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.

First isolation of Francisella halioticida strains from blue mussel (Mytilus edulis) in Normandy, France.

Mass mortality events affecting the blue mussels Mytilus edulis have been observed in France since 2014. The DNA of the bacterium Francisella halioticida, reported as pathogen of giant abalone (Haliotis gigantea) and Yesso scallop (Mizuhopecten yessoensis) has been detected recently in mussels from areas suffering mortalities. Isolation of this bacterium was attempted from individuals collected during mortality events. Identification was performed by 16S rRNA gene sequencing, real-time specific PCR and MALDI-ToF using spectra produced from the strain 8472-13A isolated from diseased Yesso scallop in Canada. Five isolates were identified as F. halioticida by real-time specific PCR and 16S rRNA sequencing. MALDI-ToF allowed the direct identification of four isolates (FR22a,b,c,d) which had 100% identity on the 16S rRNA gene with the known strains. On the other hand, one isolate (FR21) was not recognized by MALDI-ToF and had 99.9% identity on the 16S rRNA gene. The FR22 isolates showed difficult growth and required media optimization, which was not the case with the FR21 isolate. For these reasons, it was hypothesized that two type strains are present on French coasts, named FR21 and FR22. The FR21 isolate was selected for phenotypic analysis (growth curve, biochemical characteristics, electron microscopy), phylogenetic analysis and an experimental challenge. This isolate showed distinct differences compared to published F. halioticida strains, both at phenotypic and genotypic levels. Experimental infections of adult mussels led to 36% mortalities in 23 days following intramuscular injection with 3 × 107 CFU while a lower dose (3 × 103 CFU) did not lead to significant mortalities. In the context of this study, the strain FR21 was not virulent towards adult mussels.

PepTraq: a toolbox for in silico data mining and fast sequence filtering.

The development of de novo sequencing tools has led to the massive production of genomes and transcriptomes from many unconventional animal models. To tackle this huge flow of data, PepTraq brings together many functionalities generally scattered in multiple tools, so that sequences can be filtered on the basis of multiple criteria. It is particularly suitable for the identification of non-annotated transcripts, re-annotation, extraction of secretomes, neuropeptidomes, targeted search for peptides and proteins, preparing specific proteomics/peptidomics fasta files for mass spectrometry (MS) applications, MS data processing, etc. PepTraq is developed in Java, and is available as a desktop application that can be downloaded from https://peptraq.greyc.fr . It is also available as a web application at the same URL for processing small files (10-20 MB). The source code is open under a CeCILL-B licence.

Structural and Functional Characterization of Orcokinin B-like Neuropeptides in the Cuttlefish (Sepia officinalis).

The cuttlefish (Sepia officinalis) is a Cephalopod mollusk that lives in the English Channel and breeds in coastal spawning grounds in spring. A previous work showed that the control of egg-laying is monitored by different types of regulators, among which neuropeptides play a major role. They are involved in the integration of environmental cues, and participate in the transport of oocytes in the genital tract and in the secretion of capsular products. This study addresses a family of neuropeptides recently identified and suspected to be involved in the control of the reproduction processes. Detected by mass spectrometry and immunocytochemistry in the nerve endings of the accessory sex glands of the females and ovary, these neuropeptides are also identified in the hemolymph of egg-laying females demonstrating that they also have a hormone-like role. Released in the hemolymph by the sub-esophageal mass, a region that innervates the genital tract and the neurohemal area of the vena cava, in in vitro conditions these neuropeptides modulated oocyte transport and capsular secretion. Finally, in silico analyses indicated that these neuropeptides, initially called FLGamide, had extensive structural homology with orcokinin B, which motivated their name change.

Multifaceted roles of the egg perivitelline layer in avian reproduction: Functional insights from the proteomes of chicken egg inner and outer sublayers.

