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Paramyosin is a key component of thick filaments in invertebrate muscles. In this study, we isolated the full length cDNA of paramyosin from Pacific oyster (Crassostrea gigas), and determined its pattern of expression during myogenesis. The full length paramyosin (CgPM) cDNA contains an open reading frame (ORF) of 2586 bp encoding a 861-amino acid protein. Sequence analysis revealed an assembly competence domain (ACD) and a heptad repeat (d-e-f-g-a-b-c) with 28-residue repeat zones in the CgPM primary structure, a characteristic of coiled-coil protein. Quantitative analysis of CgPM expression revealed a sharp increase in trochophore stage, and peaked at the D-shaped stage. Strong CgPM expression was found in smooth adductor muscle, followed by striated adductor muscle and mantle tissue. Whole-mount in situ hybridization (WISH) showed a restricted pattern of CgPM expression in adductor muscle, larval velum retractor and foot muscles at the umbo and eyed larval stages. These data indicate that CgPM is strongly expressed during larval myogenesis in C. gigas, which provides the basis for further functional studies of paramyosin in oyster to better understand the molecular and cellular mechanisms of muscle formation in mollusks., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In many spiralians, asymmetry in the first two cleavages is achieved through the formation of a polar lobe (PL), which transiently constricts to sequester vegetal cytoplasm into the CD and D blastomeres. While microtubules and actin filaments are required for polar lobe formation, little else is known regarding the structural and functional similarities with the contractile ring, or how the PL constriction is able to form perpendicular to the cleavage plane. Examination of scallop embryos revealed that while activated myosin II could be detected in both the cleavage furrow and early PL constriction, astral or central spindle microtubules were not observed associated with the PL neck until the constriction was nearly complete. Further, inhibition of Aurora B had no effect on polar lobe initiation, but blocked both contractile ring ingression and PL constriction beyond phase II. The cortex destined for PL sequestration was marked by enrichment of the Arp2/3 complex, which was first detected during meiosis and remained enriched at the vegetal pole through the first two cleavages. Inhibition of Arp2/3 affected PL formation and partitioning of cytoplasm into the two daughter cells, suggesting that Arp2/3 plays a functional role in defining the zone of cortex to be sequestered into the polar lobe. Together, these data offer for the first time a mechanism by which a cytoskeletal specialization defines the polar lobe in this atypical form of asymmetric cell division., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

