Category Archives: Clock Zoo

Circadian clock without DNA–History and the power of metaphor

ResearchBlogging.org Last week, two intriguing and excellent articles appeared in the journal Nature, demonstrating that the transcription and translation of genes, or even the presence of DNA in the cell, are not necessary for the daily (“circadian”) rhythms to occur (O’Neill & Reddy 2011, O’Neill et al., 2011). (Scientific American is part of Nature Publishing Group.)

The two papers received quite a lot of media coverage, and deservedly so, but very few science bloggers attempted to write in-depth blog posts about them, placing them in a broader historical, theoretical and methodological context. I had a feeling that everyone was waiting for me to do so. Which is why you are now reading this. I know it is a long “Explainer” (which is all the rage in science journalism these days) but I hope you have patience for it and that you find it informative and rewarding.

What I intend to do is to, first, briefly describe and explain the research in these two papers, though the press release and media coverage were quite accurate this time. Diana Gitig did the best job of it at Ars Technica – I highly recommend you read her piece for clear background information.

Then I will try to give you a historical perspective so you can get a feel for the context in which this research was performed. This look at the history will bring into sharp relief how powerful the scientific metaphors are in guiding the questions that researchers try to answer in their laboratories. Finally, a look at the media coverage will show that the lay audience (including journalists) is guided by other metaphors – not always the same ones that are used by researchers.

What did they do?

In each of the two papers, the researchers chose an unusual laboratory model for their study. What is common to both models is that a) they are both Eukaryotic cells and b) there is no DNA transcription or RNA translation going on inside the cells.

In the first study (O’Neill & Reddy 2011), they used human red blood cells (photo left) as these cells have no nucleus, thus no DNA at all.

In the second study (O’Neill et al. 2011), the model of choice was a small protist, Ostreococcus tauri which has an interesting property – when kept in constant darkness, there is no DNA transcription or RNA translation that can be detected.

The starting point of both studies was detection of peroxiredoxins in the cytoplasm. Peroxiredoxins are enzymes (thus chemically proteins) that protect the cells from damage from strongly oxidizing molecules (often refered to as “free radicals”). The process of neutralizing such oxidants temporarily changes the chemical structure of the peroxiredoxin, which then reverts to its native state again – thus the molecule is constantly switching between the two states. This oscillation between the two states follows a daily (~24h) cycle synchronized to the day-night cycle of the environment.

In both studies, peroxiredoxins were detected using antibodies (“immunoblotting”). One of the chemical states of the molecule can be detected with this method, while the other state is indirectly detected by the comparative lack of signal. Thus a circadian rhythm would be seen as an alternating series of rises and falls of the detected signal with a period close to 24 hours.

And this is exactly what they discovered in both cases – there was a clear circadian rhythm of peroxiredoxins state-switching both in cultured red blood cells (above right) and in the cultured Ostreococcus tauri (below).

Furthermore, in the protist study, they used measurement of light emitted by luciferase added to the sea-water solution as a marker of DNA transcription and translation. While the cells were kept in constant darkness no light emitted due to presence of luciferase could be detected.

But at the onset of environmental light, luciferase-induced light measurements indicated that the transcription started at the phase predicted from the state of the clock before the cells were placed in the dark. This means that the circadian rhythm of DNA transcription did not start at some “Phase Zero,” triggered by switching on the light, but that it was driven by a clock that was operating all along while the organism was kept in the dark – a clock that does not require DNA transcription and translation.

Detecting a 24-hour rhythm is not sufficient to ensure that the rhythm is actually circadian. For a biological cycle to be considered circadian, it has to satisfy a number of criteria, e.g., it has to be endogeonous (generated inside the cell, not forced onto it by the environment), it has to persist for several cycles, it has to be unaffected by temperature levels (i.e., the period of the rhythm should be the same regardless of the level of environmental temperature kept constant in the laboratory), it has to be entrainable (synchronizable) by imposed cycles in the environment, etc.

In both model systems, the researchers performed (either in these or prior studies) the entire battery of standard experimental protocols to demonstrate that yes, these are indeed circadian rhythms in both laboratory models.

Use of temperature cycles instead of light-dark cycles to demosntrate entrainment in the first experiment makes sense as human red blood cells do not experience (and cannot detect) light, while they are normally exposed to daily fluctuations of body temperature. The difference between the dawn minimum and evening maximum temperature inside the human body can be as large as 1 degree Celsius, more than sufficient for entrainment – some entire cold-blooded animals like lizards, insulated from the environment by skin and scales, can entrain their rhythms to temperature cycles with the high-low difference as small as 2 degrees Celsius and in some individuals as small as 0.1 degrees (Underwood and Calaban 1987).

In the red blood cell paper, the researchers went further. They also detected circadian rhythms in a few other biochemical processes. For example, hemoglobin, the molecule that transports oxygen from the lungs to the cells, and carbon dioxide from the cells to the lungs, can exist in two different forms inside red blood cells. It is a complex protein molecule, built of four almost-identical units. In this form, hemoglobin can perform its normal function. But there is also a two-unit form which cannot perform the function in gas exchange. Furthermore, the two-unit form produces the oxidizing small molecules – exactly the kinds of molecules that peroxiredoxins have evolved to scavenge and neutralize. It is not surprising that the switching between two-unit and four-unit forms of hemoglobin was also seen to be circadian – and in sync with the peroxiredoxin rhythm.

Likewise in the protist paper (see O.tauri in the photo at right), the researchers performed a whole suite of additional experiments designed to eliminate a variety of potential alternative hypotheses. For example, pharmacological suppression of DNA transcription and translation did not eliminate the circadian rhythm in peroxiredoxin chemistry, but instead demonstrated a complex interplay between the clock driven by transcription of genes and the clock driven by spontaneous biochemical reactions in the cytoplasm.

In summary, the two papers are very solid, the experiments are well designed and performed, the results are persuasive, and the names of authors give me confidence that the data can be trusted.

What does this all mean?

The results of both papers demonstrated that transcription of DNA and translation of RNA is not necessary for the generation of circadian rhythms in two different types of eukaryotic cells belonging to evolutionarily very distant relatives – protists and mammals.

In the case of red blood cells, the result is clear – there is no DNA or RNA in these cells. Thus, the circadian rhythms in these cells have to be generated in the cytoplasm.

In the case of O.tauri, the picture is a little bit more complex: the cells have a nucleus which has DNA. There is a clock driven by transcription and translation of canonical “clock genes.” Yet, when this mechanism is supressed – either by constant darkness or by chemicals – the cells still exhibit circadian rhythms generated by the molecules residing in the cytoplasm (and some of those molecules, at least during the first day or two, may be strands of RNA transcribed earlier).

Furthermore, the phase at which the DNA-centered clock starts its cycle is determined by the phase of the cytoplasmic clock, not the other way round, i.e., the cytoplasmic clock is dominant over the nuclear clock.

Why is this so exciting?

Depends who you ask!

When the articles were first published I did not yet have time to read them carefully. But I have e-mail notifications set up so every time Google detects a news article or blog post containing the word “circadian” I get a message. Thus I read a number of media articles about these studies before I read the studies themselves.

The media accounts (see some examples) tended to emphasize two reasons why these studies are important.

The first one was a surprise that both humans and protists have the same molecules doing the same thing. Their surprise was a surprise to me! Circadian clocks are found everywhere. Peroxiredoxins are found in almost all living organisms on Earth. Just like the structure of cell membrane, the processes of DNA transcription and RNA translation, the genetic code, or the use of ATP (adenosine triphosphate) as the energy currency of the cell, peroxiredoxins are ubiqutous molecules in almost all of life on this planet.

Those are life’s universals, something that is expected as we have understood the unity of life even before Darwin. These universals are, to a biologist, usually deemed pretty useless and boring – the background. What gets a biologist excited is variation – the exceptions to the universals. If most organisms use a particular molecule for a particular function but one organism does not – now that is exciting! Why is this? How and why did this organism evolve this switch in function? What was the initial mutation, what was the selective pressure? Those are useful questions in biology that help us understand evolution.

My hunch is that the journalists, either by being lay audience themselves or by targeting their articles to the lay audience as they understand it, focus on the universals – the “Unity Of Life” – as something that in their minds is evidence for evolution and a counter-argument against non-scientific ideas about life (e.g. intelligent design creationism). While biologists find surprise and delight in exceptions, which are useful entries into detailed studies of evolutionary mechanisms, many in the lay audience are still surprised by the plain fact of the unity of life as it evolved from a single common ancestor.

The second reason given in MSM (mainstream media) articles as to why these studies are important is their novelty. For example, Chemical & Engineering News states that this “…involves a previously unknown cycle of posttranslational modifications, in addition to the transcription of a well-known handful of clock genes…”

There is a sense, reading all the coverage, that this is so novel, creative and revolutionary, it must have been the very first time anyone has ever thought of this! And even better – this is a great “conflict” story, in which a single study puts into question an entire field! A small group of young geniuses proved the entire old establishment wrong!

Not so fast!

Both of these studies have been done before. Several times.

I quickly went to the PDFs of the two papers and yes, the references to the old studies are there. The authors are aware of the history of the field, the giants on whose shoulders they climbed in order to see further. Of course, these are Nature papers with severe constraints on space and on the number of references. With so many experiments and so much to explain about their methods and results, they could not spend enough time on the history of the idea and on the work of their predecessors. And they could not cite all of the preceeding studies. But they chose the key ones and noted them briefly in the text – not prominently, but they are there.

So why did the MSM articles not pick up on this? First, by stressing the novelty – and the “conflict” story of geniuses proving the establishment wrong – they make the new studies seem more newsworthy, thus more likely to be approved by the editors to get into print.

Second, the MSM articles are limited by space and there is only so much one can put in 500 words. Thus, as usual, it is the context that gets left out and the novelty-factor that remains in the piece.

Finally, even if a dilligent journalist wanted to follow up on the background he or she would bump into the dreaded paywalls.

Nobody expects a journalist to know as much about the field as its practitioners do. I knew exactly what to look for and even I needed a few days to collect all the papers and read them – which is why you are reading this post now instead of last week.

As a member of the Society for Research on Biological Rhythms I have free access to the Journal of Biological Rhythms, the premier journal in the field. Once logged in, I knew exactly which five articles to look for. Those five articles (all cited below) contained the references to all the other papers I was interested in. As none of those are Open Access, I had to go to Twitter and, by using the hashtag #ICanHazPDF ask my followers to find me and send me PDFs of all of these papers. I got most of them (though some are not available or not even digitized yet as many of them are quite old). Then I had to spend some time reading them.

This all takes time, and I had the advantage of knowing where to start…perhaps even the advantage of being aware, to begin with, that such studies exist. Even Allison Brager who is in the field of chronobiology, did not note on her blog Dormivigilia the existence of prior research and exclaimed with excitement that “….the clinical and scientific relevance of this work are HUGE!!!!….” It is not always stressed hard enough to graduate students how important it is to read the historical literature of one’s field.

So, let’s quickly go through some of the aspects of the history of circadian research that are most relevant to the understanding of the context in which these two papers appeared.

Brief history of clock research

While the observations of daily rhythms in plants and animals go back to the antiquity, the first experiment in the field was performed in 1729 by Jean-Jacques d’Ortous de Mairan who observed the rise and fall of leaves of a Mimosa plant kept in constant darkness. Much early research was done in the 19th century, mostly on plants, but also a little bit on insects and humans.

In the early 20th century a number of people started studying rhythmic phenomena in living organisms. They came from very different scientific disciplines, e.g., botany (Bunning), ecology (DeCoursey), animal behavior (Kramer, Beling, Sauer), protozoology (Hastings), evolutionary biology (Pittendrigh), mammalian physiology (Richter), human biology and medicine (Aschoff, Halberg), and agriculture (Garner and Allard). It took them a few decades to discover each other’s work and to recognize that they are dealing with the same phenomenon regardless of the organism they were studying, be it fiddler crabs, starlings, tobacco or humans.

The founding moment of the field was the 1960 meeting at Cold Spring Harbor. The book of Proceedings from the Meeting (Symposia on Quantitative Biology, Vol.XXV) is a founding document of the field: I own three copies, strategically placed in three different spots in the house so at least one copy has a chance to be saved in case of fire. And you can bet I have read it over and over again during my 10 years in grad school (and after).

In 1960, structure of DNA was very new, and comparatively little was known about the inner workings of a cell. But most researchers were eager to break into the ‘black box’ and start investigating how the circadian clock ticks inside of the cells. A number of conceptual models were proposed, some focusing on cell membranes, others on DNA. Thus, experiments were started to separate the two parts of the cell and to test the role of nucleus and DNA in the clock mechanism.

Roenneberg and Merrow (2005) provide an excellent timeline of the research since then, especially the emergence of molecular and genetic techniques and subsequent findings. But briefly, over the next two decades or so, circadian mutants were discovered in a protist (Chlamidomonas), insect (Drosophila) and mammal (Golden hamster). The first canonical clock gene – the Drosophila period gene – was sequenced shrtly after.

Around 1995, clock genetics explodes. New clock genes were discovered left and right in several different organisms, from cyanobacteria to humans. There was a sudden influx of people into the field from other areas of genetics, and they required a few years to catch up on the field’s history and theory before they stopped making beginners’s mistakes in their experimental designs (though see Dunlap 2008 for different perspective on that history). But there were many of them, they had plenty of funding, they were excited and creative, had powerful new techniques, and they worked fast, so every week, or so it seemed at the time, there was a new discovery of genes involved in circadian rhytms.

Here is a timeline of the key events in this aspect of the history of the field:

What emerged from all of this activity is the transcription/translation model (Hardin, Hall & Rosbash 1990) for the circadian mechanism: a suite of canonical clock genes get transcribed and translated, and their protein products, after some delay, inhibit the transcription and translation of those same genes. Day in and day out. Those genes and their products then also regulate expression of all the other genes that the cell uses in its daily function.

