Differential analysis of the haemolymph proteome of Carcinus maenas parasitized by Sacculina carcini (Cirripeda, Rhizocephala) reveals potential mechanisms of parasite control.

Sacculina carcini is an endoparasite of the green crab, Carcinus maenas. This parasite induces behavioural changes in its host and affects its metabolism by inhibiting moulting and reproduction. Using a proteomic approach in mass spectrometry, we studied the haemolymph proteomes of healthy and parasitized wild green crabs from Brittany, France to identify proteins that are differentially expressed as a consequence of parasitization. We also investigated specific proteins involved in reproduction, moulting, and immunity. We detected 77 proteins for females and 53 proteins for males that were differentially present between the healthy and parasitized crabs, some of which were sex-specific. Detection of these differentially expressed proteins suggests that the parasite can inhibit and promote different aspects of the immune response of the host. Sacculina appears to inhibit host melanisation for self-protection, while promoting the presence of immune factors, such as antimicrobial peptides to cope with possible bacterial superinfections. Moreover, one protein, juvenile hormone esterase-like carboxylesterase, was 17-times more abundant in parasitized crabs than in healthy crabs and may be responsible for inhibiting moulting and reproduction in parasitized crabs, thus ensuring the success of Sacculina reproduction.

Molecular and physiological characterization of a crustacean cardioactive signaling system in a lophotrochozoan - the Pacific oyster (Crassostrea gigas): a role in reproduction and salinity acclimation.

The crustacean cardioactive peptide (CCAP) is an important neuropeptide involved in the regulation of a variety of physiological processes in arthropods. Although this family of peptides has an ancestral origin, its function remains poorly understood among protostome species - apart from arthropods. We functionally characterized three G protein-coupled receptors (GPCRs) in the oyster Crassostrea gigas, phylogenetically related to ecdysozoan CCAP receptors (CCAPRs) and to chordate neuropeptide S receptors (NPSRs). Cragi-CCAPR1 and Cragi-CCAPR2 were specifically activated by the Cragi-CCAP1 and Cragi-CCAP2 peptides, respectively, both derived from the same CCAP precursor. In contrast, Cragi-CCAPR3 was only partially activated by CCAP1 and CCAP2 at high concentrations. The Cragi-CCAPR1 and Cragi-CCAPR2 genes were expressed in various adult tissues. They are both most expressed in the gills, while Cragi-CCAPR3 is mainly expressed in the visceral ganglia (VG). Cragi-CCAP precursor transcripts are higher in the VG, the labial palps and the gills. Receptor and ligand-encoding transcripts are more abundantly expressed in the gonads in the first stages of gametogenesis, while the Cragi-CCAP precursor is upregulated in the VG in the last stages of gametogenesis. This suggests a role of the CCAP signaling system in the regulation of reproductive processes. A role in water and ionic regulation is also supported considering the differential expression of the CCAP signaling components in oysters exposed to brackish water.

Identification and structural characterization of the factors involved in vitellogenesis and its regulation in the African Osteoglossiforme of aquacultural interest Heterotis niloticus (Cuvier, 1829).

The African bonytongue (Heterotis niloticus) is an excellent candidate for fish farming because it has outstanding biological characteristics and zootechnical performances. However, the absence of sexual dimorphism does not favor its reproduction in captivity or the understanding of its reproductive behavior. Moreover, no molecular data related to its reproduction is yet available. This study therefore focuses on the structural identification of the different molecular actors of vitellogenesis expressed in the pituitary gland, the liver and the ovary of H. niloticus. A transcriptomic approach based on de novo RNA sequencing of the pituitary gland, ovary and liver of females in vitellogenesis led to the creation of three transcriptomes. In silico analysis of these transcriptomes identified the sequences of pituitary hormones such as prolactin (PRL), luteinizing hormone (LH) and follicle-stimulating hormone (FSH) and their ovarian receptors (PRLR, FSHR, LHR). In the liver and ovary, estrogen receptors (ER) beta and gamma, liver vitellogenins (VtgB and VtgC) and their ovarian receptors (VLDLR) were identified. Finally, the partial transcript of an ovarian Vtg weakly expressed compared to hepatic Vtg was identified based on structural criteria. Moreover, a proteomic approach carried out from mucus revealed the presence of one Vtg exclusively in females in vitellogenesis. In this teleost fish that does not exhibit sexual dimorphism, mucus Vtg could be used as a sexing biomarker based on a non-invasive technique compatible with the implementation of experimental protocols in vivo.

