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Registros recuperados: 32
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A European epidemiological survey of Vibrio splendidus clade shows unexplored diversity and massive exchange of virulence factors ArchiMer
Nasfi, H.; Travers, Marie-agnes; De Lorgeril, Julien; Habib, C.; Sannie, T.; Sorieul, L.; Gerard, J.; Avarre, J. C.; Haffner, Philippe; Tourbiez, Delphine; Renault, Tristan; Furones, D.; Roque, A.; Pruzzo, C.; Cheslett, D.; Gdoura, R.; Vallaeys, T..
The Vibrio splendidus clade has previously been associated with epidemic outbreaks of various aquatic animals, as in the case of the cupped oyster, Crassostrea gigas. To investigate whether involved strains could present a clonal origin and to identify possible alternative background carriage animals or zooplankton, a large epidemiological survey was conducted on isolates of the splendidus clade. For this purpose, Vibrio strains were isolated from various samples including oysters, mussels, sediments, zooplankton, and sea water on the basis of a North/South gradient of the European sea water zone (Ireland, The Netherlands, France, Italy, and Spain). A total of 435 isolates were successfully associated to the V. splendidus clade using real time polymerase...
Tipo: Text Palavras-chave: VNTR; Vibrio splendidus; Epidemiology; Vsm; OmpU; Crassotrea giga; Mortality.
Ano: 2015 URL: http://archimer.ifremer.fr/doc/00255/36666/35350.pdf
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A hemocyte gene expression signature correlated with predictive capacity of oysters to survive Vibrio infections ArchiMer
Rosa, Rafael Diego; De Lorgeril, Julien; Tailliez, Patrick; Bruno, Roman; Piquemal, David; Bachere, Evelyne.
Background: The complex balance between environmental and host factors is an important determinant of susceptibility to infection. Disturbances of this equilibrium may result in multifactorial diseases as illustrated by the summer mortality syndrome, a worldwide and complex phenomenon that affects the oysters, Crassostrea gigas. The summer mortality syndrome reveals a physiological intolerance making this oyster species susceptible to diseases. Exploration of genetic basis governing the oyster resistance or susceptibility to infections is thus a major goal for understanding field mortality events. In this context, we used high-throughput genomic approaches to identify genetic traits that may characterize inherent survival capacities in C. gigas. Results:...
Tipo: Text Palavras-chave: Marine invertebrate; Mollusk bivalve; Mass mortality; Transcriptome-wide analysis; Digital gene expression; Microfluidic qPCR; Survival signature; Polymorphism; Gene copy number; Survival predictive biomarkers.
Ano: 2012 URL: http://archimer.ifremer.fr/doc/00095/20624/18262.pdf
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A relationship between antimicrobial peptide gene expression and capacity of a selected shrimp line to survive a Vibrio infection ArchiMer
De Lorgeril, Julien; Gueguen, Yannick; Goarant, Cyrille; Goyard, Emmanuel; Mugnier, Chantal; Fievet, Julie; Piquemal, D; Bachere, Evelyne.
Understanding of antimicrobial defence mechanisms of penaeid shrimp should help in the design of efficient strategies for the management and disease control in aquaculture. In this study, we have specifically analysed the expression in circulating hemocytes of antimicrobial peptides (AMPs) encoding genes, such as PEN2 and PEN3, ALF, crustin, lysozyme and a putative cysteine-rich peptide. We evidenced a relationship between the level of expression of some AMPs and the successful response of the shrimp, Litopenaeus stylirostris, to circumvent a pathogenic Vibrio penaeicida infection. Additionally, significant differences in some AMP transcript amounts are evidenced between control, non-selected shrimp line and the third generation breeding of shrimp selected...
Tipo: Text Palavras-chave: Real time PCR; Cysteine rich peptide; Crustin; Anti LPS factor; Lysozyme; Penaeidins; Immune response; Vibrio penaeicida; Penaeid; Decapoda; Crustacean.
Ano: 2008 URL: http://archimer.ifremer.fr/doc/2008/publication-4524.pdf
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Amélioration génétique expérimentale de la crevette d'élevage de Nouvelle-Calédonie : Sélection d'une population de L. stylirostris résistante à la bactérie pathogène Vibrio penaeicida. Rapport final pour le Ministère de l'Outre-Mer ArchiMer
Goyard, Emmanuel; Goarant, Cyrille; Bachere, Evelyne; De Lorgeril, Julien; Mugnier, Chantal; Ansquer, Dominique; Broutoi, Francis; Brun, Pierre; Imbert, Frederic; Justou, Carole; Maillez, Jean-rene; Patrois, Jacques; Pham, Dominique; Peignon, Jean-marie.
