Browsing by Author "Saavedra, Carlos"
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- A Microarray study of Carpet-Shell Clam (Ruditapes decussatus) shows common and organ-specific growth-related gene expression Differences in gills and digestive glandPublication . Saavedra, Carlos; Milan, Massimo; Leite, Ricardo B.; Cordero, David; Patarnello, Tomaso; Leonor Cancela, M.; Bargelloni, LucaGrowth rate is one of the most important traits from the point of view of individual fitness and commercial production in mollusks, but its molecular and physiological basis is poorly known. We have studied differential gene expression related to differences in growth rate in adult individuals of the commercial marine clam Ruditapes decussatus. Gene expression in the gills and the digestive gland was analyzed in 5 fast-growing and five slow-growing animals by means of an oligonucleotide microarray containing 14,003 probes. A total of 356 differentially expressed genes (DEG) were found. We tested the hypothesis that differential expression might be concentrated at the growth control gene core (GCGC), i. e., the set of genes that underlie the molecular mechanisms of genetic control of tissue and organ growth and body size, as demonstrated in model organisms. The GCGC includes the genes coding for enzymes of the insulin/ insulin-like growth factor signaling pathway (IIS), enzymes of four additional signaling pathways (Raf/ Ras/ Mapk, Jnk, TOR, and Hippo), and transcription factors acting at the end of those pathways. Only two out of 97 GCGC genes present in themicroarray showed differential expression, indicating a very little contribution of GCGC genes to growth-related differential gene expression. Forty eight DEGs were shared by both organs, with gene ontology (GO) annotations corresponding to transcription regulation, RNA splicing, sugar metabolism, protein catabolism, immunity, defense against pathogens, and fatty acid biosynthesis. GO termenrichment tests indicated that genes related to growth regulation, development and morphogenesis, extracellular matrix proteins, and proteolysis were overrepresented in the gills. In the digestive gland overrepresented GO terms referred to gene expression control through chromatin rearrangement, RAS-related small GTPases, glucolysis, and energy metabolism. These analyses suggest a relevant role of, among others, some genes related to the IIS, such as the ParaHox gene Xlox, CCAR and the CCN family of secreted proteins, in the regulation of growth in bivalves.
- Increasing genomic information in bivalves through new EST collections in four species: Development of new genetic markers for environmental studies and genome evolutionPublication . Tanguy, A.; Bierne, N.; Saavedra, Carlos; Pina, B.; Bachere, E.; Kube, M.; Bazin, E.; Bonhomme, F.; Boudry, P.; Boulo, V.; Boutet, I.; Cancela, Leonor; Dossat, C.; Favrel, P.; Huvet, A.; Jarque, S.; Jollivet, D.; Klages, Sven; Lapegue, S.; Leite, Ricardo; Moal, J.; Moraga, D.; Reinhardt, Richard; Samain, J. F.; Zouros, E.; Canario, Adelino V. M.The generation of EST information is an essential step in the genomic characterisation of species. In the context of the European Network Marine Genomics, a common goal was to significantly increase the amount of ESTs in commercial marine mollusk species and more specifically in the less studied but ecologically and commercially important groups, such as mussel and clam genera. Normalized cDNA libraries were constructed for four different relevant bivalves species (Crassostrea gigas, Mytilus edulis, Ruditapes decussatus and Bathymodiolus azoricus), using numerous tissues and physiological conditions. In this paper, we present the analysis of the 13,013 expressed sequence tags (ESTs) generated. Each EST library was independently assembled and 1300–3000 unique sequences were identified in each species. For the different species, functional categories could be assigned to only about 16 to 27% of ESTs using the GO annotation tool. All sequences have been incorporated into a publicly available database and form the basis for subsequent microarray design, SNP detection and polymorphism analysis, and the placement of novel markers on genetic linkage maps.
