Molecular Characterization of Retinoic Acid Receptor CgRAR in Pacific Oyster (Crassostrea gigas).

Retinoic acid (RA) signaling pathways mediated by RA receptors (RARs) are essential for many physiological processes such as organ development, regeneration, and differentiation in animals. Recent studies reveal that RARs identified in several mollusks, including Pacific oyster Crassostrea gigas, have a different function mechanism compared with that in chordates. In this report, we identified the molecular characteristics of CgRAR to further explore the mechanism of RAR in mollusks. RT-qPCR analysis shows that CgRAR has a higher expression level in the hemocytes and gonads, indicating that CgRAR may play roles in the processes of development and metabolism. The mRNA expression level of both CgRAR and CgRXR was analyzed by RT-qPCR after injection with RA. The elevated expression of CgRAR and CgRXR was detected upon all-trans-RA (ATRA) exposure. Finally, according to the results of Yeast Two-Hybrid assay and co-immunoprecipitation analysis, CgRAR and CgRXR can interact with each other through the C-terminal region. Taken together, our results suggest that CgRAR shows a higher expression level in gonads and hemocytes. ATRA exposure up-regulates the expression of CgRAR and CgRXR. Besides, CgRAR can interact with CgRXR to form a heterodimer complex.

Nanopore Sequencing and De Novo Assembly of a Black-Shelled Pacific Oyster (Crassostrea gigas) Genome.

The Pacific oyster, Crassostrea gigas, belongs to one of the most species-rich phyla and provides important ecological and economical services. Here we present a genome assembly for a variety of this species, black-shelled Pacific oyster, using a combination of 61.8 Gb Nanopore long reads and 105.6 Gb raw BGI-seq short reads. The genome assembly comprised 3,676 contigs, with a total length of 587 Mb and a contig N50 of 581 kb. Annotation of the genome assembly identified 283 Mb (48.32%) of repetitive sequences and a total of 26,811 protein-coding genes. A long-term transposable element active, accompanied by recent expansion (1 million years ago), was detected in this genome. The divergence between black-shelled and the previous published Pacific oysters was estimated at about 2.2 million years ago, which implies that species C. gigas had great intraspecific genetic variations. Moreover, we identified 148/188 specifically expanded/contracted gene families in this genome. We believe this genome assembly will be a valuable resource for understanding the genetic breeding, conservation, and evolution of oysters and bivalves.

Immune-related molecular and physiological differences between black-shelled and white-shelled Pacific oysters Crassostrea gigas.

The black-and-white traits on shells and mantle edges of the Pacific oyster, Crassostrea gigas, are inheritable and correlated, and black shells (melanin pigmentation) are usually found in the Pacific oysters. Based on differentially expressed genes from RNA-Seq and physiological characteristics, in this study, Black-shelled Pacific oysters (BSO) and White-shelled Pacific oysters (WSO) were selected to determine the molecular differences between oysters with obviously different melanin content. The differences in the process of immune recognition and modulation indicated that BSO may be more sensitive to the immune substances. There might have different modulation mode of apoptosis and phagocytosis between BSO and WSO, and caspase-3 might have played a key role in the apoptotic process of BSO. Different oxidation-related pathways were enriched in both BSO and WSO, suggesting the different response strategies of BSO and WSO to oxidative stress. The physiological evidences showed that, compared with WSO, in BSO, the tyrosinase content, the caspase-3 activity and the suppression of hydroxyl radical increased, and the reactive oxygen species concentration decreased. Therefore, immune-related molecular and physiological differences were found between BSO and WSO.

PiggyBac Transposon-Mediated Transgenesis in the Pacific Oyster (Crassostrea gigas) - First Time in Mollusks.

As an effective method of transgenesis, the plasmid of PiggyBac transposon containing GFP (PiggyBac) transposon system has been widely used in various organisms but not yet in mollusks. In this work, piggyBac containing green fluorescent protein (GFP) was transferred into the Pacific oyster (Crassostrea gigas) by sperm-mediated gene transfer with or without electroporation. Fluorescent larvae were then observed and isolated under an inverted fluorescence microscope, and insertion of piggyBac was tested by polymerase chain reaction (PCR) using genomic DNA as template. Oyster larvae with green fluorescence were observed after transgenesis with or without electroporation, but electroporation increased the efficiency of sperm-mediated transgenesis. Subsequently, the recombinant piggyBac plasmid containing gGH (piggyBac-gGH) containing GFP and a growth hormone gene from orange-spotted grouper (gGH) was transferred into oysters using sperm mediation with electroporation, and fluorescent larvae were observed and isolated. The insertion of piggyBac-gGH was tested by PCR and genome walking analysis. PCR analysis indicated that piggyBac-gGH was transferred into oyster larvae; genome walking analysis further showed the detailed location where piggyBac-gGH was inserted in the oyster genome. This is the first time that piggyBac transposon-mediated transgenesis has been applied in mollusks.

A Rhodopsin-Like Gene May Be Associated With the Light-Sensitivity of Adult Pacific Oyster Crassostrea gigas.

