A tidal clock

The molecular basis of 24 hour circadian rhythms in terrestrial organisms is well understood and represents one of the major advances in the study of gene regulation of complex characters. However, the predominant rhythms in marine species that live on the coast in the intertidal zone is 12. 4 hours, reflecting the ebb and flow of the tides which are determined by the gravitational pull of the Moon and Sun on the Earth. For decades, scientists have speculated whether tidal rhythms are also related to circadian rhythms and whether they share some or all of the underlying molecular components of the 24 hour clock. We have been studying the specked sea louse, Eurydice pulchra, which shows both circadian rhythms in pigment dispersion and clear tidal rhythms in its swimming behaviour. We have identified all the main circadian clock genes in Eurydice and we can divide them up into their function. There are the genes that encode the positive regulators CLOCK and BMAL1, and these activate the genes for the negative regulators TIM, CRY2 and PER, which then feed back in a loop to deactivate the function of the positive regulators in a 24 hour cycle. This is called the negative feedback loop and explains how rhythms in gene transcription and translation of clock gene products can generate 24 hour molecular cycles. We have discovered that tidal rhythms share the positive factors but not the negative factors of the circadian clock. Furthermore we have identified putative circadian neurons and putative tidal cells in the Eurydice brain. These are major insights into how tidal clocks work.

We have assembled a draft genome for Eurydice and it contains several additional genes whose products act to regulate the positive factors and the negative factors. We would expect to find these in the corresponding tidal and circadian cells, so we shall localise the expression of these additional clock genes in the brain to see whether they are found in tidal or circadian cells, or both, or even other neurons, using a very sensitive technique called RNAscope. We shall also knock down the expression of these genes in Eurydice and examine whether they show changes in tidal or circadian behaviour thereby associating specific clock genes with specific types of rhythmic behaviour, tidal or circadian, or both. One of the positive factors that is important for tidal rhythms is BMAL1. This protein is known as the circadian transcription factor and with its partner CLOCK, binds to genes and activates them (see above). Consequently whatever DNA sequence BMAL1 binds, is potentially a control region for a circadian or tidal gene. We shall use a technique called ChIPseq to identify the DNA sequences and corresponding genes to which BMAL1 binds by referring to our Eurydice draft genome. Some of the genes under BMAL1 control may be switched on rhythmically with a tidal period and we shall compare these genes to those we already know are activated in 12 h cycles. Any that cross-match are candidates for being tidal output genes or the tidal regulators. We imagine that the crucial tidal regulators will also physically interact with BMAL1 in the same way that the circadian regulators like PER-TIM-CRY2 interact with the positive factors to generate circadian rhythms, so we shall compare the identity of proteins that interact with BMAL1 (using two techniques called co-IP and yeast-two hybrid) and again crossmatch any interactors with the genes we know bind BMAL1 or cycle with 12 h periods. In this way we hope to generate candidate genes for the elusive tidal regulators. When we have these candidate genes, we shall study where they are expressed in the brain and also knock down their expression levels to see whether they disrupt tidal behaviour. Our strategies will converge on the important genes that generate tidal rhythms and will perhaps provide a general model as to how these lunar-related rhythms are regulated in the animal kingdom.