The New-Caledonian shrimp industry is based on the controlled reproduction of the shrimp Litopenaeus stylirostris, a species which was introduced in the 80s. The major difficulty to which the industry has been faced for 10 years is the occurrence of the "syndrome 93", which corresponds to mortality phases when the temperature falls down in April-May-June. This mortality is associated to the pathogenic bacteria Vibrio penaecida and is expressed at different levels which are variable from year ta year and from pond to pond. No resistance to this pathology has been developed spontaneously. This is likely due to the protocole used to rear spawners, which does not allow to implement an efficient selective pressure at each generation An experimental selection...
Tipo: Text Palavras-chave: Amélioration génétique; P. styliroslris; Résistance; Syndrome 93; Lyzozyme; Genetic improvement; P. styliroslris; Résistance; Syndrome 93; Lyzozyme.
Ano: 2003 URL: http://archimer.ifremer.fr/doc/00109/22020/19643.pdf
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Can selection for resistance to OsHV-1 infection modify susceptibility to Vibrio aestuarianus infection in Crassostrea gigas? First insights from experimental challenges using primary and successive exposures ArchiMer
Azema, Patrick; Travers, Marie-agnes; De Lorgeril, Julien; Tourbiez, Delphine; Degremont, Lionel.
Since 2008, the emergent virus OsHV 1μvar has provoked massive mortality events in Crassostrea gigas spat and juveniles in France. Since 2012, mortality driven by the pathogenic bacteria Vibrio aestuarianus has stricken market sized adults. A hypothesis to explain the sudden increase in mortality observed in France since 2012 is that selective pressure due to recurrent viral infections could have led to a higher susceptibility of adults to Vibrio infection. In our study, two OsHV-1-resistant lines (AS and BS) and their respective controls (AC and BC) were experimentally challenged in the laboratory to determine their level of susceptibility to V. aestuarianus infection. At the juvenile stage, the selected lines exhibited lower mortality (14 and 33%) than...
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Ano: 2015 URL: http://archimer.ifremer.fr/doc/00299/41054/40215.pdf
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Cg-TIMP, un inhibiteur de metalloproteinase isolé chez Crassostrea gigas : étude de son rôle dans la réparation des lésions et dans les mécanismes de défense ArchiMer
Escoubas, Jean-michel; Montagnani, Caroline; Le Roux, Frédérique; De Lorgeril, Julien; Berthe, Franck; Bachere, Evelyne.
We isolated complementary DNA (Cg-TIMP) from Crassostrea gigas that codes for a protein highly homologous to vertebrate tissue inhibitors of metalloproteinases (TIMPs). In vertebrates, TIMPs are multifunctional proteins that play a key role in extracellular matrix metabolism. They are involved in physiological processes (e.g. embryonic development, damage repair, angiogenesis) and pathological processes (e.g. inflammatory reactions, cancer).
Tipo: Text Palavras-chave: Proteine; Cg TIMP; ADN; Crassostrea gigas.
Ano: 2001 URL: http://archimer.ifremer.fr/doc/2001/acte-3268.pdf
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Cloning and expression analysis of allograft inflammatory factor type 1 in coelomocytes of antarctic sea urchin (Sterechinus neumayeri) ArchiMer
Ovando, Fernanda; Gimpel, Carla; Cardenas, Constanza; Machado Cunha Da Silva, Jose Roberto; De Lorgeril, Julien; Gonzalez, Marcelo.
We have cloned and characterized for the first time an allograft inflammatory factor 1 (Sn-AIF-1) from the Antarctic sea urchin. We report the cloning of Sn-AIF-1 cDNA and the characterization of its expression in coelomocytes after a bacterial challenge. The cDNA Sn-AIF-1 has a size of 608 bp and encodes a polypeptide of 151 aa. The deduced amino acid sequence has a putative size of 17.430 Da, an isoelectric point of 4.92, and shows 2 elongation factor handlike motifs that normally bind calcium ions. BLAST analysis revealed close matches with other known AIF-1. The deduced amino acid sequence of Sn-AIF-1 showed high homology with AIF-1 in vertebrates such as fish, mice, and humans; and in the case of invertebrates, the major degree of identity (55%) was...
