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

Abstract 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.This work was partially supported by a grant from European Union-funded Network of Excellence "Marine Genomics Europe". CS wishes to acknowledge additional funding from the Ministry of Education and Science (Spain) through grant AGL2007-60049. MM had a PhD scholarship from the University of Florence, Italy. RL was recipient of PhD fellowship SFRH/BD/30112/2006, from the Portuguese Science and Technology Foundation (FCT) and LC and RL acknowledge a grant from FCT project ISOPERK (PTDC/CVT/72083/2006).Peer Reviewe

Environmental effects of anticholinesterasic therapeutic drugs on a crustacean species, Daphnia magna

The presence of pharmaceutical drugs in the environment is an important field of toxicology, since such residues can cause deleterious effects on exposed biota. This study assessed the ecotoxicological acute and chronic effects of two anticholinesterasic drugs, neostigmine and pyridostigmine in Daphnia magna. Our study calculated 48 h-EC50 values for the immobilization assay of 167.7 μg L(-1) for neostigmine and 91.3 μg L(-1) for pyridostigmine. In terms of feeding behavior, we calculated a 5 h-EC50 for filtration rates of 7.1 and 0.2 μg L(-1) for neostigmine and pyridostigmine, respectively; for the ingestion rates, the calculated EC50 values were, respectively, 7.5 and 0.2 μg L(-1) for neostigmine and pyridostigmine. In the reproduction assay, the most affected parameter was the somatic growth rate (LOECs of 21.0 and 2.9 μg L(-1) for neostigmine and pyridostigmine, respectively), followed by the fecundity (LOECs of 41.9 and 11.4 μg L(-1) for neostigmine and pyridostigmine, respectively). We also determined a 48 h-IC50 for cholinesterase activity of 1.7 and 4.5 μg L(-1) for neostigmine and pyridostigmine, respectively. These results demonstrated that both compounds are potentially toxic for D. magna at concentrations in the order of the μg L(-1)