José L. Sánchez

Lipid classes and fatty acid composition in the female gonad of Pecten maximus in relation to reproductive cycle and environmental variables

Abstract Lipids in the female gonad of Pecten maximus from Ria de Arousa (N.W. Spain) were analysed for lipid class composition and fatty acid composition of triacylglycerols and phospholipids and their variations in relation to gametogenic cycle were studied for 16 months. The female condition index and stereological studies showed the existence of two principal spawning periods, winter and late spring-early summer. No sexual resting period was found and oocyte lysis was high throughout the year. Ovary lipid levels (16–21.5% dry weight) displayed clear seasonal changes linked to the gametogenic cycle. The acylglycerols (20–65% total lipids)and phospholipids (26–35%) were the major lipid classes, sterols (4–8%) were present in minor amounts. The acylglycerols and free sterols showed a seasonal variation similar to that of the mean oocyte diameter. The remainder of lipid classes showed no clear seasonal variation. The 20:5n-3 levels were higher than that of 22:6n-3 in the triacylglycerols, contrary to that of phospholipids. The polyunsaturated fatty acids (PUFA) were more abundant in the triacylglycerols and the series n-3 was clearly predominant. The Σn-3 and the principal n-3 fatty acids (mainly in phospholipids) showed a seasonal variation clearly related to the reproductive cycle.

Seasonal changes in lipid classes and fatty acid composition in the digestive gland of Pecten maximus.

Seasonal variations in lipid classes and fatty acid composition of triacylglycerols and phospholipids in the digestive gland of Pecten maximus were studied over a period of 16 months. Acylglycerols predominated (19-77% of total lipids), in accordance with the role of the digestive gland as an organ for lipid storage in scallops. Seasonal variations were mainly seen in the acylglycerol content, while phospholipids (2.5-10.0% of total lipids) and sterols (1.9-7.4% of total lipids) showed only minor changes. The most abundant fatty acids were 14:0, 16:0, 18:0, 16:1(n-7), 18:1(n-9), 18:1(n-7), 18:4(n-3), 20:5(n-3) and 22:6(n-3) and these showed similar seasonal profiles in both, triacylglycerol and phospholipid fractions. In contrast to the phospholipid fraction, the triacylglycerol fraction contained more 20:5(n-3) than 22:6(n-3). In three phospholipid samples we noted a high percentage of a 22-2-non-methylene-interrupted fatty acid, previously described to have a structural role in several bivalve species. The main polyunsaturated fatty acids displayed important seasonal variations parallel to those of the acylglycerols, suggesting good nutritional conditions. A positive correlation existed between the level of saturated fatty acids and temperature, whereas the levels of polyunsaturated fatty acids correlated negatively with temperature.

Settlement Behavior of Black Scallop Larvae (Chlamys varia, L.) in Response to GABA, Epinephrine and IBMX

Abstract Competent larvae of the black scallop Chlamys varia (L.) were treated with the neurotransmitter gamma-amino-n-butyric acid (GABA), the catecholamine epinephrine, and 3-isobutyl-1-methylxanthine (IBMX), at different concentrations and times of exposure, to test the effects of exposure to the compounds on larval settlement. After 24 h, maximum percentage of settlement (>30%) was induced by 10−5 M epinephrine. After 48 h, maximum settlement rate was also achieved by 10−5 M epinephrine (>54%). Exposure to 10−6 M epinephrine and to 10−6 M GABA also induced significantly higher larval settlement than in control larvae. By contrast, IBMX failed to induce significantly higher settlement rates than in the control larvae. Epinephrine and GABA were identified as active inducers of settlement in C. varia, and were not toxic to black scallop larvae. This is the first report in which the involvement of epinephrine and GABA in the settlement of C. varia larvae is described.

Conservation of Gbx genes from EHG homeobox in bivalve molluscs

Homeobox-containing genes encode a set of transcription factors that have been shown to control spatial patterning mechanisms in bilaterian organism development. The homeobox gene Gbx, included in the EHGbox cluster, is implicated in the development of the nervous system. In this study, we surveyed five different families of Bivalvia for the presence of Gbx genes by means of PCR with degenerate primers. We were able to recover seven Gbx gene fragments from five bivalve species: Solen marginatus, Mimachlamys varia, Venerupis pullastra, Ostrea edulis and Mytilus galloprovincialis (the derived amino acid sequence were designated Sma-Gbx, Cva-Gbx, Vpu-Gbx, Oed-Gbx and Mga-Gbx, respectively). These genes are orthologous to various Gbx genes present in bilaterian genomes. The Gbx genes in four Bivalvia families, namely Solenidae, Veneridae, Ostreidae and Mytilidae, are newly reported here and we also showed additional information of the Gbx genes of Pectinidae. The phylogenetic analyses by neighbour-joining, UPGMA, maximum parsimony and Bayesian analysis clearly indicated that the Gbx sequences formed a well supported clade and assigned these Gbx genes to the Gbx family. These data permit to confirm that the homeodomain of the Gbx family is highly conserved among these five distinct families of bivalve molluscs.