The avian egg perivitelline layer (PL) is a proteinaceous structure that encloses the egg yolk. It consists of the inner and the outer perivitelline layers (IPL and OPL, respectively) that are assumed to play distinct roles in bird reproduction. To gain insight into their respective function, we analyzed the proteome of IPL and OPL in chicken unfertilized eggs after mechanical separation, using a GeLC-MS/MS strategy. Of the 412 proteins identified, 173 proteins were uniquely recovered in IPL and 98 proteins in OPL, while 141 proteins were identified in both sublayers. Genes coding the most abundant proteins were shown to be expressed either in the liver/ovary (IPL formation) or in the oviduct (OPL formation), but rarely in both. The presence of oviduct-specific proteins (including LYZ, VMO1, AvBD11, PTN, OVAL and LOC10175704) in IPL strongly suggests that they participate in the physical association of IPL to OPL, whose tight attachment was further evidenced by analyses of IPL/OPL interfaces (by scanning electron microscopy). Functional annotation of identified proteins revealed functions associated with fertilization and early development for IPL, while OPL would rather participate in egg defense and embryogenesis. Collectively, our data highlight the complementary functions of IPL and OPL that are major determinants of bird reproductive success. SIGNIFICANCE: The present study unveils for the first time the individual proteomes of the two sublayers composing the chicken egg perivitelline layer (PL), which allowed to assign their respective putative biological roles in avian reproduction. The combination of proteomics with gene expression and ultrastructural analyses provides insightful data on the structure and biochemistry of the avian PL. The functional annotation of PL proteins highlights the multifaceted biological functions of this structure in reproduction including fertilization, embryonic development, and antimicrobial protection. This work will stimulate further research to validate predicted functions and to compare the physiology and the functional specificities of PL in egg-laying species.

Involvement of RFamide neuropeptides in polyp contraction of the adult scleractinian corals Euphyllia ancora and Stylophora pistillata.

The distribution and functions of neurons in scleractinian corals remain largely unknown. This study focused on the Arg-Phe amide family of neuropeptides (RFamides), which have been shown to be involved in a variety of biological processes in animals, and performed molecular identification and characterization in the adult scleractinian coral Euphyllia ancora. The deduced amino acid sequence of the identified RFamide preprohormone was predicted to contain 20 potential neuropeptides, including 1 Pro-Gly-Arg-Phe (PGRF) amide and 15 Gln-Gly-Arg-Phe (QGRF) amide peptides. Tissue distribution analysis showed that the level of transcripts in the tentacles was significantly higher than that in other polyp tissues. Immunohistochemical analysis with the FMRFamide antibody showed that RFamide neurons were mainly distributed in the epidermis of the tentacles and mouth with pharynx. Treatment of E. ancora polyps with synthetic QGRFamide peptides induced polyp contraction. The induction of polyp contraction by QGRFamide peptide treatment was also observed in another scleractinian coral, Stylophora pistillata. These results strongly suggested that RFamides play a role in the regulation of polyp contraction in adult scleractinians.

Marine Transcriptomics Analysis for the Identification of New Antimicrobial Peptides.

Antimicrobial peptides (AMPs) participate in the immune system to avoid infection, are present in all living organisms and can be used as drugs. Fish express numerous AMP families including defensins, cathelicidins, liver-expressed antimicrobial peptides (LEAPs), histone-derived peptides, and piscidins (a fish-specific AMP family). The present study demonstrates for the first time the occurrence of several AMPs in lionfish (Pterois volitans). Using the lionfish transcriptome, we identified four transcript sequences encoding cysteine-rich AMPs and two new transcripts encoding piscidin-like peptides. These AMPs are described for the first time in a species of the Scorpaenidae family. A functional approach on new pteroicidins was carried out to determine antimicrobial sequences and potential uses, with a view to using some of these AMPs for human health or in aquaculture.

In-Depth In Silico Search for Cuttlefish (Sepia officinalis) Antimicrobial Peptides Following Bacterial Challenge of Haemocytes.

Cuttlefish (Sepia officinalis) haemocytes are potential sources of antimicrobial peptides (AMPs). To study the immune response to Vibrio splendidus and identify new AMPs, an original approach was developed based on a differential transcriptomic study and an in-depth in silico analysis using multiple tools. Two de novo transcriptomes were retrieved from cuttlefish haemocytes following challenge by V. splendidus or not. A first analysis of the annotated transcripts revealed the presence of Toll/NF-κB pathway members, including newly identified factors such as So-TLR-h, So-IKK-h and So-Rel/NF-κB-h. Out of the eight Toll/NF-κB pathway members, seven were found up-regulated following V. splendidus challenge. Besides, immune factors involved in the immune response were also identified and up-regulated. However, no AMP was identified based on annotation or conserved pattern searches. We therefore performed an in-depth in silico analysis of unannotated transcripts based on differential expression and sequence characteristics, using several tools available like PepTraq, a homemade software program. Finally, five AMP candidates were synthesized. Among them, NF19, AV19 and GK28 displayed antibacterial activity against Gram-negative bacteria. Each peptide had a different spectrum of activity, notably against Vibrio species. GK28-the most active peptide-was not haemolytic, whereas NF19 and AV19 were haemolytic at concentrations between 50 and 100 µM, 5 to 10 times higher than their minimum inhibitory concentration.

-Omic Analysis of the Sepia officinalis White Body: New Insights into Multifunctionality and Haematopoiesis Regulation.

