Establishment of killer whale (Orcinus orca) primary fibroblast cell cultures and their transcriptomic responses to pollutant exposure.

Populations of killer whale (Orcinus orca) contain some of the most polluted animals on Earth. Yet, the knowledge on effects of chemical pollutants is limited in this species. Cell cultures and in vitro exposure experiments are pertinent tools to study effects of pollutants in free-ranging marine mammals. To investigate transcriptional responses to pollutants in killer whale cells, we collected skin biopsies of killer whales from the Northern Norwegian fjords and successfully established primary fibroblast cell cultures from the dermis of 4 out of 5 of them. Cells from the individual with the highest cell yield were exposed to three different concentrations of a mixture of persistent organic pollutants (POPs) that reflects the composition of the 10 most abundant POPs found in Norwegian killer whales (p,p'-DDE, trans-nonachlor, PCB52, 99, 101, 118, 138, 153, 180, 187). Transcriptional responses of 13 selected target genes were studied using digital droplet PCR, and whole transcriptome responses were investigated utilizing RNA sequencing. Among the target genes analysed, CYP1A1 was significantly downregulated in the cells exposed to medium (11.6 µM) and high (116 µM) concentrations of the pollutant mixture, while seven genes involved in endocrine functions showed a non-significant tendency to be upregulated at the highest exposure concentration. Bioinformatic analyses of RNA-seq data indicated that 13 and 43 genes were differentially expressed in the cells exposed to low and high concentrations of the mixture, respectively, in comparison to solvent control. Subsequent pathway and functional analyses of the differentially expressed genes indicated that the enriched pathways were mainly related to lipid metabolism, myogenesis and glucocorticoid receptor regulation. The current study results support previous correlative studies and provide cause-effect relationships, which is highly relevant for chemical and environmental management.

Transcriptomic changes in relation to early-life events in the gilthead sea bream (Sparus aurata).

BACKGROUND: Teleosts are exposed to a broad range of external stimuli, which may be either of acute or chronic nature. The larval phase of certain fish species offer a unique opportunity to study the interactions between genes and environmental factors during early life. The present study investigates the effects of early-life events, applied at different time points of early ontogeny (first feeding, flexion and development of all fins; Phase 1) as well as on the subsequent juvenile stage after the application of an additional acute stressor (Phase 2) in the gilthead sea bream (Sparus aurata), a commercially important European aquaculture species. Animal performance, the cortisol response and gene expression patterns during early development as well as on the subsequent phases (juveniles) after the application of additional acute stressors were investigated. RESULTS: Significant differences on fish performance were found only for juveniles exposed to early-life events at the phase of the formation of all fins. On the transcriptome level distinct expression patterns were obtained for larvae as well as for juveniles with the most divergent expression pattern found to be again at the phase of the development of all fins, which showed to have also an impact later on in the acute stress response of juveniles. CONCLUSIONS: The present study showed that applying an early-life protocol, characterized by the unpredictable, variable and moderate intensity of the applied stimuli provides a relative realistic model to evaluate the impact of daily aquaculture practices on fish performance. In addition, the power of investigating global gene expression patterns is shown, providing significant insights regarding the response of early-life events during development and as juveniles after the application of extra acute stressors.