It has been proposed that animals have a pattern of developmental evolution resembling an hourglass because the most conserved development stage-often called the phylotypic stage-is always in midembryonic development. Although the topic has been debated for decades, recent studies using molecular data such as RNA-seq gene expression data sets have largely supported the existence of periods of relative evolutionary conservation in middevelopment, consistent with the phylotypic stage and the hourglass concepts. However, so far this approach has only been applied to a limited number of taxa across the tree of life. Here, using established phylotranscriptomic approaches, we found a surprising reverse hourglass pattern in two molluscs and a polychaete annelid, representatives of the Spiralia, an understudied group that contains a large fraction of metazoan body plan diversity. These results suggest that spiralians have a divergent midembryonic stage, with more conserved early and late development, which is the inverse of the pattern seen in almost all other organisms where these phylotranscriptomic approaches have been reported. We discuss our findings in light of proposed reasons for the phylotypic stage and hourglass model in other systems., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Pacific oyster (Crassostrea gigas) is a sessile bivalve living in the intertidal zone. It has become an attractive model for developmental studies due to its metamorphic transition from a mobile planktonic larvae to a sessile adults. To determine the effect of metamorphosis on muscle development in oyster larvae, we characterized myogenesis during larval development and metamorphosis by phalloidin staining which labels filamentous actin filaments. Our data revealed a dynamic pattern of myogenesis during embryonic and larval development. It appears that simple "U-shaped" muscle ring first developed at the trochophore stage. This was followed by a more complex musculature including an anterior adductor, velum ventral retractors at the veliger stage, and the addition of posterior adductors and foot retractors at the veliger and pediveliger stages. During metamorphosis, muscle structures in the anterior adductor, velum retractors and ventral retractors were degenerated. At the same time, mantle and gill musculature appeared and became the primary muscle system in juveniles together with the posterior adductor. In addition, indirect immunofluorescence with the monoclonal antibody against C. gigas muscle proteins (myosin heavy chains (MYHC) and α-actinin) were used to monitor changes in the developing musculature at different larval stages. The immunofluorescence staining results of muscle proteins were consistent with phalloidin staining. The expression locations of two muscle proteins were similar and mainly located in larval velum retractor and adductor muscle. The α-actinin expression positions were located in Z-lined of velum striated retractors. Data from these studies provide a comprehensive description of myogenesis in C. gigas embryos and larvae. Moreover, our data showed that metamorphosis has a significant impact on remolding the musculature after transition from a mobile planktonic larvae to a sessile mollusk, associated with certain muscle group degradation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In the marine environment, most bivalve species base their reproduction on external fertilization. Hence, gametes and young stages face many threats, including exposure to plastic wastes which represent more than 80% of the debris in the oceans. Recently, evidence has been produced on the presence of nanoplastics in oceans, thus motivating new studies of their impacts on marine life. Because no information is available about their environmental concentrations, we performed dose-response exposure experiments with polystyrene particles to assess the extent of micro/nanoplastic toxicity. Effects of polystyrene with different sizes and functionalizations (plain 2-μm, 500-nm and 50-nm; COOH-50 nm and NH 2 -50 nm) were assessed on three key reproductive steps (fertilization, embryogenesis and metamorphosis) of Pacific oysters (Crassostrea gigas). Nanoplastics induced a significant decrease in fertilization success and in embryo-larval development with numerous malformations up to total developmental arrest. The NH 2 -50 beads had the strongest toxicity to both gametes (EC 50  = 4.9 μg/mL) and embryos (EC 50  = 0.15 μg/mL), showing functionalization-dependent toxicity. No effects of plain microplastics were recorded. These results highlight that exposures to nanoplastics may have deleterious effects on planktonic stages of oysters, presumably interacting with biological membranes and causing cyto/genotoxicity with potentially drastic consequences for their reproductive success., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Oysters are a diverse group of marine bivalves that inhabit coastal systems of the world's oceans, providing a variety of ecosystem services, and represent a major socioeconomic resource. However, oyster reefs have become inevitably impacted from habitat destruction, overfishing, pollution and disease outbreaks that have pushed these structures to the break of extinction. In addition, the increased frequency of climate change related events promise to further challenge oyster species survival worldwide. Oysters' early embryonic development is likely the most vulnerable stage to climate change related stressors (e.g. salinity and temperature shifts) as well as to pollutants (e.g. arsenic), and therefore can represent the most important bottleneck that define populations' survival in a changing environment. In light of this, the present study aimed to assess two important oyster species, Crassostrea angulata and Crassostrea gigas embryo-larval development, under combinations of salinity (20, 26 and 33), temperature (20, 24 and 28 °C) and arsenic (As) exposure (0, 30, 60, 120, 240, 480, 960 and 1920 μg. As L -1 ), to infer on different oyster species capacity to cope with these environmental stressors under the eminent threat of climate change and increase of pollution worldwide. Results showed differences in each species range of salinity and temperature for successful embryonic development. For C angulata, embryo-larval development was successful at a narrower range of both salinity and temperature, compared to C. gigas. Overall, As induced higher toxicity to C. angulata embryos, with calculated EC50 values at least an order of magnitude lower than those calculated for C. gigas. The toxicity of As (measured as median effective concentration, EC50) showed to be influenced by both salinity and temperature in both species. Nonetheless, salinity had a greater influence on embryos' sensitivity to As. This pattern was mostly noticed for C. gigas, with lower salinity inducing higher sensitivity to As. Results were discussed considering the existing literature and suggest that C. angulata populations are likely to become more vulnerable under near future predictions for temperature rise, salinity shifts and pollution., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Nanos gene plays an important role in germline development in animals. However, the molecular mechanisms involved in germline development in Mollusca, the second largest animal phylum, are still poorly understood. Here we identified the Nanos orthologue from the Pacific oyster Crassostrea gigas (Cg-Nanos-like), and investigated the expression patterns of Nanos during gametogenesis and embryogenesis in C. gigas. Tissue expression analysis showed that Cg-Nanos-like was specifically expressed in female gonads. During the reproductive cycle, the expression of Cg-Nanos-like mRNA increased matching the seasonal development of the ovarian tissues in diploids, while the expression levels were significantly lower in the ovaries of sterile triploids compared to diploids. High expression of Cg-Nanos-like transcripts were detected in early embryonic stages, while the expression significantly dropped at gastrulation and was barely detectable in veliger stages. In situ hybridization showed that Cg-Nanos-like was expressed at different stages of developing oocytes, whereas positive signals were detected only in spermatogonia during the spermatogenic cycle. These findings indicated that Cg-Nanos-like was involved in the development of germ cells, and maintenance of oocyte maturation. In early embryogenesis, the transcripts were broadly expressed; following gastrulation, the expression was restricted to two cell clumps, which might be the putative primordial germ cells (PGCs) or their precursors. Based on the results, the formation of the PGCs in C. gigas was consistent with the model of transition from epigenesis to preformation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The combined effects of different salinity and temperature levels on the toxicity of Arsenic (As) were studied on the embryonic development of the oyster Crassostrea gigas. A standardized embryotoxicity test was performed to assess the interactive effects of these stressors, in a full factorial design experiment including a range of salinities (15, 19, 24, 28 and 32), temperatures (16, 20, 24, 28 and 32°C) and As concentrations (100, 300, 600, 1200, 2400µgL -1 ). The embryotoxicity endpoint was about the determination of normal larvae development rates at various conditions, and median effect concentration (EC 50 ) determination for each As exposure condition. Results showed that toxicity induced by As was characterized by retardation of embryonic development observing toxic effects at lower concentrations than previously reported studies. The presence of As in seawater resulted in a narrower range of tolerance to both salinity and temperature. These findings bring new insights on the impacts of a common contaminant on an important shellfish species having a planktonic early life stage development, with potential implications for population survival and ecosystem functioning in a changing environment., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Spiralians, including molluscs, annelids and platyhelminths, share a unique development process that includes the typical geometry of early cleavage and early segregation of cell fate in blastomeres along the animal-vegetal axis. However, the molecular mechanisms underlying this early cell fate segregation are largely unknown. Here, we report spiralian-specific expansion of the three-amino-acid loop extension (TALE) class of homeobox genes. During early development, some of these TALE genes are expressed in staggered domains along the animal-vegetal axis in the limpet Nipponacmea fuscoviridis and the polychaete Spirobranchus kraussii. Inhibition or overexpression of these genes alters the developmental fate of blastomeres, as predicted by the gene expression patterns. These results suggest that the expansion of novel TALE genes plays a critical role in the establishment of a novel cell fate segregation mechanism in spiralians.

