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Paillard Christine - One of the best experts on this subject based on the ideXlab platform.
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Relation between Biofilm Condition and Virulence in Vibrio tapetis: A Transcriptomic Study
'MDPI AG', 2018Co-Authors: Rodrigues Sophie, Paillard Christine, Van Dillen Sabine, Tahrioui Ali, Berjeaud Jean-marc, Dufour Alain, Bazire AlexisAbstract:International audienceMarine pathogenic bacteria are able to form biofilms on many surfaces, such as mollusc shells, and they can wait for the appropriate opportunity to induce their virulence. Vibrio tapetis can develop such biofilms on the inner surface of shells of the Ruditapes philippinarum clam, leading to the formation of a brown Conchiolin deposit in the form of a ring, hence the name of the disease: Brown Ring Disease. The virulence of V. tapetis is presumed to be related to its capacity to form biofilms, but the link has never been clearly established at the physiological or genetic level. In the present study, we used RNA-seq analysis to identify biofilm- and virulence-related genes displaying altered expression in biofilms compared to the planktonic condition. A flow cell system was employed to grow biofilms to obtain both structural and transcriptomic views of the biofilms. We found that 3615 genes were differentially expressed, confirming that biofilm and planktonic lifestyles are very different. As expected, the differentially expressed genes included those involved in biofilm formation, such as motility- and polysaccharide synthesis-related genes. The data show that quorum sensing is probably mediated by the AI-2/LuxO system in V. tapetis biofilms. The expression of genes encoding the Type VI Secretion System and associated exported proteins are strongly induced, suggesting that V. tapetis activates this virulence factor when living in biofilm
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Immune responses of phenoloxidase and superoxide dismutase in the manila clam Venerupis philippinarum challenged with Vibrio tapetis – Part II: Combined effect of temperature and two V. tapetis strains
'Elsevier BV', 2015Co-Authors: Richard Gaëlle, Le Bris Cédric, Lambert Christophe, Guérard Fabienne, Paillard ChristineAbstract:International audienceManila clams, Venerupis philippinarum (Adams and Reeve, 1850), were experimentally infected with two different bacterial strains and challenged with two different temperatures. Bacterial strains used in this study were Vibrio tapetis strain CECT4600T, the causative agent of Brown Ring Disease (BRD) and V. tapetis strain LP2, supposed less virulent to V. philippinarum. V. tapetis is considered to proliferate at low temperatures, i.e. under 21 °C. In a global warming context we could hypothesize a decrease of mass mortalities caused by V. tapetis but these thermal changes could also directly impact the immune system of the host V. philippinarum. Thus, the aim of this study was to investigate the effects of the extrapallial injection with V. tapetis combined with temperature challenge on two enzymes activities in V. philippinarum. More precisely, after infection, phenoloxidase (PO) and superoxide dismutase (SOD), two major enzymes involved in immune response, were studied for 30 days in two compartments : the mantle and the hemolymph. Conchyolin Deposit Stages (CDS) and Shell Repair Stages (SRS) were also determined 30 days post-injection as a proxy of the virulence of the tested strains. In this study, we highlighted that hostepathogen interaction in a varying environment affects the enzymatic response of the host. The coupled effect of V. tapetis injection and temperature challenge was detected 30 days post injection and resulted in virulence differences. These findings were supported by CDS and SRS determination in clams and lead to the conclusion that clam’s immunity could be enhanced at 22 °C while V. tapetis virulence is lowered at this temperature. Another result of our study was the increase of PO and SOD basal activities as clams are exposed to warmer temperature
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Differential expression of genes involved in immunity and biomineralization during Brown Ring Disease development and shell repair in the Manila clam, Ruditapes philippinarum.
