oysters (Ostreidae), ecosystem engineers in many estuaries, influence water quality, construct habitat, and provide food for humans and wildlife. In North America's Chesapeake Bay, once-thriving eastern oyster (Crassostrea virginica) populations have declined dramatically, making their restoration and conservation extremely challenging. Here we present data on oyster size and human harvest from Chesapeake Bay archaeological sites spanning ∼3,500 y of Native American, colonial, and historical occupation. We compare oysters from archaeological sites with Pleistocene oyster reefs that existed before human harvest, modern oyster reefs, and other records of human oyster harvest from around the world. Native American fisheries were focused on nearshore oysters and were likely harvested at a rate that was sustainable over centuries to millennia, despite changing Holocene climatic conditions and sea-level rise. These data document resilience in oyster populations under long-term Native American harvest, sea-level rise, and climate change; provide context for managing modern oyster fisheries in the Chesapeake Bay and elsewhere around the world; and demonstrate an interdisciplinary approach that can be applied broadly to other fisheries.

Shells are one of the most notable features of the majority of mollusks. In addition, the shell is also considered a key characteristic during molluscan evolution and development. However, although the morphological changes during larval shell formation have been well described, the underlying molecular mechanisms remain poorly understood. In this study, we focused on the potential involvement of a GATA gene in shell formation because GATA genes are often downstream genes of BMP (bone morphogenetic protein) signaling pathways, which have been suggested to participate in molluscan shell formation. In the Pacific oyster Crassostrea gigas, we observed that the expression of a GATA2/3 homolog (cgi-gata2/3) was clearly restricted to the edge of the shell field in early larval stages (trochophore and D-veliger). This expression pattern supports the notion that cgi-gata2/3 gene plays conserved roles in bilaterian ectodermal development. It is possible that cgi-gata2/3 is one shell-formation gene under the regulation of BMP signaling pathways. In addition, cgi-gata2/3 was also detected in the ventral side of embryos. The expression of cgi-gata2/3 away from the shell field may be involved in hematopoiesis. Our results provide fundamental support for studies into the molecular mechanisms of larval shell formation and the functions of molluscan GATA genes.

MicroRNAs (miRNAs) play important roles in regulatory processes in various organisms. To date many studies have been performed in the investigation of miRNAs of numerous bilaterians, but limited numbers of miRNAs have been identified in the few species belonging to the clade Lophotrochozoa. In the current study, deep sequencing was conducted to identify the miRNAs of Crassostrea gigas (Lophotrochozoa) at a genomic scale, using 21 libraries that included different developmental stages and adult organs. A total of 100 hairpin precursor loci were predicted to encode miRNAs. Of these, 19 precursors (pre-miRNA) were novel in the oyster. As many as 53 (53%) miRNAs were distributed in clusters and 49 (49%) precursors were intragenic, which suggests two important biogenetic sources of miRNAs. Different developmental stages were characterized with specific miRNA expression patterns that highlighted regulatory variation along a temporal axis. Conserved miRNAs were expressed universally throughout different stages and organs, whereas novel miRNAs tended to be more specific and may be related to the determination of the novel body plan. Furthermore, we developed an index named the miRNA profile age index (miRPAI) to integrate the evolutionary age and expression levels of miRNAs during a particular developmental stage. We found that the swimming stages were characterized by the youngest miRPAIs. Indeed, the large-scale expression of novel miRNAs indicated the importance of these stages during development, particularly from organogenetic and evolutionary perspectives. Some potentially important miRNAs were identified for further study through significant changes between expression patterns in different developmental events, such as metamorphosis. This study broadened the knowledge of miRNAs in animals and indicated the presence of sophisticated miRNA regulatory networks related to the biological processes in lophotrochozoans.