The HOX Gene Cluster in the Bivalve Mollusc Mytilus galloprovincialis

The clustered Hox genes play a central role in the regulation of development in bilaterian animals. In this study, we analyzed the homeobox-containing genes in a bivalve mollusc, the mussel Mytilus galloprovincialis, an unsegmented spiralian lophotrochozoan. We isolated and characterized four Hox cluster genes using the polymerase chain reaction with specific primers. Molecular alignments and phylogenetic analysis indicate that these mussel genes are homologs of the anterior group (pb ortholog), paralog group 3, and central group (PG4/Dfd and PG5/Scr) genes. The putative homeodomain sequences were designated Mgox1, Mgox2, Mgox3, and Mgox4.

The Proposed Dropping of the Genus Crassostrea for All Pacific Cupped Oysters and Its Replacement by a New Genus Magallana: A Dissenting View

B. L. BAYNE,* M. AHRENS, S. K. ALLEN, M. ANGL ES D AURIAC, T. BACKELJAU, P. BENINGER, R. BOHN, P. BOUDRY, J. DAVIS, T. GREEN, X. GUO, D. HEDGECOCK, A. IBARRA, P. KINGSLEY-SMITH, M. KRAUSE, C. LANGDON, S. LAP EGUE, C. LI, D. MANAHAN, R. MANN, L. PEREZ-PARALLE, E. N. POWELL, P. D. RAWSON, D. SPEISER, J.-L. SANCHEZ, S. SHUMWAY AND H. WANG Edinburgh, UK; Universidad Jorge Tadeo Lozano, Colombia; Virginia Institute of Marine Sciences; Norsk Institutt for Vannforskning, Norway; Royal Belgian Institute of Natural Sciences, Belgium; Universit e de Nantes, France; Maryland Department of Natural Resources; IFREMER, Plouzan e, France; Baywater Shellfish Farm; Macquarie University, Australia; Rutgers University; University of Southern California; CentroDe Investigaciones Biologicas Del Noreste,Mexico; Marine Resources Research Institute; Hofstra University; Oregon State University; IFREMER, Sete, France; Chinese Academy of Sciences, China; Universidade de Santiago de Compostela, Spain; Gulf Coast Research Laboratory; University ofMaine; University of South Carolina; University of Connecticut

Purification and properties of 6-phosphogluconate dehydrogenase fromMytilus galloprovincialis digestive gland

Abstract 1. The enzyme 6-phosphogluconate dehydrogenase from digestive gland of Mytilus galloprovincialis was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis. 2. The enzyme was purified 229-fold with a final specific activity of 2.3 μmol of NADP + reduced/min per mg of protein and overall yield of 10%. 3. The molecular weight of the native enzyme is estimated to be 100, 000 from gel-filtration studies. 4. The influence of pH and MgCl 2 concentration on enzyme activity have been studied and the results have been compared to those reported by other authors for enzymes from different sources. 5. The K m values for 6-phosphogluconate and NADP + at room temperature (20°C) are approx. 20 and 40 μM respectively. 6. NADPH was an inhibitor strictly competitive with respect to NADP + ( K i = 14 μM) and non-competitive with respect to 6-phosphogluconate ( K i = 45 μM).

Transcriptional response after exposure to domoic acid-producing Pseudo-nitzschia in the digestive gland of the mussel Mytilus galloprovincialis

