Abstract:
A Phase Response Curve (PRC) maps the phase shifts, either advances or delays, of a biological clock oscillator. The phase shifts are induced by perturbations at different phases of the clock. For circadian chronobiologists, PRCs have played a major role in improving their understanding in the mechanisms of circadian oscillators that produced the daily rhythms. On the other hand, the mechanisms that control tidal clock, responsible for 12.4 h rhythms, are still not well understood. In this thesis, I test the internal clock hypothesis for the tidal clock in the Cirolanid isopod species, Eurylana cookii and describe the effect of tidal stimuli on this clock by constructing a tidal phase response curve. The endogenous properties of clock that controls the overt tidal rhythms in E. cookii were tested in two experiments. The E. cookii displayed two tidal activity rhythms per lunar day that persisted for some time in constant conditions with average free-running period of 12.5 h. The initial peaks of the activity rhythm in the laboratory coincided with the local high tide times on the home beach. After five days in constant conditions, the period of the tidal rhythm often lengthened and sometimes became indistinguishable from the circadian rhythms. The tidal rhythms could be entrained by a tidal stimulus provided by physical water agitation and air bubbling. Three experiments were undertaken to obtain the duration of water agitation stimuli that could effectively entrain the activity rhythms. A two hour water agitation pulse could adequately entrain the rhythm and was used in all the phase shift experiments. An Aschoff Type I protocol was conducted with isopods kept in constant conditions for four days followed before a single 2 h stimulus and another four day’s free run. No clear pattern emerged in the phase shifts over the 12.4 h cycle. Data variation from this protocol was due to the lost of rhythm’s phase relationship with tidal cycles and the disappearance of tidal rhythms in constant conditions. It was also found that water agitation alone could not entrain the tidal rhythms. Method improvement was undertaken in order to produce a tidal PRC. Feeding was added alongside with water agitation as tidal zeitgeber. An Aschoff Type II protocol was performed where the isopods were entrained to five high tide cycles by the new tidal stimuli followed by a 2 h intervention at a desired tidal clock time. A 12:12 h light-dark (LD) cycle was added after it has been proven to improve activity rhythm during the day. The interaction between the circadian clock that controls pigment dispersal and tidal clock that controls activity rhythm had retained the two tidal components per lunar day in the laboratory. Addition of sediment mimicked the natural habitat of sandy beach. Regular tidal cycles at the beginning of each Aschoff Type II experiment produced more consistent rhythm onsets. Phase shift data from this new method have successfully produced a tidal PRC with an area of advances and delays over a 12.4 h time scale. From the tidal PRC, a perturbation pulse given within 2 h after the onset of activity (TT0 to TT1) and within the last 5 h of the tidal clock (TT8 to TT12.4) only caused small phase shifts to the clock. The phase shift for most of these tidal times (TT) did not exceed 0.5 h advances or delays. This might be the typical swimming and feeding time for the isopod which coincides with the high tide time. However, the clock shifted at larger magnitude, which were more than 0.5 h advances when perturbation occurred at TT2 until TT7. The greatest phase shift was achieved when perturbation occurred at TT5 with average phase advance of around 1.91 h. The tidal signal at these times is against its normal burrowing time where the isopods are supposed to be inactive. The maximum phase delay was about 0.52 h that occurred when perturbation was given at TT10. The range of entrainment for the stimuli fell in between 10.89 h to 13.32 h. Swimming and feeding occur vigorously during high tide due to the high dissolved oxygen level during this part of the tidal cycle. The wave action during high tide improves aeration of seawater. In past studies using other isopods and amphipods, swimming and feeding have been speculated to increase the oxygen uptake. Therefore, these two behaviours should happen during high tide. When burrowing, the isopod might have reduced its metabolism to conserve energy. When the tidal cues occur during its typical burrowing time, the tidal clock would signal these two behaviours to start earlier in the next cycle to take advantage on the high tide. Application of the tidal PRC developed in this study has been illustrated in the estimation of tidal cycles that are required to adjust the misalignment of tidal clock when E. cookii from Okahu Bay is transferred to a new intertidal area. It may take about six tidal cycles to get a normal phase relationship with the high tide times in the new environment that is 3 hours later than the home habitat. The tidal PRC developed from this study will provide a basis to test the hypothesis about the mechanism of tidal clock in the future. In conclusion, Aschoff Type II using simultaneous water agitation and feeding as perturbation pulse has yielded a tidal PRC from the overt tidal rhythm of E. cookii.