Introduction
Circadian rhythms or ‘clocks’ are the visual expression of endogenous oscillatory expression of genes and proteins lasting approximately 24-hours , which synchronises biochemical, physiological, and behavioural responses enabling organisms to respond to diel environmental changes
. They can be directly linked to altered metabolic, physiological and behavioural traits and influence most biological processes
. The circadian mechanism is highly conserved across organisms however, our understanding of core clock mechanisms and circadian control of fish physiology remains limited
. There is an unusually large complement of clock genes in salmonids as a direct result of the two rounds of whole genome duplication (WGD) events (Ts3R, teleost specific and Ss4R salmonid specific) resulting in an abundance of circadian related genes
. As such deciphering the circadian clock mechanism in salmonids is complex and the functional divergence remains largely unknown
.
The aim of this study were to describe the effects of the salmonid specific WGD on clock gene diversity in Salmo salar . Through phylogenetic analysis, core clock gene ohnologs were classified and renamed . This was confirmed by transcriptomic analyses during early freshwater development of salmon reared under controlled lighting regimes including photoperiodic manipulations and narrow bandwidth lights. New findings provide a tool for further circadian clock research in salmon.
Materials and Methods
The genomes of five species of salmonids (S. salar, Salvelinus alpinus, Oncorhynchus mykiss, Oncorhynchus kisutch, and Oncorhynchus tshawytscha ) were interrogated using Danio rerio clock genes [clock, arntl, period, cryptochrome, nr1d, ror and csnk1e/d ] and Esox lucius [ a sister lineage which did not undergo the Ss4R WGD ] to further explore the effect of the salmonid specific WGD Ss4R on the clock genes in salmonids. Th e transcriptomic isoforms with the highest percentage identity to the reference were selected and aligned. Maximum Likelihood trees were inferred to identify the relationship between identified clock genes and those of the late st common ancestors. Identified putative clock genes were re- classified based on the zebrafish nomenclature and were renamed from their predicted names after zebrafish orthologues.
Two separate cohorts of S. salar at different life stages [ early development (UiB) and pre-smolt juveniles (UoS)] were kept under controlled lighting regimes and sampled over 24-hours at 4 hrs intervals. A range of lighting conditions were tested during egg incubation including photoperiod (14L:10D-LD , continuous darkness DD, and continuous light-LL), intensity (0.01 W/m2, 0.1 W/m2 and 1.0 W/m2 ), and spectra (blue, green, red, and white) and at the juvenile/parr stage ( 12:12 LD vs. LL). RNA was extracted from dissected embryos or tissues (brain and liver) and sent away for RNA sequencing. RNA sequences were aligned to the published genome and gene expression patterns were statistically analysed.
Results
Most clock genes identified in salmonids are duplicated as a direct result of the salmonid specific WGD event Ss4R. However, there are some individual gene families in which some or all salmonids display gene retention or reciprocal gene loss when compared to E. lucius and D. rerio. RNA sequencing identified that all S. salar putative core clock genes identified and classified in silico were expressed at both life stages sampled . Expression varied throughout early development from eye pigmentation through to first feeding (255dd – 690dd) . Despite noise caused by individual variance two clock genes displayed significantly rhythmic expression patterns over a 24-hour sampling period pre-first feeding (690dd). Exposure to DD appeared to significantly down regulate s ome clock genes, whereas exposure to LL appeared to significantly up regulate clock gene expression of several clock genes when compared to those on the LD treatment. Spectral composition appeared to significantly influence the regulation of several clock genes. With red and green light treatments appearing to significantly down regulate a few of the identified genes when compared to white light during the midpoint of the light and dark periods. In late fresh water developmental stages (UoS) 262 genes were significantly rhythmically expressed in S. salar parr amongst which 13 of the putative core clock genes were identified . Further results will be presented on the effect of the two photoperiods in parr , the analysis and comparatives are currently being undertaken.
Discussion/ Conclusion
Clock genes are expressed from early development in S. salar . Whilst there appears to be variance between individual fish relating to the time at which clock genes peak and trough, it appears that as the salmon develop there is an increase in the number of putative core clock genes which are significantly rhythmically expressed. This is indicating the maturation of the ‘ biological clock’ or circadian mechanism in S. salar throughout freshwater developmental stages. There was a greater influence of the LL photoperiod on the significant regulation of clock genes than DD when compared to the LD group, indicating that the presence of light signals had a greater influence over clock gene regulation than the absence of lighting cues during early development. Spectral composition to a lesser extent than photoperiod also influenced the regulation of clock gene expression. However, the effect of intensity at early developmental stages was negligible. The role of feeding from first feeding in entraining the circadian system can be hypothesised. Through the identification and classification of clock gene orthologues in salmonids this study has provided a tool for better characterising the circadian mechanism in S. salar by providing a comprehensive characterisation of clock genes.
References
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