ABSTRACT Bivalve molluscs are filter feeding species that can accumulate biotoxins in their body tissues during harmful algal blooms. Amnesic Shellfish Poisoning (ASP) is caused by species of the diatom genus Pseudo‐nitzschia, which produces the toxin domoic acid. The Mytilus galloprovincialis digestive gland transcriptome was de novo assembled based on the sequencing of 12 cDNA libraries, six obtained from control mussels and six from mussels naturally exposed to domoic acid‐producing diatom Pseudo‐nitzschia australis. After de novo assembly 94,727 transcripts were obtained, with an average length of 1015 bp and a N50 length of 761 bp. The assembled transcripts were clustered (homology > 90%) into 69,294 unigenes. Differential gene expression analysis was performed (DESeq2 algorithm) in the digestive gland following exposure to the toxic algae. A total of 1158 differentially expressed unigenes (absolute fold change > 1.5 and p‐value < 0.05) were detected: 686 up‐regulated and 472 down‐regulated. Several membrane transporters belonging to the family of the SLC (solute carriers) were over‐expressed in exposed mussels. Functional enrichment was performed using Pfam annotations obtained from the genes differentially expressed, 37 Pfam families were found to be significantly (FDR adjusted p‐value < 0.1) enriched. Some of these families (sulfotransferases, aldo/keto reductases, carboxylesterases, C1q domain and fibrinogen C‐terminal globular domain) could be putatively involved in detoxification processes, in the response against of the oxidative stress and in immunological processes. Protein network analysis with STRING algorithm found alteration of the Notch signaling pathway under the action of domoic acid‐producing Pseudo‐nitzschia. In conclusion, this study provides a high quality reference transcriptome of M. galloprovincialis digestive gland and identifies potential genes involved in the response to domoic acid. Graphical abstract Figure. No Caption available. HighlightsMussels were naturally exposed to domoic acid‐producing Pseudo‐nitzschia.1158 unigenes were differentially expressed.37 Pfam families were found to be significantly enriched.Some of these families could be involved in detoxification, response against oxidative stress and/or immunological processes.Protein network analysis found alteration of the Notch signaling pathway.

Validation of Reference Genes in Mussel Mytilus galloprovincialis Tissues under the Presence of Okadaic Acid

ABSTRACT Blooms of toxic microalgae and the accumulation of their toxic compounds in bivalve molluscs are a regular worldwide problem to both consumers and producers. To select populations that accumulate a less amount of toxins is very important to understand the metabolism of biotoxins. The study of gene expression patterns by real-time quantitative polymerase chain reaction (RT-qPCR) may provide insights into the genes involved in detoxification metabolic pathways. One of the critical steps using RT-qPCR is that the expression results have to be normalized using internal reference genes. A systematic evaluation of reference genes in mussels has not been done yet and genes are frequently used as reference genes without validation. In this study, eight commonly used candidate reference genes have been tested as suitable in the mussel Mytilus galloprovincialis and their expression in mussels exposed to a toxic tide was studied by RT-qPCR. Their expression stabilities were evaluated using three different Excel applets (geNorm, NormFinder, and BestKeeper) which produced highly comparable results. The most suitable combination of reference genes for the normalization was glyceraldehyde-3-phosphate dehydrogenase (gapdh), 40S ribosomal protein S4 (rps4), and cytochrome c oxidase subunit 1 in digestive gland and gill and gapdh, rps4, and 40S ribosomal protein S27 in mantle. The M. galloprovincialis mrp1 and mrp2 genes were used to assess the quality of these reference genes. Our results show that some of the most widely used reference genes are not always suitable. This work underlies the importance of the validation of reference genes for each experimental situation and will be useful for the identification of genes involved in detoxification pathways in M. galloprovincialis.

HOX, PARAHOX, EHGBOX, AND NK GENES IN BIVALVE MOLLUSCS: EVOLUTIONARY IMPLICATIONS

ABSTRACT Molluscs belong to one of the two protostome superphyla, the Lophotrochozoa. Among molluscs, bivalves show a characteristic shell morphology separated bilaterally into two plates. The connection between the modification of the development and evolution of the molluscan body plan can be study identifying and obtaining information on the genes that regulate the development of bivalves. Homeobox genes are involved in body plan formation and in the regulation of many developmental processes in bilateria. For this reason, the evolutionary history of Hox, ParaHox, EHGbox, and NK homeobox gene families could be crucial to understand the evolution of molluscan body plans and phylogeny. The aim of this work was to compare these genes from different Bivalvia families. In this study, 22 homeobox gene fragments from five bivalve species were identified, and then these homeodomain sequences were compared with those available from other bivalves. The Hox cluster in bivalve molluscs has 11 genes. Regarding the ParaHox cluster, current data suggest that the ParaHox genes are also conserved in bivalves. Two EHGbox genes are well conserved in bivalves (en and Gbx); however, there are no data about the presence of the third one (Mnx). The NK cluster has not been examined in depth in Bivalvia; however, the conservation of Tlx and NK2 genes in different species can be confirmed. The study of the genes reported here might contribute to the understanding of their evolutionary history within the phylum and the involvement of these genes in the body plan of bivalves.