Almost all organisms have an internal biological clock that synchronizes their behavior with the environment in which they live. Endogenous biological clocks follow the major cyclical rhythms: the solar-influenced 24-hour transition of day and night, the tidal 12.4 hour rising and falling of the tides that is governed by the lunar cycle, and the annual seasonal changes.
Organisms that live in shallow waters are presumably influenced by the lunar and tidal cycles to a greater extent than the solar ones and therefore mostly exhibit circatidal, rather than circadian rhythms. As its name implies, the circa (about) tidal rhythmicity is a ~12.4-hour rhythm following the tidal oscillation with its high and low tides that alternate approximately every six hours. Consequently, intertidal organisms are typically covered in water for part of the day and exposed the rest of the time. Having evolved in this type of environment has presumably been a very strong driving force for them to adapt an endogenous tidal clock.
Countless studies conducted over the past century have helped establish a comprehensive understanding of how the circadian clock works, and importantly, which genes are involved in its ticking. These studies have been predominantly conducted in the usual biological model organisms but have expanded in recent years into further 'real world' species, such as tropical corals, arboreal monkeys and many others from wide ranging habitats.
Life on earth began its journey at sea, presumably influenced to a great extent by the tidal cycle, which perhaps went on to evolve into a 24-hour cycle. So in order to understand how timing works, it is important to first understand how it works in the sea, particularly in its shallow waters.
In a first-of-its-kind, transcriptomic study conducted over four years, researchers at Bar-Ilan University set out to broadly examine the rhythmicity of Cellana rota, an intertidal limpet. The aim of this study was to use C. rota to understand the temporal landscape of a world manifested by two strong exogenous rhythms, circadian and tidal cycles, and their impact on the biological clock/s of the organism. To date, very few studies have been conducted in this realm and still little to nothing is known about the molecular basis of tidal rhythmicity. The results of their findings were published today in the journal Scientific Reports.
Throughout the course of the study, the researchers collected hundreds of thousands of images through a customized camera setup deployed on the sea shore of Eilat in southern Israel. The camera setup monitored a limpet population for several years, day and night. Few studies have conducted such a long-term, high-resolution sampling of animal behavior in such a challenging environment as the tidal zone. Portions of this data were quantified, showing that these limpets have a robust tidal rhythmicity, and curiously only exhibiting a circadian component to their behavior only at one particular time of the year.
The researchers then removed the limpets from their natural habitat of high and low tides and brought them into the lab, where they were held under constant conditions - i.e., no tidal or circadian cues, in order to establish that they indeed possess an internal clock and are not just behaving in tune with the rising and falling of the tides. The researchers correctly predicted that if the organism has an internal clock it would continue to behave in the tidal manner in the lab, and so they did. The limpets were then kept under these constant conditions for longer periods of time in order to desynchronize their rhythmicity. The researchers then set up an aquarium with a novel mechanism that sprayed them with water every 12.4 hours, mimicking the tidal cycle. This procedure once again entrained the limpets to a rhythm mimicking which they are exposed to in nature.
"We established that limpets have a tidal rhythm. Under laboratory conditions, they didn't take the day and night cycle into account at all," says Yisrael Schnytzer, of the Mina and Everard Goodman Faculty of Life Sciences at Bar-Ilan University, who conducted the research as part of his doctoral dissertation under the supervision of Prof. Yair Achituv and Prof. Oren Levy.
The researchers subsequently returned to Eilat and sampled limpets off a boulder every four hours over the course of 48 hours on two separate occasions. They wanted to obtain a high-resolution sampling that would aid in deciphering the pattern of the limpets' gene expression over the course of time.
The researchers worked in collaboration with Dr. Mali Salmon-Divon at Ariel University, as well as Dr. Hiba Waldman Ben Asher at Bar-Ilan University who assembled the transcriptome. They further collaborated with Prof. Michael Hughes And Dr. Jiajia Li, at the Washington University School of Medicine, who conducted the rhythmicity analysis of the transcriptome. "We found that far more genes are expressed in a tidal rather than a circadian rhythm, which is not surprising based upon what we saw at sea and in the lab," says Schnytzer. This is in contrast to previous studies which have suggested that even in the tidal zone the circadian cycle is the dominant one. More importantly, the researchers found that none of the core circadian clock genes exhibited a typical circadian rhythm. They were mostly arrhythmic, an observation which has been seen by others studying non model marine organisms, particularly those residing in the tidal zone. They did, however, find that some genes which are known to have a connection to the circadian clock, albeit not at its core, exhibited a tidal rhythmicity.
"This leads us to believe that the tidal and circadian clocks are either one and the same and that there is a certain plasticity in how these genes are expressed under different environmental conditions, or that at the very least some of the "putative" circadian clock genes are involved in both timing mechanisms, yet the core of the tidal clock still evades us....," says Schnytzer.
To date these researchers are the first to conduct such a comprehensive study combining long-term observations in nature, as well as the lab supported by a transcriptomic investigation. They have provided further clues, including a checklist of genes possibly connected to the tidal "clock", that bring us a step closer to understanding how this evasive mechanism works which, as stated above, perhaps predates our own clock.