In molluscs, muscle contraction is triggered by a direct binding of Ca 2+ to myosin. The myosin essential light chain (MELC) provides the Ca 2+ binding site and is of importance for motor function and regulation. In this study, the complete cDNA sequence of MELC gene of the Pacific oyster, Crassostrea gigas, was obtained, and the expression profiles were performed in different tissues and different embryo-larval development stages. The results showed that the full length of C. gigas MELC (CgMELC) cDNA was 659bp, containing a 5'-untranslated region of 73 bp, a 3'-untranslated region of 112bp. The open reading frame encoded a 157 amino acid peptide. The protein sequence of CgMELC contained a conserved EF-hand calcium binding motif, and showed a high sequence identity (68.4-100%) with other bivalves. Quantitative analysis of CgMELC mRNA in tissues indicated that CgMELC was expressed at the highest level in the striated adductor muscle, followed by the smooth adductor muscle and mantle. During the development of embryos and larvae, quantitative analysis and whole-mount in situ hybridization revealed that CgMELC was expressed starting from the blastula stage and abundantly expressed in trochophore and D-shaped larvae, indicating that CgMELC might also be involved in regulation of larval muscle system development. Our data provided valuable information for further researches on the function of MELC and regulation of muscle contraction in oysters., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Persistent organic pollutants (POPs) are ubiquitous and coexisted in the aquatic environment. Individual and combined toxic effects of benzo[a]pyrene (BaP) and 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) on embryogenesis, and larval survival of the Pacific oyster were investigated. The EC 50 values of BaP, BDE-47 and their mixture on embryogenesis were 18.4, 203.3 and 72.0 µg/L respectively, while the LC 50 values for 96 h larval mortality were 26.8, 244.5 and 108.9 µg/L respectively. The Marking-Dawson additive toxicity indices were -0.02 and -0.19, indicating an additive effect with a trend to antagonism. In addition, DNA strand breaks were also observed in oyster embryos after exposure. Our study suggests that BaP and BDE-47 exposure can cause developmental abnormalities, DNA damage and larval mortality. Furthermore, the toxicity of the mixture is slightly lower than individual pollutant. These data will be helpful to predict the toxicity of organic pollutants, and provide criteria for marine water quality standards.

Phenanthnere (PHE) is a polycyclic aromatic hydrocarbon continuously discarded in the marine environment and bioavailable to many aquatic species. Although studies about PHE toxicity have been documented for adult oysters, the effects on early developmental stages are poorly characterized in bivalves. In this study, the effects of PHE (0.02 and 2.0μg.L -1 ) were evaluated on the embryogenesis and larval development of Crassostrea gigas. Toxicity bioassays, growth and deformities assessment, analysis of shell calcium abundance and transcript levels of genes related to xenobiotic biotransformation (CYP2AU2, CYP30C1), immune system (Cg-Tal) and tissue growth and shell formation (Ferritin, Insulin-like, Cg-Try, Calmodulin and Nacrein) were assayed in D-shape larvae after 24h of PHE exposure. At the highest concentration (2.0μg.L -1 ), PHE decreased the frequency of normal development (19.7±2.9%) and shell size (53.5±2.8mm). Developmental deformities were mostly related to abnormal mantle and shell formation. Lower calcium levels in oyster shells exposed to PHE 2.0μg.L -1 were observed, suggesting effects on shell structure. At this same PHE concentration, CYP30C1, Cg-Tal, Cg-Tyr, Calmodulin were upregulated and CYP2AU2, Ferritin, Nacrein, and Insulin-Like were downregulated compared to control larvae. At the lowest PHE concentration (0.02μg.L -1 ), it was observed a minor decrease in normal larval development (89,6±6%) and the remaining parameters were not affected. This is the first study to provide evidences that exposure to PHE can affect early oyster development at the molecular and morphological levels, possibly threatening this bivalve species., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Though several bilaterian animals use a conserved BMP2/4-Chordin antagonism to pattern the dorsal-ventral (DV) axis, the only lophotrochozoan species in which early DV patterning has been studied to date, the leech Helobdella robusta, appears to employ BMP5-8 and Gremlin. These findings call into question the conservation of a common DV patterning mechanism among bilaterian animals. To explore whether the unusual DV patterning mechanism in H. robusta is also used in other lophotrochozoan species, we investigated the expression of orthologous genes in the early embryo of a bivalve mollusk, Crassostrea gigas. Searching of the genome and phylogenetic analysis revealed that C. gigas possesses single orthologs of BMP2/4, Chordin, and BMP5-8 and no Gremlin homolog. Whole mount in situ hybridization revealed mRNA localization of BMP2/4 and Chordin on the opposite sides of embryos, suggesting the potential involvement of a BMP2/4-Chordin antagonism in DV patterning in this species. Furthermore, universal BMP5-8 expression and the absence of a Gremlin homolog in the C. gigas genome called into question any major contribution by BMP5-8 and Gremlin to early DV patterning in this species. Additionally, we identified seven genes showing asymmetric expression along the DV axis, providing further insight into DV patterning in C. gigas. We present the first report of a Chordin gene in a lophotrochozoan species and of the opposite expression of BMP2/4 (dorsal) and Chordin (ventral) along the D/V axis of a lophotrochozoan embryo. The findings of this study further the knowledge of axis formation in lophotrochozoan species and provide insight into the evolution of the animal DV patterning mechanism.

Thyroid hormones (THs) play important roles in development, metamorphosis, and metabolism in vertebrates. During the past century, TH functions were regarded as a synapomorphy of vertebrates. More recently, accumulating evidence has gradually convinced us that TH functions also occur in invertebrate chordates. To date, however, TH-related studies in non-chordate invertebrates have been limited. In this study, THs were qualitatively detected by two reliable methods (HPLC and LC/MS) in a well-studied molluscan species, the Pacific oyster Crassostrea gigas. Quantitative measurement of THs during the development of C. gigas showed high TH contents during embryogenesis and that oyster embryos may synthesize THs endogenously. As a first step in elucidating the TH signaling cascade, an ortholog of vertebrate TH receptor (TR), the most critical gene mediating TH effects, was cloned in C. gigas. The sequence of CgTR has conserved DNA-binding and ligand-binding domains that normally characterize these receptors. Experimental results demonstrated that CgTR can repress gene expression through binding to promoters of target genes and can interact with oyster retinoid X receptor. Moreover, CgTR mRNA expression was activated by T4 and the transcriptional activity of CgTR promoter was repressed by unliganded CgTR protein. An atypical thyroid hormone response element (CgDR5) was found in the promoter of CgTR, which was verified by electrophoretic mobility shift assay (EMSA). These results indicated that some of the CgTR function is conserved. However, the EMSA assay showed that DNA binding specificity of CgTR was different from that of the vertebrate TR and experiments with two dual-luciferase reporter systems indicated that l-thyroxine, 3,3',5-triiodothyronine, and triiodothyroacetic acid failed to activate the transcriptional activity of CgTR. This is the first study to functionally characterize TR in mollusks. The presence of THs and the functions of CgTR in mollusks contribute to better understanding of the evolution of the TH system.