'Elsevier BV', 2013Co-Authors: Jeffroy Fanny, Brulle Franck, Paillard ChristineAbstract:International audienceSevere drop in Manila clams production in French aquacultured fields since the end of the 1980's is associated to Brown Ring Disease (BRD). This disease, caused by the bacteria Vibrio tapetis, is characterized by specific symptoms on the inner face of the shell. Diseased animals develop Conchiolin deposit to enrobe bacteria and form new calcified layers on the shell. Suppression subtractive hybridization was performed to identify genes differentially expressed during the early interaction of V. tapetis and Ruditapes philippinarum. Results revealed 301 unique genes differentially expressed during V. tapetis challenge. Several candidates involved in immune and biomineralization processes were selected from libraries. Transcriptional expression of selected candidates was determined in hemolymph and mantle tissues and revealed spatial and temporal variations. At 56days after infection, when clams were in phase of shell repair, transcripts of galectin and ferritin in hemocytes showed higher expression. Ca-like and serpin transcripts were specifically expressed in mantle and could contribute to defense against BRD
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Occurrence of Brown Ring Disease in Carpet Shell Clams Ruditapes decussatus from the Gulf of Gabes (Tunisia, Central Mediterranean Sea)
'National Shellfisheries Association', 2011Co-Authors: El Bour Monia, Paillard Christine, Dellali Mohamed, Boukef Imene, Lakhal Fatma, Mraouna Radhia, Attia El Hili Hedia, Klena JohnAbstract:International audienceThe occurrence and spread of brown ring disease (BRD) to several northern Mediterranean coasts is described. We report the results of a 6-y surveillance of BRD in natural populations of carpet shell clams (Ruditapes decussatus) in 14 zones along the Gulf of Gabes in Tunisia (southern Mediterranean Sea). BRD symptoms in adult animals resulting from Conchiolin deposits in inner parts of carpet shell were observed in all zones surveyed. Infestation rates within each site ranged from 65-100%, and BRD prevalence varied from 1-58% of clams in winter and from 1-33% in summer. Positive correlations were demonstrated between BRD prevalence and Vibrio spp. concentrations in clams. Vibrio tapetis was not identified among the bacterial organisms, suggesting that other marine Vibrio species are capable of causing BRD-like illness in carpet shell clams
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Handling enhances the development of brown ring disease signs in Ruditapes philippinarum
'National Shellfisheries Association', 2011Co-Authors: Jean Fred, Flye-sainte-marie Jonathan, Oudard Clémence, Paillard ChristineAbstract:International audienceBrown ring disease (BRD) in the Manila clam is characterized by the formation of a brown deposit of Conchiolin on the inner surface of the shell that gives the disease its name. The development of the signs of BRD may be favored by the entry of bacteria in the extrapallial compartments via mechanical disruptions of the periostracal lamina and/or chipping of the shell margin. In order to test this hypothesis, we conducted an experiment simulating clam handling under aquaculture conditions and we checked for prevalence of BRD signs. Our results assess that rough handling of R. philippinarum in presence of the bacterium V. tapetis significantly increase the prevalence of BRD signs. As a consequence our results show that minimizing manipulations and transfers of clams during culture is beneficial to avoid the development of BRD signs
Taylor, John D. - One of the best experts on this subject based on the ideXlab platform.
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Figure 6 from: Glover EA, Taylor JD (2016) Pleurolucina from the western Atlantic and eastern Pacific Oceans: a new intertidal species from Curaçao with unusual shell microstructure (Mollusca, Bivalvia, Lucinidae). ZooKeys 620: 1-19. https://doi.org/
2016Co-Authors: Glover, Emily A., Taylor, John D.Abstract:Figure 6 - Shell microstructure of other species Pleurolucina hendersoni, Pleurolucina undata, Lucina pensylvanica and Cardiolucina quadrata. A Pleurolucina hendersoni Guadeloupe, fractured section with prominent calcified Conchiolin layer, periostracum at base. Scale bar = 20 µm B Pleurolucina hendersoni, detail of Conchiolin layer with lines of calcareous spherulites. Scale bar = 20 µm C Pleurolucina undata Baja California, fractured section with thin Conchiolin layer Scale bar = 200 µm D Pleurolucina undata, detail of Conchiolin layer with spherulites. Scale bar = 20 µm E Pleurolucina undata, single spherulites embedded in Conchiolin. Scale bar = 3 µm F Lucina pensylvanica Florida Keys, calcified Conchiolin layer. Scale bar = 20 µm G Lucina pensylvanica, single spherulite. Scale bar = 2 µm H Lucina pensylvanica, section of periostracum with calcareous granules. Shell interior to top. Scale bar = 20 µm I Cardiolucina quadrata Philippines, fractured section with Conchiolin layer. Scale bar = 200 µm J Cardiolucina quadrata detail of Conchiolin layer with calcareous aggregates. Scale bar = 50 µm K Cardiolucina quadrata detail of calcareous aggregate. Scale bar = 10 µm