An antimicrobial polypeptide was purified from an acidified gill extract of Pacific oyster (Crassostrea gigas) by C(18) reversed-phase HPLC. The purified polypeptide had a molecular weight of 8471Da containing 74 amino acid residues. Comparison of the obtained N-terminal sequences with those of others revealed that it was identical to ubiquitin reported from other species and named cgUbiquitin. cgUbiquitin showed broad potent antimicrobial activity against Gram-positive and -negative bacteria including Streptococcus iniae and Vibrio parahemolyticus (minimal effective concentrations, 7.8 and 9.8μg/mL), respectively, without hemolytic activity. The cgUbiquitin cDNA was identified from an expressed sequence tag (EST) library of oyster gill as a precursor form, encoding ubiquitin consisting of 76 amino acids fused to ribosomal protein of S27. Although the cgUbiquitin precursor mRNA was expressed at the intermediate level in the gill, the mRNA was significantly up-regulated at 48h post injection with Vibrio sp. Analysis of the cgUbiquitin C-terminus by carboxypeptidase B treatment and comparison of the retention times revealed that cgUbiquitin lacks the terminal Gly-Gly doublet and ends in an C-terminal Arg residue which might be related to antimicrobial activity. Study of the kinetics of killing and membrane permeabilization showed that this peptide was not membrane permeable and acted through a bacteriostatic process. According to the homology modeling, this peptide is composed of three secondary structural motifs including three α-helices and four β-strands separated by 7 loops regions. Our results indicate that cgUbiquitin might be related to the innate immune defenses in the Pacific oyster and this is the first report for antimicrobial function of ubiquitin isolated from any oyster species., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Rising levels of atmospheric CO(2) lead to acidification of the ocean and alter seawater carbonate chemistry, which can negatively impact calcifying organisms, including mollusks. In estuaries, exposure to elevated CO(2) levels often co-occurs with other stressors, such as reduced salinity, which enhances the acidification trend, affects ion and acid-base regulation of estuarine calcifiers and modifies their response to ocean acidification. We studied the interactive effects of salinity and partial pressure of CO(2) (P(CO2)) on biomineralization and energy homeostasis in juveniles of the eastern oyster, Crassostrea virginica, a common estuarine bivalve. Juveniles were exposed for 11 weeks to one of two environmentally relevant salinities (30 or 15 PSU) either at current atmospheric P(CO2) (∼400 μatm, normocapnia) or P(CO2) projected by moderate IPCC scenarios for the year 2100 (∼700-800 μatm, hypercapnia). Exposure of the juvenile oysters to elevated P(CO2) and/or low salinity led to a significant increase in mortality, reduction of tissue energy stores (glycogen and lipid) and negative soft tissue growth, indicating energy deficiency. Interestingly, tissue ATP levels were not affected by exposure to changing salinity and P(CO2), suggesting that juvenile oysters maintain their cellular energy status at the expense of lipid and glycogen stores. At the same time, no compensatory upregulation of carbonic anhydrase activity was found under the conditions of low salinity and high P(CO2). Metabolic profiling using magnetic resonance spectroscopy revealed altered metabolite status following low salinity exposure; specifically, acetate levels were lower in hypercapnic than in normocapnic individuals at low salinity. Combined exposure to hypercapnia and low salinity negatively affected mechanical properties of shells of the juveniles, resulting in reduced hardness and fracture resistance. Thus, our data suggest that the combined effects of elevated P(CO2) and fluctuating salinity may jeopardize the survival of eastern oysters because of weakening of their shells and increased energy consumption.

Abnormal thickening and chambering in Crassostrea gigas oysters have been adopted for many years as bioindicators of available tributyltin (TBT) in coastal waters. Nevertheless, since natural causes can also induce the formation of multiple chambers, a field study and laboratory experimentation has been conducted with 72 examples of C. gigas in successive culture media. This work has enabled differences to be established between natural fine sediment-induced characteristics and the influence of TBT on the shells. External shell deformities have been assessed using three biometric indices, shell thickness index, weight index and volume index. Internal differences have been observed in longitudinal sections of the shell: retraction of growth, stagnation of the adductor muscle scar and thinning of the chambers in the TBT-polluted shell secretion. A new index, the opening chambers index, has been proposed, with a value of less than 1 in the TBT-polluted environment and greater than 1 in shells secreted in an unpolluted production site. These conclusions should be borne in mind when C. gigas is used in biomonitoring programmes.