Tipo: Text Palavras-chave: Antarctica; Sea urchin; Sterechinus neumayeri; Coelomocytes; Gene expression; AIF-1.
Ano: 2012 URL: http://archimer.ifremer.fr/doc/00098/20955/18572.pdf
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Comprendre et prédire les maladies multifactorielles de bivalves marins : le cas de l’huître ArchiMer
De Lorgeril, Julien; Gueguen, Yannick; Mitta, Guillaume.
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Ano: 2019 URL: https://archimer.ifremer.fr/doc/00487/59821/62964.pdf
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DEDUCTION : A research project for shrimp farming sustainability in New-Caledonia ArchiMer
Beliaeff, Benoit; Chim, Liet; Della Patrona, Luc; Goyard, Emmanuel; Herlin, Jose; Labreuche, Yannick; Walling, Emilie; Ansquer, Dominique; Brun, Pierre; Castex, Mathieu; Coatanea, Denis; Courties, Claude; De Lorgeril, Julien; Dufour, Robert; Frappier, Julie; Goarant, Cyrille; Huber, Matthias; Lemaire, Pierrette; Lemonnier, Hugues; Loubersac, Lionel; Le Roux, Frederique; Lucas, Ronan; Patrois, Jacques; Peignon, Jean-marie; Pham, Dominique; Ramage, Yannick; Soulard, Benoit; Vic, Marlene; Vourey, Elodie; Wabete, Nelly.
New Caledonian shrimp farming feasibility studies started in the mid-seventies. Today ca. 2000 tons of Litopenaeus stylirostris, introduced in 1978, are semi-intensively produced each year. Since fifteen years, production has been impacted by two seasonal vibriosis, the “Summer” syndrome caused by Vibrio nigripulchritudo in relation to pond bottom and water column quality, and the “Winter Syndrome” caused by Vibrio penaeicida in relation to drastic temperature drops . With the ambition of elucidating processes possibly leading to mortality outbreaks in ponds, Ifremer has been conducting a research program since 2003 under the DESANS project (2003-2006) and the present DEDUCTION project, equally funded by the South and North Provinces and by the Government...
Tipo: Text Palavras-chave: Shrimp farming; Technical and scientific support; Vibriosis; Water quality; Pathology; Ecophysiology; Genetics; Zoosanitary monitoring and procedures; Multifactorial and multidisciplinary approach.
Ano: 2009 URL: http://archimer.ifremer.fr/doc/00143/25438/23591.pdf
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Distinct immune responses of juvenile and adult oysters (Crassostrea gigas) to viral and bacterial infections ArchiMer
Green, Timothy J.; Vergnes, Agnes; Montagnani, Caroline; De Lorgeril, Julien.
Since 2008, massive mortality events of Pacific oysters (Crassostrea gigas) have been reported worldwide and these disease events are often associated with Ostreid herpesvirus type 1 (OsHV-1). Epidemiological field studies have also reported oyster age and other pathogens of the Vibrio genus are contributing factors to this syndrome. We undertook a controlled laboratory experiment to simultaneously investigate survival and immunological response of juvenile and adult C. gigas at different time-points post-infection with OsHV-1, Vibrio tasmaniensis LGP32 and V. aestuarianus. Our data corroborates epidemiological studies that juveniles are more susceptible to OsHV-1, whereas adults are more susceptible to Vibrio. We measured the expression of 102...
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Ano: 2016 URL: http://archimer.ifremer.fr/doc/00348/45875/45575.pdf
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Expression, tissue localization and synergy of antimicrobial peptides and proteins in the immune response of the oyster Crassostrea gigas ArchiMer
Schmitt, Paulina; De Lorgeril, Julien; Gueguen, Yannick; Destoumieux-garzon, Delphine; Bachere, Evelyne.