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Figure 4 from: Glover EA, Taylor JD (2016) Pleurolucina from the western Atlantic and eastern Pacific Oceans: a new intertidal species from Curaçao with unusual shell microstructure (Mollusca, Bivalvia, Lucinidae). ZooKeys 620: 1-19. https://doi.org/
2016Co-Authors: Glover, Emily A., Taylor, John D.Abstract:Figure 4 - Shell microstructure of Pleurolucina harperae. A Fractured section of shell margin showing major notch growth halt and Conchiolin layer. Scale bar = 400 µm B Fractured section showing succession of shell layers. Shell exterior at top. Scale bar = 100 µm C Conchiolin layer with regular bands of spherulites. Scale bar = 40 µm D Individual spherulite. Scale bar = 2 µm E Adjacent spherulites embedded in Conchiolin with narrow channels between layers. Scale bar = 5 µm F Single spherulites with channels below and above. Scale bar = 5 µm. cl crossed lamellar layer co Conchiolin layer cp composite prismatic layer ip irregular prismatic layer p periostracum sp spherulitic prismatic layer
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Pleurolucina from the western Atlantic and eastern Pacific Oceans: a new intertidal species from Curaçao with unusual shell microstructure (Mollusca, Bivalvia, Lucinidae)
2016Co-Authors: Glover, Emily A., Taylor, John D.Abstract:A new shallow water species of the lucinid bivalve Pleurolucina is described from Curaçao in the southern Caribbean Sea and compared with known species of the genus from the western Atlantic and eastern Pacific Oceans. Although confused with the Floridian species P. leucocyma, it is most similar to the eastern Pacific P. undata. As in all studied lucinids, the new species possesses symbiotic bacteria housed in the ctenidia. The shell microstructure is unusual with repeated and intercalated Conchiolin layers that have sublayers of ‘tulip-shaped’ calcareous spherules. Predatory drillings by naticid gastropods frequently terminate at the Conchiolin layers
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Figure 5 from: Glover EA, Taylor JD (2016) Pleurolucina from the western Atlantic and eastern Pacific Oceans: a new intertidal species from Curaçao with unusual shell microstructure (Mollusca, Bivalvia, Lucinidae). ZooKeys 620: 1-19. https://doi.org/10.3897/zookeys.620.9569
2016Co-Authors: Glover, Emily A., Taylor, John D.Abstract:Figure 5 - Failed and multiple drill holes in shells of Pleurolucina harperae. A L = 6.8 mm B L = 9.8 mm C L =7.8 mm D SEM of failed drill hole terminating at Conchiolin layer. Scale bar = 1.0 mm
Paul C Southgate - One of the best experts on this subject based on the ideXlab platform.
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mantle regeneration in the pearl oysters pinctada fucata and pinctada margaritifera
Aquaculture, 2005Co-Authors: Hector Acostasalmon, Paul C SouthgateAbstract:Abstract Mantle tissue in pearl oysters (Pteriidae) is responsible for secreting the mother-of-pearl or nacre lining the shell. When grafted into another oyster, excised mantle tissue, commonly called ‘saibo’, is responsible for cultured pearl production. This study was undertaken to assess the process of mantle regeneration in the Akoya pearl oyster Pinctada fucata and the blacklip pearl oyster Pinctada margaritifera following mantle excision. Prior to saibo excision, oysters were anaesthetized with 2 mL L −1 propylene phenoxetol. Saibo tissue was excised from 50 P . fucata and 10 P. margaritifera . After excision, all oysters were returned to culture conditions. All oysters were maintained in panel (pocket) nets on a long-line at Magnetic Island, north Queensland, Australia. Oysters were anaesthetized and sacrificed 3, 6, 9, 12, 15, 20, 30, 45, 60 and 90 days after saibo excision to assess mantle regeneration using histological and histochemical techniques. Survival over the 90-day study was 100% and 70% for P. fucata and P. margaritifera , respectively. After excision, the mantle tissue healed within the first three days and began growing as connective tissue. Muscular development was seen between days 60 and 90 after excision. Shell formation abilities were recovered by day 15 when secretory cells and Conchiolin secretions were first observed. Both P. fucata and P. margaritifera regenerated mantle to its original extent within the 90-day study. This is the first description of in vivo mantle regeneration in pearl oysters and the results have major implications for the lucrative cultured pearl industry. Our results indicate that mantle tissue donors need not be killed for pearl production. Those producing saibo that results in good quality pearls could be used as future parent-stock in breeding programs or for subsequent saibo donation and pearl production.