But not all were happy with it.

If you read Dunlap 2008 you will certainly detect a tension between researchers who studied whole organisms (always with evolution in the backs of their minds) and treated the clock as a ‘black box’ for decades before the genetic explosion, and the geneticists who came into the field in the mid-1990s. The former regarded the latter as arrogant and simplistic in their complete focus on DNA. The latter regarded the former as out-dated holists who don’t understand the magnificient power of DNA and treated them like Lysenko by those who do not understand Lysenko.

You need to remember that this was in the middle of the Human Genome Project hype, when there was a huge overselling of crude genocentric and gene-deterministic ideas, many of which were fully embraced by the geneticists at the time (they have learned better since then).

I do not want to exaggerate the tension – it was mostly muted. Geneticists were mainly welcome into the field. After all, they were bringing in their tools and skills to do what the field was hoping to do all along – crack open the “black box” and peer inside. The two groups treated each other with respect, and soon started collaborating. The old guard of chronobiology was impressed by the speed and capability of the genetics labs, the rate at which new techniques were developed and improved, and the rapidity of discoveries. They learned (or sent their students to learn) the techniques, and started thinking how to employ them to study circadian phenomena that prior research has already shown occured at higher levels of organization.

The hope was that the genetics and molecular approaches will quickly discover all the core clock genes and the way they interract with each other so the focus can shift back to explaining the things that really matter – properties of ensembles of clock cells and the behaviors of whole organisms (after all, DNA is invisible to selection – phenotype of the whole organism is what ecology and evolution can see to act upon).

The discovery of clock genes required the use of a limited number of laboratory models that are amenable to genetic dissection: mouse as the model for all vertebrates, fruitfly representing all invertebrates, Neurospora crassa standing in for all fungi, Arabidopsis being “the plant” and Synechococcus being used as the only bacteirum known (at the time) to possess a circadian clock. In the early years, a few protists were also used – Paramecium, Euglena, Gonyalax, Acetabularia and Chlamydomonas, but they were later largely abandoned, while new models, like Xenopus and zebrafish entered the arena.

This was a highly unusual state for the field – chronobiology was always extremely comparative, with thousands of species of organisms being studied over the years. The hope was that, once the genes are discovered in model organisms, the findings can be applied to other creatures for a more comprehensive and comparative research program.

Likewise, the focus on genes was also seen as temporary, something to be “waited out” until the findings can be applied to other levels of organization in a more integrative approach. Thus, entire lines of research were reduced or have essentially stopped – tidal, lunar and circannual rhythms, Sun-compass orientation, photoperiodism, development, ecology and evolution – waiting for new techniques and new findings that will enable them to re-start.

And that is exactly what happened – a decade or so later, the field concluded that all the core clock genes were discovered and that the transcription/translation feedback loop model is good enough. The study of previously semi-abandoned topics (and organisms) started with new zeal and gusto.

Persisting problems

At the time of the Cold Spring Harbor symposium in 1960, there were two main lines of thinking about the cellular mechanism of the circadian clock. One focused on the nucleus and the DNA (Ehret and Trucco 1966). The other focused on the cell membrane (Njus et al. 1974).

How does one go about figuring out which one of the two models is right, using techniques available at the time?

One approach is to use cells that do not normally possess a nucleus or any DNA – like mammalian red blood cells – to see if they have circadian rhythms. If yes – nuclues is not important, membrane (or cytoplasm) is. Studies were difficult and results not always clear, but most could detect rhythms in red blood cells (Cornelius and Rensing 1976, Mabood et al. 1978, Ohm-Schradera et al. 1980, Peleg et al. 1990a,b)

Second approach is to use very large cells that can survive long enough once the nucleus is removed – in comes the protist Acetabularia (Sweeney and Haxo 1961, Schweiger et al. 1964, Vanden Driesche 1966,Terborgh and McLeod 1967, Vanden Driesche and Bonotto 1969, Sweeney 1974, Mergenhagen and Schweiger 1975a,b, Hartwig et al. 1985, Woolum 1991, Runft, Linda and Mandoli 1996). These studies showed that clock operates after the nucleus is removed, and, once the nucleus is reintroduced, it is the clock in the cytoplasm that determines the phase, entraining the nuclear clock.

The third approach is to pharmacologically block DNA transcription and RNA translation. This was, over the years, performed in a number of organisms, including Acetabularia (photo on the right) and, much more recently, the sea-slug Bulla gouldiana (Page 2000). Again, rhythms persisted in the absence of DNA transcription.

Fourth approach is to find single-cell organisms that reproduce or divide more often than once a day and see if the circadian phase is preserved during the process – there is no DNA transcription during cell division. This was initially done in the protist Paramecium (Barnett 1966), but later it was cyanobacteria that were used in this approach (Mori et al. 1996, Kondo et al. 1997). Circadian phase is preserved during reproduction in Paramecium and cell-division in bacteria.

Fifth approach is to find organisms that have circadian rhythms but do not have clock genes. Yeast (Saccharomyces cerevisiae) is one such organism. In the nematode Caenorhabditis elegans, which shows circadian rhythms, the genes usually used for circadian timing are instead used for developmental timing (so-called heterochronic genes).

Sixth approach is to study the rhythms in either the cell membranes (for example in the protist Gonyalax polyedra, Adamich et al. 1976, or fruiftly, Nitabach et al. 2005) or elements of the cytoplasm directly, in a dish (using bacterial clock proteins, Tomita et al. 2004, Mehra et al. 2006, Mori et al, 2007). Again, the isolated cell membrane cycles, and blocking the membrane processes also blocks overt rhythms in whole organisms. Bacterial clock proteins (not DNA) kaiA, kaiB and kaiC, when placed in a test tube, spontaneously oscillate in a circadian fashion.

Finally, one can genetically affect the clock: mutating, deleting, shutting down or overexpressing (forcing expression at high levels at all times with no cycling) canonical clock genes and see if any residual rhytmicity remains. This was done in the fruitfly (Helfrich-Förster 2000), where morning peak of activity is eliminated when the clock gene cycling is stopped, but the evening bout of activity in male flies persists nonetheless. Sometimes a genetics paper would triumphantly state that a deletion of a gene rendered half of the flies arrhythmic, just to be met with a question “so, how do the other half of the flies still cycle without it?”

This research program started with enthusiasm immediatelly after the symposium, yielding troves of interesting data over the years. But, once the geneticists entered the fray, these results were forgotten or ignored. They did not conform to the DNA-based model. The easiest way to make a circadian geneticist in the mid-1990s angry at a conference was to utter the word “Acetabularia” – this was “noise” to be ignored and swept under the rug.

You can see the timeline of the history of this “shadow research program” here:

Why did this research persist despite the victorious run of the transcription/translation model?

The earliest studies in this area were a direct outgrowth of the ideas discussed at Cold Spring Harbor. They all yielded the data suggesting that DNA is not the only part of the clock mechanism. Yet, once genetics work took off, these results were ignored. At least some of the people in the field were worried that genetic work is ignoring something potentially important.

Who in the field was worried about this depended on their own background and experience? First, people who worked on organisms that yielded unusual experimental data throughout the history of circadian research, including the fungi and the protists (especially Gonyaulax polyedra, recently renamed Lingulodinium polyedrum but you are unlikely to find many circadian papers using the new name, and the systematics may still be in flux) were one such group.

People working on non-mammalian vertebrates (fish, amphibians, reptiles and birds) were cognisant of the complexity of circadian organization – same clock genes, expressed in different tissues, resulted in clocks of different properties. The clock in the pineal organ, the clock in the retina, the clock in the SCN (suprachiasmatic nucleus of the hypothalamus), the peripheral clocks in all the other tissues – each of those behaved differently despite using the exact same molecular machinery. So the properties must have been modified by something else in the cell, or by the interactions between cells in the tissue.

On top of that, many phenomena, e.g., photoperiodism or sleep, are not properties of individual cells but of interactions between ensembles of cells in the tissues, or even interactions between the organism and its environment. The simplistic “this gene is for clocks” model just could not explain the complexity of observed reality.

Once all the clock genes were deemed discovered, the critiques started popping up (Roenneberg and Merrow 1998, 1999, 2005, Lakin-Thomas 2000, 2006), trying to move the circadian research up the levels of organization to the interplay between cells, tissues, organs and organisms. Most of these calls for the return to the organism were reviews of all the studies showing that DNA is not enough – somewhat like this article is. The two Nature articles last week are just the latest research results in this tradition.

The power of metaphor

Where does this fundamental misunderstanding between molecular and organismal biologists come from? They are both biologists, right? So they should be expected to operate from the same basic principles.

But they don’t. Geneticists come from a tradition starting with Schroedinger’s 1944 book What is Life? This is a linear, hierarchical view of life, with upward causation: genes cause and control everything else. Also, gene is the only level on which natural selection acts (Dawkins 1976). The reigning metaphor of this worldview is the “program.”

On the other hand, biologists coming from the study of evolution, ecology and animal behavior have a “systems” view of life in which many interacting elements, none of them with a primacy, determine the behavior of the entire system. There is no single element in control. The phenomena are a result of interactions, not of dominance of any particular actor.

The causation is downward (natural selection). DNA is just one of the elements in the system. Selection acts simultanously at several levels, including whole organisms and groups (Brandon 1996, Gannett 1976, Godfrey-Smith 1999, Griffiths and Gray 1994, Hubbard and Wald 1993, Keller 1995, Lewontin 1992, Nijhout 1990, Nelkin and Lindee 2004, Kitcher, P. 1999, Rose et al. 1990, are just a tip of the iceberg of the literature analyzing and criticizing the hierarchical DNA-first worldview). The reigning metaphor of this worldview is “the tangled bank”.

Circadian field is not the only area of biology in which these two worldviews clashed. But it is worth noting here that the studies of clock genes ignored everything else, while the studies that questioned DNA supremacy never just shifted the control to some other element – all of those studies say that DNA is not sufficient, not that it is replaced by another controller.

Let’s look at the “program” as a metaphor. A program is a term from information theory. It is a deterministic algorithm leading to a particular result. But what is reading that program? What is the “computer” that runs it? The cell?

And where is the person using the computer, the one who decides to run the program and decides if the program is useful or not? Where is natural selection?

Look at all the terminology of molecular biology: transcription, translation…those are all terms from information theory, which is linear, deterministic and hierarchical – there is a cause that controls the effect.

Even the “News and Views” article accompanying last week’s two papers (Bass and Takahashi 2011) re-frames the results of the papers into information theory metaphor. All the stuff that is happening in the cytoplasm is referred to as “post-translational” as if it was just something more that DNA “caused,” perhaps a little further downstream than usual.

But it is not. The cycles in the cytoplasm are not caused by anything any piece of DNA did. When in sync, the genetic feedback loops and cytoplasmic clocks work synergistically. But when placed in opposition, the cytoplasmic clock dominates (e.g., determines the phase, period, etc.).

The centrality of the gene in much of biological thinking led to another error that these two papers in Nature just fixed. Because different kingdoms of life (bacteria, protista, plants, fungi and animals) have different clock genes, it was assumed, despite the identical mechanistic logic of the mechanism, that the clock evolved independently several times. Identity of players trumped the mechanism of interaction between them.

But if, as the papers show, all organisms have cytoplasmic clocks based on anti-oxidant enzymes, then this cytoplasmic clock is the scaffolding, the base which allows evolution and replacement of all sorts of clock genes in different groups. As clock genes come and go, they can always latch onto the ever-present cytoplasmic clock. And the organism can keep on ticking regardless of the evolving stage in which any particular clock gene may be. This argues for a single origin of the circadian clock, due to universally adaptive nature of the clock, as postulated by Colin Pittendrigh decades ago.

The clock metaphor

The theory of biological rhythms has benefited immensely from the use of the clock as metaphor. Thinking of biological rhythms in terms of oscillatory theory (borrowed from physics) has allowed us to understand how the biological clock works, how it gets synchronized with the environment (entrainment), and how systems with multiple clocks can act together to produce higher-order phenomena (e.g., photoperiodism – measurement of seasonal changes in daylength).

The clock metaphor was also a key for understanding the mechanism as a collection of interacting cogs and wheels. This was crucial for the discovery of clock genes later on.

But once the number of clock genes was determined to be very small, and the interlocking feedback loops between them became the dominant paradigm for the mechanism, the meaning of the clock metaphor shifted – instead of looking at all the potential cogs and wheels, only those made of or from DNA counted. There is nothing wrong with counting everything – genes, and cytoplasmic elements, and the cell membrane, and the interactions between clock cells in a tissue – as cogs and wheels of the biological clock, but somehow, somewhere, we forgot that and settled for a DNA-only view.

Every metaphor that scientists invent has a heuristic value. The information theoretical thinking about genes sped up the research in genetics and molecular biology. The clock metaphor sped up the circadian research.

But it is always a good idea to sometimes step back and consider if the dominant metaphor is constraining in some ways, if it limits the imagination. I have argued before that an occasional switch to a different circadian metaphor – perhaps player-piano, or endless tape recorder, or Rube-Goldberg Machine, or camshaft, or Moebius strip – can be a good way to look at the problem from a new angle. This can be a very productive endeavor, opening one’s eyes to new angles, starting new avenues of research. Every field of science has its metaphors, and it is always a good idea to sometimes analyze them, and sometimes replace them once they outlive their usefulness.

What metaphors are used by lay audience and the media?

There is a difference between metaphors used by scientists to guide their research programs, and metaphors used by journalists to explain research to lay audiences.

The clock metaphor, for example, means ‘interlocking cogs and wheels to study’ for researchers, but ‘timepiece in your brain that tells you when to wake up and when to fall asleep’ for the audience.

