RNA profiling identifies novel, photoperiod-history dependent markers associated with enhanced saltwater performance in juvenile Atlantic salmon

Abstract

Atlantic salmon migrate to sea following completion of a developmental process known as smolting, which establishes a seawater (SW) tolerant phenotype. Smolting is stimulated by exposure to long photoperiod or continuous light (LL) following a period of exposure to short photoperiod (SP), and this leads to major changes in gill ion exchange and osmoregulatory function. Here, we performed an RNAseq experiment to discover novel genes involved in photoperiod-dependent remodeling of the gill. This revealed a novel cohort of genes whose expression rises dramatically in fish transferred to LL following SP exposure, but not in control fish maintained continuously on LL or on SP. A follow-up experiment revealed that the SP-history dependence of LL induction of gene expression varies considerably between genes. Some genes were inducible by LL exposure after only 2 weeks exposure to SP, while others required 8 weeks prior SP exposure for maximum responsiveness to LL. Since subsequent SW growth performance is also markedly improved following 8 weeks SP exposure, these photoperiodic history-dependent genes may be useful predictive markers for full smolt development.

Fig 1. RNA profiling of photoperiodic history-dependent changes in gill gene expression in juvenile Atlantic salmon.

A) Schematic presentation of experiment 1. Sampling time-points are indicated by black dots. B) Plasma osmolality (mOsm kg^-1) following a 24-h SW challenge; data are mean ± SEM of n = 6 fish per sampling point. ** significantly higher osmolality than in LL and SPLL fish at the same sampling point, p<0.01. Where error bars do not appear, errors lie within the symbol. C) Heatmap showing genes that are highly differentially expressed (FDR<0.01, logFC >|2|) between experimental groups over the three latter sampling points of exp.1. Hierarchical clustering has been used to generate six clusters. D) The averaged expression profile (z score) of the six clusters of DEGs, data are mean ± SEM, n = 6.

Fig 2. Temporal expression profiling of selected genes from cluster 3 in experiment 1.

Data are presented as normalized (TMM) counts and are mean± SEM of n = 6 fish, except for SPLL on day 68 where n = 5. *, ** significantly higher expression than LL and SP values at the corresponding time point, p<0.05, 0.01, respectively; # significantly lower expression than at day 1, p<0.05. Where error bars do not appear, errors lie within the symbol.

Fig 3. Effect of SP exposure duration on smolting performance parameters.

(A) Experimental design for experiment 2. (B) Plasma osmolality after 24-h SW challenge tests at the indicated sampling points. Data are mean ± SEM of n = 9–10 fish per sample point. **, significantly higher values than at day 1 and four and eight weeks after return to LL, p<0.01. (C) Gill Na⁺, K⁺- ATPase activity; data are mean ± SEM of n = 6–10 fish per sampling point. *, **, significantly higher activity than at day 1 of the experiment, p<0.05, 0.01, respectively. Where error bars do not appear, errors lie within the symbol. The dashed line represents the SP control group.

Fig 4. qPCR profiling of effect of SP exposure duration on selected cluster 3 genes.

Data are normalized mRNA abundance, mean ± SEM of n = 6 fish per sampling point. *, ** significantly higher expression than LL and SP values at the corresponding time point, p<0.05, 0.01, respectively; # significantly lower expression than at day 1, p<0.05. Where error bars do not appear, errors lie within the symbol. The dashed line represents the SP control group.