Kuwait is a country with low rainfall and highly concentrated industrial and domestic effluents entering its coastal waters. These can be both treated and untreated. In this study we sampled a series of coastal and open-sea sites and used a variety of analyses to identify those sites requiring the most attention. We used a high throughput GC-MS screen to look for over 1000 chemicals in the samples. Estrogen and androgen screens assessed the potential to disrupt endocrine activity. An oyster embryo development screen was used to assess biological effect potential. The chemical screen identified sites which had high numbers of identified industrial and domestic chemicals. The oyster screen showed that these sites had also caused high levels of developmental abnormalities with 100% of embryos affected at some sites. The yeast screen showed that estrogenic chemicals were present in outfalls at 2-3 ng/l E2 equivalent, and detectable even in some open water sites., (Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.)

The explosion of the Deepwater Horizon (DWH) oil platform resulted in large amounts of crude oil and dispersant Corexit 9500A® released into the Gulf of Mexico and coincided with the spawning season of the oyster, Crassostrea virginica. The effects of exposing gametes and embryos of C. virginica to dispersant alone (Corexit), mechanically (HEWAF) and chemically dispersed (CEWAF) DWH oil were evaluated. Fertilization success and the morphological development, growth, and survival of larvae were assessed. Gamete exposure reduced fertilization (HEWAF: EC201h=1650μg tPAH50L(-1); CEWAF: EC201h=19.4μg tPAH50L(-1); Corexit: EC201h=6.9mgL(-1)). CEWAF and Corexit showed a similar toxicity on early life stages at equivalent nominal concentrations. Oysters exposed from gametes to CEWAF and Corexit experienced more deleterious effects than oysters exposed from embryos. Results suggest the presence of oil and dispersant during oyster spawning season may interfere with larval development and subsequent recruitment., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Lightsticks are artifacts used as attractors in a type of commercial fishery, known as surface longline gear. Despite the excessive use, the contamination risks of these devices have not yet been properly investigated. This research aimed to fill up this gap by determining the chemical composition and the toxicity of lightsticks recently activated, compared to those one year after activation and to the ones collected on the beaches. The analyzes were carried out by Gas Chromatography coupled with Mass Spectrometry (GC-MS). Additionally, the variations in composition and the toxicity of their sea Water Soluble Fractions (WSF) were evaluated based on the WSF-effects of Crassostrea rhizophorae embryonic development. The GC-MS analysis made possible the identification of nineteen substances in the water soluble fraction of the lightsticks, such as dibutyl phthalate (DBP) and dimethyl phthalate (DMP). The value of the WSF-effective concentration (EC50) was in an average of 0.35%. After one year of the lightsticks activation, the toxicity was even higher (0.65%). Furthermore, other substances, also present in the lightsticks-WSF caused persistent toxicity even more dangerous to the environment than DBP and DMP. This essay discusses their toxicity effects and possible environment damages., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

DNA methylation within promoter regions (PRDM) controls vertebrate early gene transcription and thereby development, but is neglected outside this group. However, epigenetic features in the oyster Crassostrea gigas suggest functional significance of PDRM in invertebrates. To investigate this, reporter constructs containing in vitro methylated oyster Hox gene promoters were transfected into oyster embryos. The influence of in vivo methylation was studied using bisulfite sequencing and DNA methyltransferase inhibition during development. Our results demonstrate that methylation controls the transcriptional activity of the promoters investigated, unraveling a functional role for PRDM in a lophotrochozoan, an important finding regarding the evolution of epigenetic regulation., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