Likewise, the gene-control metaphor is something that is easy to understand for the audience that may be used to a hierachical worldview of top-down control (in society, family, religion, politics, or simplistic mechanics of everyday life). A systems-worldview requires a little bit more tolerance for ambiguity (which not everyone has) and a little more sophisticated understanding as to how complex systems work (i.e., how complex behaviors emerge out of interactions between multiple elements, in which the nature of interactions is more important than the identity and behavior of individual elements).

This is probably why the media reports could not capture the complexity of the findings. It provided an or option instead of an and option – the lay reader is probably going to think that DNA has nothing to do with the clock, instead of understanding that both DNA and other elements of the cell are partners. Still, in the media saturated with “gene for X” stories, an occasional “not in our genes” story is a positive event.

On the other hand, since the early 2000s (once the hype over Human Genome Project died down a little bit), the geneticists have moved away from the gene-control metaphor to some extent. Yes, they still sometimes slip up to old habits of mind (and their terminology shows it) – like when they use the term “post-translational” for everything that does not involve DNA in the cell – but the results of their own studies, from quantitative genetics to bumping into walls in some areas of research, have moved them to a more systems-like thinking. They are reinventing Physiology and calling it Systems Biology. And we are all better off for it. It is a more complete Physiology, with the ‘black box’ now wide open.

Another way that gene-primacy seeps into coverage of science is when new studies using molecular techniques are said to have confirmed the old studies using more traditional methods. For a recent example, see how the hypothesis of butterfly migration and speciation by Nabokov was said to have been confirmed by a recent molecular study. But molecular techniques are new, still being tested, calibrated and evaluated. The Nabokov story is really about well done work from the past using tried and tested old reliable techniques, that was strengthened by the new study and in turn validated the molecular method. Comparative anatomy is what validated the genetic method, not the other way round.

Likewise, in this example in clocks, it is very nice that new techniques repeated the old results. Each strengthens the other. The new study does not confirm the old as much as they all confirm each other. But for those enamored with molecules (or those who always think that new is better than old), this duo of papers will seal the deal if the old papers did not.

Conclusion

To summarize, the publication of these two studies in Nature last week is, in my opinion, quite a milestone in the field. First, it showed how it was possible for the clock to originate only once on Earth yet evolve a number of different molecular elements – the cytoplasmic clock was there all along, keeping time while the genes swapped.

Second, it re-framed the discussion of the mechanism. It forcefully demonstrated what many prior studies did in small increments, but this time with modern techniques we love and with enormous power. By reminding the people in the field that DNA is an important but not sufficient element of the clock, it will hopefully guide future research in a new direction, with a more complete view of the clock, and perhaps may even allow some people to venture out and try other productive metaphors instead.

References:

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Hastings, Michael H., Elizabeth S. Maywood and John S. O’Neill (2008) Cellular Circadian Pacemaking and the Role of Cytosolic Rhythms, Current Biology 18, R805–R815, DOI 10.1016/j.cub.2008.07.021

Helfrich-Förster, Charlotte (2000) Differential Control of Morning and Evening Components in the Activity Rhythm of Drosophila melanogaster—Sex-Specific Differences Suggest a Different Quality of Activity, J Biol Rhythms, vol. 15 no. 2 135-154, doi: 10.1177/074873040001500208

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Kondo, T., Mori, T., Lebedeva, N.V., Aoki, S., Ishiura, M., and Golden, S.S. (1997). Circadian rhythms in rapidly dividing cyanobacteria. Science 275, 224–227.

Lakin-Thomas, Patricia L., (2006) Transcriptional Feedback Oscillators: Maybe, Maybe Not…, J Biol Rhythms, vol. 21, 2: pp. 83-92.

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Image credits: Red blood cells, Wikimedia Commons; Ostreococcus tauri, The Joint Genome Institute; Acetabularia crenulata, The College of Exploration; graphs – from O’Neill JS, & Reddy AB (2011) and O’Neill JS. et al. 2011; and Transcription-translation Feedback Loop model, Nature Reviews Neuroscience

Me and the copperheads–or why we still don’t know if snakes secrete melatonin at night

It seems this is a week of venom here at the Guest Blog! First it was Rachel Nuwer on Monday who looked at the U.S. death statistics at the hands (Okay, fangs and stingers) of venomous animals. Then yesterday David Manly explained how snakes bite and how their venom evolved (and still evolves). So, I thought I should complete a trifecta, and tell you a brief story about my one and only close encounter with venomous snakes (also recounted in a very old post of mine).

For background, I did my graduate studies in the laboratory of Dr. Herbert Underwood studying circadian biology – how animals’ brains measure the time of day and time of year. Dr. Underwood is one of the pioneers in the field and has, over the years, done research on several species of lizards and birds. By the time I joined the lab, it has somewhat shifted in focus from a comparative breadth to a more focused depth of research on circadian physiology in Japanese quail.

While most of our work was done in quail, we were also allowed to make brief forays into studies of other species, including some colaborative work on turkeys, a small study on crayfish by me, and an interesting study on three species of geckos by my lab-mate Chris Steele. Chris came to our lab from a herpetology background, so I was not surprised when he suggested we team up with another colleague of ours, Jim Green who studied the evolution of snake venom, for a small study on snakes’ circadian rhythm in melatonin synthesis and secretion.

What is important to know is that melatonin is a hormone secreted by all vertebrates (and many invertebrates and even plants) only at night. The main place where melatonin is produced in vertebrates is the pineal gland. Apart from producing melatonin, the pineal of non-mammalian vertebrates (e.g., lampreys, fish, amphibians, reptiles and birds) is also a photosensitive organ – it detects the intensity of environmental light. Light is quite capable of penetrating through the skin and skull of animals and the pineal is located on the very top of the brain, often under a spot where the skull bone is thinner and more transparent.

Detection of light directly by the pineal entrains (synchronizes) the circadian clock to the light-dark changes of the environment. In many non-mammalian vertebrates, pineal is also the place where the circadian clock is located, thus it makes sense that it is directly sensitive to light. Rhythmic secretion of melatonin then synchronizes other body functions to the day-night cycles in the environment.

In mammals, there are no photoreceptors in the pineal. There is also no circadian clock in the pineal. It serves only as a melatonin source in mammals….and in snakes – at least according to anatomy. Snakes are the only non-mammalian vertebrates that have pineal glands that, under the microscope, look like they came from mammals.

What we wanted to do is to see if snakes have melatonin at all, and if so, if it shows a daily rhythm in concentration like it does in other Vertebrates (believe it or not, nobody’s done that yet). We wanted to see if snakes, like all other vertebrates, secrete melatonin only at night. And, as this was Jim Green’s research animal, the copperheads were the only snakes we had at our disposal (once the project was greenlighted by the Animal Care committee), about ten of them, each in its own terrarium in a tiny shed outside of campus.

We needed to take blood samples at noon (hypothesizing that we would not be able to detect any melatonin in the middle of the day) and, after a few days of recovery, again at midnight (testing the notion that the snake’s pineal secretes melatonin at night). So, we went in at noon one day. Jim would pick up a snake and hold it by its head. Chris was holding the body of the snake. Jim’s advisor Hal Heatwole was taking the blood samples straight from the heart, and I was the “nurse assistant” taking care of needles, syringes, anticoagulant, test-tubes, etc. The whole experiment, sampling blood from 10 snakes, took perhaps an hour or so and worked out perfectly without any glitches.

About a week later, when we came for a repeat session at midnight, we were starkly reminded that copperheads are nocturnal animals. They were active. And I mean ACTIVE! To avoid the acute, direct effect of light on stopping all melatonin synthesis and release, we had to take samples in the dark, aided only by a very dim red pen-light, with some highly uncooperative snakes. The process took hours!

At one point one of the snakes, a large male, got loose in the room and, since the room was essentially dark I could not see where it was underneath the cages. So I said, “OK, you snake guys figure out where it is and call me back once you have it under control,” and I slid out of the door. I got teased for this act of cowardice for years afterwards.

Unfortunately, the melatonin essay did not work and we did not have enough blood volume to try with a new kit, so the study was never completed. The snakes got used in other experiments, Jim finished and defended his thesis and left town and nobody else wanted to try to do a repeat of this experiment. I hope one day someone will. Perhaps with a non-venomous snake species for a change – makes midnight sampling much safer and easier!

Image from (now sadly defunct) Backyard Jungle.

References:

Kunkel, B. W., The paraphysis and pineal region of the garter snake, The Anatomical Record, Volume 9, Issue 8, pages 607–636, August 1915

Ralph, C. L., Evolution of Pineal Control of Endocrine Function in Lower Vertebrates. Amer. Zool. (1983) 23 (3): 597-605. doi: 10.1093/icb/23.3.597

Kalsow C. M., Greenhouse S. S., Gern W., Adamus G., Hargrave P. A., Lang L. S., Donoso L. A. Photoreceptor cell specific proteins of snake pineal. J Pineal Res. 1991 Sep;11(2):49-56.

Evolutionary Medicine: Does reindeer have a circadian stop-watch instead of a clock?

ResearchBlogging.orgWhenever I read a paper from Karl-Arne Stokkan’s lab, and I have read every one of them, no matter how dense the scientese language I always start imagining them running around the cold, dark Arctic, wielding enormous butterfly nets, looking for and catching reindeer (or ptarmigans, whichever animal the paper is about) to do their research.

