Transcriptome sequencing and microarray development for the Manila clam, Ruditapes philippinarum: genomic tools for environmental monitoring

Abstract

BackgroundThe 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.ResultsA 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.ConclusionsThe 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.

Key metabolic pathways involved in xenobiotic biotransformation and stress responses revealed by transcriptomics of the mangrove oyster Crassostrea brasiliana

“…Furthermore, while a recent study has shown a clear association between increased expression of SULTs enzymes and PAHs in the channel catfish Ictalurus punctatus (Gaworecki et al, 2004), contradictory results in SULT mRNA levels have also been detected in mice treated with microsomal enzyme inducers (Alnouti and Klaasen, 2008). Thus, the SULTs inducibility is yet to be confirmed for several species, including fish and molluscs (Janer et al, 2005;Milan et al, 2011;Roméo and Wirgin, 2011). However, the presence of putative SULTs, a key component of phase II metabolism of endogenous and exogenous compounds, in C. brasiliana suggests a role in the detoxification or endocrine metabolism of oysters, as previously suggested for mussels (Janer et al, 2005;Lavado et al, 2006).…”

“…Most of the identified GSTs were assigned to the sigma (6 sequences), followed by the omega class (4 sequences), members of which are known to play a role in the xenobiotic detoxification and in the protection against oxidative stress (Board et al 2000;Fonseca et al, 2010;Milan et al, 2011). Indeed, omega GST has been reported to be useful as a biomarker for hydrocarbon and domestic sewage exposure in oysters (Boutet et al, 2004;Lüchmann et al, 2014;.…”

“…9.9% in the razor clam Sinonovacula constricta (Niu et al, 2013), 12% in the blue mussel Mytilus galloprovincialis (Craft et al, 2010), 17% in the Antarctic bivalve Laternula elliptica (Clark et al, 2010;Meyer et al, 2009), 24% in the Manila clam Ruditapes philippinarum (Milan et al, 2011), 25% in the krill Euphausia superba (Clark et al, 2011), 28% in the Yesso scallop Pactinopecten yessoensis (Hou et al, 2011) and 39% in the sea cucumber Apostichopus japonicus (Du et al, 2012). This is almost certainly due to the genome sequencing and annotation of a close relative; the Pacific oyster, Crassostrea gigas (Zhang et al, 2012a).…”

Deep sequencing of the mantle transcriptome of the great scallop Pecten maximus

“…Putative annotation based on sequence similarity searching could be assigned to approximately 23.5% of the contigs (Table 2). These data generated from the mantle of P. maximus form a valuable addition to those generated from hemocytes of the same species (Pauletto et al, 2014) and, in a more general context, to the transcriptomes generated for other molluscs including the Yesso scallop Patinopecten yessoensis (Hou et al, 2011), Mytilus galloprovincialis (Craft et al, 2010), Laternula elliptica (Clark et al, 2010), Meretrix meretrix (Huan et al, 2012), Ruditapes philippinarum (Milan et al, 2011), Haliotis midae (Franchini et al, 2011), several pearl oysters (Huang et al, 2013) and the oyster genome data , thus increasing the sequence resource available for commercially important shellfish species and for researchers investigating shell deposition processes in molluscs.…”

Gene expression profile analysis of Manila clam (Ruditapes philippinarum) hemocytes after a Vibrio alginolyticus challenge using an immune-enriched oligo-microarray

“…Before 2011, less than 6,000 nucleotide sequences were available for this species. Our two groups released 457,717 and 975,190 raw reads from adult/larval tissues and hemocytes, respectively [20,21]. Concurrently, studies by Ghiselli et al on gonad tissue yielded approximately 90 million raw sequences [22].…”