Comparative transcriptome analysis reveals molecular damage associated with cryopreservation in Crassostrea angulata D-larvae rather than to cryoprotectant exposure.

BACKGROUND: The Portuguese oyster Crassostrea angulata, a bivalve of significant economic and ecological importance, has faced a decline in both production and natural populations due to pathologies, climate change, and anthropogenic factors. To safeguard its genetic diversity and improve reproductive management, cryopreservation emerges as a valuable strategy. However, the cryopreservation methodologies lead to some damage in structures and functions of the cells and tissues that can affect post-thaw quality. Transcriptomics may help to understand the molecular consequences related to cryopreservation steps and therefore to identify different freezability biomarkers. This study investigates the molecular damage induced by cryopreservation in C. angulata D-larvae, focusing on two critical steps: exposure to cryoprotectant solution and the freezing/thawing process. RESULTS: Expression analysis revealed 3 differentially expressed genes between larvae exposed to cryoprotectant solution and fresh larvae and 611 differentially expressed genes in cryopreserved larvae against fresh larvae. The most significantly enriched gene ontology terms were "carbohydrate metabolic process", "integral component of membrane" and "chitin binding" for biological processes, cellular components and molecular functions, respectively. Kyoto Encyclopedia of Genes and Genomes enrichment analysis identified the "neuroactive ligand receptor interaction", "endocytosis" and "spliceosome" as the most enriched pathways. RNA sequencing results were validate by quantitative RT-PCR, once both techniques presented the same gene expression tendency and a group of 11 genes were considered important molecular biomarkers to be used in further studies for the evaluation of cryodamage. CONCLUSIONS: The current work provided valuable insights into the molecular repercussions of cryopreservation on D-larvae of Crassostrea angulata, revealing that the freezing process had a more pronounced impact on larval quality compared to any potential cryoprotectant-induced toxicity. Additionally, was identify 11 genes serving as biomarkers of freezability for D-larvae quality assessment. This research contributes to the development of more effective cryopreservation protocols and detection methods for cryodamage in this species.

Expression and functional analysis of Fushi Tarazu transcription factor 1 (FTZ-F1) in the regulation of steroid hormones during the gonad development of Fujian Oyster, Crassostrea angulata.

Crassostrea angulata, a major shellfish cultivated in Southern China, has experienced a notable surge in commercial value in recent years. Understanding the molecular mechanisms governing their reproductive processes holds significant implications for advancing aquaculture practices. In this study, we cloned the orphan nuclear receptor gene, Fushi Tarazu transcription factor 1 (FTZ-F1), of C. angulata and investigated its functional role in the gonadal development. The full-length cDNA of FTZ-F1 spans 2357 bp and encodes a protein sequence of 530 amino acids. Notably, the amino acid sequence of FTZ-F1 in C. angulata shares remarkable similarity with its homologues in other species, particularly in the DNA-binding region (>90%) and ligand-binding region (>44%). In C. angulata, the highest expression level of FTZ-F1 was observed in the ovary, exhibiting more than a 200-fold increase during the maturation stage compared to the initiation stage (P < 0.001). Specifically, FTZ-F1 was mainly expressed in the follicular cells surrounding the oocytes of C. angulata. Upon inhibiting FTZ-F1 gene expression in C. angulata through RNA interference (RNAi), a substantial reduction in the expression of genes involved in the synthesis of sex steroids in the gonads, including 3ß-HSD, Cyp17, and follistatin, was observed. In addition, estradiol (E2) and testosterone (T) levels also showed a decrease upon FTZ-F1 silencing, resulting in a delayed gonadal development. These results indicate that FTZ-F1 acts as a steroidogenic factor, participating in the synthesis and regulation of steroid hormones and thus playing an important role in the reproductive and endocrine systems within oysters.

Comparison of the effects of reef and anthropogenic soundscapes on oyster larvae settlement.