Characterization of an evolutionarily conserved calcitonin signalling system in a lophotrochozoan, the Pacific oyster (Crassostrea gigas).

In Protostoma, the diuretic hormone 31 (DH31) signalling system was long considered as the orthologue of the chordate calcitonin (CT) signalling system. Using the Pacific oyster (Crassostrea gigas) transcriptomic database GigaTON, we characterized seven G-protein-coupled receptors (GPCRs) named Cragi-CTR1-7 and phylogenetically related to chordate CT receptors (CTRs) and to protostome DH31 receptors. Two CT precursors (Cragi-CTP1 and Cragi-CTP2) containing two CT-type peptides and encoded by two distinct genes with a similar organization were also characterized. These oyster neuropeptides (Cragi-CT1/2) exhibit the two N-terminal paired cysteine residues and, except CTP2-derived peptide (Cragi-CTP2dp), show the C-terminal proline-amide motif typical of deuterostome CT-type peptides. All mature Cragi-CTs except Cragi-CTP2dp were detected in visceral ganglion extracts using mass spectrometry. Cell-based assays revealed that the previously characterized oyster receptors Cg-CT-R and Cragi-CTR2 were specifically activated by Cragi-CT1b and Cragi-CT2, respectively. This activation does not require the co-expression of receptor activity-modifying proteins (RAMPs). Thus, oyster CT signalling appears functionally more closely related to vertebrate CT/CTR signalling than to calcitonin gene-related peptide/calcitonin receptor-like receptor (CGRP/CLR) signalling. Gene expression profiles in different adult tissues and in oysters acclimated to brackish water suggest the potential implication of both Cg-CT-R/Cragi-CT1b and Cragi-CTR2/Cragi-CT2 in water and ionic regulations, although with apparently opposite effects. The present study represents the first comprehensive characterization of a functional CT-type signalling system in a protostome and provides evidence for its evolutionarily ancient origin and its early role in osmotic homeostasis.

Emergence of a cholecystokinin/sulfakinin signalling system in Lophotrochozoa.

Chordate gastrin/cholecystokinin (G/CCK) and ecdysozoan sulfakinin (SK) signalling systems represent divergent evolutionary scenarios of a common ancestral signalling system. The present article investigates for the first time the evolution of the CCK/SK signalling system in a member of the Lophotrochozoa, the second clade of protostome animals. We identified two G protein-coupled receptors (GPCR) in the oyster Crassostrea gigas (Mollusca), phylogenetically related to chordate CCK receptors (CCKR) and to ecdysozoan sulfakinin receptors (SKR). These receptors, Cragi-CCKR1 and Cragi-CCKR2, were characterised functionally using a cell-based assay. We identified di- and mono-sulphated forms of oyster Cragi-CCK1 (pEGAWDY(SO3H)DY(SO3H)GLGGGRF-NH2) as the potent endogenous agonists for these receptors. The Cragi-CCK genes were expressed in the visceral ganglia of the nervous system. The Cragi-CCKR1 gene was expressed in a variety of tissues, while Cragi-CCKR2 gene expression was more restricted to nervous tissues. An in vitro bioassay revealed that different forms of Cragi-CCK1 decreased the frequency of the spontaneous contractions of oyster hindgut. Expression analyses in oysters with contrasted nutritional statuses or in the course of their reproductive cycle highlighted the plausible role of Cragi-CCK signalling in the regulation of feeding and its possible involvement in the coordination of nutrition and energy storage in the gonad. This study confirms the early origin of the CCK/SK signalling system from the common bilaterian ancestor and delivers new insights into its structural and functional evolution in the lophotrochozoan lineage.

Characterization of a tachykinin signalling system in the bivalve mollusc Crassostrea gigas.