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Mantle regeneration in the pearl oysters Pinctada fucata and Pinctada margaritifera
Aquaculture, 2005Co-Authors: Héctor Acosta-salmón, Paul C SouthgateAbstract:Mantle tissue in pearl oysters (Pteriidae) is responsible for secreting the mother-of-pearl or nacre lining the shell. When grafted into another oyster, excised mantle tissue, commonly called 'saibo', is responsible for cultured pearl production. This study was undertaken to assess the process of mantle regeneration in the Akoya pearl oyster Pinctada fucata and the blacklip pearl oyster Pinctada margaritifera following mantle excision. Prior to saibo excision, oysters were anaesthetized with 2 mL L-1 propylene phenoxetol. Saibo tissue was excised from 50 P. fucata and 10 P. margaritifera. After excision, all oysters were returned to culture conditions. All oysters were maintained in panel (pocket) nets on a long-line at Magnetic Island, north Queensland, Australia. Oysters were anaesthetized and sacrificed 3, 6, 9, 12, 15, 20, 30, 45, 60 and 90 days after saibo excision to assess mantle regeneration using histological and histochemical techniques. Survival over the 90-day study was 100% and 70% for P. fucata and P. margaritifera, respectively. After excision, the mantle tissue healed within the first three days and began growing as connective tissue. Muscular development was seen between days 60 and 90 after excision. Shell formation abilities were recovered by day 15 when secretory cells and Conchiolin secretions were first observed. Both P. fucata and P. margaritifera regenerated mantle to its original extent within the 90-day study. This is the first description of in vivo mantle regeneration in pearl oysters and the results have major implications for the lucrative cultured pearl industry. Our results indicate that mantle tissue donors need not be killed for pearl production. Those producing saibo that results in good quality pearls could be used as future parent-stock in breeding programs or for subsequent saibo donation and pearl production. © 2005 Elsevier B.V. All rights reserved
Jeffroy Fanny - One of the best experts on this subject based on the ideXlab platform.
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Differential expression of genes involved in immunity and biomineralization during Brown Ring Disease development and shell repair in the Manila clam, Ruditapes philippinarum.
'Elsevier BV', 2013Co-Authors: Jeffroy Fanny, Brulle Franck, Paillard ChristineAbstract:International audienceSevere drop in Manila clams production in French aquacultured fields since the end of the 1980's is associated to Brown Ring Disease (BRD). This disease, caused by the bacteria Vibrio tapetis, is characterized by specific symptoms on the inner face of the shell. Diseased animals develop Conchiolin deposit to enrobe bacteria and form new calcified layers on the shell. Suppression subtractive hybridization was performed to identify genes differentially expressed during the early interaction of V. tapetis and Ruditapes philippinarum. Results revealed 301 unique genes differentially expressed during V. tapetis challenge. Several candidates involved in immune and biomineralization processes were selected from libraries. Transcriptional expression of selected candidates was determined in hemolymph and mantle tissues and revealed spatial and temporal variations. At 56days after infection, when clams were in phase of shell repair, transcripts of galectin and ferritin in hemocytes showed higher expression. Ca-like and serpin transcripts were specifically expressed in mantle and could contribute to defense against BRD
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Production et caractérisation de familles de palourdes japonaises, Ruditapes philippinarum, résistantes à la maladie de l'anneau brun
HAL CCSD, 2011Co-Authors: Jeffroy FannyAbstract:The culture of Manila clams in Brittany has faded from the 80 years after the onset of the disease called vibriosis of the Brown Ring (MAB). This disease is caused by the presence of the bacterium Vibrio tapetis causes a characteristic Conchiolin deposit on the inner edge of the valves of the clams. The animals are able to repair the shell at the Conchiolin deposits by a phenomenon of recalcification. Broodstock was selected according to their stage of disease development after inoculation of bacteria into the pallial cavity or following their physiological response in the hemolymph. Families of clams were produced and comparisons of the response to the disease were made between families in order to differentiate with the least susceptible individuals to this disease. Thus, these studies have to distinguish two families showing different characteristics of the immune level and hemocyte parameters in the development of the MAB. A