Expanding the Repertoire of Spongian-16-One Derivatives in Australian Nudibranchs of the Genus Goniobranchus and Evaluation of Their Anatomical Distribution

Extracts of the mantle and viscera of the Indo-Pacific nudibranchs Goniobranchus aureopurpureus and Goniobranchus sp. 1 afforded 11 new diterpenoids (1–11), all of which possess a tetracyclic spongian-16-one scaffold with extensive oxidation at C-6, C-7, C-11, C-12, C-13, and/or C-20. The structures and relative configuration were investigated by NMR experiments, while X-ray crystallography provided the absolute configuration of 1, including a 2′S configuration for the 2-methylbutanoate substituent located at C-7. Dissection of animal tissue revealed that the mantle and viscera tissues differed in their metabolite composition with diterpenes 1–11 present in the mantle tissue of the two nudibranch species.

Transcription of Genes Involved in Biomineralization in Two Mantle Morphs of Pearl Oyster, Pinctada Persica

A few species of mollusks display color variation in their soft tissues. In pearl oysters, the color polymorphism in mantle tissue is associated with the color and radiance of shell and pearl. The study of biomineralization related genes in mantle tissue of pearl oysters can be used as a suitable approach to better identify the molecular mechanisms that influence shell and pearl quality and color variations. In this study, we investigated the transcription of biomineralization-related genes in black and orange mantle morphotypes of pearl oyster, Pinctada persica in both warm and cool seasons using quantitative real-time PCR. Our results showed that the genes involved in biomineralization of the prismatic and nacre layer, i.e.; ASP, KRMP, MRNP34, SHELL, SHEM1B, LINKINE, PIF, SHEM5, NACREIN, and in pigmentation (TYR2A) were significantly highly expressed in orange phenotype compared to those of black one, suggesting the existence of different genetic processes between two color morphs of mantle tissue and the more active role of genes in orange morphotype. In black mantle phenotype, ASP, KRMP, SHEM5 and PIF and in the orange phenotype, only KRMP and PRISM showed difference in seasonal expression. This study provides an accurate understanding of the mantle trait of P. persica.

Independent adoptions of a set of proteins found in the matrix of the mineralized shell-like eggcase of Argonaut octopuses

The Argonaut octopus, commonly called the paper nautilus, has a spiral-coiled shell-like eggcase. As the main characteristics, the eggcase has no internal septum, is composed entirely of calcite with chitosan being the main polycarbonate and is reportedly formed by organic materials secreted from the membranes of the arms. Meanwhile, the biomineralized external "true" shells of the Mollusks, which includes the Cephalopods, are secreted from the mantle tissue. Therefore, the histological origin of the two shells is completely different. The question of how the Argonauts, which phylogenetically diverged from the completely shell-less octopuses, could form a converging shell-like external structure has thus intrigued biologists for a long time. To answer this question, we performed a multi-omics analysis of the transcriptome and proteome of the two congeneric Argonaut species, Argonauta argo and A. hians. Our result indicates that the shell-like eggcase is not a homolog of the shell, even at the protein level, because the Argonauts apparently recruited a different set of protein repertoires to as eggcase matrix proteins (EcMPs). However, we also found the homologs of three shell matrix proteins (SMPs) of the Conchiferan Mollusks, Pif-like, SOD, and TRX, in the eggcase matrix. The proteins were also found in the only surviving shelled Cephalopods, the nautiloid Nautilus pompilius. Phylogenetic analysis revealed that homologous genes of the Conchiferan SMPs and EcMPs were found in the draft genome of shell-less octopuses. Our result reported here thus suggests that the SMP-coding genes are conserved in both shelled and shell-less Cephalopods. Meanwhile, the Argonauts adopted some of the SMP-coding genes and other non-SMP-coding genes, to form a convergent, non-homologous biomineralized external structure, the eggcase, which is autapomorphic to the group.

Transcriptomic analysis of shell repair and biomineralization in the blue mussel, Mytilus edulis

Background Biomineralization by molluscs involves regulated deposition of calcium carbonate crystals within a protein framework to produce complex biocomposite structures. Effective biomineralization is a key trait for aquaculture, and animal resilience under future climate change. While many enzymes and structural proteins have been identified from the shell and in mantle tissue, understanding biomieralization is impeded by a lack of fundamental knowledge of the genes and pathways involved. In adult bivalves, shells are secreted by the mantle tissue during growth, maintenance and repair, with the repair process, in particular, amenable to experimental dissection at the transcriptomic level in individual animals. Results Gene expression dynamics were explored in the adult blue mussel, Mytilus edulis, during experimentally induced shell repair, using the two valves of each animal as a matched treatment-control pair. Gene expression was assessed using high-resolution RNA-Seq against a de novo assembled database of functionally annotated transcripts. A large number of differentially expressed transcripts were identified in the repair process. Analysis focused on genes encoding proteins and domains identified in shell biology, using a new database of proteins and domains previously implicated in biomineralization in mussels and other molluscs. The genes implicated in repair included many otherwise novel transcripts that encoded proteins with domains found in other shell matrix proteins, as well as genes previously associated with primary shell formation in larvae. Genes with roles in intracellular signalling and maintenance of membrane resting potential were among the loci implicated in the repair process. While haemocytes have been proposed to be actively involved in repair, no evidence was found for this in the M. edulis data. Conclusions The shell repair experimental model and a newly developed shell protein domain database efficiently identified transcripts involved in M. edulis shell production. In particular, the matched pair analysis allowed factoring out of much of the inherent high level of variability between individual mussels. This snapshot of the damage repair process identified a large number of genes putatively involved in biomineralization from initial signalling, through calcium mobilization to shell construction, providing many novel transcripts for future in-depth functional analyses.