Although tachykinin-like neuropeptides have been identified in molluscs more than two decades ago, knowledge on their function and signalling has so far remained largely elusive. We developed a cell-based assay to address the functionality of the tachykinin G-protein coupled receptor (Cragi-TKR) in the oyster Crassostrea gigas. The oyster tachykinin neuropeptides that are derived from the tachykinin precursor gene Cragi-TK activate the Cragi-TKR in nanomolar concentrations. Receptor activation is sensitive to Ala-substitution of critical Cragi-TK amino acid residues. The Cragi-TKR gene is expressed in a variety of tissues, albeit at higher levels in the visceral ganglia (VG) of the nervous system. Fluctuations of Cragi-TKR expression is in line with a role for TK signalling in C. gigas reproduction. The expression level of the Cragi-TK gene in the VG depends on the nutritional status of the oyster, suggesting a role for TK signalling in the complex regulation of feeding in C. gigas.

Molecular characterization of an adipokinetic hormone-related neuropeptide (AKH) from a mollusk.

Adipokinetic hormones (AKH) are key regulators of energy mobilization in insects. With the growing number of genome sequence available, the existence of genes encoding AKH related peptides has now been established in protostomes. Here we investigated the occurrence of a mature AKH-like neuropeptide (Cg-AKH) in the oyster Crassostrea gigas. We unambiguously elucidated the primary structure of this neuropeptide by mass spectrometry from peptidic extracts of oyster visceral ganglia. Cg-AKH mature peptide (pQVSFSTNWGS-amide) represents an additional member of the AKH family of peptides. The organization of Cg-AKH encoding gene and its corresponding transcript is also described. Cg-AKH gene was found to be expressed in the nervous system though at extremely low levels compared to other neuropeptide encoding genes such as the oyster GnRH gene. Although both reproduction and feeding are known to affect the energy balance in oysters, no significant differential expression of Cg-AKH gene could be evidenced in relation with the nutritional status or along the reproductive cycle. The possible involvement of Cg-AKH in the regulation of energy balance in oyster remains an open question.

Functional characterization of a short neuropeptide F-related receptor in a lophotrochozoan, the mollusk Crassostrea gigas.

Members of the short neuropeptide F (sNPF) family of peptides and their cognate receptors play key roles in a variety of physiological processes in arthropods. In silico screening of GigasDatabase, a specific expressed sequence tag database from the Pacific oyster Crassostrea gigas, resulted in the identification of a receptor (Cg-sNPFR-like) phylogenetically closely related to sNPF receptors (sNPFRs) of insects. A reverse endocrinology approach was undertaken to identify the peptide ligand(s) of this orphan receptor. Though structurally distinct from insect sNPFs, three RFamide peptides derived from the same precursor, i.e. GSLFRFamide, SSLFRFamide and GALFRFamide, specifically activate the receptor in a dose-dependent manner, with respective EC50 values (half-maximal effective concentrations) of 1.1, 2.1 and 4.1 µmol l(-1). We found that both Cg-sNPFR-like receptor and LFRFamide encoding transcripts are expressed in the oyster central nervous system and in other tissues as well, albeit at lower levels. Mass spectrometry analysis confirmed the wide distribution of LFRFamide mature peptides in several central and peripheral tissues. The Cg-sNPFR-like receptor was more abundantly expressed in ganglia of females than of males, and upregulated in starved oysters. In the gonad area, highest receptor gene expression occurred at the start of gametogenesis, when storage activity is maximal. Our results suggest that signaling of LFRFamide peptides through the Cg-sNPFR-like receptor might play a role in the coordination of nutrition, energy storage and metabolism in C. gigas, possibly by promoting storage at the expense of reproduction.

Gametogenesis in the Pacific oyster Crassostrea gigas: a microarrays-based analysis identifies sex and stage specific genes.