Cephalopods, like other protostomes, lack an adaptive immune system and only rely on an innate immune system. The main immune cells are haemocytes (Hcts), which are able to respond to pathogens and external attacks. First reports based on morphological observations revealed that the white body (WB) located in the optic sinuses of cuttlefish was the origin of Hcts. Combining transcriptomic and proteomic analyses, we identified several factors known to be involved in haematopoiesis in vertebrate species in cuttlefish WB. Among these factors, members of the JAK-STAT signaling pathway were identified, some of them for the first time in a molluscan transcriptome and proteome. Immune factors, such as members of the Toll/NF-κB signaling pathway, pattern recognition proteins and receptors, and members of the oxidative stress responses, were also identified, and support an immune role of the WB. Both transcriptome and proteome analyses revealed that the WB harbors an intense metabolism concurrent with the haematopoietic function. Finally, a comparative analysis of the WB and Hct proteomes revealed many proteins in common, confirming previous morphological studies on the origin of Hcts in cuttlefish. This molecular work demonstrates that the WB is multifunctional and provides bases for haematopoiesis regulation in cuttlefish.

Involvement of GLWamide neuropeptides in polyp contraction of the adult stony coral Euphyllia ancora.

The existence and function of neurons remain largely unexplored in scleractinian corals. To gain a better understanding of neuronal functions in coral physiology, this study focused on Glycine-Leucine-Tryptophan-amide family neuropeptides (GLWamides), which have been shown to induce muscle contraction and larval metamorphosis in other cnidarians. Molecular identification and functional characterization of GLWamides in the adult stony coral Euphyllia ancora were performed. We successfully elucidated the full-length cDNA of GLWamide preprohormone in E. ancora (named EaGLW preprohormone). The deduced amino acid sequence was predicted to contain six potential GLWamide peptides. Tissue distribution analysis demonstrated that transcripts of EaGLW preprohormone were mainly expressed in the mouth (including the pharynx) and tentacles of the polyps. Immunodetection with an anti-GLWamide monoclonal antibody revealed that GLWamide neurons were mainly distributed in the epidermis of the mouth region and tentacle, in agreement with the distribution patterns of the transcripts. Treatment of the isolated mouth and tentacles with synthetic GLWamide peptides induced the contraction of these isolated tissues. Treatment of polyps with synthetic GLWamide peptides induced the contraction of polyps. These results suggest that GLWamides are involved in polyp contraction (myoactivity) in adult scleractinians. Our data provide new information on the physiological function of neuropeptides in scleractinians.

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.

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.

Design of antimicrobial peptides from a cuttlefish database.

No antimicrobial peptide has been identified in cephalopods to date. Annotation of transcriptomes or genomes using basic local alignment Search Tool failed to yield any from sequence identities. Therefore, we searched for antimicrobial sequences in the cuttlefish (Sepia officinalis) database by in silico analysis of a transcriptomic database. Using an original approach based on the analysis of cysteine-free antimicrobial peptides selected from our Antimicrobial Peptide Database (APD3), the online prediction tool of the Collection of Anti-Microbial Peptides (CAMPR3), and a homemade software program, we identified potential antibacterial sequences. Nine peptides less than 25 amino acids long were synthesized. The hydrophobic content of all nine of them ranged from 30 to 70%, and they could form alpha-helices. Three peptides possessed similarities with piscidins, one with BMAP-27, and five were totally new. Their antibacterial activity was evaluated on eight bacteria including the aquatic pathogens Vibrio alginolyticus, Aeromonas salmonicida, or human pathogens such as Salmonella typhimurium, Listeria monocytogenes, or Staphylococcus aureus. Despite the prediction of an antimicrobial potential for eight of the peptides, only two-GR21 and KT19-inhibited more than one bacterial strain with minimal inhibitory concentrations below 25 µM. Some sequences like VA20 and FK19 were hemolytic, while GR21 induced less than 10% of hemolysis on human blood cells at a concentration of 200 µM. GR21 was the only peptide derived from a precursor with a signal peptide, suggesting a real role in cuttlefish immune defense.

Dietary aquaculture by-product hydrolysates: impact on the transcriptomic response of the intestinal mucosa of European seabass (Dicentrarchus labrax) fed low fish meal diets.