Reindeer_bw.GIFlepidopterist.gif

If I was not so averse to cold, I’d think this would be the best career in science ever!
It is no surprise that their latest paper – A Circadian Clock Is Not Required in an Arctic Mammal (press release) – was widely covered by the media, both traditional and blogs, See, for example, The Scientist, BBC, Scientific American podcast and Wired Science.
Relevant, or just cool?
It is hard to find a science story that is more obviously in the “that’s cool” category, as opposed to the “that’s relevant” category. For the background on this debate, please read Ed Yong, David Dobbs, DeLene Beeland, Colin Schultz, and the series of Colin’s interviews with Carl Zimmer, Nicola Jones, David Dobbs, Jay Ingram, Ferris Jabr, Ed Yong and Ed Yong again.
I agree, it is a cool story. It is an attention-grabbing, nifty story about charismatic megafauna living in a strange wilderness. I first saw the work from the lab in a poster session at a conference many years ago, and of all the posters I saw that day, it is the reindeer one that I still remember after all these years.
Yet, the coolness of the story should not hide the fact that this research is also very relevant – both to the understanding of evolution and to human medicine. Let me try to explain what they did and why that is much more important than what a quick glance at the headlines may suggest. I did it only part-way a few years ago when I blogged about one of their earlier papers. But let me start with that earlier paper as background, for context.
Rhythms of Behavior
In their 2005 Nature paper (which was really just a tiny subset of a much longer, detailed paper they published elsewhere a couple of years later), Stokkan and colleagues used radiotelemetry to continuously monitor activity of reindeer – when they sleep and when they roam around foraging.
You should remember that up in the Arctic the summer is essentially one single day that lasts several months, while the winter is a continuous night that lasts several months. During these long periods of constant illumination, reindeer did not show rhythms in activity – they moved around and rested in bouts and bursts, at almost unpredictable times of “day”. Their circadian rhythms of behavior were gone.
But, during brief periods of spring and fall, during which there are 24-hour light-dark cycles of day and night, the reindeer (on the northern end of the mainland Norway, but not the population living even further north on Svaldbard which remained arrhythmic throughout), showed daily rhythms of activity, suggesting that this species may possess a circadian clock.
Rhythms of Physiology
In a couple of studies, including the latest one, the lab also looked into a physiological rhythm – that of melatonin synthesis and secretion by the pineal gland. Just as in activity rhythms, melatonin concentrations in the blood showed a daily (24-hour) rhythm only during the brief periods of spring and fall. Furthermore, in the latest paper, they kept three reindeer indoors for a couple of days, in light-tight stalls, and exposed them to 2.5-hour-long periods of darkness during the normal light phase of the day. Each such ‘dark pulse’ resulted in a sharp rise of blood melatonin, followed by just as abrupt elimination of melatonin as soon as the lights went back on.
reindeer melatonin.jpg
Rhythms of gene expression
Finally, in this latest paper, they also looked at the expression of two of the core clock genes in fibroblasts kept in vitro (in a dish). Fibroblasts are connective tissue cells found all around the body, probably taken out of reindeer by biopsy. In other mammals, e.g., in rodents, clock genes continue to cycle with a circadian period for a very long time in a dish. Yet, the reindeer fibroblasts, after a couple of very weak oscillations that were roughly in the circadian range, decayed into complete arrhytmicity – the cells were healthy, but their clocks were not ticking any more.
reindeer fibroblasts.jpg
What do these results suggest?
There is something fishy about the reindeer clock. It is not working the same way it does in other mammals studied to date. For example, seals and humans living in the Arctic have normal circadian rhythms of melatonin. Some other animals show daily rhythms in behavior. But in reindeer, rhythms in behavior and melatonin can be seen only if the environment is rhythmic as well. In constant light conditions, it appears that the clock is not working. But, is it? How do we know?
During the long winter night and the long summer day, the behavior of reindeer is not completely random. It is in bouts which show some regularity – these are ultradian rhythms with the period much shorter than 24 hours. If the clock is not working in reindeer, i.e., if there is no clock in this species, then the ultradian rhythms would persist during spring and fall as well. Yet we see circadian rhythms during these seasons – there is an underlying clock there which can be entrained to a 24-hour light-dark cycle.
This argues for the notion that the deer’s circadian clock, unless forced into synchrony by a 24 external cycle, undergoes something called frequency demultiplication. The idea is that the underlying cellular clock runs with a 24-hour period but that is sends signals downstream of the clock, triggering phenotypic (observable) events, several times during each cycle. The events happen always at the same phases of the cycle, and are usually happening every 12 or 8 or 6 or 4 or 3 or 2 or 1 hours – the divisors of 24. Likewise, the clock can trigger the event only every other cycle, resulting in a 48-hour period of the observable behavior.
If we forget for a moment the metaphor of the clock and think instead of a Player Piano, it is like the contraption plays the note G several times per cycle, always at the same moments during each cycle, but there is no need to limit each note to appear only once per cycle.
On the other hand, both the activity and melatonin rhythms appear to be driven directly by light and dark – like a stop-watch. In circadian parlance this is called an “hourglass clock” – an environmental trigger is needed to turn it over so it can start measuring time all over again. Dawn and dusk appear to directly stop and start the behavioral activity, and onset of dark stimulates while onset of light inhibits secretion of melatonin. An “hourglass clock” is an extreme example of a circadian clock with a very low amplitude.
While we mostly pay attention to period and phase, we should not forget that amplitude is important. Yes, amplitude is important. It determines how easy it is for the environmental cue to reset the clock to a new phase – lower the amplitude of the clock, easier it is to shift. In a very low-amplitude oscillator, onset of light (or dark) can instantly reset the clock to Phase Zero and start timing all over again – an “hourglass” behavior.
The molecular study of the reindeer fibroblasts also suggests a low-amplitude clock – there are a couple of weak oscillations to be seen before the rhythm goes away completely.
There may be other explanations for the observed data, e.g., masking (direct effect of light on behavior bypassing the clock) or relative coordination (weak and transient entrainment) but let’s not get too bogged down in arcane circadiana right now. For now, let’s say that the reindeer clock exists, that it is a very low-amplitude clock which entrains readily and immediately to light-dark cycles, while it fragments or demultiplies in long periods of constant conditions.
Why is this important to the reindeer?
During long night of the winter and the long day of the summer it does not make sense for the reindeer to behave in 24-hour cycles. Their internal drive to do so, driven by the clock, should be overpowered by the need to be flexible – in such a harsh environment, behavior needs to be opportunistic – if there’s a predator in sight: move away. If there is food in sight – go get it. If you are full and there is no danger, this is a good time to take a nap. One way to accomplish this is to de-couple the behavior from the clock. The other strategy is to have a clock that is very permissive to such opportunistic behavior – a very low-amplitude clock.
But why have clock at all?
Stokkan and colleagues stress that the day-night cycles in spring help reindeer time seasonal events, most importantly breeding. The calves/fawns should be born when the weather is the nicest and the food most plentiful. The reindeer use those few weeks of spring (and fall) to measure daylength (photoperiod) and thus time their seasonality – or in other words, to reset their internal calendar: the circannual clock.
But, what does it all mean?
All of the above deals only with one of the two hypotheses for the adaptive function (and thus evolution) of the circadian clock. This is the External Synchronization hypothesis. This means that it is adaptive for an organism to be synchronized (in its biochemistry, physiology and behavior) with the external environment – to sleep when it is safe to do so, to eat at times when it will be undisturbed, etc. In the case of reindeer, since there are no daily cycles in the environment for the most of the year, there is no adaptive value in keeping a 24-hour rhythm in behavior, so none is observed. But since Arctic is highly seasonal, and since the circadian clock, through daylength measurement (photoperiodism) times seasonal events, the clock is retained as an adaptive structure.
This is not so new – such things have been observed in cave animals, as well as in social insects.
What the paper does not address is the other hypothesis – the Internal Synchronization hypothesis for the existence of the circadian clock – to synchronize internal events. So a target cell does not need to keep producing (and wasting energy) to produce a hormone receptor except at the time when the endocrine gland is secreting the hormone. It is a way for the body to temporally divide potentially conflicting physiological functions so those that need to coincide do so, and those that conflict with each other are separated in time – do not occur simultaneously. In this hypothesis, the clock is the Coordination Center of all the physiological processes. Even if there is no cycle in the environment to adapt to, the clock is a necessity and will be retained no matter what for this internal function, though the period now need not be close to 24 hours any more.
What can be done next?
Unfortunately, reindeer are not fruitflies or mice or rats. They are not endangered (as far as I know), but they are not easy to keep in the laboratory in large numbers in ideal, controlled conditions, for long periods of time.
Out in the field, one is limited as to what one can do. The only output of the clock that can be monitored long-term in the field is gross locomotor activity. Yet, while easiest to do, this is probably the least reliable indicator of the workings of the clock. Behavior is too flexible and malleable, too susceptible to “masking” by direct effects of the environment (e.g., weather, predators, etc,). And measurement of just gross locomotor activity does not tell us which specific behaviors the animals are engaged in.
It would be so nice if a bunch of reindeer could be brought into a lab and placed under controlled lighting conditions for a year at a time. One could, first, monitor several different specific behaviors. For example, if feeding, drinking and defecation are rhythmic, that would suggest that the entire digestive system is under circadian control: the stomach, liver, pancreas, intestine and all of their enzymes. Likewise with drinking and urination – they can be indirect indicators of the rhytmicity of the kidneys and the rest of the excretory system.
In a lab, one could also continuously monitor some physiological parameters with simple, non-invasive techniques. One could, for example monitor body temperature, blood pressure and heart-rate, much more reliable markers of circadian output. One could also take more frequent blood samples (these are large animals, they can take it) and measure a whole plethora of hormones along with melatonin, e.g., cortisol, thyroid hormones, progesterone, estrogen, testosterone, etc (also useful for measuring seasonal responses). One could measure metabolites in urine and feces and also gain some insight into rhythms of the internal biochemistry and physiology. All of that with no surgery and no discomfort to the animals.
Then one can place reindeer in constant darkness and see if all these rhythms persist or decay over time. Then one can make a Phase-Response Curve and thus test the amplitude of the underlying oscillator (or do that with entrainment to T-cycles, if you have been clicking on links all along, you’ll know what I’m talking about). One can test their reproductive response to photoperiod this way as well.
Finally, fibroblasts are peripheral cells. One cannot expect the group to dissect suprachiasmatic nuclei out of reindeer to check the state of the master pacemaker itself. And in a case of such a damped circadian system, testing a peripheral clock may not be very informative. Better fibroblasts than nothing, but there are big caveats about using them.
Remember that the circadian system is distributed all around the body, with each cell containing a molecular clock, but only the pacemaker cells in the suprachiasmatic nucleus are acting as a network. In a circadian system like the one in reindeer, where the system is low-amplitude to begin with, it is almost expected that peripheral clocks taken out of the body and isolated in a dish will not be able to sustain rhythms for very long. Yet those same cells, while inside of the body, may be perfectly rhythmic as a part of the ensemble of all the body cells, each sending entraining signals to the others every day, thus the entire system as a whole working quite well as a body-wide circadian clock. This can be monitored in real-time in transgenic mice, but the technology to do that in reindeer is still some years away.
Finally, one could test a hypothesis that the reindeer clock undergoes seasonal changes in its organization at the molecular level by comparing the performance of fibroblasts (and perhaps some other peripheral cells) taken out of animals at different times of year.
What’s up with this being medically relevant?
But why is all this important? Why is work on mice not sufficient and one needs to pay attention to a strange laboratory animal model like reindeer?
First, unlike rodents, reindeer is a large, mostly diurnal animal. Just like us.
a1 reindeer.jpg
Second, reindeer normally live in conditions that make people sick, yet they remain just fine, thank you. How do they do that?
Even humans who don’t live above the Arctic Circle (or in the Antarctica), tend to live in a 24-hour society with both light and social cues messing up with our internal rhythms.
We have complex circadian systems that are easy to get out of whack. We work night-shifts and rotating shifts and fly around the globe getting jet-lagged. Jet-lag is not desynchronization between the clock and the environment, it is internal desynchronization between all the cellular clocks in our bodies.
In the state of almost permanent jet-lag that many of us live in, a lot of things go wrong. We get sleeping disorders, eating disorders, obesity, compromised immunity leading to cancer, problems with reproduction, increase in psychiatric problems, the Seasonal Affective Disorder, prevalence of stomach ulcers and breast cancer in night-shift nurses, etc.
Why do we get all that and reindeer don’t? What is the trick they evolved to stay healthy in conditions that drive us insane and sick? Can we learn their trick, adopt it for our own medical practice, and use it? Those are kinds of things that a mouse and a rat cannot provide answers to, but reindeer can. I can’t think of another animal species that can do that for us. Which is why I am glad that Stokkan and friends are chasing reindeer with enormous butterfly nets across Arctic wasteland in the darkness of winter ;-)
Lu, W., Meng, Q., Tyler, N., Stokkan, K., & Loudon, A. (2010). A Circadian Clock Is Not Required in an Arctic Mammal Current Biology, 20 (6), 533-537 DOI: 10.1016/j.cub.2010.01.042

Yes, Archaea also have circadian clocks!

ResearchBlogging.orgIf you ever glanced at the circadian literature, you have probably encountered the statement that “circadian rhythms are ubiquitous in living systems”. In all of my formal and informal writing I qualified that statement somewhat, stating something along the lines of “most organisms living on or near the Earth’s surface have circadian rhythms”. Why?
In the earliest days of chronobiology, it made sense to do most of the work on readily available organisms: plants, insects, mammals and birds. During the 20th century, thousands of species of animals, fungi, protists and plants – all living on the planet’s surface – were tested for the possession of the circadian clock, and one was always found. Hence the “ubiquitous” statement seen in so many papers.
But, as it was later discovered, for some marine organisms moon cycles are more important than day-night cycles so they have evolved lunar clocks in addition or instead of circadian clocks (see sponges and cnidaria, for some examples). In the intertidal zone, the tides are more important for survival than the daily rhythms, so the organisms living there have evolved tidal clocks. Animals that live in caves have lost circadian rhythms, at least in behavioral output (a clock may still be operating underneath, driving metabolic or developmental rhythms). In the polar regions, rhythmicity may be seasonal. In subterranean animals, like Blind Naked mole-rats, most individuals are without rhythms, but young males that leave the colony in order to join another one develop rhythmicity during their adventurous journey. In social insects, only the individuals that go outside the hive to forage exhibit daily rhythms.
How does one figure out if an organism has a clock? You need to pick a good output and a way to continuously monitor it. Then you put the organism in constant conditions of light, temperature, air pressure, sound etc., and monitor the output for many days. If you do the statistics on the data at the end of the experiment and see that there is a periodicity in the data (for at least the first 2-3 days)that is reasonably close to 24 hours (between 16 and 32 hours is usually thought to be the limits), you know that your organism of choice has a circadian clock.
In a related experiment, you expose the organism to an environmental periodicity – usually a light-dark cycle, as it is usually the strongest cue, as the evolution of circadian clocks and light-detecting mechanisms is closely intertwined – to see if the rhythmicity of the organism can be synchronized (entrained) to the environmental cycle, indicating that it is a biological function and not the chance quirk in your data. Without these two experiments providing positive data it does not make sense to do any further investigations into mechanisms of entrainment, anatomical location of the clock or the cellular mechanism of the clock.
The trick is to find a good output to monitor. It is easy for rodents – they will run in running wheels (so will cockroaches). Songbirds will jump from perch to perch. Lizards will walk around the cage and tilt the floor from one side to another. And while behavioral output – the general locomotor activity – is not the most reliable (it is very prone to masking effects, so for instance mice will generally not run in wheels in bright light, while rats will), it is usually the easiest and cheapest to monitor and, in most cases (see an example where it failed, while monitoring body temperature worked) will be sufficient.
But what do you do when the organism does not have a measurable behavioral output, especially one that can be continuously monitored by machines? You start thinking very, very hard. And you come up with an alternative. You may be able to implant radiotransmitters and monitor body temperature. Or you may record vocalizations. Or you may take small blood samples several times per day and assay for something like melatonin.
The technological constrains limited our ability to discover circadian clocks in bacteria until the 1990s. Until then, the existence of such clocks was a mystery (one that everyone in the field was eager to see solved). I have written several posts about the discoveries of clocks in bacteria: Circadian Clocks in Microorganisms, Clocks in Bacteria I: Synechococcus elongatus, Clocks in Bacteria II: Adaptive Function of Clocks in Cyanobacteria, Clocks in Bacteria III: Evolution of Clocks in Cyanobacteria, Clocks in Bacteria IV: Clocks in other bacteria, Clocks in Bacteria V: How about E.coli? The understanding of the way bacterial clocks work (more like a relay or a switch than a clock) made us rethink the clock metaphor we have been using for almost a century.
So it appears that most Eukaryotes have clocks and at least some bacteria have them as well. But the other large group – the Third Domain: Archaea – eluded us thus far. After all, Archaea are notoriously difficult to culture in the laboratory and it took some time to figure out how to keep them alive outside of their natural extreme environments.
Do Archaea have clocks? We did not know. Until now. A couple of weeks ago, PLoS ONE published a paper that is the first to demonstrate the daily rhythms in an Archaeon: Diurnally Entrained Anticipatory Behavior in Archaea by Kenia Whitehead, Min Pan, Ken-ichi Masumura, Richard Bonneau and Nitin S. Baliga. Here is the text of the Abstract:

By sensing changes in one or few environmental factors biological systems can anticipate future changes in multiple factors over a wide range of time scales (daily to seasonal). This anticipatory behavior is important to the fitness of diverse species, and in context of the diurnal cycle it is overall typical of eukaryotes and some photoautotrophic bacteria but is yet to be observed in archaea. Here, we report the first observation of light-dark (LD)-entrained diurnal oscillatory transcription in up to 12% of all genes of a halophilic archaeon Halobacterium salinarum NRC-1. Significantly, the diurnally entrained transcription was observed under constant darkness after removal of the LD stimulus (free-running rhythms). The memory of diurnal entrainment was also associated with the synchronization of oxic and anoxic physiologies to the LD cycle. Our results suggest that under nutrient limited conditions halophilic archaea take advantage of the causal influence of sunlight (via temperature) on O2 diffusivity in a closed hypersaline environment to streamline their physiology and operate oxically during nighttime and anoxically during daytime.