Settlement is a critical period in the life cycle of marine invertebrates with a planktonic larval stage. For reef-building invertebrates such as oysters and corals, settlement rates are predictive for long-term reef survival. Increasing evidence suggests that marine invertebrates use information from ocean soundscapes to inform settlement decisions. Sessile marine invertebrates with a planktonic stage are particularly reliant on environmental cues to direct them to ideal habitats. As gregarious settlers, oysters prefer to settle amongst members of the same species. It has been hypothesized that oyster larvae from species Crassostrea virginica and Ostrea angasi use distinct conspecific oyster reef sounds to navigate to ideal habitats. In controlled laboratory experiments we exposed Pacific Oyster Magallana gigas larvae to anthropogenic sounds from conspecific oyster reefs, vessels, combined reef-vessel sounds as well as off-reef and no speaker controls. Our findings show that sounds recorded at conspecific reefs induced higher percentages of settlement by about 1.44 and 1.64 times compared to off-reef and no speaker controls, respectively. In contrast, the settlement increase compared to the no speaker control was non-significant for vessel sounds (1.21 fold), combined reef-vessel sounds (1.30 fold), and off-reef sounds (1.18 fold). This study serves as a foundational stepping stone for exploring larval sound feature preferences within this species.

A 26-year time series of mortality and growth of the Pacific oyster C. gigas recorded along French coasts.

We used a compiled data set from a monitoring network of oyster production coordinated by IFREMER (the French Research Institute for the Exploitation of the Sea). This network monitors the growth and mortality of the Pacific oyster Crassostrea gigas along French coasts since 1993. The archive, although publicly available, has been challenging to use due to changes in protocols and little information on metadata. Here, we describe data collection for almost 30 years, cleaning and processing. For 13 locations, we modeled growth and mortality of spat (less than one-year-old individuals) and half-grown oysters (between one and two-year-old individuals) as a function of time to cope with changes in data acquisition frequency, and produced standardized annual growth and cumulative mortality indicators to improve data usability. This improved database is expected to be used by ecologists interested in the evolution of life-cycle indicators of a marine species under the influence of climate change. It can also be valuable for epidemiologists because mortality data traces the emergence and spread of a massive epizootic.

Shell Biosynthesis and Pigmentation as Revealed by the Expression of Tyrosinase and Tyrosinase-like Protein Genes in Pacific Oyster (Crassostrea gigas) with Different Shell Colors.

The widely recognized color polymorphisms of molluscan shell have been appreciated for hundreds of years by collectors and scientists, while molecular mechanisms underlying shell pigmentation are still poorly understood. Tyrosinase is a key rate-limiting enzyme for the biosynthesis of melanin. Here, we performed an extensive multi-omics data mining and identified two tyrosinase genes, including tyrosinase and tyrosinase-like protein 2 (Tyr and Typ-2 respectively), in the Pacific oyster Crassostrea gigas, and investigated the expression patterns of tyrosinase during adults and embryogenesis in black and white shell color C. gigas. Tissue expression analysis showed that two tyrosinase genes were both specifically expressed in the mantle, and the expression levels of Tyr and Typ-2 in the edge mantle were significantly higher than that in the central mantle. Besides, Tyr and Typ-2 genes were black shell-specific compared with white shell oysters. In situ hybridization showed that strong signals for Tyr were detected in the inner surface of the outer fold, whereas positive signals for Typ-2 were mainly localized in the outer surface of the outer fold. In the embryos and larvae, the high expression of Tyr mRNA was detected in eyed-larvae, while Typ-2 mRNA was mainly expressed at the trochophore and early D-veliger. Furthermore, the tyrosinase activity in the edge mantle was significantly higher than that in the central mantle. These findings indicated that Tyr gene may be involved in shell pigmentation, and Typ-2 is more likely to play critical roles not only in the formation of shell prismatic layer but also in shell pigmentation. In particular, Typ-2 gene was likely to involve in the initial non-calcified shell of trochophores. The work provides valuable information for the molecular mechanism study of shell formation and pigmentation in C. gigas.

Genome Structural Variation Landscape and Its Selection Signatures in the Fast-growing Strains of the Pacific Oyster, Crassostrea gigas.

