Circadian clocks in two intertidal species, the American Horseshoe crab and the mummichog

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Reilly, Brendan Michael
Although the molecular mechanism of animal circadian clocks has been outlined in a few model species, much less is known in other animals. In fruit flies and mice, the molecular clock is composed of a transcription translation feedback loop that is composed of a set of core and accessory proteins. However, since even these two clocks differ in a number of fundamental ways, an analysis of non-model species may help to uncover both common and novel circadian clock mechanisms. The American Horseshoe crab (Limulus polyphemus) and the mummichog (Fundulus heteroclitus) are intertidal organisms that both exhibit circadian rhythms. In addition, because they live in the intertidal zone, these animals may also have another clock that serves to synchronize them to the twice per day tidal changes. The two goals of this thesis were to identify putative circadian clock proteins in the mummichog using publicly available data and to quantify potential changes in abundance of putative circadian proteins in the horseshoe crab. Bioinformatical analysis of the mummichog proteome demonstrated the presence of putative proteins of all five of the core vertebrate circadian proteins as well as 17 accessory proteins. This is the first study to identify core, accessory, input and output clock proteins in a single fish and this work will help to establish ground work for future circadian work in fish. In addition, this work allows for comparative bioinformatics work on intertidal species and may help to provide insights on the molecular mechanisms of the clock system that controls circatidal rhythms. In Horseshoe crabs, three putative circadian proteins (CLOCK, CYCLE1, and photoreceptive CRYPTOCHROME) were detected in the central nervous system using western blotting and authentic antibodies. Interestingly, although these proteins have been shown to cycle by time of day and circadian time in other species, there was a lack of cycling in the abundance of these proteins. While this may suggest that post-translational modifications of core proteins by accessory proteins may drive their circadian clock, other core proteins may instead be responsible for driving circadian rhythms in horseshoe crabs. Overall, the results presented in this thesis will provide the basis for further investigations into the circadian systems of these species.