C1q domain-containing (C1qDC) proteins are regarded as important players in the innate immunity of bivalve mollusks and other invertebrates and their highly adaptive binding properties indicate them as efficient pathogen recognition molecules. Although experimental studies support this view, the molecular data available at the present time are not sufficient to fully explain the great molecular diversification of this family, present in bivalves with hundreds of C1q coding genes. Taking advantage of the fully sequenced genome of the Pacific oyster Crassostrea gigas and more than 100 transcriptomic datasets, we: (i) re-annotated the oyster C1qDC loci, thus identifying the correct genomic organization of 337 C1qDC genes, (ii) explored the expression pattern of oyster C1qDC genes in diverse developmental stages and adult tissues of unchallenged and experimentally treated animals; (iii) investigated the expansion of the C1qDC gene family in all major bivalve subclasses. Overall, we provide a broad description of the functionally relevant features of oyster C1qDC genes, their comparative expression levels and new evidence confirming that a gene family expansion event has occurred during the course of Bivalve evolution, leading to the diversification of hundreds of different C1qDC genes in both the Pteriomorphia and Heterodonta subclasses., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In many groups, epigenetic mechanisms influence developmental gene regulation under environmental inputs. The Pacific oyster Crassostrea gigas belongs to lophotrochozoans and its larval development is highly dependent on temperature, but the role of epigenetic mechanisms in this context is unknown despite high levels of the recently characterized Jumonji histone demethylase (JHDM) orthologues (Cg_Jumonji) suggesting a physiological relevance of histone methylation in the oyster development. Because in other species alterations of the histone methylation pattern have deleterious outcomes, we investigated the influence of temperature during the oyster larval life on histone methylation and JHDM expression. To shed light on this point, oyster embryonic and early larval development experiments were carried out at different temperatures (18 °C, 25 °C and 32 °C). Histone methylation levels were investigated using fluorescent ELISA at 6 and 24h post-fertilization. When compared to the 25 °C group, at 18 °C H3K4, H3K9 and H3K27 residues were hypomethylated at 6h post fertilization (hpf) and hypermethylated at 24 hpf. In contrast, at 32 °C, 6hpf animals present a dramatic hypermethylation (ca. 4-fold) of all examined residues, which is minored but sustained at 24 hpf. RT-qPCR investigations of the mRNA expression of the nine oyster JHDMs, showed gene- and stage-specific temperature sensitivities throughout the early life of oysters. This study provides evidence of the biological significance of histone methylation during development in a lophotrochozoan species. Our results also indicate that temperature influences histone methylation, possibly through the expression level of putative actors of its regulation, which might participate in developmental control. To our knowledge, this is the first report indicating a direct relationship between an epigenetic mark and an environmental parameter in marine molluscs. Such investigations could help better understand the molecular mechanisms of development and adaptation in lophotrochozoans., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The brevetoxin-producing dinoflagellate, Karenia brevis, adversely affects many shellfish species including the commercially and ecologically important bivalve molluscs, the northern quahog (=hard clam) Mercenaria mercenaria and eastern oyster Crassostrea virginica, in the Gulf of Mexico, USA. This study assessed the effects of exposure of these bivalves to K. brevis during their early development. In separate experiments, embryos of 2-4 cell stage of M. mercenaria and C. virginica were exposed to both whole and lysed K. brevis cells isolated from Manasota Key, Florida. Low bloom concentrations of 500 to 3000 cells mL(-1) were simulated for 96 h. Shell length, percent abnormality (and normality), and percent mortality of resulting larvae were measured. Percentages were recorded after 6, 24, and 96 h of exposure; larval shell length was measured at 24 and 96 h. For both quahogs and oysters, the effects of exposing embryos to K. brevis on all larval responses were generally dose- and time-dependent. Percent mortalities and abnormalities of both clam and oyster embryos increased significantly after only 6h of exposure to whole cells of K. brevis. For clams, these parameters were significantly higher in whole and lysed treatments (at 3000 cells mL(-1)) than in controls. Percent mortalities of oysters were significantly higher in the whole-cell treatment (3000 cells mL(-1)) than under control conditions. After 24h of exposure, mean larval shell length of both bivalve species was significantly reduced relative to controls. This was evident for clam larvae in both the lysed treatment at 1500 cells mL(-1) and in whole and lysed treatments at 3000 cells mL(-1), and for oyster larvae in the lysed treatment at 3000 cells mL(-1). After 96 h, both species exposed to the lysed cell treatment at 3000 cells mL(-1) had significantly smaller larvae compared to those in the control. Overall, lysed cells of K. brevis had a more pronounced effect on shell length, percent abnormality, and mortality in both clams and oysters than did whole cells. Given the fact that blooms of K. brevis overlap with the spawning periods of these two bivalves, and that cells of this naked dinoflagellate are readily lysed by wave action, these results suggest that exposure to K. brevis during the early life history stages of clams and oysters could adversely affect their population recruitment. Further, the presence of whole or lysed cells of K. brevis in hatcheries could have a major negative impact on production., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Multiple ferritin subunit genes are reported in mollusks, but they have not been systematically classified. Based on the recently published whole genome sequence, we screened out the four ferritin subunit genes (cgi-fer1-cgi-fer4) from the Pacific oyster Crassostrea gigas. The four genes were predicted to encode two non-secretory and two secretory peptides. Further phylogenetic analyses revealed two groups of non-secretory and secretory ferritin subunits in mollusks. This differs dramatically from the situation in mammals or insects, which contain only non-secretory or secretory ferritin subunits. These results emphasize the evolution of molluscan ferritin subunit genes. The expression patterns of the four genes during early development exhibited dramatic differences, indicating the functional diversity of these genes. Among them, cgi-fer2 was the only gene expressed prevalently and is thus suggested to be the major house-keeping ferritin subunit gene. The expression of the other three genes was tissue-specific beginning in the D-veliger stage. Based on their expression patterns, we inferred important functions of cgi-fer2 in ciliated tissues and of the other three genes in the digestive system. Moreover, our results indicated potentially different roles of ferritin subunit genes during larval shell formation in gastropods and bivalves, which may be helpful in understanding the molecular mechanisms that cause the different shells of gastropods and bivalves. In addition, we conducted a further semi-quantitative analysis of the four genes in four major developmental stages and five adult tissues. The results also revealed dramatically different expression patterns of the genes, which brought additional functional indications. This work may promote studies on molluscan ferritins and shed light on the evolution of ferritin subunit genes among different animal groups., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Surfactants used in herbicide formulations are generally considered inert with no toxic effects on animals. Polyethoxylated tallow amines (POEAs) are non-ionic surfactants used in many herbicide formulations to promote the penetration of the active matter into plant cuticles. The present study aimed to assess the toxicity of a POEA surfactant system, the Genamin T-200®, on two larval stages of the Pacific oyster, Crassostrea gigas. The embryotoxicity of Genamin T-200® was quantified after 36 hr of exposure, considering both arrested development and abnormalities in D-shaped larvae. The ability of pediveliger larvae to metamorphose was studied after 24 hr exposure to Genamin T-200®. According to the European toxicity classification, the present results suggest that Genamin T-200® could be considered very toxic to embryo larval development, with an EC₅₀ of 262 µg/l, and toxic to metamorphosis processes with an EC₅₀ of 3,027 µg/l. The high toxicity of glyphosate-based formulations compared to the active ingredient and its by-product appears to be due primarily to surfactants.