The Pacific oyster (Crassostrea gigas) genome is highly polymorphic and affluent in structural variations (SVs), a significant source of genetic variation underlying inter-individual differences. Here, we used two genome assemblies and 535 individuals of genome re-sequencing data to construct a comprehensive landscape of structural variations in the Pacific oyster. Through whole-genome alignment, 11,087 short SVs and 11,561 copy number variations (CNVs) were identified. While analysis of re-sequencing data revealed 511,170 short SVs and 979,486 CNVs, a total of 63,100 short SVs and 58,182 CNVs were identified in at least 20 samples and regarded as common variations. Based on the common short SVs, both Fst and Pi ratio statistical methods were employed to detect the selective sweeps between 20 oyster individuals from the fast-growing strain and 20 individuals from their corresponding wild population. A total of 514 overlapped regions (8.76 Mb), containing 746 candidate genes, were identified by both approaches, in addition with 103 genes within 61 common CNVs only detected in the fast-growing strains. The GO enrichment and KEGG pathway analysis indicated that the identified candidate genes were mostly associated with apical part of cell and were significantly enriched in several metabolism-related pathways, including tryptophan metabolism and histidine metabolism. This work provided a comprehensive landscape of SVs and revealed their responses to selection, which will be valuable for further investigations on genome evolution under selection in the oysters.

Tolerance of northern Gulf of Mexico eastern oysters to chronic warming at extreme salinities.

The eastern oyster, Crassostrea virginica, provides critical ecosystem services and supports valuable fishery and aquaculture industries in northern Gulf of Mexico (nGoM) subtropical estuaries where it is grown subtidally. Its upper critical thermal limit is not well defined, especially when combined with extreme salinities. The cumulative mortalities of the progenies of wild C. virginica from four nGoM estuaries differing in mean annual salinity, acclimated to low (4.0), moderate (20.0), and high (36.0) salinities at 28.9 °C (84 °F) and exposed to increasing target temperatures of 33.3 °C (92 °F), 35.6 °C (96 °F) or 37.8 °C (100 °F), were measured over a three-week period. Oysters of all stocks were the most sensitive to increasing temperatures at low salinity, dying quicker (i.e., lower median lethal time, LT50) than at the moderate and high salinities and resulting in high cumulative mortalities at all target temperatures. Oysters of all stocks at moderate salinity died the slowest with high cumulative mortalities only at the two highest temperatures. The F1 oysters from the more southern and hypersaline Upper Laguna Madre estuary were generally more tolerant to prolonged higher temperatures (higher LT50) than stocks originating from lower salinity estuaries, most notably at the highest salinity. Using the measured temperatures oysters were exposed to, 3-day median lethal Celsius degrees (LD50) were estimated for each stock at each salinity. The lowest 3-day LD50 (35.1-36.0 °C) for all stocks was calculated at a salinity of 4.0, while the highest 3-day LD50 (40.1-44.0 °C) was calculated at a salinity of 20.0.

A Novel Dop2/Invertebrate-Type Dopamine Signaling System Potentially Mediates Stress, Female Reproduction, and Early Development in the Pacific Oyster (Crassostrea gigas).

The dopaminergic signaling pathway is involved in many physiological functions in vertebrates, but poorly documented in protostome species except arthropods. We functionally characterized a novel dopamine receptor in the Pacific oyster (Crassostrea gigas), activated by dopamine and tyramine with different efficacy and potency orders. This receptor - Cragi-DOP2R - belongs to the D1-like family of receptors and corresponds to the first representative of the Dop2/invertebrate-type dopamine receptor (Dop2/INDR) group ever identified in Lophotrochozoa. Cragi-DOP2R transcripts were expressed in various adult tissues, with higher expression levels in the visceral ganglia and the gills. Following an experiment under acute osmotic conditions, Cragi-DOP2R transcripts significantly increased in the visceral ganglia and decreased in the gills, suggesting a role of dopamine signaling in the mediation of osmotic stress. Furthermore, a role of the Cragi-DOP2R signaling pathway in female gametogenesis and in early oyster development was strongly suggested by the significantly higher levels of receptor transcripts in mature female gonads and in the early embryonic stages.

