Regulation of growth-related genes by nutrition in paralarvae of the common octopus (Octopus vulgaris).

The common octopus (Octopus vulgaris) is a species of great interest to the aquaculture industry. However, the high mortalities registered during different phases of the octopus lifecycle, particularly the paralarvae stage, present a challenge for commercial aquaculture. Improvement of diet formulation is seen as one way to reduce mortality and improve growth. Molecular growth-markers could help to improve rearing protocols and increase survival and growth performance; therefore, over a hundred orthologous genes related to protein balance and muscle growth in vertebrates were identified for the common octopus and their suitability as molecular markers for growth in octopus paralarvae explored. We successfully amplified 14 of those genes and studied their transcription in paralarvae either fed with artemia, artemia + zoea diets or submitted to a short fasting-refeeding procedure. Paralarvae fed with artemia + zoea had higher growth rates compared to those fed only with artemia, as well as a significant increase in octopus mtor (mtor-L) and hsp90 (hsp90-L) transcription, with both genes also up-regulated during refeeding. Our results suggest that at least mtor-L and hsp90-L are likely linked to somatic growth in octopus paralarvae. Conversely, ckip1-L, crk-L, src-L and srf-L had expression patterns that did not match to periods of growth as would be expected based on similar studies in vertebrates, indicating that further research is needed to understand their function during growth and in a muscle specific context.

Global impact of diet and temperature over aquaculture of Octopus vulgaris paralarvae from a transcriptomic approach.

Common octopus, Octopus vulgaris, is an economically important cephalopod species. However, its rearing under captivity is currently challenged by massive mortalities previous to their juvenile stage due to nutritional and environmental factors. Dissecting the genetic basis and regulatory mechanism behind this mortality requires genomic background knowledge. A transcriptomic sequencing of 10 dph octopus paralarvae from different experimental conditions was constructed via RNA-seq. A total of 613,767,530 raw reads were filtered and de novo assembled into 363,527 contigs of which 82,513 were annotated in UniProt carrying also their GO and KEGG information. Differential gene expression analysis was carried out on paralarvae reared under different diet regimes and temperatures, also including wild paralarvae. Genes related to lipid metabolism exhibited higher transcriptional levels in individuals whose diet includes crustacean zoeas, which had an impact over their development and immune response capability. High temperature induces acclimation processes at the time that increase metabolic demands and oxidative stress. Wild individuals show an expression profile unexpectedly similar to Artemia fed individuals. Proteomic results support the hypothesis revealed by transcriptional analysis. The comparative study of the O. vulgaris transcriptomic profiles allowed the identification of genes that deserve to be further studied as candidates for biomarkers of development and health. The results obtained here on the transcriptional variations of genes caused by diet and temperature will provide new perspectives in understanding the molecular mechanisms behind nutritional and temperature requirements of common octopus that will open new opportunities to deepen in paralarvae rearing requirements.

A pilot study to elucidate effects of artificial selection by size on the zebrafish (Danio rerio) fast skeletal muscle transcriptome.

Fish size is a complex trait determined by the interaction of environmental and genetic factors. While evidence exists that fish final length and size are partially determined by muscle fibre structure. However, the molecular basis behind final body size and muscle fibre composition remains yet to be fully understood. Here we use the fish model zebrafish (Danio rerio) to explore the molecular mechanisms involved in muscle fibre number, muscle growth and their role on animal size determination. In order to do so, we used two lines of zebrafish artificially selected by Amaral and Johnston 2012 for body length to obtain large (L) and small (S) genetic lineages. After selection animals from the L-lineage were significantly larger than individuals from the S-lineage (+6.6%, +6.7% and + 5.5% for standard, total and fork lengths) and had larger and higher number of muscle fibres (+15% and + 24% on average fibre area and fibre number respectively) while individuals from S-lineage showed a higher fibre density (+17%). RNAseq analysis on fast skeletal muscle revealed differences in gene expression, splice variants (SV) abundance and single nucleotide polymorphism (SNPs) retention between lineages, indicating the complexity of the molecular processes involved in the determination of the trait. We found that animals from the L-lineage showed an enrichment in genes related to the dystroglycan complex (GO:0016011) while genes related to the proteasome (GO:0022624), sarcomere (GO:0030017), muscle homeostasis (GO:0046716) and response to stress (GO:0006950) were enriched on the S-lineage. At individual gene level we also found differences in expression, SV or/and SNPs retention for some key genes involved in muscle development such as mef2ca, mef2cb, stac3, map14a and components of the circadian network (per1a, per1b, per3 and cry2b). These preliminary results provide an initial insight of the molecular changes induced by artificial selection for size, that could aid future studies related to determination of fish growth.

IGF-I binding and receptor signal transduction in primary cell culture of muscle cells of gilthead sea bream: changes throughout in vitro development.

We examined the possibility of culturing muscle cells of gilthead sea bream in vitro and assessed variations in insulin-like growth factor-I (IGF-I) binding during myocyte development. The viability of the cell culture was determined by fluorescence-activated cell-sorting analysis, which showed that the percentage of dead cells decreased with cell differentiation. The intracellular reduction of MTT into formazan pigment was preferentially carried out as cells differentiated (from day 4) indicating an increase in metabolic activity. IGF-I-binding assays demonstrated that the number of receptors increased from 190 +/- 0.09 fmol/mg protein in myocytes at day 5 to 360 +/- 0.09 fmol/mg protein in myotubes at day 12. The affinity of IGF-I receptors did not change significantly during cell development (from 0.89 +/- 0.09 to 0.98 +/- 0.09 nM). The activation of various kinase (ERK 1/2 MAPK and Akt/PKB) proteins by IGFs and insulin was studied by means of Western blot analysis. Levels of MAPK-P increased after IGF and insulin treatment during the first stages of cell culture, with a low response being observed at day 15, whereas IGFs displayed a stimulatory effect on Akt-P throughout the cell culture period, even on day 15. This study thus shows that (1) gilthead sea bream myocytes can be cultured, (2) they express functional IGF-I receptors that increase in number as they differentiate in vitro; (3) IGF signalling transduction through IGF-I receptors stimulates the MAPK and Akt pathways, depending on the development stage of the muscle cell culture.