The combined effects of temperature and ammonia concentration on the percent fertilization and percent hatching in Crassostrea ariakensis were examined under laboratory conditions using the central composite design and response surface methodology. The results indicated: (1) The linear effects of temperature and ammonia concentration on the percent fertilization were significant (P<0.05), and the quadratic effects were highly significant (P<0.01). The interactive effect between temperature and ammonia concentration on the percent fertilization was not significant (P>0.05). (2) The linear effect of temperature on the percent hatching was highly significant (P<0.01), and that of ammonia concentration was nonsignificant (P>0.05). The quadratic effects of temperature and ammonia concentration on the percent hatching were highly significant (P<0.01). The interaction on the percent hatching was not significant (P>0.05). Temperature was more important than ammonia in influencing the fertilization and hatching in C. ariakensis. (3) The model equations of the percent fertilization and hatching towards temperature and ammonia concentration were established, with the coefficients of determination R(2)=99.4% and 99.76%, respectively. Through the lack-of-fit test, these models were of great adequacy. The predictive coefficients of determination for the two model equations were as high as 94.6% and 98.03%, respectively, showing that they could be used for practical projection. (4) Via the statistical simultaneous optimization technique, the optimal factor level combination, i.e., 25°C/0.038mgmL(-1), was derived, at which the greatest percent fertilization 95.25% and hatching 83.26% was achieved, with the desirability being 97.81%. Our results may provide advantageous guidelines for the successful reproduction of C. ariakensis., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Jumonji (Jmj) proteins are histone demethylases, which control the identity of stem cells. Jmj genes were characterized from plants to mammals where they have been implicated in the epigenetic regulation of development. Despite the Pacific oyster Crassostrea gigas representing one of the most important aquaculture resources worldwide, the molecular mechanisms governing the embryogenesis and reproduction of this lophotrochozoan species remain poorly understood. However, annotations in the C. gigas EST library suggested the presence of putative Jumonji genes, raising the question of the conservation of this family of histone demethylases in the oyster. Using Primer walking, 5'-RACE PCR and in silico analyses, we characterized nine Jumonji orthologues in the oyster, called Cg-Jmj, bearing conserved domains critical for putative histone demethylase activity. Phylogenic analyses revealed that oyster Jumonji cluster into two distinct groups: 'single-domain Jmj' and 'multi-domain Jmj', and define 8 subgroups corresponding to each cognate orthologues in metazoans. RT-qPCR investigations showed specific regulations of Cg-Jmj mRNAs during the early development and along the reproduction cycle. Furthermore, in situ and in toto hybridizations indicate that oyster Jumonji genes are transcribed mostly within the gonad in adult oysters whereas they display a ubiquitous expression during embryonic and larval development. Our study demonstrates the presence of nine Jumonji orthologues in the oyster C. gigas. Their domain conservation and their expression profile suggest an implication during reproduction and development, questioning about the epigenetic regulation by histone methylation in lophotrochozoans., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Bivalve larvae use catch muscles for rapid shell closure and maintenance of the closed condition. We used specific antibodies against the muscle proteins together with phalloidin and neuronal markers, FMRFamide and serotonin (5-HT), to analyze mutual distribution of muscle and neuronal elements in larvae of the mussel, Mytilus trossulus, and the oyster, Crassostrea gigas. At trochophore and early veliger stages no anatomical connections between muscular and nervous system were detected. By the pediveliger stage the 5-HT innervation of the anterior adductor developed in oyster only, while rich FMRFa innervation of the adductor muscles developed in both species. Possible roles and mechanisms of FMRFamide and serotonin in the regulation of the catch state are discussed.

In the oyster Crassostrea gigas, a successive hermaphrodite, Cg-Foxl2, an ortholog of Foxl2, is suspected to be involved in vitellogenesis or female sex determination. The existence of a natural antisense transcript (NAT) of this factor has been suspected in gonads but needs to be confirmed to better understand the early events of the gonadic differentiation. The occurrence of this NAT was studied by orientation-specific RT-PCR. The NAT and its mRNA expressions were investigated during the development and in adults by real-time PCR and in situ hybridization. The presence of stable in vivo RNA-RNA duplexes was also explored by RNase protection-based approach. This work is the first evidence of characterization of a NAT in the gonads of mollusks and Lophotrochozoa. This NAT named Cg-Foxl2os is supposed to be polyadenylated and forms RNA-RNA duplexes with its mRNA. Cg-Foxl2os is significantly more expressed than Cg-Foxl2 in 2-month-old spats and in mature males. It is co-localized with the mRNA in the cytoplasm of spermatogonia and spermatocytes. The results of this study demonstrate the existence of a NAT of Cg-Foxl2 in the gonads of C. gigas. It may regulate its mRNA expression through formation of cytoplasmic RNA-RNA duplexes during the oyster gonadic differentiation., (Copyright © 2012 S. Karger AG, Basel.)

In September 2008, organotin (Ot) compounds were prohibited from being used worldwide. From 1997 onward in Japan, the production of paints containing TBT (tributylin) compounds was prohibited, and thus alternatives to Ot antifoulants have been used since then. It has been said that the decomposition characteristics of these materials are better than those of Ot compounds. The toxicity of alternative Ot antifoulants (e.g., diuron, irgarol 1051®, and Sea-Nine 211®) and Ot compounds (TBT and TPT (triphenyltin)), using oysters that inhabit a large area of Hiroshima Bay, were evaluated. The results showed that the toxicity of diuron and irgarol 1051 is very low, and the toxicity of Sea-Nine 211 is almost the same as that of TPT. Sea-Nine 211's effect was stronger on oysters than other shellfish, causing concern about the extent of Sea-Nine 211's impact on oyster development.