BACKGROUND: The Pacific oyster Crassostrea gigas (Mollusca, Lophotrochozoa) is an alternative and irregular protandrous hermaphrodite: most individuals mature first as males and then change sex several times. Little is known about genetic and phenotypic basis of sex differentiation in oysters, and little more about the molecular pathways regulating reproduction. We have recently developed and validated a microarray containing 31,918 oligomers (Dheilly et al., 2011) representing the oyster transcriptome. The application of this microarray to the study of mollusk gametogenesis should provide a better understanding of the key factors involved in sex differentiation and the regulation of oyster reproduction. METHODOLOGY/PRINCIPAL FINDINGS: Gene expression was studied in gonads of oysters cultured over a yearly reproductive cycle. Principal component analysis and hierarchical clustering showed a significant divergence in gene expression patterns of males and females coinciding with the start of gonial mitosis. ANOVA analysis of the data revealed 2,482 genes differentially expressed during the course of males and/or females gametogenesis. The expression of 434 genes could be localized in either germ cells or somatic cells of the gonad by comparing the transcriptome of female gonads to the transcriptome of stripped oocytes and somatic tissues. Analysis of the annotated genes revealed conserved molecular mechanisms between mollusks and mammals: genes involved in chromatin condensation, DNA replication and repair, mitosis and meiosis regulation, transcription, translation and apoptosis were expressed in both male and female gonads. Most interestingly, early expressed male-specific genes included bindin and a dpy-30 homolog and female-specific genes included foxL2, nanos homolog 3, a pancreatic lipase related protein, cd63 and vitellogenin. Further functional analyses are now required in order to investigate their role in sex differentiation in oysters. CONCLUSIONS/SIGNIFICANCE: This study allowed us to identify potential markers of early sex differentiation in the oyster C. gigas, an alternative hermaphrodite mollusk. We also provided new highly valuable information on genes specifically expressed by mature spermatozoids and mature oocytes.

Identification of three singular glycosyl hydrolase family 18 members from the oyster Crassostrea gigas: Structural characterization, phylogenetic analysis and gene expression.

Glycoside hydrolase family 18 (GH18) includes chitinases and non-enzymatic chitinase-like proteins (CLPs) with representatives among eukaryotes (animals and plants), prokaryotes and viruses. In Lophotrochozoa, one of the three clades of bilaterian animals, only three members (Cg-Clp1, Cg-Clp2 and Cg-Chit) have been reported from the bivalve mollusc Crassostrea gigas. Here, we describe the cloning and the characterization of two additional chitinases (Cg-Chit2 and Cg-Chit3) and a new CLP (Cg-Clp3) from this species. Cg-Chit2 presents an atypical C-terminal hydrophobic region acting probably as a GPI-anchor signal for plasma membrane attachment. On the contrary, Cg-Chit3 displays a C-terminal truncated structure leading to a possible sequestration in lysosomes. Phylogenetic analyses suggest that CLPs have appeared independently in the three main branches of bilaterian animals, as a result of convergent evolution. Gene expression profiles analyzed by quantitative RT-PCR support the involvement of Cg-Clp3 in embryonic development, adult oyster growth and tissue remodelling during metamorphosis and gonadal restructuring.

Alternative splicing of a single precursor mRNA generates two subtypes of Gonadotropin-Releasing Hormone receptor orthologues and their variants in the bivalve mollusc Crassostrea gigas.

Despite their economic importance, only very little information is available regarding (neuro)endocrine regulation of reproduction in bivalve molluscs. To gain insights into the molecular control of gonadic development of these animals, G protein-coupled receptors (GPCR) specifically expressed in the gonad of the pacific oyster Crassostrea gigas were investigated. One such receptor, Cg-GnRH-R, an oyster GPCR orthologue of vertebrate GnRH receptors clearly involved in the control of oyster gametogenesis was first identified [Rodet, F., Lelong, C., Dubos, M.P., Costil, K. and Favrel, P., 2005. Molecular cloning of a molluscan Gonadotropin-Releasing Hormone receptor orthologue specifically expressed in the gonad. Biochim Biophys Acta 1730 187-95.]. We report here the characterization of multiple transcripts encoding GnRH-R orthologues (Cg-GnRH-RII-L/Cg-GnRH-RII-S) including a truncated receptor (Cg-GnRH-R-TF) and demonstrate they are generated by the alternative splicing of a single mRNA precursor. The differential structure of these receptors suggests that Cg-GnRH-R on one hand and Cg-GnRH-RII-L/Cg-GnRH-RII-S on the other hand constitute two receptor subtypes with regard to ligand specificity. Pattern of expression of these transcripts suggests that Cg-GnRH-R cognate ligand is specifically involved in the control of gametogenesis while Cg-GnRH-RII-L and Cg-GnRH-RII-S ones likely do not control reproductive functions specifically. Hypothesis on the involvement of this family of receptors in signalling both GnRH and APGWamide in molluscs is discussed.

