Achievements for each user project

Katerina Moutou

Access to CCMAR infrastructure was provided for 28 days and controlled experimental trials were conducted to establish the relationship between muscle growth in sea bream of different ages and feeding regime (starvation vs feeding) and the transcriptional profile of myosin light chain 2 (MLC2A) transcripts. MLC2 is a structural muscle protein that appears in two isoforms, MLC2A and MLC2B, with differential developmental and tissue expression. In addition, MLC2A is the product of three transcripts generated by alternative polyadenylation.
A successful fish trial was conducted that generated series of valuable samples for real-time RT-PCR, histology and in situ hybridization. In the duration of the study, all samples of white and red muscle collected were processed for histological analysis and the applicant gained valuable experience in the generation of probes and in situ hybridization as well as in immunohistochemistry for the differentiation of muscle fibers. RNA was extracted and transcribed to cDNA for real-time PCR analysis. As a result of the excellent organization of the host institute, the study was extended to test the effect of refeeding on MLC2 expression, simulating in this way the natural feeding conditions. Analysis of samples has been continued in the home institute and it is expected to provide a thorough insight to a molecular mechanism of transcript turnover and a multi-leveled growth mechanism with important ramifications for aquaculture.
Finally, there was an attempt to establish primary muscle cultures and construct the developmental profile of transcript appearance, which will be transferred to the home institute and provide the basis for detailed future studies.

Oleg Simakov

Faro - we assessed general polychaete diversity and took oceanographic measurements in Ria Formosa. We found only few Platynereis individuals in Ria, suggesting that strong currents and low salinity are prohibiting establishment of a stable population. However, outside of Ria (near Vilamoura), we identified Platynereis population and collected spawning individuals. RNA of these individuals will be sequenced and will give us linking information between Platynereis populations around the Northern European Atlantic coast (Oban, Kristineberg), and Mediterranean (Ischia). This data will help us investigate speciation/migrations rates and will shed light on adaptation to local environments (large variations in water temperatures, salinity, substrate, and benthic flora/fauna).

Marisol Izquierdo

To understand the effect of polyunsaturated fatty acids, and particularly of docosahexaenoic acid (DHA), on bone cells proliferation, differentiation and mineralization, VSa16 cells developed from seabream vertebra were cultured with different media containing graded concentrations of DHA and four different inhibitors of particular enzymatic pathways regulated by this fatty acid. Morphological studies live, conventionally and immune stained cells were combined with analysis of the culture medium, biochemical and gene expression studies. Cell culture studies lasted for four weeks which was the duration of the funding and allowed to optimize the experimental design and trial conditions as well as to obtain preliminary results. The results of this four weeks study have showed that DHA effect on bone cells is dose dependant and that only particular DHA metabolites and pathways seem to be involved. These results are the first evidences in teleost fish regarding the implication of some enzymatic pathways assayed on bone cells. Our results complement previous studies conducted on murine and human bone cell lines. Further experiments will be conducted during September in order to complete the information. The results will be published as soon as possible in scientific journals with a wide impact funding by ASSEMBLE to initiate this new cooperation will be acknowledged. The project has allowed to establish a new research co-operation between CCMAR, the University of Las Palmas de Gran Canaria and Ghent University.