BACKGROUND: Aquaculture production is expected to double by 2030, and demands for aquafeeds and raw materials are expected to increase accordingly. Sustainable growth of aquaculture will require the development of highly nutritive and functional raw materials to efficiently replace fish meal. Enzymatic hydrolysis of marine and aquaculture raw materials could bring new functionalities to finished products. The aim of this study was to determine the zootechnical and transcriptomic performances of protein hydrolysates of different origins (tilapia, shrimp, and a combination of the two) in European seabass (Dicentrarchux labrax) fed a low fish meal diet (5%), for 65 days. RESULTS: Results were compared to a positive control fed with 20% of fish meal. Growth performances, anterior intestine histological organization and transcriptomic responses were monitored and analyzed. Dietary inclusion of protein hydrolysates in the low fish meal diet restored similar growth performances to those of the positive control. Inclusion of dietary shrimp hydrolysate resulted in larger villi and more goblet cells, even better than the positive control. Transcriptomic analysis of the anterior intestine showed that dietary hydrolysate inclusion restored a pattern of intestinal gene expression very close to the pattern of the positive control. However, as compared to the low fish meal diet and depending on their origin, the different hydrolysates did not modulate metabolic pathways in the same way. Dietary shrimp hydrolysate inclusion modulated more metabolic pathways related to immunity, while nutritional metabolism was more impacted by dietary tilapia hydrolysate. Interestingly, the combination of the two hydrolysates enhanced the benefits of hydrolysate inclusion in diets: more genes and metabolic pathways were regulated by the combined hydrolysates than by each hydrolysate tested independently. CONCLUSIONS: Protein hydrolysates manufactured from aquaculture by-products are promising candidates to help replace fish meal in aquaculture feeds without disrupting animal metabolism and performances.

Identification of a moronecidin-like antimicrobial peptide in the venomous fish Pterois volitans: Functional and structural study of pteroicidin-α.

The present study characterizes for the first time an antimicrobial peptide in lionfish (Pterois volitans), a venomous fish. Using a peptidomic approach, we identified a mature piscidin in lionfish and called it pteroicidin-α. We detected an amidated form (pteroicidin-α- CONH2) and a non-amidated form (pteroicidin-α-COOH), and then performed their functional and structural study. Interestingly, the two peptides displayed different antibacterial and hemolytic activity levels. Pteroicidin-α-CONH2 was bactericidal on human pathogens like Staphylococcus aureus or Escherichia coli, as well as on the fish pathogen Aeromonas salmonicida, while pteroicidin-α-COOH only inhibited their growth. Furthermore, the two peptides induced hemolysis of red blood cells from different vertebrates, namely humans, sea bass and lesser-spotted dogfish. Hemolysis occurred with low concentrations of pteroicidin-α-CONH2, indicating greater toxicity of the amidated form. Circular dichroism analysis showed that both peptides adopted a helical conformation, yet with a greater α-helix content in pteroicidin-α-CONH2. Overall, these results suggest that amidation strongly influences pteroicidin-α by modifying its structure and its physico-chemical characteristics and by increasing its hemolytic activity.

Crustacean cardioactive peptides: Expression, localization, structure, and a possible involvement in regulation of egg-laying in the cuttlefish Sepia officinalis.

The cuttlefish (Sepia officinalis) is a cephalopod mollusk distributed on the western European coast, in the West African Ocean and in the Mediterranean Sea. On the Normandy coast (France), cuttlefish is a target species of professional fishermen, so its reproduction strategy is of particular interest in the context of stock management. Egg-laying, which is coastal, is controlled by several types of regulators among which neuropeptides. The cuttlefish neuropeptidome was recently identified by Zatylny-Gaudin et al. (2016). Among the 38 neuropeptide families identified, some were significantly overexpressed in egg-laying females as compared to mature males. This study is focused on crustacean cardioactive peptides (CCAPs), a highly expressed neuropeptide family strongly suspected of being involved in the control of egg-laying. We investigated the functional and structural characterization and tissue mapping of CCAPs, as well as the expression patterns of their receptors. CCAPs appeared to be involved in oocyte transport through the oviduct and in mechanical secretion of capsular products. Immunocytochemistry revealed that the neuropeptides were localized throughout the central nervous system (CNS) and in the nerve endings of the glands involved in egg-capsule synthesis and secretion, i.e. the oviduct gland and the main nidamental glands. The CCAP receptor was expressed in these glands and in the subesophageal mass of the CNS. Multiple sequence alignments revealed a high level of conservation of CCAP protein precursors in Sepia officinalis and Loligo pealei, two cephalopod decapods. Primary sequences of CCAPs from the two species were fully conserved, and cryptic peptides detected in the nerve endings were also partially conserved, suggesting biological activity that remains unknown for the time being.

Neuropeptidome of the Cephalopod Sepia officinalis: Identification, Tissue Mapping, and Expression Pattern of Neuropeptides and Neurohormones during Egg Laying.