What does that mean? What did they do?
First, they picked a good candidate species – Halobacterium salinarum. Why is it a good candidate? Because it lives near the Earth’s surface, in salty lakes and ponds (like this one, in Africa):
salinarium in a lake.gif
Many Archaea live in places where no light ever penetrates: deep inside the rock or ice or the oceanic floor. Some Archaea are exposed to light in cyclical fashion but not a 24-hour cycle – I have written somewhere before that I expect the Archaea living in the waters of the Old Faithful geiser in Yellowstone National Park to have a 45-minute clock instead. But Halobacterium salinarum is exposed to the natural periodicity of the day-night cycle on the surface and is thus a good candidate for an Archaeon that may have evolved a circadian clock. This is how the Halobacterium salinarum look like under the microscope:
salinarium micrograph.gif
There is another reason this is a good candidate. The light-dark cycle has a potential adaptive consequence to the critter. Water that is saturated with salt will have a high variation of its oxygen content and this variation is dependent on the environmental temperature: when it is colder outside, oxygen can more readily disolve in the salty water. When it is warm, it cannot.
The environment where Halobacterium salinarum lives is cold during the night and hot during the day. But the temperature changes are much more gradual and slow than changes in illumination (as well as less dependable: there are colder and warmer days), so being in tune with the light is a better way to synchronize one’s activities than measuring temperature (or oxygen content) directly. By entraining to a ligh-dark cycle, these organisms can make switches in their oxygen-dependent metabolism in a more timely (and thus more energy-efficient) fashion: by predicting instead of reacting to the changes in temperature over the course of 24 hours.
So, Whitehead et al placed some Halobacterium salinarum in light-dark cycles and subsequently released them into constant darkness. But what did they measure? Archaea are known to be lousy wheel-runners!
In bacteria, much of the work is done by measuring bioluminescence coming from the expression of the luciferase gene inserted next to one of the clock gene promoters. But here, we don’t know which if any gene is a clock gene and we do not have the technology ready yet. But, these days microarrays are cheaper and easier to use then some years ago when I started grad school. And remember that Everything Important Cycles!
So they took samples of the organism six times per day and ran them on microarrays, comparing the expression of all the genes between the sampling times, both during entrainment to LD cycles and in the subsequent DD (constant dark) environment:
archaea microarrays.JPG
What they discovered is that about 12% of the genes cycle with the period of 24 hours in LD cycles and continue to cycle in DD with a circadian period of around 21.6 hours:
archaea periods.JPG
What is most interesting is that the genes that cycle are the genes that are involved in oxygen (or oxygen-dependent) metabolism – exactly the kinds of genes that are expected to cycle in this organism. Some of these genes are also know to be directly regulated by oxygen. Now we know they can also be regulated – directly and/or indirectly through a clock – by light, inducing expression in preparation for the changes in oxygen concentration, not just in direct response. In this way, the cell is ready to use oxygen a little bit ahead of time. No time wasted.
I am very excited about this finding. This opens up a whole avenue of future research, something that the authors also realize:

Indeed, further detailed experimentation is necessary to ascertain precise phasing, temperature compensation, adaptability to different periods of entrainment etc. to ascertain the mechanistic underpinnings of this diurnal entrainment and its physiological implications.

Once we know there is a clock in Archaea – and now we do due to this paper – we can start studying it in detail.
Furthermore, this finding has big implications for the study of the evolutionary origins of the circadian clock (and light-reception associated with it). The molecular mechanism of the clock is very different between Bacteria and Eukaryotes, leading the field to conclude that the clock evolved independently in these two groups (and perhaps more – some people think that protist, plant, fungal and animal clocks evolved independently of each other as well). Now we can try to figure out how Archaea measure time. Is their mechanism similar to that in Bacteria? Or in Eukaryotes? Or something completely different, indicating another independent evolutionary origin? Or something in-between Bacteria and Eukaryotes, containing some elements of both, suggesting that perhaps there was only one evolutionary origin for clocks in all the life on Earth. The authors note that this last scenario is a strong contender:

Finally, the discovery of diurnal entrainment of gene expression in an archaeon also raises important questions regarding the origin of light-responsive clock mechanisms. This is because archaeal information processing machinery is assembled from components that share ancestry with eukaryotic (general transcription factors and RNA polymerase) and bacterial (sequence-specific transcription regulators) counterparts [44]. Furthermore, components of both bacterial [45,46] and eukaryotic [47] clocks are encoded in its genome [6,32].

Of course, since this is an Open Access article, you can and should read it yourself to get more details. And post ratings, notes and comments while you are there.
Whitehead, K., Pan, M., Masumura, K., Bonneau, R., & Baliga, N. (2009). Diurnally Entrained Anticipatory Behavior in Archaea PLoS ONE, 4 (5) DOI: 10.1371/journal.pone.0005485
Update: see comment thread for more. Unfortunately, scientists still at this day and age do not report everything and keep data secret. Apparently, this was the case in the question posed by this study. I hear from trusted sources that there is still not evidence for a clock in Archaea beyond the direct effects of light on gene expression and O2 metabolism.

Why social insects do not suffer from ill effects of rotating and night shift work?

ResearchBlogging.orgMost people are aware that social insects, like honeybees, have three “sexes”: queens, drones and workers.
Drones are males. Their only job is to fly out and mate with the queen after which they drop dead.
Female larvae fed ‘royal jelly’ emerge as queens. After mating, the young queen takes a bunch of workers with her and sets up a new colony. She lives much longer than other bees and spends her life laying gazillions of eggs continuously around the clock, while being fed by workers.
Female larvae not fed the ‘royal jelly’ emerge as workers.
Workers perform a variety of jobs in the hive. Some are hive-cleaners, some are ‘nurses’ (they feed the larvae), some are queen’s chaperones (they feed the queen), some are guards (they defend the hive and attack potential enemies) and some are foragers (they collect nectar and pollen from flowers and bring it back to the hive).
What most people are not aware of, though, is that there is a regular progression of ‘jobs’ that each worker bee goes through. The workers rotate through the jobs in an orderly fashion. They all start out doing generalized jobs, e.g., cleaning the hive. Then they move up to doing a more specialized job, for instance being a nurse or taking care of the queen. Later, they become guards, and in the end, when they are older, they become foragers – the terminal phase.
This pattern of behavioral development is called “age polyethism” (poly = many, ethism = expression of behavior), or sometimes “temporal polyethism” (image from BeeSpotter):
Age polyethism.jpg
This developmental progression in behavior is accompanied by changes in brain structure, patterns of neurotransmitter and hormone synthesis and secretion, and patterns of gene expression in the central nervous system.
Some years ago (as in “more than ten years ago”) Gene Robinson and his students started looking at daily patterns of activity in honeybees. The workers in their early stages are doing jobs inside the hive, where it is always dark. They clean the hive, take care of the eggs and pupae, and feed the larvae and the queen around the clock. Each individual bee sometimes works and sometimes sleeps, without any semblance of a 24-hour pattern. Different individuals work and sleep at different, apparently random times. The hive as a whole is thus constantly busy – there is always a large subset of workers performing their duties, day and night.
As they get older, they start doing the jobs, like being guards, that expose them to the outside of the hive, thus to the light-dark and temperature cycles of the outside world.
Finally, the foragers only go out during the daytime and have clear and distinct daily rhythms. Furthermore, the foragers have to consult an internal clock in order to orient towards the Sun in their travels, as well as to be able to communicate the distance and location of flowers to their mates in the hive using the ‘waggle dance’. As bees are social insects, it is difficult to keep individuals in isolation for longer periods of time, but it has been done successfully and, in such studies, foragers exhibit both freerunning (in constant darkness) and entrained (in light-dark cycles) circadian rhythms, while younger workers do not.
In the Robinson lab, then PhD student Dan Toma and postdoc Guy Bloch did much of the early and exciting work on figuring out how the rhythmicity develops in individual worker bees as they pass through the procession of ‘jobs’.
In an early study, they measured levels of expression of mRNA of the core clock gene Period (Per). The gene was expressed at low levels and no visible daily rhythm in early-stage workers, but at much higher levels and in a circadian fashion in foragers.
As the levels of expression were measured crudely – in entire bee brains – it was impossible at the time to be sure which of the two potential mechanisms were operating: 1) the celluular clock did not work until the bee became a forager, or 2) the cellular clocks were working, but different cells were not synchronized with each other, producing a collectively arrhythmic output: both as measured by gene expression of the entire brain and as measured by behavior of the live bee.
Either way, the study showed correlation: the appearance of the functional circadian clock coincided with other changes in the brain structure, brain chemistry and bee behavior. They could not say at the time what causes what, or even if the syncronicity of changes was purely coincidental. They needed to go beyond correlation and for that they needed to experimentally change the timing to see if various processes can be dissociated or if they are tightly bound to each other.
And there is a clever way to do this! First, they took some hives and removed all the foragers from it. This disrupted the harmony of the division of labor in the hive – too many cleaners and nurses, but nobody is bring the food home. When that happens, the behavioral development of other workers speeds up dramatically – in no time, some nurses and guards develop into foragers. And, lo and behold, the moment they became foragers, they developed rhythms in behavior and rhythms of the Per gene expression in the brain. So, as the development is accelerated, everything about it is accelerated at the same rate: gene expression, brain structure, neurochemistry, and behavioral rhythmicity.
Nice, but then they did something even better. They removed most of the cleaners and nurses from some hives. Again, the balance of the division of labor was disrupted – plenty of food is arriving into the hive but there is not enough bees inside to take care of that food, process it, feed the larvae, etc. What happened then? Well, some of the foragers went back into the hive and started performing the house-keeping duties instead of flying out and about. And, interestingly, their brain structure and chemistry reverted its development to resemble that of cleaners and nurses. They lost behavioral rhythmicity and started working randomly around the clock. And the rhythm of clock-gene expression disappeared as well.
So, genetic, neural, endocrine, circadian and behavioral changes all go together at all times. Social structure of the colony, through the patterns of pheromones present in the hive, affects the gene expression, brain development and function, and behavior of individual bees. Just like the gene expression and behavioral patterns, the patterns of melatonin synthesis and secretion in honeybee brains is low and arrhythmic in young workers and becomes greater and rhythmic in foragers. With the recent sequencing of the honeybee genome, the potential for future research in honeybee chronobiology looks promising and exciting.
But are these findings generalizable or are they specific to honeybees? How about other species of bees or other social insects, like wasps, ants and termites? Are they the same?
Other species of socials insects have been studied in terms of age polyethism as well. The earliest study I am aware of (let me know if there is an older one) studying behavioral rhytmicity in relation to behavioral development was a 2004 Naturwissenschaften paper by Sharma et al. on harvester ants. In that study, different castes of worker ants exhibited different patterns – some were strongly diurnal, some nocturnal, some had strange shifts in period, and some were arrhythmic. Those with rhythms could entrain to light-dark cycles as well as display freerunning rhythms in constant darkness.
Just last month, a new paper on harvester ants came out in BMC Ecology (Open Access). In it, Ingram et al. show that foragers have circadian rhythms (both in constant darkness and entrained to LD cycles) in expression of Period gene (as well as behavioral rhythms), while ants working on tasks inside the hive do not exhibit any rhythms either in clock-gene expression or in behavior, suggesting that the connection between age polyethism and the development of the circadian clock may be a universal property of all social insects.
We know that in humans, night-shift and rotating-shift schedules are bad for health as the body is in the perpetual state of jet-lag: the numerous clocks in our bodies are not synchronized with each other. We have evolved to be diurnal animals, entrained to environmental light cycles and not traveling over many time zones within hours, or working around the clock. Social insects have evolved a different strategy to deal with the potentially ill effects of shift-work: switch off the clock entirely until one develops far enough that time-keeping becomes a requirement.
Yang, L., Qin, Y., Li, X., Song, D., & Qi, M. (2007). Brain melatonin content and polyethism in adult workers of Apis mellifera and Apis cerana (Hym., Apidae) Journal of Applied Entomology, 131 (9-10), 734-739 DOI: 10.1111/j.1439-0418.2007.01229.x
Sharma, V., Lone, S., Goel, A., & Chandrashekaran, M. (2004). Circadian consequences of social organization in the ant species Camponotus compressus Naturwissenschaften, 91 (8) DOI: 10.1007/s00114-004-0544-6
Ingram, K., Krummey, S., & LeRoux, M. (2009). Expression patterns of a circadian clock gene are associated with age-related polyethism in harvester ants, Pogonomyrmex occidentalis BMC Ecology, 9 (1) DOI: 10.1186/1472-6785-9-7

Circadian Rhythm of Aggression in Crayfish

ResearchBlogging.orgLong-time readers of this blog remember that, some years ago, I did a nifty little study on the Influence of Light Cycle on Dominance Status and Aggression in Crayfish. The department has moved to a new building, the crayfish lab is gone, I am out of science, so chances of following up on that study are very low. And what we did was too small even for a Least Publishable Unit, so, in order to have the scientific community aware of our results, I posted them (with agreement from my co-authors) on my blog. So, although I myself am unlikely to continue studying the relationship between the circadian system and the aggressive behavior in crayfish, I am hoping others will.
And a paper just came out on exactly this topic – Circadian Regulation of Agonistic Behavior in Groups of Parthenogenetic Marbled Crayfish, Procambarus sp. by Abud J. Farca Luna, Joaquin I. Hurtado-Zavala, Thomas Reischig and Ralf Heinrich from the Institute for Zoology, University of Gottingen, Germany:

Crustaceans have frequently been used to study the neuroethology of both agonistic behavior and circadian rhythms, but whether their highly stereotyped and quantifiable agonistic activity is controlled by circadian pacemakers has, so far, not been investigated. Isolated marbled crayfish (Procambarus spec.) displayed rhythmic locomotor activity under 12-h light:12-h darkness (LD12:12) and rhythmicity persisted after switching to constant darkness (DD) for 8 days, suggesting the presence of endogenous circadian pacemakers. Isogenetic females of parthenogenetic marbled crayfish displayed all behavioral elements known from agonistic interactions of previously studied decapod species including the formation of hierarchies. Groups of marbled crafish displayed high frequencies of agonistic encounters during the 1st hour of their cohabitation, but with the formation of hierarchies agonistic activities were subsequently reduced to low levels. Group agonistic activity was entrained to periods of exactly 24 h under LD12:12, and peaks of agonistic activity coincided with light-to-dark and dark-to-light transitions. After switching to DD, enhanced agonistic activity was dispersed over periods of 8-to 10-h duration that were centered around the times corresponding with light-to-dark transitions during the preceding 3 days in LD12:12. During 4 days under DD agonistic activity remained rhythmic with an average circadian period of 24.83 ± 1.22 h in all crayfish groups tested. Only the most dominant crayfish that participated in more than half of all agonistic encounters within the group revealed clear endogenous rhythmicity in their agonistic behavior, whereas subordinate individuals, depending on their social rank, initiated only between 19.4% and 0.03% of all encounters in constant darkness and displayed no statistically significant rhythmicity. The results indicate that both locomotion and agonistic social interactions are rhythmic behaviors of marbled crayfish that are controlled by light-entrained endogenous pacemakers.