Diverse families of antimicrobial peptides and proteins have been described in oysters. We investigated here how antimicrobials are involved in the immune response against a pathogenic strain of Vibrio splendidus. Oyster antimicrobials were shown to display a wide variety of expression profiles in hemocyte populations and tissues. Oyster defensins are constitutively expressed in specific tissues such as mantle (Cg-Defm) or hemocytes (Cg-Defhs), while Cg-BPI is inducible and Cg-Prp appears down-regulated in hemocytes upon infection. The migratory behavior of hemocytes that express the different antimicrobials was found to be involved in the oyster response to a pathogenic Vibrio infection. Indeed, it contributes to colocalize several antimicrobials that...
Tipo: Text Palavras-chave: Pacific oyster; Bivalve mollusk; Innate immunity; Bactericidal/permeability-increasing protein; Proline-rich peptide; Defensin; Vibrio.
Ano: 2012 URL: http://archimer.ifremer.fr/doc/00093/20382/18267.pdf
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First evidence of the activation of Cg-timp, an immune response component of pacific oysters, through a damage-associated molecular pattern pathway ArchiMer
Montagnani, Caroline; Avarre, Jean-christophe; De Lorgeril, Julien; Quiquand, M; Boulo, Viviane; Escoubas, Jean-michel.
In a previous work, we characterized a Crassostrea gigas cPNA (Cg-timp) encoding a protein which presents all the features of vertebrate tissue inhibitor of metalloproteinase (TIMP). The expression pattern of this gene led us to propose that Cg-timp is an important factor in oyster wound heating and defense mechanisms. Here we describe the analysis of Cg-timp expression in oysters challenged by live or dead bacteria as well as by bacterial secretory/excretory products and metalloproteinase. Surprisingly, bacterial secretory/excretory products activate Cg-timp gene expression whereas heat-inactivated ones do not. To address the question of the signal transduction pathway involved in Cg-timp gene activation, we isolated and sequenced Cg-timp promoter and...
Tipo: Text Palavras-chave: Damage associated molecular pattern; Tissue inhibitor of metalloproteinase; Metalloproteinase; Mollusk immunity; Crassostrea gigas; Oyster.
Ano: 2007 URL: http://archimer.ifremer.fr/doc/2007/publication-2094.pdf
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Functional Divergence in Shrimp Anti-Lipopolysaccharide Factors (ALFs): From Recognition of Cell Wall Components to Antimicrobial Activity ArchiMer
Rosa, Rafael Diego; Vergnes, Agnes; De Lorgeril, Julien; Goncalves, Priscila; Perazzolo, Luciane Maria; Saune, Laure; Romestand, Bernard; Fievet, Julie; Gueguen, Yannick; Bachere, Evelyne; Destoumieux-garzon, Delphine.
Antilipopolysaccharide factors (ALFs) have been described as highly cationic polypeptides with a broad spectrum of potent antimicrobial activities. In addition, ALFs have been shown to recognize LPS, a major component of the Gram-negative bacteria cell wall, through conserved amino acid residues exposed in the four-stranded beta-sheet of their three dimensional structure. In penaeid shrimp, ALFs form a diverse family of antimicrobial peptides composed by three main variants, classified as ALF Groups A to C. Here, we identified a novel group of ALFs in shrimp (Group D ALFs), which corresponds to anionic polypeptides in which many residues of the LPS binding site are lacking. Both Group B (cationic) and Group D (anionic) shrimp ALFs were produced in a...
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Ano: 2013 URL: http://archimer.ifremer.fr/doc/00160/27077/25236.pdf
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Generation and analysis of a 29,745 unique Expressed Sequence Tags from the Pacific oyster (Crassostrea gigas) assembled into a publicly accessible database: the GigasDatabase ArchiMer
Fleury, Elodie; Huvet, Arnaud; Lelong, Christophe; De Lorgeril, Julien; Boulo, Viviane; Gueguen, Yannick; Bachere, Evelyne; Tanguy, Arnaud; Moraga, Dario; Fabioux, Caroline; Lindeque, Penelope; Shaw, Jenny; Reinhardt, Richard; Prunet, Patrick; Davey, Grace; Lapegue, Sylvie; Sauvage, Christopher; Corporeau, Charlotte; Moal, Jeanne; Gavory, Frederick; Wincker, Patrick; Moreews, Francois; Klopp, Christophe; Mathieu, Michel; Boudry, Pierre; Favrel, Pascal.