Characterization of chitinase-like proteins (Cg-Clp1 and Cg-Clp2) involved in immune defence of the mollusc Crassostrea gigas.

Chitinase-like proteins have been identified in insects and mammals as nonenzymatic members of the glycoside hydrolase family 18. Recently, the first molluscan chitinase-like protein, named Crassostrea gigas (Cg)-Clp1, was shown to control the proliferation and synthesis of extracellular matrix components of mammalian chondrocytes. However, the precise physiological roles of Cg-Clp1 in oysters remain unknown. Here, we report the cloning and the characterization of a new chitinase-like protein (Cg-Clp2) from the oyster Crassostrea gigas. Gene expression profiles monitored by quantitative RT-PCR in adult tissues and through development support its involvement in tissue growth and remodelling. Both Cg-Clp1- and Cg-Clp2-encoding genes were transcriptionally stimulated in haemocytes in response to bacterial lipopolysaccharide challenge, strongly suggesting that these two close paralogous genes play a role in oyster immunity.

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.

The phylogenetically conserved molluscan chitinase-like protein 1 (Cg-Clp1), homologue of human HC-gp39, stimulates proliferation and regulates synthesis of extracellular matrix components of mammalian chondrocytes.

Members of chitinase-like proteins (CLPs) have attracted much attention because of their ability to promote cell proliferation in insects (imaginal disc growth factors) and mammals (YKL-40). To gain insights into the molecular processes underlying the physiological control of growth and development in Lophotrochozoa, we report here the cloning and biochemical characterization of the first Lophotrochozoan CLP from the oyster Crassostrea gigas (Cg-Clp1). Gene expression profiles monitored by real time quantitative reverse transcription-PCR in different adult tissues and during development support the involvement of this protein in the control of growth and development in C. gigas. Recombinant Cg-Clp1 demonstrates a strong affinity for chitin but no chitinolytic activity, as was described for the HC-gp39 mammalian homolog. Furthermore, transient expression of Cg-Clp1 in primary cultures of rabbit articular chondrocytes as well as the use of both purified recombinant protein and conditioned medium from Cg-Clp1-expressing rabbit articular chondrocytes established that Cg-Clp1 stimulates cell proliferation and regulates extracellular matrix component synthesis, showing for the first time a possible involvement of a CLP on type II collagen synthesis regulation. These observations together with the fact that Cg-Clp1 gene organization strongly resembles that of its mammalian homologues argue for an early evolutionary origin and a high conservation of this class of proteins at both the structural and functional levels.

Molecular cloning of a molluscan gonadotropin-releasing hormone receptor orthologue specifically expressed in the gonad.

Despite their economic importance, only very little information is available regarding (neuro)endocrine mechanisms of reproduction in bivalve molluscs. To gain insights into the molecular control of gonadic development of these animals, G protein-coupled receptors (GPCR) expressed in the gonad of the pacific oyster Crassostrea gigas were investigated. One such receptor was cloned by RT-PCR using oligonucleotide primers derived from consensus sequences of various vertebrate (neuro)peptide receptors. This receptor named Cg-GnRH-related receptor (Cg GnRH-R) exhibits a high degree of amino acid sequence identity with both vertebrate GnRH receptors and insect AKH receptors. Quantitative RT-PCR shows a specific expression of Cg-GnRH-R in both male and female gonads during the reproductive cycle. This demonstrates for the first time the plausible involvement of a GnRH receptor orthologue in the control of reproduction in a protostomian invertebrate.

Molecular and physiological characterization of an invertebrate homologue of a calcitonin-related receptor.

Calcitonin is a key hormone involved in the regulation of calcium metabolism in vertebrates. Using oligonucleotide primers derived from consensus sequences of vertebrate calcitonin receptors, we have cloned and characterized the first representative of an invertebrate calcitonin receptor from the bivalve mollusc Crassostrea gigas. This receptor named Cg CT-R exhibits 39% amino acid sequence identity with both human calcitonin and calcitonin gene-related precursor receptors. Cg CT-R is expressed mainly in the gills and the mantle edge as well as at lower levels in muscles, digestive gland, heart, and labial palps. Transfer of animals from seawater to brackish water resulted in a significant decrease of Cg CT-R transcript levels in the gills, thus suggesting a role for ionic balance in molluscs.