We have compelling evidence that circatidal, 12.4 h rhythms share positive but not the negative molecular components with the 24 h circadian clock in the marine isopod, Eurydice pulchra and that there are dedicated tidal and circadian neurons in the brain. Specifically, we have demonstrated that BMAL1, the positive regulator of the circadian clock is involved in generating circatidal cycles. We shall
1. ... use a sensitive in situ method we have successfully trialled to identify the neurons in which the components of the intersected circadian feedback loops are expressed. We predict that the transcripts of the negative regulators (per, tim, cry2, cwo) will be expressed in circadian cells but the transcripts of the positive regulators, bmal1/clk, and Pdp1e/vri and Ror/Rev-erba, identified in our draft Eurydice genome, will be expressed in circadian and circatidal cells.
2. ... knock down these additional BMAL1 regulators and assess circadian and circatidal phenotypes
3. ...use Chipseq to identify BMAL1 target genes by referring to our draft genome. We will compare the target promoters to our circatidal 12 h cycling transcriptome, identifying promising circatidal candidates via bioinformatic analysis. Candidate circatidal regulators will be knocked down to assess phenotypes and in situ hybridisation will determine their cellular brain expression patterns.
4. ...perform co-immunoprecipitations using BMAL1 antibodies to pull down any interacting proteins and confirm their identity with Mass spectroscopy. We shall also use the yeast-two hybrid assay using BMAL1 as bait to identify interacting proteins from a Eurydice cDNA library. We predict that the circatidal negative regulator will physically interact with BMAL1-CLK
5. ... about 10% of transcripts show circatidal 12 h cycling and we shall annotate these and crossmatch any to the corresponding circatidal candidate genes identified in 3 and 4.

Our approaches should converge onto the tidal mechanism

The question we are trying to answer is a big one, 'how are tidal rhythms generated at the molecular level?' so the impact of our work will be felt beyond the immediate area of chronobiology, and include evolutionary biologist, neurobiologists, genome biologists and ecologists who are interested in shoreline biology. Tidal rhythms are the result of lunar/solar interactions with the earth, and lunar rhythms have been well documented in some forms of mental illness, so even clinicians might be interested in our work. Our findings may find themselves covered in biology textbooks, given the general interest in this area of biological timing, so the impact on future undergraduate and graduate education should not be underestimated.

From a commercial perspective there is a $15 billion industry in crustacean farming, particularly of shrimps and crabs, which provides a significant level of protein to the underdeveloped world in Asia and south America. Both species show lunar and tidal rhythms, and both have been devastated by various diseases. As the immune system cycles with the prevalent biological rhythm of the organism understanding how these lunar-related rhythms work could have beneficial effects on farming practises. The intertidal ragworm industry is worth $6 billion and supplies fish feed and fish-bait worldwide. Again understanding how the tidal clock works may have beneficial effects on the way the worms are farmed. We have already been consulting for Seabait which farms ragworms in the north of England.

The general public are fascinated by 'biorhythms' and clock biology is often in the national press or television/radio. Indeed our publications on Eurydice has been featured on television and on the BBC website. Within the Genetics Department in Leicester, we are extremely fortunate to have a national CETL (Centre of Excellence in Teaching and Learning) for GENIE (Genetics, Education, Networking, Innovation and Excellence), who main activity is outreach. Its website attract thousands of hits every month to its Virtual Genetics Education Centre for schools and colleges, higher education centres, the general public, as well as health professionals and policymakers. GENIE conducts about 35 meetings/workshops per year, and CPK, Lin Zhang and Matthew Blades have regularly contributed to GENIE functions on an ad hoc basis. We shall maintain our efforts in keeping the BIORHYTHMS section of the website updated with our tidal work and we shall use the many opportunities offered to us and organised by GENIE, to present our work to various groups, both local and national as we have been doing for years. We present our work to health professionals, schools, women's groups, rotary clubs, science clubs, open days, public lectures etc.

Postscript - Finally, to underscore impact, the 2017 Nobel Prize for Physiology or Medicine was just announced earlier this morning and it was won by Jeffrey Hall, Michael Rosbash (both at Brandeis University) and Michael Young (Rockefeller University). I had the good fortune to work with Hall and Rosbash first as a postdoc in Hall's laboratory from 1978 where I initiated the work with clock genes as a side project - until 1997 when I published my last circadian paper with Rosbash. All that work that I did with Hall/Rosbash (~20 papers worth) was funded by BBSRC (or SERC as it was called at the time). Consequently, the impact of SERC/BBSRC funding has been immense in this case and I think council should pat itself on the back for funding UK clock research, with its many groups, for the last three decades. The UK punches way above its weight in this field. Hopefully working out how the tidal clock works will have a smaller, but still significant impact in international science.