Enchytraeus albidus microarray: enrichment, design, annotation and database (EnchyBASE)

Abstract

Enchytraeus albidus (Oligochaeta) is an ecologically relevant species used as standard test organisms for risk assessment. Effects of stressors in this species are commonly determined at the population level using reproduction and survival as endpoints. The assessment of transcriptomic responses can be very useful e.g. to understand underlying mechanisms of toxicity with gene expression fingerprinting. In the present paper the following is being addressed: 1) development of suppressive subtractive hybridization (SSH) libraries enriched for differentially expressed genes after metal and pesticide exposures; 2) sequencing and characterization of all generated cDNA inserts; 3) development of a publicly available genomic database on E. albidus. A total of 2100 Expressed Sequence Tags (ESTs) were isolated, sequenced and assembled into 1124 clusters (947 singletons and 177 contigs). From these sequences, 41% matched known proteins in GenBank (BLASTX, e-value ≤ 10(-5)) and 37% had at least one Gene Ontology (GO) term assigned. In total, 5.5% of the sequences were assigned to a metabolic pathway, based on KEGG. With this new sequencing information, an Agilent custom oligonucleotide microarray was designed, representing a potential tool for transcriptomic studies. EnchyBASE (http://bioinformatics.ua.pt/enchybase/) was developed as a web freely available database containing genomic information on E. albidus and will be further extended in the near future for other enchytraeid species. The database so far includes all ESTs generated for E. albidus from three cDNA libraries. This information can be downloaded and applied in functional genomics and transcription studies.

Figure 1. Exposure experimental setup.

Schematic representation of the exposures performed and pools of RNA used for the SSH library development: A) Library enriched for genes differentially expressed after metal exposures; B) Library enriched for genes differentially expressed after pesticide exposures. CT = Control; EC50 = Concentration that induces 50% reduction in the number of juveniles (50% effect concentration on reproduction); EC90 = Concentration that induces 90% reduction in the number of juveniles (90% effect concentration on reproduction).

Figure 2. Expressed Sequence Tags distribution over the 177 contigs.

Figure 3. Cluster overlaps between the three different libraries.

The common clusters represent contigs assembled from ESTs with different library origins. Normalized: normalized library; SSH Metals: Library enriched for genes differentially expressed after metal exposures; SSH Pesticides: Library enriched for genes differentially expressed after pesticide exposures.