relationship between families and ornamentation/color of the shell has been detected and enabled, in the thesis, to differentiate families in addition to the microsatellite tool. Moreover, it was shown that the V. tapetis induces production of nitric oxide in vitro in hemocytes of Manila clams. Moreover, when NO production is inhibited, the number of adherent cells decreases. Finally, molecular approaches have helped to highlight the specific expression of two gene transcripts (carbonic anhydrase like and serpin) in the mantle related to the development of the MAB.La culture de la palourde japonaise, Ruditapes philippinarum, en Bretagne s'est estompée à partir des années 80 après l'apparition de la vibriose appelée Maladie de l'Anneau Brun (MAB). Cette maladie est causée par la présence de la bactérie Vibrio tapetis qui provoque un dépôt caractéristique de conchyoline sur le bord interne des valves des palourdes. Les animaux parviennent à réparer la coquille au niveau des dépôts de conchyoline par un phénomène de recalcification. Des géniteurs et des familles de palourdes ont été étudiés afin de différencier les individus les moins sensibles à cette maladie. Ainsi, ces études ont permis de discriminer deux familles montrant des caractères de défense immunitaires différents au niveau des paramètres hémocytaires et au niveau du développement de la MAB. De plus, il a été démontré que le V. tapetis induit une production de l'oxyde nitrique in vitro dans les hémocytes de la palourde japonaise. Enfin, les approches moléculaires ont permis de mettre en évidence l'expression spécifique de deux transcrits de gènes (carbonique anhydrase like et inhibiteur de sérine protéases (serpine)) dans le manteau en lien avec le développement de la MAB
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Production et caractérisation de familles de palourdes japonaises, Ruditapes philippinarum, résistantes à la maladie de l'anneau brun
2011Co-Authors: Jeffroy Fanny, Paillard ChristineAbstract:La culture de la palourde japonaise, Ruditapes philippinarum, en Bretagne s est estompée à partir des années 80 après l apparition de la vibriose appelée Maladie de l Anneau Brun (MAB). Cette maladie est causée par la présence de la bactérie Vibrio tapetis qui provoque un dépôt caractéristique de conchyoline sur le bord interne des valves des palourdes. Les animaux parviennent à réparer la coquille au niveau des dépôts de conchyoline par un phénomène de recalcification. Des géniteurs et des familles de palourdes ont été étudiés afin de différencier les individus les moins sensibles à cette maladie. Ainsi, ces études ont permis de discriminer deux familles montrant des caractères de défense immunitaires différents au niveau des paramètres hémocytaires et au niveau du développement de la MAB. De plus, il a été démontré que le V. tapetis induit une production de l oxyde nitrique in vitro dans les hémocytes de la palourde japonaise. Enfin, les approches moléculaires ont permis de mettre en évidence l expression spécifique de deux transcrits de gènes (carbonique anhydrase like et inhibiteur de sérine protéases (serpine)) dans le manteau en lien avec le développement de la MAB.The culture of Manila clams in Brittany has faded from the 80 years after the onset of the disease called vibriosis of the Brown Ring (MAB). This disease is caused by the presence of the bacterium Vibrio tapetis causes a characteristic Conchiolin deposit on the inner edge of the valves of the clams. The animals are able to repair the shell at the Conchiolin deposits by a phenomenon of recalcification. Broodstock was selected according to their stage of disease development after inoculation of bacteria into the pallial cavity or following their physiological response in the hemolymph. Families of clams were produced and comparisons of the response to the disease were made between families in order to differentiate with the least susceptible individuals to this disease. Thus, these studies have to distinguish two families showing different characteristics of the immune level and hemocyte parameters in the development of the MAB. A relationship between families and ornamentation/color of the shell has been detected and enabled, in the thesis, to differentiate families in addition to the microsatellite tool. Moreover, it was shown that the V. tapetis induces production of nitric oxide in vitro in hemocytes of Manila clams. Moreover, when NO production is inhibited, the number of adherent cells decreases. Finally, molecular approaches have helped to highlight the specific expression of two gene transcripts (carbonic anhydrase like and serpin) in the mantle related to the development of the MAB.BREST-SCD-Bib. electronique (290199901) / SudocSudocFranceF
Glover, Emily A. - One of the best experts on this subject based on the ideXlab platform.