Background: Although bivalves are among the most-studied marine organisms because of their ecological role and economic importance, very little information is available on the genome sequences of oyster species. This report documents three large-scale cDNA sequencing projects for the Pacific oyster Crassostrea gigas initiated to provide a large number of expressed sequence tags that were subsequently compiled in a publicly accessible database. This resource allowed for the identification of a large number of transcripts and provides valuable information for ongoing investigations of tissue-specific and stimulus-dependant gene expression patterns. These data are crucial for constructing comprehensive DNA microarrays, identifying single nucleotide...
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Ano: 2009 URL: http://archimer.ifremer.fr/doc/2009/publication-6770.pdf
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HSP70 from the Antarctic sea urchin Sterechinus neumayeri: molecular characterization and expression in response to heat stress ArchiMer
Gonzalez-aravena, Marcelo; Calfio, Camila; Mercado, Luis; Morales-lange, Byron; Bethke, Jorn; De Lorgeril, Julien; Cardenas, Cesar A..
Background Heat stress proteins are implicated in stabilizing and refolding denatured proteins in vertebrates and invertebrates. Members of the Hsp70 gene family comprise the cognate heat shock protein (Hsc70) and inducible heat shock protein (Hsp70). However, the cDNA sequence and the expression of Hsp70 in the Antarctic sea urchin are unknown. Methods We amplified and cloned a transcript sequence of 1991 bp from the Antarctic sea urchin Sterechinus neumayeri, experimentally exposed to heat stress (5  and 10 °C for 1, 24 and 48 h). RACE-PCR and qPCR were employed to determine Hsp70 gene expression, while western blot and ELISA methods were used to determine protein expression. Results The sequence obtained from S. neumayeri showed high identity with Hsp70...
Tipo: Text Palavras-chave: Hsp70; Coelomocytes; Antarctica; Echinoderms; Climate change.
Ano: 2018 URL: http://archimer.ifremer.fr/doc/00435/54619/56019.pdf
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Immune response of the Antarctic sea urchin Sterechinus neumayeri: cellular, molecular and physiological approach ArchiMer
Gonzalez-aravena, Marcelo; Perez-troncoso, Carolina; Urtubia, Rocio; Branco, Paola; Machado Cunha Da Silva, Jose Roberto; Mercado, Luis; De Lorgeril, Julien; Bethke, Jorn; Paschke, Kurt.
In the Antarctic marine environment, the water temperature is usually between 2 and - 1.9 degrees C. Consequently, some Antarctic species have lost the capacity to adapt to sudden changes in temperature. The study of the immune response in Antarctic sea urchin (Sterechinus neumayeri) could help us understand the future impacts of global warming on endemic animals in the Antarctic Peninsula. In this study, the Antarctic sea urchins were challenged with lipopolysaccharides and Vibrio alginolitycus. The cellular response was evaluated by the number of coelomocytes and phagocytosis. A significant increase was observed in red sphere cells and total coelomocytes in animals exposed to LPS. A significant rise in phagocytosis in animals stimulated by LPS was also...
Tipo: Text Palavras-chave: Sea urchin; Antarctica; Sterechinus neumayeri; Coelomocytes; Phagocytosis; Gene expression.
Ano: 2015 URL: http://archimer.ifremer.fr/doc/00439/55012/56441.pdf
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Immune-suppression by OsHV-1 viral infection causes fatal bacteraemia in Pacific oysters ArchiMer
De Lorgeril, Julien; Lucasson, Aude; Petton, Bruno; Toulza, Eve; Montagnani, Caroline; Clerissi, Camille; Vidal-dupiol, Jeremie; Chaparro, Cristian; Galinier, Richard; Escoubas, Jean Michel; Haffner, Philippe; Degremont, Lionel; Charriere, Guillaume; Lafont, Maxime; Delort, Abigail; Vergnes, Agnes; Chiarello, Marlene; Faury, Nicole; Rubio, Tristan; Leroy, Marc; Perignon, Adeline; Regler, Denis; Morga, Benjamin; Alunno-bruscia, Marianne; Boudry, Pierre; Le Roux, Frederique; Destoumieux-garzon, Delphine; Gueguen, Yannick; Mitta, Guillaume.
Infectious diseases are mostly explored using reductionist approaches despite repeated evidence showing them to be strongly in fluenced by numerous interacting host and environmental factors. Many diseases with a complex aetiology therefore remain misunderstood. By developing a holistic approach to tackle the complexity of interactions, we decipher the complex intra-host interactions underlying Pacific oyster mortality syndrome affecting juveniles of Crassostrea gigas, the main oyster species exploited worldwide. Using experimental infections reproducing the natural route of infection and combining thorough molecular analyses of oyster families with contrasted susceptibilities, we demonstrate that the disease is caused by multiple infection with an initial...