I think the best way for me to explain what they did in this study is to do a head-to-head comparison between our study and their study – it is striking how the two are complementary! On one hand, there is no overlap in methods at all (so no instance of scooping for sure), yet on the other, both studies came up with similar results, thus strengthening each other’s findings. You may want to read my post for the introduction to the topic, as I explain there why studying aggression in crayfish is important and insightful, what was done to date, and what it all means, as well as the standard methodology in the field. So, let’s see how the two studies are similar and how the two differ:
1) We were sure we used the Procambarus clarkii species. They are probably not exactly sure what species they had, so they denoted it as Procambarus sp., noting in the Discussion that it was certainly NOT the Procambarus clarkii, which makes sense as our animals were wild-caught in the USA and theirs in Germany. As both studies got similar results, this indicates that this is not a single-species phenomenon, but can be generalizable at least to other crayfish, if not broader to other crustaceans, arhtropods or all invertebrates.
2) We used only males in our study. They used only females. In crayfish, both sexes fight. It is nice, thus, to note that other aspects of the behavior are similar between sexes.
3) We used the term ‘aggression’. They use the term ‘agonistic behavior’, which is scientese for ‘aggression’, invented to erase any hints of anthropomorphism. Not a bad strategy, generally, as assumed aggression in some other species has been later shown to be something else (e.g., homosexual behavior), but in crayfish it is most certainly aggression: they meet, they display, they fight, and if there is no place to escape, one often kills the other – there is no ‘loving’ going on there, for sure.
4) The sizes of animals were an order of magnitude different between the two studies. Their crayfish weighed around 1-2g while ours were 20-40g in body mass. This may be due to species differences, but is more likely due to age – they used juveniles while we used adults. Again, it is nice to see that results in different age groups are comparable.
5) We did not measure general locomotor activity of our animals in isolation. We, with proper caveats, used aggressive behavior of paired animals as a proxy for general locomotor activity, and were straightforward about it – we measured aggressive behavior alone in a highly un-natural setup. As Page and Larimer (1972) have done these studies before, we did not feel the need to replicate those with our animals.
The new study, however, did monitor gross locomotor activity of isolated crayfish. Their results, confirming what Page and Larimer found out, demonstrate once again that activity rhythms are a poor marker of the underlying circadian pacemaker (which is why Terry Page later focused on the rhythm of electrical activity of the eye, electroretinogram – ERR – in subsequent studies) in crayfish. Powerful statistics tease out rhythmicity in most individuals, but this is not a rhythm I would use if I wanted to do more complex studies, e.g., analysis of entrainment to exotic LD cycles or to build and interpret a Phase-Response Curve. Just look at their representative example (and you know this is their best):
crayfish image 1.JPG
You can barely make out the rhythm even in the light-dark cycle (white-gray portion of the actograph) and the rhythms in constant darkness (solid gray) are even less well defined – thus only statistical analysis (bottom) can discover rhythms in such records. The stats reveal a peak of activity in the early night and a smaller peak of activity at dawn, similarly to what Page and Larimer found in their study, and similar to what we saw during our experiments.
6) They used an arena of a much larger size than ours. We did it on purpose – we wanted to ‘force’ the animals to fight as much as possible by putting them in tight quarters where they cannot avoid each other, as we were interested in physiology and wanted it intensified so we could get clearly measurable (if exaggerated) results. Their study is, thus, more ecologically relevant, but one always has to deal with pros and cons in such decisions: more realistic vs. more powerful. They chose realism, we chose power. Together, the two approaches reinforce and complement each other.
7) As I explained in my old post – there are two methodological approaches in this line of research:

Two standard experimental practices are used in the study of aggression in crustaceans. In one, two or more individuals are placed together in an aquarium and left there for a long period of time (days to weeks). After the initial aggressive encounters, the social status of an individual can be deduced from its control of resources, like food, shelter and mates.
In the other paradigm, two individuals are allowed to fight for a brief period of time (less than an hour), after which they are isolated again and re-tested the next day at the same time of day.

They used the first method. We modified the second one (testing repeatedly, every 3 hours over 24 hours, instead of just once a day).
What they did was place 6 individuals in the aquarium, a couple of hours before lights-off, then monitor their aggressive behavior over several days. What they found, similar to us, is that the most intense fights resulting in a stable social hierarchy occur in the early portion of the night:
crayfish image 2.JPG
Once the social hierarchy is established on that first night, the levels of aggression drop significantly, and occasional bouts of fights happen at all times, with perhaps a slight increase at the times of light switches: both off and on. Released into constant darkness, the pattern continues, with the most dominant individual initiating aggressive encounters a little more often during light-transitions then between them. The other five animals had no remaining rhythm of agonistic behavior: they just responded to attacks by the Numero Uno when necessary.
In our study we tried to artificially elevate the levels of aggression by repeatedly re-isolating and re-meeting two animals at a time. And even with that protocol, we saw the most intense fights at early night, and most conclusive fights, i.e., those that resulted in stable social hierarchy, also occuring at early nights, while the activity at other time of the day or night were much lower.
8) The goals of two studies differed as well, i.e., we asked somewhat different questions.
Our study was designed to provide some background answers that would tell us if a particular hypothesis is worth testing: winning a fight elevates serotonin in the nervous system; elevated serotonin correlated with the hightened aggression in subsequent fights, more likely leading to subsequent victories; crayfish signal dominance status to each other via urine; melatonin is a metabolic product of serotonin; melatonin is produced only during the night with a very sharp and high peak at the beginning of the night; if there is more serotonin in the nervous system, there should be more melatonin in the urine; perhaps melatonin may be the signature molecule in the urine indicating social status.
In order to see if this line of thinking is worth pursuing, we needed to see, first, if the most aggressive bouts happen in the early night and if the most decisive fights (those that lead to stable hiararchy) happen in the early night. This is what we found, indicating that our hypothesis is worth testing in the future.
They asked a different set of questions:
Is there a circadian rhythm of locomotor activity? They found: Yes.
Is there a circadian rhythm of aggression? They found: Yes.
Do the patterns of general activity and aggressive activity correlate with each other? They found: Yes.
Does the aggression rhythm persist in constant darkness conditions? They found: Yes.
Do all individuals show circadian rhythm of aggression? They found: No. Only the most dominant individual does. The others just defend themselves when attacked.
Is there social entrainment in crayfish, i.e., do they entrain their rhythms to each other in constant conditions? They found: No. All of them just keep following their own inherent circadian periods and drift apart after a while.
Is there a pattern of temporal competitive exclusion, i.e., do submissive individuals shift their activity patterns so as not to have to meet The Badassest One? They found: No. All of them just keep following their own inherent circadian periods.
So, a nice study overall, the first publication I know of that attempts to connect the literature on circadian rhythms in crayfish to the literature on aggressive behavior in crayfish.
Except….

Continue reading

To Equine Things There is a Season (guest post by Barn Owl)

As I announced this morning, there will be several guest posts here over the next several weeks. The first one, by Barn Owl of the lovely Guadalupe Storm-Petrel blog, is likely to appeal to a lot of my readers as it combines several of my own interests:
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ResearchBlogging.orgfriendlyfoal.jpgIn this guest-post for A Blog Around the Clock, I’ll combine three things that Coturnix especially likes: horses, circadian biology, and an Open Access research paper. For the equestrian, there are two main seasonal issues, controlled primarily by photoperiod, or day length, which must be considered, especially if one shows the horse or competes in various events and games. Perhaps the most obvious seasonal changes are in the horse’s coat, with shedding cycles in the spring and in the fall. In the Northern Hemisphere, the fall shedding cycle is relatively inconsequential for equestrian activities (though it is important, as always, to groom your horse frequently to remove the shed hair), but the spring shedding cycle can be a nightmare, so to speak. The horse hair permeates your saddle blankets, your riding clothes, the interior of your car, and your respiratory tract. Birds gather around eagerly when you groom your horse, to fly away with tufts of hair for their nests. One of my friends used to mark “the lying down of the grey horse” each year, i.e. the spring day on which her big flea-bitten grey gelding rolled and rubbed off much of his shed winter coat, in the form of a large “hair angel”.
Although humans have selected for horse coat colors and patterns that they find attractive and interesting, the horse’s coat evolved to complement the thermoregulatory functions of the other skin components. The long outer, or “guard”, hairs of a horse’s coat are equipped with piloerector muscles, allowing a layer of insulating air to be trapped between the raised shafts. Our winters here in South Texas are relatively mild, so we rarely need to blanket our horses for more than a day or two at a time. A light blanket is sufficient, and is a good thing to take along to early-season polocrosse tournaments, when nighttime temperatures can dip below freezing, and the horses cannot move about sufficiently in their small temporary pens to warm themselves. Even in colder climates, horses are capable of staying warm during rough weather, as long as they have plenty of fodder, some shelter from wind, rain, and ice, and can move around to generate heat. In fact, a heavy winter coat can cause a performance or show horse to sweat excessively, and so some equestrians “body clip” their equine athletes, and blanket them when they are not exercising or performing.
nursingfoal.jpgSeasonal hair growth and pigmentation cycles have been studied extensively in sheep, goats, mink, arctic fox, and mice, and it is clear that they are responsive to photoperiod and melatonin levels. As Coturnix has described previously in his blog, melatonin is a multifunctional lipophilic molecule, primarily produced by the pineal gland in response to noradrenergic stimulation from sympathetic neurons. Numerous brain areas have high levels of melatonin receptors, but cells in other organs, including the skin, are also responsive to melatonin. The transcription levels of genes encoding melatonin receptors appear to be correlated with the hair cycle phases of telogen (resting) and anagen (growth). Hair follicle activity and hair shaft elongation are very responsive to melatonin, with both “overcoat” and “undercoat” fur affected; these effects of melatonin on hair growth have been demonstrated in sheep, goats, mink, ferrets, dogs, and red deer (Fischer et al., 2008). The autumn and spring molt, or shedding, phases in the horse are likely to reflect changing melatonin levels, and could perhaps be modified by dietary melatonin supplements, as has been achieved in cashmere goats and merino sheep. However, another important melatonin target organ is the hypothalamus, which in turn regulates secretion of hormones by the anterior pituitary.
Of course the involvement of the anterior pituitary indicates that a second major seasonal cycle for horses is reproduction. Melatonin inhibits production of gonadotropin-releasing hormone (GnRH), which causes secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) by the anterior pituitary. During the breeding season, a mare will cycle from ovulation to ovulation approximately every 21 days, and the changing levels of FSH and LH will in turn alter levels of estrogen and progesterone. A fertile mare “in season” (in estrus) will show distinctive behavior that is recognizable to every other horse, and to every horse owner as well. She will raise her tail and hold it to one side, she will squat and urinate small amounts frequently, and she will lean or rub against other horses, fences, trailers, and humans. She will be friendly and flirtatious to geldings that she normally disdains, and needless to say, this can be a very frustrating time for the geldings. I had a Thoroughbred mare who once sneaked out of a gate that was open for a split second, to make a beeline for a Quarter Horse stallion on a farm half a mile away; I caught her prancing and “showing” and trying desperately to figure out how to get into his paddock. And I currently have a Thoroughbred gelding who loves flirtatious mares of any breed, and will crawl under electobraid, or swim across stock ponds, to pay a gentlemanly visit to the ladies.
suspiciousfoal.jpgIn the winter, when day length is short, most mares will enter a non-cycling phase, with an inactive reproductive tract. Lower levels of GnRH mean less FSH to induce the maturation of oocyte-containing follicles, and insufficient LH to induce ovulation. There is a transitional phase between the non-cycling (anestrus) and cycling (estrus) phases, during which the ovarian follicles will mature, but not undergo ovulation, and it is this phase that some horse breeders will attempt to manipulate with prolonged light exposure. In the US, all Thoroughbred foals, regardless of which month they are born, will have their first birthday on their first January 1. On average, gestation in the horse is 340 days, so ideally, the mare should be bred in late winter or early spring, such that her foal is delivered in January, February, or early March of the following year. This is important in the racing industry, to produce a bigger colt at a given racing age, but of course there are other reasons to alter a mare’s breeding cycle, particularly if she is a show or performance horse.
The desire to manipulate equine breeding and the timing of foal delivery has led to a substantial amount of physiological research on fluctuating hormone and melatonin levels, and on light cycle responses, in horse reproduction. For a nice comparison of circadian melatonin levels, temperature, locomotor activity, and blood chemistry in the Thoroughbred mare and the Comisana ewe, see a recent paper by Piccione and colleagues; in the horse, melatonin levels peak after midnight (middle of dark phase), whereas locomotor activity peaks in the middle of the light phase. Since the late 1940s, it has been recognized that the photoperiod is the major signal (zeitgeber) that controls the timing of estrus in mares. By the 1980s, it was generally accepted that the reproductive cycle for most long-lived mammals was optimally synchronized with seasonal changes, through an interaction of the environmental photoperiod and endogenous melatonin levels. However, subsequent measurements of fluctuations in melatonin levels, in horses and in other seasonal breeders, made it clear that the model of suppression of the reproductive cycle by increased melatonin levels was overly simplistic (though the simplistic model still persists on equine information websites).
piccionefig4.jpg
In a 1995 paper, Guerin and colleagues reported plasma melatonin levels in mixed breed mares, under conditions of natural photoperiod, and though there was a clear circadian pattern to melatonin secretion, the peak values and duration of elevated levels of this molecule did not differ significantly between seasons. The observation that about 15-20% of mares continue to cycle throughout the nonbreeding period, i.e. fail to enter the anestrus phase during the shorter day-lengths of winter, led to the identification of other signals that influence the hypothalamus-pituitary control of the breeding cycle. Fitzgerald and McManus (2000) found that this continuous reproductive activity, throughout the winter months, is much more common in mature mares, than in young mares. Continuous treatment with melatonin, through an implant under the skin, did not suppress the estrus cycle in these mature mares. Instead, energy availability, as measured by weight, percent body fat, and circulating leptin levels, seems to alter the occurrence of anestrus in mares. Moreover, mature mares that failed to undergo anestrus had similar winter month levels of melatonin, as did mares that ceased reproductive activity in response to shorter day length.
Finally, a little discussion of an Open Access paper, on a chronobiology issue that is relevant to the performance of elite equine athletes in the Olympics, and at other international venues. Of course the horse and rider must both travel to the competition site, and both are potentially subject to the fatigue, malaise, loss of appetite, and impaired concentration, characteristic of jet lag. To determine how horses might be affected by jet lag, Murphy and colleagues (2007) housed six healthy mares (mixed light horse breed), entrained to a 12 hour light/12 hour dark natural photoperiod, in a light-proofed barn. The researchers then advanced the light/dark cycle by ending the dark period six hours early, and measured both body temperature and serum melatonin levels over the next 11 days. In contrast to the melatonin rhythm in humans and other animals, the equine melatonin phase advance occurred within the first day after the light/dark cycle shift. Re-entrainment of the body temperature rhythm occurred more slowly, and was not complete until 3 days after the shift. Nevertheless, by the criteria of both melatonin and body temperature rhythms, horses appear to adapt much more quickly to abrupt shifts in the light-dark cycle, than do most other animals. This same group of researchers has also examined regulation of clock genes in different tissues of the horse, a paper which might make an interesting subject for another post.