Growth and survival of the native oyster Crassostrea gasar cultured under different stocking densities in two grow-out systems in tropical climate / [Crescimento e sobrevivência da ostra nativa Crassostrea gasar cultivada em diferentes densidades em dois sistemas em clima tropical]

Survival and growth of the native oyster Crassostrea gasar along the juvenile and adult phases were evaluated in three different stocking densities [low (D), medium (2D) and high (3D)] and in two grow-out systems (fixed and floating system). The fixed system consisted of a rack made with PVC, fixed from the bottom with wood sticks. The floating system consisted of floating bags suspended by a rack made with PVC and maintained submerged from the seawater surface by eight floats. Survival and shell height of oysters cultured after 30, 60 and 90 days were registered in each phase and in each grow-out system. Results showed that the grow-out system did not affect survival and growth of C. gasar in the juvenile and adult phases. The tested densities affected the survival of oysters cultured over time in both phases but did not affect oyster growth. At times analyzed, it was observed positive growth in juvenile oysters grow after 90 days of culture. However, in the adult phase, no growth was observed after 90 days of culture. Oyster yield was higher in the density 3D, in both juvenile and adult phases. These findings contributed to the development of the oyster C. gasar culture.(AU)

A sobrevivência e o crescimento da ostra nativa Crassostrea gasar nas fases juvenil e adulta foram avaliados sob três diferentes densidades de estocagem [baixa (D), média (2D) e alta (3D)] e dois sistemas de engorda (fixo e flutuante). O sistema fixo consistiu em uma mesa de PVC, fixada na parte inferior com varas de madeira. O sistema flutuante consistiu em travesseiros flutuantes suspensos por uma mesa de PVC e mantidas submersas da superfície da água do mar por oito flutuadores. Registraram-se sobrevivência e altura da concha de ostras cultivadas após 30, 60 e 90 dias, em cada fase (juvenil e adulta) e em cada sistema (fixo e flutuante). Os resultados mostraram que o sistema de engorda não afetou a sobrevivência e o crescimento de C. gasar nas fases juvenil e adulta. As densidades testadas afetaram a sobrevivência das ostras ao longo do tempo, em ambas as fases, mas não afetaram o crescimento em altura. Nos tempos analisados, ostras juvenis apresentaram crescimento após 90 dias de cultivo. Porém, na fase adulta, não foi observado crescimento após 90 dias de cultivo. A produção de ostras, foi maior na densidade 3D, nas fases juvenil e adulta. Os presentes achados contribuíram para o desenvolvimento do cultivo da ostra C. gasar.(AU)

Susceptibility variation to the main pathogens of Crassostrea gigas at the larval, spat and juvenile stages using unselected and selected oysters to OsHV-1 and/or V. aestuarianus.

French commercial hatcheries are massively producing Crassostrea gigas selected for their higher resistance to OsHV-1, and soon should also implement selection for increasing resistance to Vibrio aestuarianus. The first objective of this study was to optimize the breeding programs for dual resistance to OsHV-1 and V. aestuarianus to determine the earliest life stage for which oysters are able to develop disease resistance. Wild stocks and selected families were tested using experimental infections by both pathogens at the larval, spat and juvenile stages. Oyster families could be evaluated for OsHV-1 as soon as the larval stage by a bath method, but this only highlighted the most resistant families; those that showed the highest resistance to V. aestuarianus could be determined using the cohabitation method at the juvenile stage. The second objective of this study was to determine if selection to increase/decrease the resistance to OsHV-1 and V. aestuarianus could have an impact on other major pathogens currently detected in hatchery at the larval stage, and in nursery and field at the spat/juveniles stages (V. coralliilyticus, V. crassostreae, V. tasmaniensis, V. neptunius, V. europaeus, V. harveyi, V. chagasi). No relationship was found between mortality caused by V. aestuarianus/OsHV-1 and the mortality caused by the other virulent bacterial strains tested regardless the stages, except between OsHV-1 and V. tasmaniensis at the juvenile stage. Finally, miscellaneous findings were evidenced such as (1) bath for bacterial challenges was not adapted for spat, (2) the main pathogens at the larval stage were OsHV-1 and V. coralliilyticus using bath, while it was V. coralliilyticus, V. europaeus, and V. neptunius at the juvenile stage by injection, and (4) variation in mortality was observed among families/wild controls for all pathogens at larval and juvenile stages, except for V. harveyi for larvae.