Light-sensitivity is important for mollusc survival, as it plays a vital role in reproduction and predator avoidance. Light-sensitivity has been demonstrated in the adult Pacific oyster Crassostrea gigas, but the genes associated with light-sensitivity remain unclear. In the present study, we designed experiments to identify the genes associated with light-sensitivity in adult oysters. First, we assessed the Pacific oyster genome and identified 368 genes annotated with the terms associated with light-sensitivity. Second, the function of the four rhodopsin-like superfamily member genes was tested by using RNAi. The results showed that the highest level of mRNA expression of the vision-related genes was in the mantle; however, this finding is not true for all oyster genes. Interestingly, we also found four rhodopsin-like superfamily member genes expressed at an very high level in the mantle tissue. In the RNAi experiment, when one of rhodopsin-like superfamily member genes (CGI_1001253) was inhibited, the light-sensitivity capacity of the injected oysters was significantly reduced, suggesting that CGI_10012534 may be associated with light-sensitivity in the adult Pacific oyster.

The Molecular Differentiation of Anatomically Paired Left and Right Mantles of the Pacific Oyster Crassostrea gigas.

Left-right (L-R) asymmetry is controlled by gene regulation pathways for the L-R axis, and in vertebrates, the gene Pitx2 in TGF-ß signaling pathway plays important roles in the asymmetrical formation of organs. However, less is known about the asymmetries of anatomically identical paired organs, as well as the transcriptional regulation mechanism of the gene Pitx in invertebrates. Here, we report the molecular biological differences between the left and right mantles of an invertebrate, the Pacific oyster Crassostrea gigas, and propose one possible mechanism underlying those differences. RNA sequencing (RNA-seq) analysis indicated that the paired organs showed different gene expression patterns, suggesting possible functional differences in shell formation, pheromone signaling, nerve conduction, the stress response, and other physiological processes. RNA-seq and real-time qPCR analysis indicated high right-side expression of the Pitx homolog (cgPitx) in oyster mantle, supporting a conserved role for Pitx in controlling asymmetry. Methylation-dependent restriction-site associated DNA sequencing (MethylRAD) identified a methylation site in the promoter region of cgPitx and showed significantly different methylation levels between the left and right mantles. This is the first report, to our knowledge, of such a difference in methylation in spiralians, and it was further confirmed in 18 other individuals by using a pyrosequencing assay. The miRNome analysis and the TGF-ß receptor/Smad inhibition experiment further supported that several genes in TGF-ß signaling pathway may be related with the L/R asymmetry of oyster mantles. These results suggested that the molecular differentiation of the oyster's paired left and right mantles is significant, TGF-ß signaling pathway could be involved in establishing or maintaining the asymmetry, and the cgPitx gene as one of genes in this pathway; the different methylation levels in its promoter regions between L/R mantles was the one of possible mechanisms regulating the left-right functional differentiation.

A Preliminary Study on the Pattern, the Physiological Bases and the Molecular Mechanism of the Adductor Muscle Scar Pigmentation in Pacific Oyster Crassostrea gigas.

The melanin pigmentation of the adductor muscle scar and the outer surface of the shell are among attractive features and their pigmentation patterns and mechanism still remains unknown in the Pacific oyster Crassostrea gigas. To study these pigmentation patterns, the colors of the adductor muscle scar vs. the outer surface of the shell on the same side were compared. No relevance was found between the colors of the adductor muscle scars and the corresponding outer surface of the shells, suggesting that their pigmentation processes were independent. Interestingly, a relationship between the color of the adductor muscle scars and the dried soft-body weight of Pacific oysters was found, which could be explained by the high hydroxyl free radical scavenging capacity of the muscle attached to the black adductor muscle scar. After the transcriptomes of pigmented and unpigmented adductor muscles and mantles were studied by RNAseq and compared, it was found that the retinol metabolism pathway were likely to be involved in melanin deposition on the adductor muscle scar and the outer surface of the shell, and that the different members of the tyrosinase or Cytochrome P450 gene families could play a role in the independent pigmentation of different organs.

Molecular Cloning and Characterization of Full-Length cDNA of Calmodulin Gene from Pacific Oyster Crassostrea gigas.

The shell of the pearl oyster (Pinctada fucata) mainly comprises aragonite whereas that of the Pacific oyster (Crassostrea gigas) is mainly calcite, thereby suggesting the different mechanisms of shell formation between above two mollusks. Calmodulin (CaM) is an important gene for regulating the uptake, transport, and secretion of calcium during the process of shell formation in pearl oyster. It is interesting to characterize the CaM in oysters, which could facilitate the understanding of the different shell formation mechanisms among mollusks. We cloned the full-length cDNA of Pacific oyster CaM (cgCaM) and found that the cgCaM ORF encoded a peptide of 113 amino acids containing three EF-hand calcium-binding domains, its expression level was highest in the mantle, hinting that the cgCaM gene is probably involved in shell formation of Pacific oyster, and the common ancestor of Gastropoda and Bivalvia may possess at least three CaM genes. We also found that the numbers of some EF hand family members in highly calcified species were higher than those in lowly calcified species and the numbers of these motifs in oyster genome were the highest among the mollusk species with whole genome sequence, further hinting the correlation between CaM and biomineralization.