References:

Bastian, T. (2005) The Foal is the Goal: Managing Your Mare and Handling a Stallion. Trafalgar Square Publishing: North Pomfret, VT
Fischer, T.W., Slominski, A., Tobin, D.J., and Paus, R. (2008) Melatonin and the hair follicle. J. Pineal Res. 44, 1-15.
Fitzgerald, B.P., and McManus, C.J. (2000) Photoperiodic versus metabolic signals as determinants of seasonal anestrus in the mare. Biol. Reprod. 63, 335-340.
Guerin, M.V., Deed, J.R., Kennaway, D.J., and Matthews, C.D. (1995) Plasma melatonin in the horse: Measurements in natural photoperiod and in acutely extended darkness throughout the year. J. Pineal Res. 19, 7-15.
Murphy, B.A., Elliott, J.A., Sessions, D.R., Vick, M.M., Kennedy, E.L., Fitzgerald, B.P. (2007). Rapid phase adjustment of melatonin and core body temperature rhythms following a 6-h advance of the light/dark cycle in the horse. Journal of Circadian Rhythms, 5(1), 5. DOI: 10.1186/1740-3391-5-5
PICCIONE, G., CAOLA, G., REFINETTI, R. (2005). Temporal relationships of 21 physiological variables in horse and sheep. Comparative Biochemistry and Physiology – Part A: Molecular & Integrative Physiology, 142(4), 389-396. DOI: 10.1016/j.cbpa.2005.07.019

Rainforest Glow-worms glow at night because their clock says so

ResearchBlogging.orgGlow worms glimmer on cue:

University of Queensland researcher and lecturer Dr David Merritt has discovered that Tasmanian cave glow-worms are energy conservationists: they switch their lights off at night-time.
The discovery was made during a partially funded UQ Firstlink study, which revealed that the glow-worm’s prey-luring light output is governed by circadian rhythms, regardless of ambient light levels.
The study aimed to investigate the physiology and behaviours of cave dwelling glow-worms, which are actually the immature or larval stage of a mosquito-like fly found in Queensland, New South Wales, Victoria, Tasmania and New Zealand.
The study’s leader, Dr Merritt, says that unlike their rainforest dwelling counterparts, the cave-dwelling Tasmanian glow-worm can detect the time of day, even from the deepest stretches of their caves.

Circadian Regulation of Bioluminescence in the Prey-Luring Glowworm, Arachnocampa flava, by David J. Merritt and Sakiko Aotani, Journal of Biological Rhythms, Vol. 23, No. 4, 319-329 (2008), DOI: 10.1177/0748730408320263

The glowworms of New Zealand and Australia are bioluminescent fly larvae that generate light to attract prey into their webs. Some species inhabit the constant darkness of caves as well as the dim, natural photophase of rain-forests. Given the diversity of light regimens experienced by glowworms in their natural environment, true circadian rhythmicity of light output could be present. Consequently the light emission characteristics of the Australian subtropical species Arachnocampa flava, both in their natural rainforest habitat and in artificial conditions in the laboratory, were established. Larvae were taken from rainforest and kept alive in individual containers. When placed in constant darkness (DD) in the laboratory they maintained free-running, cyclical light output for at least 28 days, indicating that light output is regulated by an endogenous rhythm. The characteristics of the light emission changed in DD: individuals showed an increase in the time spent glowing per day and a reduction in the maximum light output. Most individuals show a free-running period greater than 24 h. Manipulation of the photophase and exposure to skeleton photoperiods showed that light acts as both a masking and an entraining agent and suggests that the underlying circadian rhythm is sinusoidal in the absence of light-based masking. Manipulation of thermoperiod in DD showed that temperature cycles are an alternative entraining agent. Exposure to a period of daily feeding in DD failed to entrain the rhythm in the laboratory. The endogenous regulation of luminescence poses questions about periodicity and synchronization of bioluminescence in cave glowworms.

Gotta love a paper in which Drosophila is used only as food for the organism under study (for the food-entrainment experiment)! Reminds me of the old departmental games of “my organism eats yours” back in grad school.
Anyway, all of the experiments in this paper were done on rainforest glow-worms, not the cave-dwelling ones. And as far as I know this is the first attempt to do any chronobiological studies on this organism, so the authors did the logical thing and performed a standard battery of tests in the lab: monitoring the glowing intensity rhythms in constant darkness (showing that the rhythm is driven endogenously, by an internal clock) and in light-dark cycles (showing that the rhythm is entrainable by light and with what phase, i.e., that the insect larvae are nocturnal, although the cave animals glow while it is light outside):
glow-worm.JPG
In addition, since they are interested in cave-dwelling organisms, they tested the ability of temperature cycles fo entrain the rhythm (it worked) as well as scheduled feeding times (this did not work).
But the impetus for the work, unlike what the media article suggests (tourism!), is evolutionary:

We conclude that glowworms exhibit true circadian
regulation of their light output. Light acts as both an
entraining agent and a masking agent. The dominant
role of light in establishing the characteristics of the
light output rhythm raises questions about the rhythmicity
and period of bioluminescence within caves
where glowworms have never been exposed to daylight.
A number of species such as A. luminosa from
New Zealand and A. tasmaniensis from Tasmania,
Australia, have large populations in caves as well as in
rainforest. Based on laboratory analyses of A. flava,
glowworms in caves would either be arrhythmic
because they have never been exposed to photic
entrainment cues, or would be rhythmic but individuals
in a colony would be asynchronous because they
have different free-running periods. It will be of interest
to establish the rhythmicity and phase of luminescence
in cave-dwelling glowworm populations. The
fact that members of the genus Arachnocampa inhabit
both photoperiodic and aphotoperiodic habitats
makes them ideal for examination of the retention of
circadian rhythmicity in cave environments where
very few circadian cues are present.

So, I expect that the authors will next attempt a comparative study – pitting the rainforest and cave-dwelling populations of the same species directly against each other in a similar battery of experiments. I am looking forward to seeing the results.
Merritt, D.J., Aotani, S. (2008). Circadian Regulation of Bioluminescence in the Prey-Luring Glowworm, Arachnocampa flava. Journal of Biological Rhythms, 23(4), 319-329. DOI: 10.1177/0748730408320263

Why do earthworms come up to the surface after the rain?

ResearchBlogging.orgBelieve it or not, this appears to have something to do with their circadian rhythms!
Back in the 1960s and early 1970s, there was quite a lot of research published on the circadian rhythms in earthworms, mostly by Miriam Bennett. As far as I can tell, nobody’s followed up on that work since. I know, from a trusted source, that earthworms will not run in running-wheels, believe it or not! The wheels were modified to contain a groove down the middle (so that the worm can go only in one direction and not off the wheel), the groove was covered with filter paper (to prevent the worm from escaping the groove) and the paper was kept moist with some kind of automated sprinkler system. Still, the earthworms pretty much stood still and the experiments were abandoned.
Dr.Bennett measured locomotion rhythms in other ways, as well as rhythms of oxygen consumption, light-avoidance behavior, etc. With one of my students, some years ago, I tried to use earthworms as well – we placed groups of worms in different lighting conditions (they were inside some soil, but not deep enough for them to completely avoid light) – the data were messy and inconclusive, except that worms kept in constant light all laid egg-cases and all died (evolutionary trade-off between longevity and fecundity, or just a last-ditch effort at reproduction before imminent death?). Worms in (short-day and long-day) LD cycles and in constant dark did not lay eggs and more-or-less survived a few days.
I intended to write a long post reviewing the earthworm clock literature, but that was before I got a job….perhaps one day. But the news today is that there is a new paper that suggests that clocks may have something to do with a behavior all of us have seen before: earthworms coming out to the surface during or after a rain.
In the paper, Role of diurnal rhythm of oxygen consumption in emergence from soil at night after heavy rain by earthworms, Shu-Chun Chuang and Jiun Hong Chen from the Institute of Zoology at National Taiwan University, compared responses of two different species of earthworms, one of which sufraces during rain and the other does not. They say:

Two species of earthworms were used to unravel why some earthworm species crawl out of the soil at night after heavy rain. Specimens of Amynthas gracilis, which show this behavior, were found to have poor tolerance to water immersion and a diurnal rhythm of oxygen consumption, using more oxygen at night than during the day. The other species, Pontoscolex corethrurus, survived longer under water and was never observed to crawl out of the soil after heavy rain; its oxygen consumption was not only lower than that of A. gracilis but also lacked a diurnal rhythm. Accordingly, we suggest that earthworms have at least two types of physical strategies to deal with water immersion and attendant oxygen depletion of the soil. The first is represented by A. gracilis; they crawl out of the waterlogged soil, especially at night when their oxygen consumption increases. The other strategy, shown by P. corethrurus, allows the earthworms to survive at a lower concentration of oxygen due to lower consumption; these worms can therefore remain longer in oxygen-poor conditions, and never crawl out of the soil after heavy rain.

So, one species has low oxygen consumption AND no rhythm of it. It survives fine, for a long time, when the soil is saturated with water. The other species has greater oxygen consumption and is thus more sensitive to depletion of oxygen when the ground is saturated with water. Furthermore, they also exhibit a daily rhythm of oxygen consumption – they consume more oxygen during the night than during the day. Thus, if it rains during the day, they may or may not surface, but if it rains as night they have to resurface pretty quickly.
Aydin Orstan describes the work in more detail on his blog Snail’s Tales, and he gets the hat-tip for alerting me to this paper.
Chuang, S., Chen, J.H. (2008). Role of diurnal rhythm of oxygen consumption in emergence from soil at night after heavy rain by earthworms. Invertebrate Biology, 127(1), 80-86. DOI: 10.1111/j.1744-7410.2007.00117.x

Clocks in Bacteria V: How about E.coli?

Clocks in Bacteria V: How about E.coli?Fifth in the five-part series on clocks in bacteria, covering more politics than biology (from May 17, 2006):

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Clocks in Bacteria IV: Clocks in other bacteria

Clocks in Bacteria IV: Clocks in other bacteriaFourth in the five-part series on clocks in bacteria (from April 30, 2006):

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Clocks in Bacteria III: Evolution of Clocks in Cyanobacteria

Clocks in Bacteria III: Evolution of Clocks in CyanobacteriaThe third installment in the five-part series on clocks in bacteria (from April 19, 2006):

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Clocks in Bacteria II: Adaptive Function of Clocks in Cyanobacteria

Clocks in Bacteria II: Adaptive Function of Clocks in CyanobacteriaSecond post in a series of five (from April 05, 2006):

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Clocks in Bacteria I: Synechococcus elongatus

Clocks in Bacteria I: Synechococcus elongatus
First in a series of five posts on clocks in bacteria (from March 08, 2006)…

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Circadian Clocks in Microorganisms

Circadian Clocks in MicroorganismsThe first in a series of posts on circadian clocks in microorganisms (from February 23, 2006)…

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Daily Rhythms in Cnidaria

Daily Rhythms in CnidariaThe origin and early evolution of circadian clocks are far from clear. It is now widely believed that the clocks in cyanobacteria and the clocks in Eukarya evolved independently from each other. It is also possible that some Archaea possess clock – at least they have clock genes, thought to have arived there by lateral transfer from cyanobacteria.[continued under the fold]

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Do sponges have circadian clocks?

