Cytoskeletal polarization and cytokinetic signaling drives polar lobe formation in spiralian embryos.

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.

Gene Expression Does Not Support the Developmental Hourglass Model in Three Animals with Spiralian Development.

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.

Combined effects of arsenic, salinity and temperature on Crassostrea gigas embryotoxicity.

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 (EC50) 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.

Expression patterns indicate that BMP2/4 and Chordin, not BMP5-8 and Gremlin, mediate dorsal-ventral patterning in the mollusk Crassostrea gigas.

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.

Individual and Combined Toxicities of Benzo[a]pyrene and 2,2',4,4'-Tetrabromodiphenyl Ether on Early Life Stages of the Pacific Oyster, Crassostrea gigas.

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 EC50 values of BaP, BDE-47 and their mixture on embryogenesis were 18.4, 203.3 and 72.0 µg/L respectively, while the LC50 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.

Effect of simultaneous variation in temperature and ammonia concentration on percent fertilization and hatching in Crassostrea ariakensis.

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.

Innervation of bivalve larval catch muscles by serotonergic and FMRFamidergic neurons.

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.

Integrated assessment of mangrove sediments in the Camamu Bay (Bahia, Brazil).

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.

Effects of organotin alternative antifoulants on oyster embryo.

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.

Characterization of paramyosin protein structure and gene expression during myogenesis in Pacific oyster (Crassostrea gigas).

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.

Developmental dynamics of myogenesis in Pacific oyster Crassostrea gigas.

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.

Fine structure of the early stages of spermatogenesis in the Pacific oyster, Crassostrea gigas (Mollusca, Bivalvia).

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.

Effects of the coastal sediment elutriates containing persistent organic pollutants (POPs) on early reproductive outputs of the Pacific oyster, Crassostrea gigas.

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

Comparative sensitivity of Crassostrea angulata and Crassostrea gigas embryo-larval development to As under varying salinity and temperature.

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.

Oocyte maturation and origin of the germline as revealed by the expression of Nanos-like in the Pacific oyster Crassostrea gigas.

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.

Nanoplastics impaired oyster free living stages, gametes and embryos.

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 NH2-50 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 NH2-50 beads had the strongest toxicity to both gametes (EC50 = 4.9 µg/mL) and embryos (EC50 = 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.

Characterization of an atypical family 18 chitinase from the oyster Crassostrea gigas: evidence for a role in early development and immunity.

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.

Expression of immune-related genes in the oyster Crassostrea gigas during ontogenesis.

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.

Effects of dissolved organic carbon on the toxicity of copper to the developing embryos of the Pacific oyster (Crassostrea gigas).

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.

Toxicity of untreated wood leachates towards two saltwater organisms (Crassostrea gigas and Artemia franciscana).

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.