Striated myosin heavy chain gene is a crucial regulator of larval myogenesis in the pacific oyster Crassostrea gigas.

Pacific oyster (Crassostrea gigas), the most productive economical bivalve mollusc, is identified as an attractive model for developmental studies due to its classical mosaic developmental pattern. Myosin heavy chain is a structural and functional component of myosin, the key muscle protein of thick filament. Here, full length cDNA of striated myosin heavy chains in C. gigas (CgSmhc) was obtained, and the expression profiles were examined in different development stage. CgSmhc had a high expression level in trochophore and D-shaped stage during embryo-larval stage. In adult, CgSmhc was a muscle-specific gene and primarily expressed in muscle tissues. Then, activity of 5' flanking region of CgSmhc were examined through an reconstructed EGFP vector. The results indicated that 3098 bp 5'-flanking region of CgSmhc owned various conserved binding sites of myogenesis-related regulatory elements, and the 2000 bp 5'-flanking sequence was sufficient to induce the CgSmhc expression. Subsequently, the CRISPR/Cas9-mediated target disruption of CgSmhc was generated by co-injection of Cas9mRNA and CgSmhc-sgRNAs into one-cell stage embryos of C. gigas. Loss of CgSmhc had a visible effect on the sarcomeric organization of thin filaments in larval musculature, indicating that CgSmhc was required during larval myogenesis to regulate the correct assembly of sarcomere.

CRISPR/Cas9 Mediated High Efficiency Knockout of Myosin Essential Light Chain Gene in the Pacific Oyster (Crassostrea Gigas).

Pacific oyster (Crassostrea gigas) is one of the most widely cultivated shellfish species in the world. Because of its economic value and complex life cycle involving drastic changes from a free-swimming larva to a sessile juvenile, C. gigas has been used as a model for developmental, environmental, and aquaculture research. However, due to the lack of genetic tools for functional analysis, gene functions associated with biological or economic traits cannot be easily determined. Here, we reported a successful application of CRISPR/Cas9 technology for knockout of myosin essential light chain gene (CgMELC) in C. gigas. C. gigas embryos injected with sgRNAs/Cas9 contained extensive indel mutations at the target sites. The mutant larvae showed defective musculature and reduced motility. In addition, knockout of CgMELC disrupted the expression and patterning of myosin heavy chain positive myofibers in larvae. Together, these data indicate that CgMELC is involved in larval muscle contraction and myogenesis in oyster larvae.

Regulation of sperm motility in Eastern oyster (Crassostrea virginica) spawning naturally in seawater with low salinity.

Oyster aquaculture is expanding worldwide, where many farms rely on seed produced by artificial spawning. As sperm motility and velocity are key determinants for fertilization success, understanding the regulation of sperm motility and identifying optimal environmental conditions can increase fertility and seed production. In the present study, we investigated the physiological mechanisms regulating sperm motility in Eastern oyster, Crassostrea virginica. Sperm motility was activated in ambient seawater with salinity 4-32 PSU with highest motility and velocity observed at 12-24 PSU. In artificial seawater (ASW) with salinity of 20 PSU, sperm motility was activated at pH 6.5-10.5 with the highest motility and velocity recorded at pH 7.5-10.0. Sperm motility was inhibited or totally suppressed in Na+, K+, Ca2+, and Mg2+-free ASW at 20 PSU. Applications of K+ (500 µM glybenclamide and 10-50 mM 4-aminopyridine), Ca2+ (1-50 µM mibefradil and 10-200 µM verapamil), or Na+ (0.2-2.0 mM amiloride) channel blockers into ASW at 20 PSU inhibited or suppressed sperm motility and velocity. Chelating extracellular Ca2+ ions by 3.0 and 3.5 mM EGTA resulted in a significant reduction and full suppression of sperm motility by 4 to 6 min post-activation. These results suggest that extracellular K+, Ca2+, and Na+ ions are involved in regulation of ionic-dependent sperm motility in Eastern oyster. A comparison with other bivalve species typically spawning at higher salinities or in full-strength seawater shows that ionic regulation of sperm motility is physiologically conserved in bivalves. Elucidating sperm regulation in C. virginica has implications to develop artificial reproduction, sperm short-term storage, or cryopreservation protocols, and to better predict how changes in the ocean will impact oyster spawning dynamics.