Pesticide run-off into the ocean represents a potential threat to marine organisms, especially bivalves living in coastal environments. However, little is known about the effects of environmentally relevant concentrations of pesticides at the individual level. In this study, the suppression subtractive hybridisation technique was used to discover the main physiological function affected by a cocktail of three pesticides (lindane, metolachlor and carbofuran) in the Pacific oyster Crassostrea gigas. Two oyster populations exposed to different pollution levels in the wild were investigated. The pesticide concentrations used to induce stress were close to those found in the wild. In a time course experiment, the expression of three genes implicated in iron metabolism and oxidative stress as well as that of two ubiquitous stress proteins was examined. No clear regulation of gene or protein expression was found, potentially due to a low-dose effect. However, we detected a strong site- and organ-specific response to the pesticides. This study thus (1) provides insight into bivalve responses to pesticide pollution at the level of the transcriptome, which is the first level of response for organisms facing pollution, and (2) raises interesting questions concerning the importance of the sites and organs studied in the toxicogenomic field.

Toxin accumulation by suspension-feeding qualifier depends on a balance between processes regulating toxin uptake (i.e. ingestion and absorption of toxic cells) and elimination (i.e. egestion, exchange among tissues, excretion, degradation and/or biotransformation) during exposure to toxic blooms. This laboratory study compares the size-specific uptake and elimination kinetics of domoic acid (DA) from Pseudo-nitzschia multiseries in two co-occurring bivalves, the oyster Crassostrea virginica and the mussel Mytilus edulis. Domoic acid concentrations were measured in visceral and non-visceral tissues of different-sized oysters and mussels during simultaneous long-term exposure to toxic P. multiseries cells in the laboratory, followed by depuration on a non-toxic algal diet. Mussels attained 7-17-fold higher DA concentrations than oysters, depending on the body size and exposure time, and also detoxified DA at higher rates (1.4-1.6 d(-1)) than oysters (0.25-0.88 d(-1)) of a comparable size. Small oysters attained markedly higher weight-specific DA concentrations (maximum=78.6 μg g(-1)) than large, market-sized individuals (≤ 13 μg g(-1)), but no clear relationship was found between body size and DA concentration in mussels (maximum=460 μg g(-1)). Therefore, differential DA accumulation by the two species was, on average, approximately 3-fold more pronounced for large bivalves. An inverse relationship between DA elimination rate and body size was established for oysters but not mussels. Elimination of DA was faster in viscera than in other tissues of both bivalves; DA exchange rate from the former to the latter was higher in oysters. The contribution of viscera to the total DA burden of mussels was consistently greater than that of other tissues during both uptake (>80%) and depuration (>65%) phases, whereas it rapidly decreased from 70-80% to 30-40% in oysters, and this occurred faster in smaller individuals. Residual DA concentrations (≤ 0.25 μg g(-1)) were detected at later depuration stages (up to 14 d), mainly in viscera of oysters and non-visceral tissues of mussels, suggesting that a second, slower-detoxifying toxin compartment exists in both species. However, a simple exponential decay model was found to adequately describe DA elimination kinetics in these bivalves. The lower capacity for DA accumulation in oysters compared to mussels can thus only be explained by the former's comparatively low toxin intake rather than faster toxin elimination., (2010. Published by Elsevier B.V. All rights reserved.)

Soluble protein (MPSP, myostracal prism soluble protein) obtained from myostracum in oyster shell (Crassostrea gigas) was characterized using biochemical and molecular biological techniques. From an analysis of secondary protein structure, it was shown that beta-structure was predominant in MPSP. And via in vitro assays, the relation of MPSP to biomineral phase and morphology was studied. SDS-PAGE revealed one major protein band of 20 kDa. An amino acid sequence of 160 amino acids was deduced for myostracum by characterization of the complementary DNA encoding the protein. The deduced protein was composed of a high proportion of Gly and Asp, typifying a calcium-binding protein for shell formation, and a relatively high proportion of Val, Ala and Ile, typifying an adhesive protein. In contrast to prevailing expectations, (Gly-Asp)n-type sequence motifs exist in MPSP, demanding a revision of previous theories of protein-mineral interactions. The cDNA sequence of myostracum is elucidated for the first time.