Molecular Characterization of Carbonic Anhydrase II (CA II) and Its Potential Involvement in Regulating Shell Formation in the Pacific Abalone, Haliotis discus hannai

Carbonic anhydrases (CAs) are a family of metalloenzymes that can catalyze the reversible interconversion of CO2/HCO3–, ubiquitously present in both prokaryotes and eukaryotes. In the present study, a CA II (designated as HdhCA II) was sequenced and characterized from the mantle tissue of the Pacific abalone. The complete sequence of HdhCA II was 1,169 bp, encoding a polypeptide of 349 amino acids with a NH2-terminal signal peptide and a CA architectural domain. The predicted protein shared 98.57% and 68.59% sequence identities with CA II of Haliotis gigantea and Haliotis tuberculata, respectively. Two putative N-linked glycosylation motifs and two cysteine residues could potentially form intramolecular disulfide bond present in HdhCA II. The phylogenetic analysis indicated that HdhCA II was placed in a gastropod clade and robustly clustered with CA II of H. gigantea and H. tuberculata. The highest level of HdhCA II mRNA expression was detected in the shell forming mantle tissue. During ontogenesis, the mRNA of HdhCA II was detected in all stages, with larval shell formation stage showing the highest expression level. The in situ hybridization results detected the HdhCA II mRNA expression in the epithelial cells of the dorsal mantle pallial, an area known to express genes involved in the formation of a nacreous layer in the shell. This is the first report of HdhCA II in the Pacific abalone, and the results of this study indicate that this gene might play a role in the shell formation of abalone.

Ocean acidification induces subtle shifts in gene expression and DNA methylation in mantle tissue of the Eastern oyster (Crassostrea virginica)

Early evidence suggests that DNA methylation can mediate phenotypic responses of marine calcifying species to ocean acidification (OA). Few studies, however, have explicitly studied DNA methylation in calcifying tissues through time. Here, we examined the phenotypic and molecular responses in the extrapallial fluid and mantle (fluid and tissue at the calcification site) in the Eastern oyster (Crassostrea virginica) exposed to experimental OA over 80 days. Oysters were reared under three experimental pCO2 treatments (‘control’, 580 μatm; ‘moderate OA’, 1000 uatm; ‘high OA’, 2800 μatm) and sampled at 6 time points (24 hours - 80 days). We found that high OA initially induced changes in the pH of the extrapallial fluid (pHEPF) relative to the external seawater, but the magnitude of this difference was highest at 9 days and diminished over time. Calcification rates were significantly lower in the high OA treatment compared to the other treatments. To explore how oysters regulate their extrapallial fluid, gene expression and DNA methylation were examined in the mantle-edge tissue of oysters from day 9 and 80 in the control and high OA treatments. Mantle tissue mounted a significant global molecular response (both in the transcriptome and methylome) to OA that shifted through time. Although we did not find individual genes that were significantly differentially expressed to OA, the pHEPF was correlated with the eigengene expression of several co-expressed gene clusters. A small number of OA-induced differentially methylated loci were discovered, which corresponded with a weak association between OA-induced changes in genome-wide gene body DNA methylation and gene expression. Gene body methylation, however, was not significantly correlated with the eigengene expression of pHEPF correlated gene clusters. These results suggest that in C. virginica, OA induces a subtle response in a large number of genes, but also indicates that plasticity at the molecular level may be limited. Our study highlights the need to re-assess the plasticity of tissue-specific molecular responses in marine calcifiers, as well as the role of DNA methylation and gene expression in mediating physiological and biomineralization responses to OA.

Developments in marine invertebrate primary culture reveal novel cell morphologies in the model bivalve Crassostrea gigas

Cell culture provides useful model systems used in a wide range of biological applications, but its utility in marine invertebrates is limited due to the lack of immortalised cell lines. Primary cell and tissue cultures are typically used but remain poorly characterised for oysters, which can cause issues with experimental consistency and reproducibility. Improvements to methods of repeatable isolation, culture, and characterisation of oyster cells and tissues are required to help address these issues. In the current study, systematic improvements have been developed to facilitate the culture of primary cells from adult Pacific oyster tissues and identify novel cell morphologies that have not been reported previously. Cultures analysed by light microscopy, qPCR, and live cell imaging demonstrated maintenance of live, metabolically active Pacific oyster cells for several weeks post-explant. Interestingly, whole hearts dissected from adult oysters were found to continue contracting rhythmically up to 8 weeks after being transferred to a tissue culture system. Mantle tissue explants were also actively moving in the culture system. These improvements in primary cell culture of bivalves may be beneficial for research in ecotoxicology, virology, immunology, and genetic resistance to disease.