Identification, characterization, and expression profiles of insulin-like peptides suggest their critical roles in growth regulation of the Pacific oyster, Crassostrea gigas.

The insulin/insulin-like growth factor signaling (IIS) pathway is well-known in regulation of cell growth and proliferation in vertebrates, while its role in invertebrates such as mollusks remains largely unknown. In this study, we performed an extensive multi-omics data mining and identified four insulin-like peptide genes, including ILP, MIRP3, MIRP3-like and ILP7, in the Pacific oyster, Crassostrea gigas. Their potential roles in growth regulation were further investigated using the selectively bred fast-growing C. gigas variety "Haida No.1". Expression profiling and in situ hybridization of these insulin-like peptides suggested their distinct tissue-specific expression pattern, with dominant expression in the neural enrichment tissues such as labial palp, visceral ganglia, adductor muscle, and digestive gland. The expressions of insulin-like peptides were significantly altered by food abundance in a gene-specific fashion. The expression of ILP was reduced during fasting and increased after re-feeding, the expressions of MIRP3 and ILP7 were generally induced during fasting and down-regulated after re-feeding, while the expression of MIRP3-like was firstly up-regulated and then down-regulated during the fasting and re-feeding process. Furthermore, the expressions of all four insulin-like peptide genes were significantly suppressed at low temperature, in accordance with the growth inhibition. These results indicated that all four insulin-like peptides would play critical but different roles in regulation of growth in the oysters. This work provides valuable information for further investigation on growth regulation mechanism in mollusks and molecular assisted breeding of growth with other production traits in the Pacific oyster.

Cloning and characterisation of NMDA receptors in the Pacific oyster, Crassostrea gigas (Thunberg, 1793) in relation to metamorphosis and catecholamine synthesis.

Bivalve metamorphosis is a developmental transition from a free-living larva to a benthic juvenile (spat), regulated by a complex interaction of neurotransmitters and neurohormones such as L-DOPA and epinephrine (catecholamine). We recently suggested an N-Methyl-D-aspartate (NMDA) receptor pathway as an additional and previously unknown regulator of bivalve metamorphosis. To explore this theory further, we successfully induced metamorphosis in the Pacific oyster, Crassostrea gigas, by exposing competent larvae to L-DOPA, epinephrine, MK-801 and ifenprodil. Subsequently, we cloned three NMDA receptor subunits CgNR1, CgNR2A and CgNR2B, with sequence analysis suggesting successful assembly of functional NMDA receptor complexes and binding to natural occurring agonists and the channel blocker MK-801. NMDA receptor subunits are expressed in competent larvae, during metamorphosis and in spat, but this expression is neither self-regulated nor regulated by catecholamines. In-situ hybridisation of CgNR1 in competent larvae identified NMDA receptor presence in the apical organ/cerebral ganglia area with a potential sensory function, and in the nervous network of the foot indicating an additional putative muscle regulatory function. Furthermore, phylogenetic analyses identified molluscan-specific gene expansions of key enzymes involved in catecholamine biosynthesis. However, exposure to MK-801 did not alter the expression of selected key enzymes, suggesting that NMDA receptors do not regulate the biosynthesis of catecholamines via gene expression.

A functional m6 A-RNA methylation pathway in the oyster Crassostrea gigas assumes epitranscriptomic regulation of lophotrochozoan development.