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Figure 6 from: Glover EA, Taylor JD (2016) Pleurolucina from the western Atlantic and eastern Pacific Oceans: a new intertidal species from Curaçao with unusual shell microstructure (Mollusca, Bivalvia, Lucinidae). ZooKeys 620: 1-19. https://doi.org/
2016Co-Authors: Glover, Emily A., Taylor, John D.Abstract:Figure 6 - Shell microstructure of other species Pleurolucina hendersoni, Pleurolucina undata, Lucina pensylvanica and Cardiolucina quadrata. A Pleurolucina hendersoni Guadeloupe, fractured section with prominent calcified Conchiolin layer, periostracum at base. Scale bar = 20 µm B Pleurolucina hendersoni, detail of Conchiolin layer with lines of calcareous spherulites. Scale bar = 20 µm C Pleurolucina undata Baja California, fractured section with thin Conchiolin layer Scale bar = 200 µm D Pleurolucina undata, detail of Conchiolin layer with spherulites. Scale bar = 20 µm E Pleurolucina undata, single spherulites embedded in Conchiolin. Scale bar = 3 µm F Lucina pensylvanica Florida Keys, calcified Conchiolin layer. Scale bar = 20 µm G Lucina pensylvanica, single spherulite. Scale bar = 2 µm H Lucina pensylvanica, section of periostracum with calcareous granules. Shell interior to top. Scale bar = 20 µm I Cardiolucina quadrata Philippines, fractured section with Conchiolin layer. Scale bar = 200 µm J Cardiolucina quadrata detail of Conchiolin layer with calcareous aggregates. Scale bar = 50 µm K Cardiolucina quadrata detail of calcareous aggregate. Scale bar = 10 µm
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Figure 4 from: Glover EA, Taylor JD (2016) Pleurolucina from the western Atlantic and eastern Pacific Oceans: a new intertidal species from Curaçao with unusual shell microstructure (Mollusca, Bivalvia, Lucinidae). ZooKeys 620: 1-19. https://doi.org/
2016Co-Authors: Glover, Emily A., Taylor, John D.Abstract:Figure 4 - Shell microstructure of Pleurolucina harperae. A Fractured section of shell margin showing major notch growth halt and Conchiolin layer. Scale bar = 400 µm B Fractured section showing succession of shell layers. Shell exterior at top. Scale bar = 100 µm C Conchiolin layer with regular bands of spherulites. Scale bar = 40 µm D Individual spherulite. Scale bar = 2 µm E Adjacent spherulites embedded in Conchiolin with narrow channels between layers. Scale bar = 5 µm F Single spherulites with channels below and above. Scale bar = 5 µm. cl crossed lamellar layer co Conchiolin layer cp composite prismatic layer ip irregular prismatic layer p periostracum sp spherulitic prismatic layer
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Pleurolucina from the western Atlantic and eastern Pacific Oceans: a new intertidal species from Curaçao with unusual shell microstructure (Mollusca, Bivalvia, Lucinidae)
2016Co-Authors: Glover, Emily A., Taylor, John D.Abstract:A new shallow water species of the lucinid bivalve Pleurolucina is described from Curaçao in the southern Caribbean Sea and compared with known species of the genus from the western Atlantic and eastern Pacific Oceans. Although confused with the Floridian species P. leucocyma, it is most similar to the eastern Pacific P. undata. As in all studied lucinids, the new species possesses symbiotic bacteria housed in the ctenidia. The shell microstructure is unusual with repeated and intercalated Conchiolin layers that have sublayers of ‘tulip-shaped’ calcareous spherules. Predatory drillings by naticid gastropods frequently terminate at the Conchiolin layers
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Figure 5 from: Glover EA, Taylor JD (2016) Pleurolucina from the western Atlantic and eastern Pacific Oceans: a new intertidal species from Curaçao with unusual shell microstructure (Mollusca, Bivalvia, Lucinidae). ZooKeys 620: 1-19. https://doi.org/10.3897/zookeys.620.9569
2016Co-Authors: Glover, Emily A., Taylor, John D.Abstract:Figure 5 - Failed and multiple drill holes in shells of Pleurolucina harperae. A L = 6.8 mm B L = 9.8 mm C L =7.8 mm D SEM of failed drill hole terminating at Conchiolin layer. Scale bar = 1.0 mm