- mRNA-Seq and microarray development for the Grooved carpet shell clam, Ruditapes decussatus: a functional approach to unravel host -parasite interactionPublication . Leite, Ricardo; Milan, Massimo; Coppe, Alessandro; Bortoluzzi, Stefania; Dos Anjos, António; Reinhardt, Richard; Saavedra, Carlos; Patarnello, T.; Cancela, Leonor; Bargelloni, LucaBackground: The Grooved Carpet shell clam Ruditapes decussatus is the autochthonous European clam and the most appreciated from a gastronomic and economic point of view. The production is in decline due to several factors such as Perkinsiosis and habitat invasion and competition by the introduced exotic species, the manila clam Ruditapes philippinarum. After we sequenced R. decussatus transcriptome we have designed an oligo microarray capable of contributing to provide some clues on molecular response of the clam to Perkinsiosis. Results: A database consisting of 41,119 unique transcripts was constructed, of which 12,479 (30.3%) were annotated by similarity. An oligo-DNA microarray platform was then designed and applied to profile gene expression in R. decussatus heavily infected by Perkinsus olseni. Functional annotation of differentially expressed genes between those two conditionswas performed by gene set enrichment analysis. As expected, microarrays unveil genes related with stress/infectious agents such as hydrolases, proteases and others. The extensive role of innate immune system was also analyzed and effect of parasitosis upon expression of important molecules such as lectins reviewed.Conclusions: This study represents a first attempt to characterize Ruditapes decussatus transcriptome, an important marine resource for the European aquaculture. The trancriptome sequencing and consequent annotation will increase the available tools and resources for this specie, introducing the possibility of high throughput experiments such as microarrays analysis. In this specific case microarray approach was used to unveil some important aspects of host-parasite interaction between the Carpet shell clam and Perkinsus, two non-model species, highlighting some genes associated with this interaction. Ample information was obtained to identify biological processes significantly enriched among differentially expressed genes in Perkinsus infected versus non-infected gills. An overview on the genes related with the immune system on R. decussatus transcriptome is also reported.
- Transcriptional profiling of populations in the clam Ruditapes decussatus suggests genetically determined differentiation in gene expression along parallel temperature gradients and between races of the Atlantic ocean and west Mediterranean seaPublication . Saavedra, Carlos; Milan, Massimo; M. Leite, Ricardo; Cordero, David; Patarnello, Tomaso; Cancela, M. Leonor; Bargelloni, LucaOngoing ocean warming due to climate change poses new challenges for marine life and its exploitation. We have used transcriptomics to find genetically based responses to increased temperature in natural populations of the marine clam Ruditapes decussatus, which lives along parallel thermal gradients in southern Europe. Clams of the Atlantic and West Mediterranean races were collected in northern (cool) and a southern (warm) localities. The animals were kept in running seawater in the warm, southern Atlantic locality for a 15-week period. During this period, water temperature was raised to typical southern European summer values. After this period, an expression profile was obtained for a total of 34 clams and 11,025 probes by means of an oligonucleotide microarray. We found distinct transcriptional patterns for each population based on a total of 552 differentially expressed genes (DEGs), indicating innate differences which probably have a genetic basis. Race and latitude contributed significantly to gene expression differences, with very different sets of DEGs. A gene ontology analysis showed that races differed mainly in the genes involved in ribosomal function and protein biosynthesis, while genes related to glutathione metabolism and ATP synthesis in the mitochondria were the most outstanding with respect to north/south transcriptional differences.
- Transcriptome sequencing and microarray development for the Manila clam, Ruditapes philippinarum: genomic tools for environmental monitoringPublication . Milan, Massimo; Coppe, Alessandro; Reinhardt, Richard; Cancela, Leonor; Leite, Ricardo; Saavedra, Carlos; Ciofi, Claudio; Chelazzi, Guido; Patarnello, T.; Bortoluzzi, Stefania; Bargelloni, LucaAbstract Background The Manila clam, Ruditapes philippinarum, is one of the major aquaculture species in the world and a potential sentinel organism for monitoring the status of marine ecosystems. However, genomic resources for R. philippinarum are still extremely limited. Global analysis of gene expression profiles is increasingly used to evaluate the biological effects of various environmental stressors on aquatic animals under either artificial conditions or in the wild. Here, we report on the development of a transcriptomic platform for global gene expression profiling in the Manila clam. Results A normalized cDNA library representing a mixture of adult tissues was sequenced using a ultra high-throughput sequencing technology (Roche 454). A database consisting of 32,606 unique transcripts was constructed, 9,747 (30%) of which could be annotated by similarity. An oligo-DNA microarray platform was designed and applied to profile gene expression of digestive gland and gills. Functional annotation of differentially expressed genes between different tissues was performed by enrichment analysis. Expression of Natural Antisense Transcripts (NAT) analysis was also performed and bi-directional transcription appears a common phenomenon in the R. philippinarum transcriptome. A preliminary study on clam samples collected in a highly polluted area of the Venice Lagoon demonstrated the applicability of genomic tools to environmental monitoring. Conclusions The transcriptomic platform developed for the Manila clam confirmed the high level of reproducibility of current microarray technology. Next-generation sequencing provided a good representation of the clam transcriptome. Despite the known limitations in transcript annotation and sequence coverage for non model species, sufficient information was obtained to identify a large set of genes potentially involved in cellular response to environmental stress.