N6 -methyladenosine (m6 A) is a prevalent epitranscriptomic mark in eukaryotic RNA, with crucial roles for mammalian and ecdysozoan development. Indeed, m6 A-RNA and the related protein machinery are important for splicing, translation, maternal-to-zygotic transition and cell differentiation. However, to date, the presence of an m6 A-RNA pathway remains unknown in more distant animals, questioning the evolution and significance of the epitranscriptomic regulation. Therefore, we investigated the m6 A-RNA pathway in the oyster Crassostrea gigas, a lophotrochozoan model whose development was demonstrated under strong epigenetic influence. Using mass spectrometry and dot blot assays, we demonstrated that m6 A-RNA is actually present in the oyster and displays variations throughout early oyster development, with the lowest levels at the end of cleavage. In parallel, by in silico analyses, we were able to characterize at the molecular level a complete and conserved putative m6 A machinery. The expression levels of the identified putative m6 A writers, erasers and readers were strongly regulated across oyster development. Finally, RNA pull-down coupled to LC-MS/MS allowed us to prove the actual presence of readers able to bind m6 A-RNA and exhibiting specific developmental patterns. Altogether, our results demonstrate the conservation of a complete m6 A-RNA pathway in the oyster and strongly suggest its implication in early developmental processes including MZT. This first demonstration and characterization of an epitranscriptomic regulation in a lophotrochozoan model, potentially involved in the embryogenesis, bring new insights into our understanding of developmental epigenetic processes and their evolution.

Bivalves are NO different: nitric oxide as negative regulator of metamorphosis in the Pacific oyster, Crassostrea gigas.

BACKGROUND: Nitric oxide (NO) is presumed to be a regulator of metamorphosis in many invertebrate species, and although NO pathways have been comparatively well-investigated in gastropods, annelids and crustaceans, there has been very limited research on the effects of NO on metamorphosis in bivalve shellfish. RESULTS: In this paper, we investigate the effects of NO pathway inhibitors and NO donors on metamorphosis induction in larvae of the Pacific oyster, Crassostrea gigas. The nitric oxides synthase (NOS) inhibitors s-methylisothiourea hemisulfate salt (SMIS), aminoguanidine hemisulfate salt (AGH) and 7-nitroindazole (7-NI) induced metamorphosis at 75, 76 and 83% respectively, and operating in a concentration-dependent manner. Additional induction of up to 54% resulted from exposures to 1H-[1,2,4]Oxadiazole[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase, with which NO interacts to catalyse the synthesis of cyclic guanosine monophosphate (cGMP). Conversely, high concentrations of the NO donor sodium nitroprusside dihydrate in combination with metamorphosis inducers epinephrine, MK-801 or SMIS, significantly decreased metamorphosis, although a potential harmful effect of excessive NO unrelated to metamorphosis pathway cannot be excluded. Expression of CgNOS also decreased in larvae after metamorphosis regardless of the inducers used, but intensified again post-metamorphosis in spat. Fluorescent detection of NO in competent larvae with DAF-FM diacetate and localisation of the oyster nitric oxide synthase CgNOS expression by in-situ hybridisation showed that NO occurs primarily in two key larval structures, the velum and foot. cGMP was also detected in the foot using immunofluorescent assays, and is potentially involved in the foot's smooth muscle relaxation. CONCLUSION: Together, these results suggest that the NO pathway acts as a negative regulator of metamorphosis in Pacific oyster larvae, and that NO reduction induces metamorphosis by inhibiting swimming or crawling behaviour, in conjunction with a cascade of additional neuroendocrine downstream responses.

Proteome of larval metamorphosis induced by epinephrine in the Fujian oyster Crassostrea angulata.

