Testing the expression of circadian clock genes in the tissues of Chinook salmon, Oncorhynchus tshawytscha

ABSTRACT

Animals have an endogenous circadian clock that temporally regulates 24 hour (h) oscillations in behavior and physiology. This highly conserved mechanism consists of two positive regulators, Bmal and Clock, and two negative regulators, Cry and Per, that run with a 24-h cycle that synchronizes itself with environmental changes in light, food, and temperature. We examined the circadian clock in Chinook salmon (Oncorhynchus tshawytscha), a non-model organism in which the function of the clock has not been studied. Recent studies indicate that clock genes in Chinook salmon play a role in its evolution of local adaptation, possibly by influencing migration timing. We designed real-time quantitative PCR (RT-qPCR) assays to quantify the transcription of components of the clock system, and validated these for PCR efficiency and specificity in detecting Chinook target genes. Chinook salmon tissue samples were collected in 3-h intervals, over the course of 24 h, from five different organs. Our data indicate that the circadian clock functions differently in each of these tissues. In the liver, positive and negative regulators exhibit anti-phasic peaking in the evening and morning, respectively. However, in the heart, these same regulators peak and trough with a different timing, indicating that the liver and heart are not synchronous. The digestive tract displays yet another difference: simultaneous phases in the expression of positive and negative clock regulators, and we do not observe significant rhythms in clock gene expression in the retina. Our data show that there is a functional clock in Chinook salmon tissues, but that this clock behaves in a tissue-specific manner, regardless of the whole animal being exposed to the same environmental cues. These results highlight the adaptive role of the clock in Chinook salmon and that it may have different positive and negative effects depending on tissue function.

KEYWORDS:

• Chinook salmon
• clock genes
• rhythms
• feeding entrainment
• peripheral clocks

Acknowledgments

We thank Dr. Trevor Pitcher for rearing and housing the Chinook salmon in these experiments at his Freshwater Restoration Ecology Centre. We thank Razagh Hashemi Shahraki and Dr. Subba Rao Chaganti for their technical aid and input for the NextGen sequencing. We also thank Nathaniel Bernardon, Sharon Lavigne, and Kyle Stokes from the Karpowicz lab for their help and support. This work was supported by a Discovery Grant (P.K. and D.H.), Engage Grant (P.K.), and Undergraduate Student Research Awards (M.T. and M.H.) from Natural Sciences and Engineering Research Council, and Canada Foundation for Innovation and Ontario Research Fund grants (Dr. Trevor Pitcher).

Additional information

Funding

This work was supported by the Natural Sciences and Engineering Research Council of Canada.