Camamu Bay, an Environmentally Protected Area, may be affected by the pressures of tourism and oil exploration in the adjacent continental platform. The current quality of the mangrove sediments was evaluated by porewater bioassays using embryos of Crassostrea rhizophorae and by an analysis of benthic macrofauna and its relationships with organic compounds, trace metals and bioavailability. Porewater toxicity varied from low to moderate in the majority of the samples, and polychaetes dominated the benthos. The Grande Island sampling station (Station 1) presented more sandy sediments, differentiated macrobenthic assemblages and the highest metal concentrations in relation to other stations and guideline values, and it was the only station that indicated a possible bioavailability of metals. The origin of the metals (mainly barium) is most likely associated with the barite ore deposits located in the Grande and Pequena islands. These results may be useful for future assessment of the impact of oil exploration in the coastal region., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Ocean acidification, due to anthropogenic CO₂ absorption by the ocean, may have profound impacts on marine biota. Calcareous organisms are expected to be particularly sensitive due to the decreasing availability of carbonate ions driven by decreasing pH levels. Recently, some studies focused on the early life stages of mollusks that are supposedly more sensitive to environmental disturbances than adult stages. Although these studies have shown decreased growth rates and increased proportions of abnormal development under low pH conditions, they did not allow attribution to pH induced changes in physiology or changes due to a decrease in aragonite saturation state. This study aims to assess the impact of several carbonate-system perturbations on the growth of Pacific oyster (Crassostrea gigas) larvae during the first 3 days of development (until shelled D-veliger larvae). Seawater with five different chemistries was obtained by separately manipulating pH, total alkalinity and aragonite saturation state (calcium addition). Results showed that the developmental success and growth rates were not directly affected by changes in pH or aragonite saturation state but were highly correlated with the availability of carbonate ions. In contrast to previous studies, both developmental success into viable D-shaped larvae and growth rates were not significantly altered as long as carbonate ion concentrations were above aragonite saturation levels, but they strongly decreased below saturation levels. These results suggest that the mechanisms used by these organisms to regulate calcification rates are not efficient enough to compensate for the low availability of carbonate ions under corrosive conditions.

This work investigates the ecotoxicological evaluation of contaminated dredged sediments from French Mediterranean navy harbour (A), commercial port (B) and two composite specimens (C) and (D) coming from the mixture of A and B with other port sediments. The toxicity of elutriates from these sediments is estimated using embryo-toxicity test, Microtox® solid phase test, LuminoTox, phytotoxicity tests and genotoxicity test. Bioassay responses are not clearly correlated with chemical contamination in the whole sediment and vary as a function of tested organisms. The highest contaminated samples (A and C) are almost always more toxic than the less contaminated samples (B and D). Among composite sediments, the mixture effect with other sediments is not efficient to decrease toxicity in sample C, suggesting that other parameters influence toxicity level such as particle size or organic matter content. These parameters should be taken into consideration in order to improve the efficiency of the mixture process and produce composite sediments with low toxicity., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Members of the Transforming Growth factor beta (TGF-beta) superfamily of cell signalling polypeptides are known to play important roles in cell proliferation and differentiation during development and in various physiological processes of most animal clades. Recent findings in the mollusc Crassostrea gigas demonstrate the occurrence of a diversity of TGF-beta signalling components including various ligands, three type I receptors but only a single type II receptor. This report describes the characterization of Cg-ActRII, a new type II receptor displaying homology with vertebrate and Drosophila Activin type II receptors. The use of zebrafish embryo as a reporter organism revealed that, in a way similar to its zebrafish counterpart, overexpression of Cg-ActRII or its dominant negative acting truncated form resulted in a dose dependent range of dorsoventral defects coupled with anterior disorders. Expression pattern of Cg-ActRII transcripts examined by real time PCR and in situ PCR in C. gigas showed high levels of Cg-ActRII transcripts in early embryonic stages and in the developing larval central nervous system. Except for a high expression in the visceral ganglia, most oyster adult tissues displayed rather low levels of transcripts. Altogether, the data suggest a high degree of conservation at both the structural and functional levels during evolution for this class of receptors.

This work assesses the ecotoxicological effects of polluted sediment after a decontamination treatment process using a new sediment washing technique. Sediment samples were collected from four sites in Marghera Port industrial channels (Venice, Italy). Ecotoxicological evaluations were performed with Vibrio fischeri and Crassostrea gigas bioassays. Whole sediment and elutriate were deemed as the most suitable environmental matrices for this study. Toxicity scores developed in the Lagoon of Venice for V. fischeri on whole sediment and for C. gigas on elutriate were considered for the final ranking of samples. Ecotoxicological results showed that the treated sediment samples presented both acute and sub-chronic toxicities, which were mainly attributed to the presence of some remaining chemicals such as metals and polyaromatic hydrocarbons. The acute toxicity ranged from low to medium, while the sub-chronic one from absent to very high, suggesting that treated sediments could not be reused in direct contact with seawater.

The aim of this study is to describe the early stages of spermatogenesis of the Pacific oyster Crassostrea gigas using both light and electron microscopy. The gonad is formed by gonadal tubules invaginated in a connective tissue constituting a storage tissue. Myoepithelial cells surround each gonadal tubule and are associated with an acellular matrix delimiting the outer part of the tubule, the inner part is composed by intragonadal somatic cells associated with germinal lineage. Two types of spermatogonia are identified, where type I spermatogonia (Spg I) are large, scarce and pale cells leaned against the base of the tubule (nuclear diameter: 5.5+/-0.5 microm). Type II spermatogonia (Spg II) are clustered and dark cells which appear smaller than type I (nuclear diameter: 4.3+/-0.3 microm). The aspect of nuage-like material in cytoplasm is described from pale spermatogonia to primary spermatocytes (nuclear diameter: pachytene 3.6+/-0.3 microm, diplotene 3.4+/-0.3 microm), while no structure related to a chromatoid body was observed in oyster spermatocytes and spermatids.

We previously found that embryonic development of the bivalve species was highly vulnerable to xenobiotic chemicals, damaging the coastal ecosystem integrity To further assess their potential damage to ecosystem, the xenobiotic composition of the sediment elutriates from two representative industrialized Korean coasts, Pohang and Ulsan, were determined with gas chromatography/mass spectrometry (GC/MS). The presumed critical dilution of the elutriate was then exposed to early life stages of the Pacific oyster (Crassostrea gigas), embryonic development and metamorphic stage to first spat, at which they were believably more vulnerable by the chemical exposure. The early life damage by the xenobiotic exposure was apparently significant by the significant degree of pollution. Here, we indicated their potential damages to the Pacific oyster

