Aquaculture Europe 2014

October 14-17, 2014

Donostia–San Sebastián, Spain

STRESS DURING EMBRYOGENESIS OF ATLANTIC SALMON (SALMO SALAR L.) - DOES IT MATTER?

H. Johnsen* and H. Tveiten
 
Nofima, Muninbakken 9-13, 9291 Tromsø, Norway
E-mail: hanne.johnsen@nofima.no

Introduction
 
In fish, development of endocrine organs and factors involved in regulation of the HPI-axis (hypothalamus-pituitary-interrenal axis) take place during embryogenesis and post hatch stages. Genes involved in regulation of the HPI-axis include paralogs of crf (corticotropin releasing factor), pomc (pro-opiomelanocortin), mc2r (melanocortin 2 receptor), gr (glucocorticoid receptor) and possibly the mr (mineralocorticoid receptor). In some species, environmental stress during embryogenesis and early post natal life can substantially change the epigenome structure, resulting in altered DNA methylation patterns and suppression or enhancement of gene expression. Such epigenetic changes can induce lifelong alterations in the phenotype of the stress exposed organism. The epigenome, or the environmental influence on the epigenome structure, have been little studied in important aquaculture species such as Atlantic salmon. This study aims to clarify how stress during early life may alter expression of genes related to HPI-axis development and if such changes may alter stress coping abilities later in life.
 
Materials and methods
 
Batches of milt and unfertilized salmon eggs were obtained from a commercial supplier (Aqua Gen, Trondheim, Norway). Fertilization was performed according to standard procedures using milt in excess at the Aquaculture research station (Tromsø, Norway). Egg/embryo incubation was performed in upwelling incubators at a temperature of 7 °C. Eggs were divided into four groups: 1) unstressed control, 2) eggs stressed during embryogenesis, 3) fry stressed during post-hatch stages, 4) embryos and post-hatch fry stressed during both stages. Each treatment group was incubated in triplicate trays containing c. 1800 eggs in each replicate. The different treatment groups were exposed to bouts of stress (cold-shock and air exposure - drop from 7 °C to 0.2 °C for 1 min, followed by air exposure (15 °C) for 1 min and then transfer back to 7 °C) either during embryogenesis (5 times from 250 to 450 day degrees (DG) or during yolk-sac stages (4 times, from 540 to 880 DG), or both. To investigate the influence of stress treatment during embryogenesis and post hatch stages on developmental timing and HPI-axis functionality of the stress response, a stress test was performed just prior to start feeding (880DG), when the HPI-axis is assumed to become functional (i.e. release of cortisol in response to stress). Stress was applied as indicated above, and fry was sampled for molecular and endocrine analysis at 0, 1, 3 and 24 hours post-stress. Quantitative PCR (qPCR) was conducted using sequence specific primers for crf1, crf2, pomcA1, pomcA2, pomcB, mc2r, mr, gr1, gr2. To evaluate possible epigenetic effects the expression of dnmt1 (DNA cytosine-5- methyltransferase 1) involved in maintenance of established methylation patterns and dnmt3a and 3b important in establishing new methylation patterns were analyzed. To further investigate epigenetic effects, pyrosequencing was conducted on HPI-axis related gene promoters using the Pyromark Q24 Advanced system (Qiagen).   
 
Results
 
Stress treatment did not result in embryo or fry mortality in any of the treated groups. Also, stress treatment did not have any major effect on weight development from hatching until week 29 post hatching. Under resting conditions, just prior to start feeding (880 DG), expression of most HPI-axis related genes were reduced in the stress treated groups compared to that of the control (Fig. 1). This was particularly evident for mc2r where all treated groups had a significant reduction in relative expression. This implies that, at least in group 1, the effect of stress during embryogenesis on gene expression was maintained for at least 2 months after the last stress exposure.
 
 
Previous stress treatment did influence HPI-axis related gene expression when fry were exposed to acute stress just prior to start feeding. For example, corticotrophin releasing factor (crf1) gene expression was clearly responsive to stress and was also influenced by previous stress exposure, resulting in a more rapid down regulation in group 2 and 3 (Fig. 2). The gene was significantly down regulated by 3 and 24 hours post-stress, irrespective of previous treatment.
 
Discussion and conclusion
In summary, it appears that stress exposure during early life stages of Atlantic salmon may have the potential to induce long term effects on gene expression which is central to the functionality of HPI-axis. Further, embryonic stress may also alter responses in gene expression and endocrine changes to acute stress during later stages of development. If such altered stress coping abilities also manifest into juvenile and adult stages, it may hold great potential for the production of a more robust salmon in the future.
 
Project number: 265957 - FP7-KBBE-2010-4
Program: COPEWELL "A new integrative framework for the study of fish welfare based on the concepts of allostasis, appraisal and coping styles".