Phylogenetic analysis of the caspase family in bivalves: implications for programmed cell death, immune response and development.

BACKGROUND: Apoptosis is an important process for an organism's innate immune system to respond to pathogens, while also allowing for cell differentiation and other essential life functions. Caspases are one of the key protease enzymes involved in the apoptotic process, however there is currently a very limited understanding of bivalve caspase diversity and function. RESULTS: In this work, we investigated the presence of caspase homologues using a combination of bioinformatics and phylogenetic analyses. We blasted the Crassostrea gigas genome for caspase homologues and identified 35 potential homologues in the addition to the already cloned 23 bivalve caspases. As such, we present information about the phylogenetic relationship of all identified bivalve caspases in relation to their homology to well-established vertebrate and invertebrate caspases. Our results reveal unexpected novelty and complexity in the bivalve caspase family. Notably, we were unable to identify direct homologues to the initiator caspase-9, a key-caspase in the vertebrate apoptotic pathway, inflammatory caspases (caspase-1, - 4 or - 5) or executioner caspases-3, - 6, - 7. We also explored the fact that bivalves appear to possess several unique homologues to the initiator caspase groups - 2 and - 8. Large expansions of caspase-3 like homologues (caspase-3A-C), caspase-3/7 group and caspase-3/7-like homologues were also identified, suggesting unusual roles of caspases with direct implications for our understanding of immune response in relation to common bivalve diseases. Furthermore, we assessed the gene expression of two initiator (Cg2A, Cg8B) and four executioner caspases (Cg3A, Cg3B, Cg3C, Cg3/7) in C. gigas late-larval development and during metamorphosis, indicating that caspase expression varies across the different developmental stages. CONCLUSION: Our analysis provides the first overview of caspases across different bivalve species with essential new insights into caspase diversity, knowledge that can be used for further investigations into immune response to pathogens or regulation of developmental processes.

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.

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.

Cloning retinoid and peroxisome proliferator-activated nuclear receptors of the Pacific oyster and in silico binding to environmental chemicals.

Disruption of nuclear receptors, a transcription factor superfamily regulating gene expression in animals, is one proposed mechanism through which pollution causes effects in aquatic invertebrates. Environmental pollutants have the ability to interfere with the receptor's functions through direct binding and inducing incorrect signals. Limited knowledge of invertebrate endocrinology and molecular regulatory mechanisms, however, impede the understanding of endocrine disruptive effects in many aquatic invertebrate species. Here, we isolated three nuclear receptors of the Pacific oyster, Crassostrea gigas: two isoforms of the retinoid X receptor, CgRXR-1 and CgRXR-2, a retinoic acid receptor ortholog CgRAR, and a peroxisome proliferator-activated receptor ortholog CgPPAR. Computer modelling of the receptors based on 3D crystal structures of human proteins was used to predict each receptor's ability to bind to different ligands in silico. CgRXR showed high potential to bind and be activated by 9-cis retinoic acid and the organotin tributyltin (TBT). Computer modelling of CgRAR revealed six residues in the ligand binding domain, which prevent the successful interaction with natural and synthetic retinoid ligands. This supports an existing theory of loss of retinoid binding in molluscan RARs. Modelling of CgPPAR was less reliable due to high discrepancies in sequence to its human ortholog. Yet, there are suggestions of binding to TBT, but not to rosiglitazone. The effect of potential receptor ligands on early oyster development was assessed after 24h of chemical exposure. TBT oxide (0.2µg/l), all-trans retinoic acid (ATRA) (0.06 mg/L) and perfluorooctanoic acid (20 mg/L) showed high effects on development (>74% abnormal developed D-shelled larvae), while rosiglitazone (40 mg/L) showed no effect. The results are discussed in relation to a putative direct (TBT) disruption effect on nuclear receptors. The inability of direct binding of ATRA to CgRAR suggests either a disruptive effect through a pathway excluding nuclear receptors or an indirect interaction. Our findings provide valuable information on potential mechanisms of molluscan nuclear receptors and the effects of environmental pollution on aquatic invertebrates.

Dynamics of nuclear receptor gene expression during Pacific oyster development.

BACKGROUND: Nuclear receptors are a highly conserved set of ligand binding transcription factors, with essential roles regulating aspects of vertebrate and invertebrate biology alike. Current understanding of nuclear receptor regulated gene expression in invertebrates remains sparse, limiting our ability to elucidate gene function and the conservation of developmental processes across phyla. Here, we studied nuclear receptor expression in the early life stages of the Pacific oyster, Crassostrea gigas, to identify at which specific key stages nuclear receptors are expressed RESULTS: We used quantitative RT-PCR to determine the expression profiles of 34 nuclear receptors, revealing three developmental key stages, during which nuclear receptor expression is dynamically regulated: embryogenesis, mid development from gastrulation to trochophore larva, and late larval development prior to metamorphosis. Clustering of nuclear receptor expression patterns demonstrated that transcriptional regulation was not directly related to gene phylogeny, suggesting closely related genes may have distinct functions. Expression of gene homologs of vertebrate retinoid receptors suggests participation in organogenesis and shell-formation, as they are highly expressed at the gastrulation and trochophore larval initial shell formation stages. The ecdysone receptor homolog showed high expression just before larval settlement, suggesting a potential role in metamorphosis. CONCLUSION: Throughout early oyster development nuclear receptors exhibited highly dynamic expression profiles, which were not confined by gene phylogeny. These results provide fundamental information on the presence of nuclear receptors during key developmental stages, which aids elucidation of their function in the developmental process. This understanding is essential as ligand sensing nuclear receptors can be disrupted by xenobiotics, a mode of action through which anthropogenic environmental pollutants have been found to mediate effects.

The nuclear receptor gene family in the Pacific oyster, Crassostrea gigas, contains a novel subfamily group.

BACKGROUND: Nuclear receptors are a superfamily of transcription factors important in key biological, developmental and reproductive processes. Several of these receptors are ligand- activated and through their ability to bind endogenous and exogenous ligands, are potentially vulnerable to xenobiotics. Molluscs are key ecological species in defining aquatic and terrestrial habitats and are sensitive to xenobiotic compounds in the environment. However, the understanding of nuclear receptor presence, function and xenobiotic disruption in the phylum Mollusca is limited. RESULTS: Here, forty-three nuclear receptor sequences were mined from the genome of the Pacific oyster, Crassostrea gigas. They include members of NR0-NR5 subfamilies, notably lacking any NR6 members. Phylogenetic analyses of the oyster nuclear receptors have been conducted showing the presence of a large novel subfamily group not previously reported, which is named NR1P. Homologues to all previous identified nuclear receptors in other mollusc species have also been determined including the putative heterodimer partner retinoid X receptor, estrogen receptor and estrogen related receptor. CONCLUSION: C. gigas contains a highly diverse set of nuclear receptors including a novel NR1 group, which provides important information on presence and evolution of this transcription factor superfamily in invertebrates. The Pacific oyster possesses two members of NR3, the sex steroid hormone receptor analogues, of which there are 9 in humans. This provides increasing evidence that steroid ligand specific expansion of this family is deuterostome specific. This new knowledge on divergence and emergence of nuclear receptors in C. gigas provides essential information for studying regulation of molluscan gene expression and the potential effects of xenobiotics.