Exploring the larval transcriptome of the common sole (Solea solea L.)

Abstract

Background: The common sole (Solea solea) is a promising candidate for European aquaculture; however, the limited knowledge of the physiological mechanisms underlying larval development in this species has hampered the establishment of successful flatfish aquaculture. Although the fact that genomic tools and resources are available for some flatfish species, common sole genomics remains a mostly unexplored field. Here, we report, for the first time, the sequencing and characterisation of the transcriptome of S. solea and its application for the study of molecular mechanisms underlying physiological and morphological changes during larval-to-juvenile transition.

Results: The S. solea transcriptome was generated from whole larvae and adult tissues using the Roche 454 platform. The assembly process produced a set of 22,223 Isotigs with an average size of 726 nt, 29 contigs and a total of 203,692 singletons. Of the assembled sequences, 75.2% were annotated with at least one known transcript/protein; these transcripts were then used to develop a custom oligo-DNA microarray. A total of 14,674 oligonucleotide probes (60 nt), representing 12,836 transcripts, were in situ synthesised onto the array using Agilent non-contact ink-jet technology. The microarray platform was used to investigate the gene expression profiles of sole larvae from hatching to the juvenile form. Genes involved in the ontogenesis of the visual system are up-regulated during the early stages of larval development, while muscle development and anaerobic energy pathways increase in expression over time. The gene expression profiles of key transcripts of the thyroid hormones (TH) cascade and the temporal regulation of the GH/IGF1 (growth hormone/insulin-like growth factor I) system suggest a pivotal role of these pathways in fish growth and initiation of metamorphosis. Pre-metamorphic larvae display a distinctive transcriptomic landscape compared to previous and later stages. Our findings highlighted the up-regulation of gene pathways involved in the development of the gastrointestinal system as well as biological processes related to folic acid and retinol metabolism. Additional evidence led to the formation of the hypothesis that molecular mechanisms of cell motility and ECM adhesion may play a role in tissue rearrangement during common sole metamorphosis.

Conclusions: Next-generation sequencing provided a good representation of the sole transcriptome, and the combination of different approaches led to the annotation of a high number of transcripts. The construction of a microarray platform for the characterisation of the larval sole transcriptome permitted the definition of the main processes involved in organogenesis and larval growth.

Figure 1

Global analyses of larval gene expression profiles. A. Principal Component Analysis (PCA) on the entire probe set. B. Sample clustering through AutoSOME. Ss: Solea solea, larval stages are indicated by number, biological replicates are distinguished by letters A, B, C and D.

Figure 2

Gene expression value over time of “Eye morphogenesis” and “Neurological system” pathways. Heatmaps representing gene expression values in each developmental stage of genes involved in A. eye and B. neurological system morphogenesis.

Figure 3

Temporal expression of “hatching” enzymes. Gene expression levels (log2), from 1 to 33 dph, of S. solea transcripts codifying hatching enzymes. A. S. solea hatching enzymes (HCE and LCE); B. S. solea homologs of astacin-like protein; C. S. solea N_isotig00480. Graphics show mean gene expression measured with microarray, bars indicate standard deviation (SD) across biological replicates. Statistical significance (p < 0.05) when comparing one larval stage against the previous one is indicated by asterisk (*).

Figure 4

Expression of TRH, TSH and TH receptors during larval development. Gene expression levels (log2), from 1 to 33 dph, of S. solea transcripts codifying key genes of the TH cascade. A.S. solea TRH, TSH and D1 transcripts; B.S. solea THRαA and THRαB transcripts. Graphics show mean gene expression measured with microarray, bars indicate standard deviation (SD) across biological replicates. Statistical significance (p < 0.05) when comparing one larval stage against the previous one is indicated by asterisk (*).

Figure 5

Expression of GH/IGFI axis genes during larval development. Gene expression levels (linear), from 1 to 33 dph, of S. solea transcripts codifying key genes of the GH/IGFI pathway. A.S. solea GH and GHRH transcripts; B.S. solea IGFI and IGFIR transcripts. Graphics show mean gene expression measured with microarray, bars indicate standard deviation (SD) across biological replicates. Statistical significance (p < 0.05) when comparing one larval stage against the previous one is indicated by asterisk (*).