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Ano: 2018 URL: https://archimer.ifremer.fr/doc/00461/57234/59228.pdf
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Inefficient immune response is associated with microbial permissiveness in juvenile oysters affected by mass mortalities on field ArchiMer
De Lorgeril, Julien; Escoubas, Jean Michel; Loubiere, Vincent; Pernet, Fabrice; Le Gall, Patrik; Vergnes, Agnes; Aujoulat, Fabien; Jeannot, Jean-luc; Jumas-bilak, Estelle; Got, Patrice; Gueguen, Yannick; Destoumieux-garzon, Delphine; Bachere, Evelyne.
Since 2008, juvenile Crassostrea gigas oysters have suffered from massive mortalities in European farming areas. This disease of complex etiology is still incompletely understood. Triggered by an elevated seawater temperature, it has been associated to infections by a herpes virus named OsHV-1 as well as pathogenic vibrios of the Splendidus clade. Ruling out the complexity of the disease, most of our current knowledge has been acquired in controlled experiments. Among the many unsolved questions, it is still ignored what role immunity plays in the capacity oysters have to survive an infectious episode. Here we show that juvenile oysters susceptible to the disease mount an inefficient immune response associated with microbial permissiveness and death. We...
Tipo: Text Palavras-chave: Host pathogen interaction; Innate immunity; Invertebrate; Mollusk; In situ mortality; Total bacteria; Crassostrea gigas.
Ano: 2018 URL: https://archimer.ifremer.fr/doc/00433/54470/55845.pdf
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Molecular characterisation of prolactin and analysis of extrapituitary expression in the European sea bass Dicentrarchus labrax under various salinity conditions ArchiMer
Boutet, Isabelle; Lorin Nebel, C; De Lorgeril, Julien; Guinand, B.
dAlthough prolactin has been demonstrated to be the main hormone involved in adaptation to dilute media in several freshwater teleosts, few studies have been conducted in marine teleosts. In the Mediterranean, the sea bass Dicentrarchus labrax inhabits environments ranging from the open sea to coastal lagoons, where salinity varies greatly. We characterised the prolactin (prl) gene and analysed its expression in two organs (gill and intestine) in D. labrax acclimated to either freshwater or seawater. A 2819 bp long sequence encompassing the prl gene and a part (282 bp) of the promoter were identified, and these comprised 5 coding exons separated by 4 introns. Prolactin was similarly expressed in fresh- and seawater adapted fish, although expression in...
Tipo: Text Palavras-chave: Prolactin; Phylogenctic analysis; MRNA expression; Osmoregulation; Intestine; Gill; Dicentrarchuslabrax.
Ano: 2007 URL: http://archimer.ifremer.fr/doc/2007/publication-2537.pdf
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Molecular characterization of penaeidins from two Atlantic Brazilian shrimp species, Farfantepenaeus paulensis and Litopenaeus schmitti ArchiMer
Barracco, Margherita; De Lorgeril, Julien; Gueguen, Yannick; Bachere, Evelyne.
We report here the molecular cloning of new members of the penaeidin family from two Atlantic penaeids from Brazil, Litopenaeus schmitti and Farfantepenaeus paulensis. The presence of penaeidins in the granular hemocytes of both shrimps was first evidenced by immunofluorescence, using polyclonal antibodies raised against L. vannamei penaeidin Litvan PEN3-1. cDNAs from the hemocytes of both Brazilian species were obtained by reverse transcription and the sequences encoding penaeidins were amplified by PCR, using primers based on penaeidin consensus sequences. Five penaeidin clones were obtained. According to the international penaeidin classification (PenBase, http://www.penbase.immunaqua.com), the deduced amino acid sequences of two clones from L. schmitti...
Tipo: Text Palavras-chave: Immuno detection; Molecular cloning; Litopenaeus schmitti; Farfantepenaeus paulensis; Brazilian penaeid shrimps; Hemocytes; Antimicrobial peptides; Penaeidins.
Ano: 2005 URL: http://archimer.ifremer.fr/doc/2005/publication-3582.pdf
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