Bioassays are routinely employed for sediment quality assessment. In order to be able to effectively use Bioassays responses in regulatory and management frameworks, toxicity scores, which rank toxicity data in defined classes that are continuous and difficult to interpret, should be reliable and suitable tools to support decisions about the presence or absence of toxicity in tested samples and on how toxic a sample is. A statistical approach is needed to define thresholds for toxicity scores. The Minimum Significance Difference (MSD) criterion allowed the evaluation of toxicity thresholds for each test-matrix and organism pair, based on large sets of experimental data. The MSD values were normalized with respect to the control, ranked in ascending order, and the 90th percentile was identified; the Toxicity Threshold (TT) was calculated by subtracting the 90th percentile from 100 and the Toxicity Limit (TL) was estimated as the percentage of control response multiplied by TT. Taking into account sample responses normalized with respect to control (S), when S > TL, the sample is considered nontoxic; when S

The effects of humic acid (HA) on copper speciation and its subsequent toxicity to the sensitive early life stages of the Pacific oyster (Crassostrea gigas) are presented. Differential pulse anodic stripping voltammetry with a hanging mercury drop electrode was used to measure the copper species as labile copper (LCu; free ion and inorganic copper complexes) and total copper (TCu) with respect to increasing HA concentration. The TCu and LCu 50% effect concentrations (EC50s) in the absence of HA were 20.77 microg/L (95% confidence interval [CI], 24.02-19.97 microg/L) and 8.05 microg/L (95% CI, 9.6-5.92 microg/L) respectively. A corrected dissolved organic carbon (DOC) concentration (HA only) of 1.02 mg/L was required to significantly increase the TCu EC50 to approximately 41.09 microg/L (95% CI, 44.27-37.52 microg/L; p < 0.05), almost doubling that recorded when DOC (as HA) was absent from the test media. In contrast, the LCu EC50 was unaffected by changes in DOC concentration and was stable throughout the corrected DOC concentration range. The absence of change in the LCu EC50, despite increased HA concentration, suggests that the LCu fraction, not TCu, was responsible for the observed toxicity to the oyster embryo. This corresponds with the current understanding of copper toxicity and supports the free-ion activity model for copper toxicity.

Wood is widely used in the development of freshwater, estuarine and marine coastlines. Timbers last according to their content of naturally occurring preservatives (mostly phenols and aldehydes), produced to prevent decay from biotic agents. When untreated woods are exposed to aquatic media, leachates are generated with likely toxic effects on the target environment. The potential impact on saltwaters of leachates from some untreated timbers of both native and tropical species has been assessed. The leaching procedure was set up considering British Standard test methods for paints and OECD guidelines for wood preservatives emission scenarios. Toxicity was monitored via the acute toxicity test with the brine shrimp Artemia franciscana and the sub-chronic embryotoxicity test with the oyster Crassostrea gigas. Brine shrimps evidenced no toxic effects while oysters discriminated well among leachates: the tropical wood species showed similar or relatively lower toxic effects than the native ones according to both leaching cycles (24 and 72 h). The ecotoxicological data have been integrated with some physical and chemical parameters.

The work presented here reports the expression of immune-related genes during ontogenesis in the oyster Crassostrea gigas. Expression patterns of 18 selected genes showed that RNAs detected in oocytes and 2-4 cell embryos are of maternal origin and that gene transcription starts early after fertilization. The expression patterns of 4 genes (Cg-timp, Cg-tal, Cg-EcSOD and Drac3) suggested that hemocytes appear in the gastrula-trochophore stages. The localization of Cg-tal expression suggested that hematopoietic cells were derived from vessels and/or artery endothelia cells. Moreover, a bacterial challenge affected the level of expression of genes. Indeed, a change in expression levels was observed for Cg-LBP/BPI, Cg-timp, Drac3 and Cg-MyD88 genes in larval stages upon exposure to non-pathogenic bacteria. In early juveniles, a modulation was also observed for Cg-LBP/BPI, Cg-timp, Cg-MyD88 and for Cg-tal, according to the concentration of bacteria. Altogether, the results showed that studying the appearance of immunocompetent cells through their ability to express immune-related genes is a tool to gain insight the ontogenesis of the oyster immune system.

Despite their physiological significance in immune and growth-controlling processes in plants and animals, no chitinolytic enzyme has been identified yet at the molecular level in Lophotrochozoa, one of the major clades of bilaterian animals. Here, we report the cloning and the characterization of a singular chitinase homologue from the bivalve mollusc Crassostrea gigas (Cg-Chit). This protein displays a modular structure including a conserved catalytic domain attached to a peritrophin-A type chitin-binding domain and an unconventional C-terminal hydrophobic sequence acting as a potential membrane anchor domain. Gene expression profiles monitored by quantitative RT-PCR in different adult tissues and during development support for the first time the involvement of such a protein in early embryonic development. Furthermore, Cg-Chit encoding gene was transcriptionally stimulated in haemocytes in response to either bacterial or LPS challenge. This suggests that Cg-Chit plays an important role as an immunity effector in molluscs.

Methodological research was carried out to evaluate the discriminatory capability of three toxicity bioassays toward different elutriation ratios (1:4, 1:20, 1:50, and 1:200 sediment:water ratios). Samples from six sampling stations of the Lagoon of Venice have been investigated. The toxicity bioassay results (sea urchin Paracentrotus lividus Lmk sperm cell and embryo toxicity bioassays and bivalve mollusk Crassostrea gigas Thunberg embryo toxicity bioassays) have shown that elutriates generated from the widely used 1:4 ratio were less toxic than those from intermediate ratios (1:20 and 1:50).

Bioassay methods currently used to assess the toxicity of effluents, transitional and marine waters measure endpoints such as larval immobilisation or mortality, however, they offer limited data regarding important sub-lethal effects, including genotoxicity. The metaphase chromosome aberration (CAb) assay is routinely used in mammalian systems for testing samples for genotoxicity. In the current study, an in vivo CAb test system has been developed and optimised for use with the early (embryo-larval) life stages of the Pacific oyster Crassostrea gigas, a species used routinely in both effluent hazard assessment and marine environmental monitoring programmes. The method was validated with two reference mutagens: Methylmethanesulfonate, a direct acting mutagen; and benzo[a]pyrene an indirect acting reference mutagen.