BACKGROUND: The Fujian oyster Crassostrea angulata is an economically important species that has typical settlement and metamorphosis stages. The development of the oyster involves complex morphological and physiological changes, the molecular mechanisms of which are as yet unclear. RESULTS: In this study, changes in proteins were investigated during larval settlement and metamorphosis of Crassostrea angulata using epinephrine induction. Protein abundance and identity were characterized using label-free quantitative proteomics, tandem mass spectrometry (MS/ MS), and Mascot methods. The results showed that more than 50% (764 out of 1471) of the quantified proteins were characterized as differentially expressed. Notably, more than two-thirds of the differentially expressed proteins were down-regulated in epinephrine-induced larvae. The results showed that "metabolic process" was closely related to the development of settlement and metamorphosis; 5 × 10- 4 M epinephrine induced direct metamorphosis of larvae and was non-toxic. Calmodulin and MAPK pathways were involved in the regulation of settlement of the oyster. Expression levels of immune-related proteins increased during metamorphosis. Hepatic lectin-like proteins, cadherins, calmodulin, calreticulin, and cytoskeletal proteins were involved in metamorphosis. The nervous system may be remodeled in larval metamorphosis induced by epinephrine. Expression levels of proteins that were enriched in the epinephrine signaling pathway may reflect the developmental stage of the larvae, that may reflect whether or not larvae were directly involved in metamorphosis when the larvae were treated with epinephrine. CONCLUSION: The study provides insight into proteins that function in energy metabolism, immune responses, settlement and metamorphosis, and shell formation in C. angulata. The results contribute valuable information for further research on larval settlement and metamorphosis.

The effects of atrazine on the microbiome of the eastern oyster: Crassostrea virginica.

Long-standing evidence supports the importance of maintaining healthy populations of microbiota for the survival, homeostasis, and complete development of marine mollusks. However, the long-term ecological effects of agricultural runoff on these populations remains largely unknown. Atrazine (6-Chloro-n-ethyl-n'-(1-methylethyl)-triazine-2,4-diamine), a prevalent herbicide in the United States, is often used along tributaries of the Chesapeake Bay where oyster breeding programs are concentrated. To investigate any potential effects atrazine maybe having on mollusk-prokaryote interactions, we used 16S rRNA gene amplicons to evaluate how microbial compositions shift in response to exposure of environmentally relevant concentrations of atrazine previously found within the Chesapeake Bay. The dominant bacterial genera found within all groups included those belonging to Pseudoalteromonas, Burkholderia, Bacteroides, Lactobacillis, Acetobacter, Allobaculum, Ruminococcus, and Nocardia. Our results support previously published findings of a possible core microbial community in Crassostrea virginica. We also report a novel finding: oysters exposed to atrazine concentrations as low as 3 µg/L saw a significant loss of a key mutualistic microbial species and a subsequent colonization of a pathogenic bacteria Nocardia. We conclude that exposure to atrazine in the Chesapeake Bay may be contributing to a significant shift in the microbiomes of juvenile oysters that reduces fitness and impedes natural and artificial repopulation of the oyster species within the Bay.

Probiotic Bacterium and Microalga Interaction on Rearing Kumamoto Oyster Crassostrea sikamea Spat.

This study assessed in vitro interaction between Bacillus bacteria and microalgae and their posterior in vivo effect on rearing Kumamoto oyster Crassostrea sikamea. The probiotic strains Bacillus licheniformis (MAt32), B. subtilis (MAt43) and B. subtilis (GAtB1) were individually inoculated in triplicate into 250 mL flasks containing 1 × 104 colony forming units (CFU) mL-1 of bacteria and 4.5 × 104 cell mL-1 of microalgae (Isochrysis galbana or Chaetoceros calcitrans) to evaluate their growth during a 7-day culture. Single cultures of microalgae or bacilli served as control. Additionally, C. sikamea spat was treated for 28 days with four single/combined bacillus treatments in triplicate at a concentration of 1 × 106 CFU mL-1 as follows: (a) control, without treatments; (b) combination of two antibiotics (10 mg L-1); (c) B. licheniformis; (d) B. subtilis; (e) B. subtilis subtilis and (f) mixed bacilli. The results showed a significantly (P < 0.05) increased growth of Bacillus strains co-cultured with microalgae, while the growth of I. galbana co-cultured with bacteria was not reduced significantly (P > 0.05) compared with the control group. C. sikamea spat treated with Bacillus showed significantly (P < 0.05) higher growth and survival than the control group. In this study, C. calcitrans microalgae were susceptible to the presence of probiotic bacteria. Nonetheless, this reduction in microalgal growth observed in vitro increased growth and survival of C. sikamea spat exposed to probiotic bacteria when compared to spat without probiotics.