Cephalopods exhibit a wide variety of behaviors such as prey capture, communication, camouflage, and reproduction thanks to a complex central nervous system (CNS) divided into several functional lobes that express a wide range of neuropeptides involved in the modulation of behaviors and physiological mechanisms associated with the main stages of their life cycle. This work focuses on the neuropeptidome expressed during egg-laying through de novo construction of the CNS transcriptome using an RNAseq approach (Illumina sequencing). Then, we completed the in silico analysis of the transcriptome by characterizing and tissue-mapping neuropeptides by mass spectrometry. To identify neuropeptides involved in the egg-laying process, we determined (1) the neuropeptide contents of the neurohemal area, hemolymph (blood), and nerve endings in mature females and (2) the expression levels of these peptides. Among the 38 neuropeptide families identified from 55 transcripts, 30 were described for the first time in Sepia officinalis, 5 were described for the first time in the animal kingdom, and 14 were strongly overexpressed in egg-laying females as compared with mature males. Mass spectrometry screening of hemolymph and nerve ending contents allowed us to clarify the status of many neuropeptides, that is, to determine whether they were neuromodulators or neurohormones.

The Toll/NF-κB pathway in cuttlefish symbiotic accessory nidamental gland.

The female genital apparatus of decapod cephalopods contains a symbiotic accessory nidamental gland (ANG) that harbors bacterial symbionts. Although the ANG bacterial consortium is now well described, the impact of symbiosis on Sepia officinalis innate immunity pathways remains unknown. In silico analysis of the de novo transcriptome of ANG highlighted for the first time the existence of the NF-κB pathway in S. officinalis. Several signaling components were identified, i.e. five Toll-like receptors, eight signaling cascade features, and the immune response target gene iNOS, previously described as being involved in the initiation of bacterial symbiosis in a cephalopod gland. This work provides a first key for studying bacterial symbiosis and its impact on innate immunity in S. officinalis ANG.

How Egg Case Proteins Can Protect Cuttlefish Offspring?

Sepia officinalis egg protection is ensured by a complex capsule produced by the female accessory genital glands and the ink bag. Our study is focused on the proteins constituting the main egg case. De novo transcriptomes from female genital glands provided essential databases for protein identification. A proteomic approach in SDS-PAGE coupled with MS unveiled a new egg case protein family: SepECPs, for Sepia officinalis Egg Case Proteins. N-glycosylation was demonstrated by PAS staining SDS-PAGE gels. These glycoproteins are mainly produced in the main nidamental glands. SepECPs share high sequence homology, especially in the signal peptide and the three cysteine-rich domains. SepECPs have a high number of cysteines, with conserved motifs involved in 3D-structure. SDS-PAGE showed that SepECPs could form dimers; this result was confirmed by TEM observations, which also revealed a protein network. This network is similar to the capsule network, and it associates these structural proteins with polysaccharides, melanin and bacteria to form a tight mesh. Its hardness and elasticity provide physical protection to the embryo. In addition, SepECPs also have bacteriostatic antimicrobial activity on GRAM- bacteria. By observing the SepECP / Vibrio aestuarianus complex in SEM, we demonstrated the ability of these proteins to agglomerate bacteria and thus inhibit their growth. These original proteins identified from the outer egg case ensure the survival of the species by providing physical and chemical protection to the embryos released in the environment without any maternal protection.

Diversity of the RFamide Peptide Family in Mollusks.

Since the initial characterization of the cardioexcitatory peptide FMRFamide in the bivalve mollusk Macrocallista nimbosa, a great number of FMRFamide-like peptides (FLPs) have been identified in mollusks. FLPs were initially isolated and molecularly characterized in model mollusks using biochemical methods. The development of recombinant technologies and, more recently, of genomics has boosted knowledge on their diversity in various mollusk classes. Today, mollusk FLPs represent approximately 75 distinct RFamide peptides that appear to result from the expression of only five genes: the FMRFamide-related peptide gene, the LFRFamide gene, the luqin gene, the neuropeptide F gene, and the cholecystokinin/sulfakinin gene. FLPs display a complex spatiotemporal pattern of expression in the central and peripheral nervous system. Working as neurotransmitters, neuromodulators, or neurohormones, FLPs are involved in the control of a great variety of biological and physiological processes including cardiovascular regulation, osmoregulation, reproduction, digestion, and feeding behavior. From an evolutionary viewpoint, the major challenge will then logically concern the elucidation of the FLP repertoire of orphan mollusk classes and the way they are functionally related. In this respect, deciphering FLP signaling pathways by characterizing the specific receptors these peptides bind remains another exciting objective.