Blogging on Peer-Reviewed Research

Do sponges have circadian clocks?Much of the biological research is done in a handful of model organisms. Important studies in organisms that can help us better understand the evolutionary relationships on a large scale tend to be hidden far away from the limelight of press releases and big journals. Here’s one example (March 30, 2006):

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Postscript to Pittendrigh’s Pet Project – Phototaxis, Photoperiodism and Precise Projectile Parabolas of Pilobolus on Pasture Poop

Postscript to Pittendrigh's Pet Project - Phototaxis, Photoperiodism and Precise Projectile Parabolas of Pilobolus on Pasture PoopWe have recently covered interesting reproductive adaptations in mammals, birds, insects, flatworms, plants and protists. For the time being (until I lose inspiration) I’ll try to leave cephalopod sex to the experts and the pretty flower sex to the chimp crew.
In the meantime, I want to cover another Kingdom – the mysterious world of Fungi. And what follows is not just a cute example of a wonderfully evolved reproductive strategy, and not just a way to couple together my two passions – clocks and sex – but also (at the very end), an opportunity to post some of my own hypotheses online.

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Mel-Mel-Mel: it’s easy to remember in snowshoe hares

It has been almost three years since I promised to write a post detailing the photoperiodic response in mammals. (Birds are more complicated).
Now Shelley gives a good example – the snowshoe hare which changes color annually: it is dark in summer and white in winter. It is pretty easy to remember – it’s all the Mel-something molecules involved. So, here is a very simplified, but essentially correct description of how this happens:
Light is detected by the photo-pigment melanopsin in the retinal ganglion cells of the eye. The cells send a signal to the clock (in the suprachiasmatic nucleus, SCN).
SCN sends a signal to the pineal gland. During the night, when it is dark, the pineal gland responds to the SCN signal by synthetizing and releasing the hormone melatonin into the bloodstream. The duration of the melatonin release is an indicator of the length of the night: long night = winter, short night = summer.
Melatonin receptors are found in the SCN, in some other places in the brain, and in some other places in the body. In the snowshoe hare, one of the targets of melatonin is the hypothalamo-pituitary system that controls the deposition of the pigment melanin into the hair follicles.
Thus, in summer, melanin gets deposited into the hair follicles and the hair that grows out of them is dark. At the onset of winter, when the clock starts detecting the shortening of the day (i.e., lengthening of the night-time melatonin signal), melanin is supressed and the dark hair is replaced with white hair (and more of it) instead.

Clocks and Migratory Orientation in Monarch Butterflies

Blogging on Peer-Reviewed Research

I had no time to read this in detail and write a really decent overview here, perhaps I will do it later, but for now, here are the links and key excerpts from a pair of exciting new papers in PLoS Biology and PLoS ONE, which describe the patterns of expression of a second type of cryptochrome gene in Monarch butterflies.
This cryptochrome (Cry) is more similar to the vertebrate Cry than the insect Cry, also present in this butterfly. The temporal and spatial patterns of expression of the two types of Cry suggest that they may be involved in the transfer of time-information from the circadian clock to the brain center involved in spatial orientation during long-distance migration.
The PLoS Biology paper looks at these patterns of expression, while the PLoS ONE paper identifies a whole host of genes potentially implicated in migratory behavior, including the Cry2. Here is the PLoS Biology paper:
Cryptochromes Define a Novel Circadian Clock Mechanism in Monarch Butterflies That May Underlie Sun Compass Navigation:

During their spectacular fall migration, eastern North American monarch butterflies (Danaus plexippus) use a time-compensated sun compass to help them navigate to their overwintering sites in central Mexico. The circadian clock plays a critical role in monarch butterfly migration by providing the timing component to time-compensated sun compass orientation. Here we characterize a novel molecular clock mechanism in monarchs by focusing on the functions of two CRYPTOCHROME (CRY) proteins. In the monarch clock, CRY1, a Drosophila-like protein, functions as a blue-light photoreceptor for photic entrainment, whereas CRY2, a vertebrate-like protein, functions within the clockwork as the major transcriptional repressor of the self-sustaining feedback loop. An oscillating CRY2-positive neural pathway was also discovered in the monarch brain that may communicate circadian information directly from the circadian clock to the central complex, which is the likely site of the sun compass. The monarch clock may be the prototype of a clock mechanism shared by other invertebrates that express both CRY proteins, and its elucidation will help crack the code of sun compass orientation.

Here is the editorial synopsis:
In Monarchs, Cry2 Is King of the Clock:

Back in the brain, the authors showed that Cry2 was also found in a few dozen cells in brain regions previously linked to time-keeping in the butterfly, and this Cry2 underwent circadian oscillation in these cells, but not in many other cells that were not involved in time keeping. By taking samples periodically over many hours, they found that nuclear localization of Cry2 coincided with maximal transcriptional repression of the clockwork, in keeping with its central role of regulating the feedback cycle. This is a novel demonstration of nuclear translocation of a clock protein outside flies.
Finally, the authors investigated Cry2′s activity in the central complex, the brain structure that is believed to house the navigational compass of the monarch. Monarchs integrate information on the position of the sun and the direction of polarized light to find their way from all over North America to the Mexican highlands, where they spend the winter. Cry2, but not the other clock proteins, was detected in parts of the central complex where it undergoes strong circadian cycling. Some cells containing Cry2 linked up with the clock cells, while others projected toward the optic lobe and elsewhere in the brain.
Along with highlighting the central importance of Cry2 in the inner workings of the monarch’s clock, the results in this study suggest that part of the remarkable navigational ability of the butterfly relies on its ability to integrate temporal information from the clock with spatial information from its visual system. This allows the monarch to correct its course as light shifts across the sky over the course of the day. Other cues used for charting its path remain to be elucidated.

This is the PLoS ONE paper:
Chasing Migration Genes: A Brain Expressed Sequence Tag Resource for Summer and Migratory Monarch Butterflies (Danaus plexippus):

North American monarch butterflies (Danaus plexippus) undergo a spectacular fall migration. In contrast to summer butterflies, migrants are juvenile hormone (JH) deficient, which leads to reproductive diapause and increased longevity. Migrants also utilize time-compensated sun compass orientation to help them navigate to their overwintering grounds. Here, we describe a brain expressed sequence tag (EST) resource to identify genes involved in migratory behaviors. A brain EST library was constructed from summer and migrating butterflies. Of 9,484 unique sequences, 6068 had positive hits with the non-redundant protein database; the EST database likely represents ~52% of the gene-encoding potential of the monarch genome. The brain transcriptome was cataloged using Gene Ontology and compared to Drosophila. Monarch genes were well represented, including those implicated in behavior. Three genes involved in increased JH activity (allatotropin, juvenile hormone acid methyltransfersase, and takeout) were upregulated in summer butterflies, compared to migrants. The locomotion-relevant turtle gene was marginally upregulated in migrants, while the foraging and single-minded genes were not differentially regulated. Many of the genes important for the monarch circadian clock mechanism (involved in sun compass orientation) were in the EST resource, including the newly identified cryptochrome 2. The EST database also revealed a novel Na+/K+ ATPase allele predicted to be more resistant to the toxic effects of milkweed than that reported previously. Potential genetic markers were identified from 3,486 EST contigs and included 1599 double-hit single nucleotide polymorphisms (SNPs) and 98 microsatellite polymorphisms. These data provide a template of the brain transcriptome for the monarch butterfly. Our “snap-shot” analysis of the differential regulation of candidate genes between summer and migratory butterflies suggests that unbiased, comprehensive transcriptional profiling will inform the molecular basis of migration. The identified SNPs and microsatellite polymorphisms can be used as genetic markers to address questions of population and subspecies structure.

Here is an article written after the press release, which, as such articles usually do, greatly overstates the extent of the findings:
Clocking monarch migration:

In previous work, Reppert and his team showed that pigment-producing genes in the monarch eye communicate with the butterfly’s circadian clock. As part of the new study, Reppert and his team also found, in an area of the monarch brain called the central complex, a definitive molecular and cellular link between the circadian clock and the monarch’s ability to navigate using the sun. Briscoe said that Reppert’s study was “really going to overturn a lot of views we had about the specific components of circadian clocks.”

The spatial and temporal patterns of expression make Cry2 the most serious candidate for the connection between the clock and the Sun-compass orientation mechanism. Much work, both at the molecular and at higher levels of organization needs to be done to figure out the exact mechanism by which this animal, during migration, compensates for the Sun’s movement across the sky during the day, and thus does not stray off course. Cry2 appears to be a good molecular “handle” for such studies.
For background, see my older post on the initial discovery of Cry2 in Monarch butterflies by the same team.

Me and the Copperheads

Me and the CopperheadsLast week I had lunch with a good old friend of mine, Jim Green. He got his degree in Zoology, then a law degree (patent law) and is now coming back for yet another degree in biological and chemical engineering. He did his research on snakes, so we reminisced and laughed about the time several years ago (that was before Kevin joined the lab, which is why I was recruited for this study in the first place) when we were taking blood samples from copperheads.

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Do sponges have circadian clocks?

Do sponges have circadian clocks?Much of the biological research is done in a handful of model organisms. Important studies in organisms that can help us better understand the evolutionary relationships on a large scale tend to be hidden far away from the limelight of press releases and big journals. Here’s one example (March 30, 2006):

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Biological Clocks in Protista

Biological Clocks in ProtistaWriting a chronobiology blog for a year and a half now has been quite a learning experience for me. I did not know how much I did not know (I am aware that most of my readers know even less, but still….). Thus, when I wrote about clocks in birds I was on my territory – this is the stuff I know first-hand and have probably read every paper in the field. The same goes for topics touching on seasonality and photoperiodism as my MS Thesis was on this topic. I feel equally at home when discussing evolution of clocks. I am also familiar with the clocks in some, but not all, arthropods. And that is all fine and well….but, my readers are anthropocentric. They want more posts about humans – both clocks and sleep – something I knew very little about. So, I have learned a lot over the past year and a half by digging through the literature and books on the subject. I was also forced to learn more about the molecular machinery of the circadian clock as most newsworthy (thus bloggable) new papers are on the clock genetics.

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Flirting under Moonlight on a Hot Summer Night, or, The Secret Night-Life of Fruitflies

Blogging on Peer-Reviewed Research

As we mentioned just the other day, studying animal behavior is tough as “animals do whatever they darned please“. Thus, making sure that everything is controlled for in an experimental setup is of paramount importance. Furthermore, for the studies to be replicable in other labs, it is always a good idea for experimental setups to be standardized. Even that is often not enough. I do not have access to Science but you may all recall a paper from several years ago in which two labs tried to simultaneously perform exactly the same experiment in mice, using all the standard equipment, exactly the same protocols, the same strain bought from the same supplier on the same date, the same mouse-feed, perhaps even the same colors of technicians’ uniforms and yet, they got some very different data!
The circadian behavior is, fortunately, not chaotic, but quite predictable, robust and easily replicable between labs in a number of standard model organisms. Part of the success of the Drosophila research program in chronobiology comes from the fact that for decades all the labs used exactly the same experimental apparatus, this one, produced by Trikinetics (Waltham, Massachusetts) and Carolina Biologicals (Burlington, North Carolina):
drosophila%20apparatus.jpg
This is a series of glass tubes, each containing a single insect. An infrared beam crosses the middle of each tube and each time the fly breaks the beam, by walking or flying up and down the tube, the computer registers one “pen deflection”. All of those are subsequently put together into a form of an actograph, which is the standard format for the visual presentation of chronobiological data, which can be further statistically analyzed.
The early fruitfly work was done mainly in Drosophila pseudoobscura. Most of the subsequent work on fruitfly genetics used D.melanogaster instead. Recently, some researchers started using the same setup to do comparative studies of other Drosophila species. Many fruitfly clock labs have hundreds, even thousands, of such setups, each contained inside a “black box” which is essentially an environmental chamber in which the temperature and pressure are kept constant, noise is kept low and constant (“white noise”), and the lights are carefully controlled – exact timing of lights-on and lights-off as well as the light intensity and spectrum.
In such a setup, with a square-wave profile of light (abrupt on and off switches), every decent D.melanogaster in the world shows this kind of activity profile:
fruitfly%20crepuscular.JPG
The activity is bimodal: there is a morning peak (thought to be associated with foraging in the wild) and an evening peak (thought to be associated with courtship and mating in the wild).
The importance of standardization is difficult to overemphasize – without it we would not be able to detect many of the subtler mutants, and all the data would be considered less trustworthy. Yet, there is something about standardization that is a negative – it is highly artificial. By controlling absolutely everything and making the setup as simple as possible, it becomes very un-representative of the natural environment of the animal. Thus, the measured behavior is also likely to be quite un-natural.
Unlike in the lab, the fruitflies out in nature do not live alone – they congregate with other members of the species. Unlike in a ‘black box’, the temperature fluctuates during the day and night in the real world. Also unlike the lab, the intensity and spectrum of light change gradually during the duration of the day while the nights are not pitch-black: there are stars and the Moon providing some low-level illumination as well. Thus, after decades of standardized work, it is ripe time to start investigating how the recorded behaviors match up with the reality of natural behavior in fruitflies.
Three recent papers address these questions by modifying the experimental conditions in one way or another, introducing additional environmental cues that are usually missing in the standard apparatus (and if you want to know what they found, follow me under the fold):

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Diurnal Rhythm of Deep-Sea Diving in Whale Sharks

Yup, that was going to be the title of this post. I got the paper and was ready to write the post when I noticed that Peter scooped me and posted about the same paper today (yup, there is just not that many cool papers on Charismatic Marine Megavertebrates to spread around this week). I have nothing to add, so just go and see his post:

The results demonstrated that a free-ranging whale shark displays ultradian, diel and circa-lunar rhythmicity of diving behaviour. Whale sharks dive to over 979.5 m, making primarily diurnal deep dives and remaining in relatively shallow waters at night.

Do whales sleep?

It is Marine Megavertebrate Week right now, so why not take a look at one of the most Mega of the Megaverts – the grey whale (Eschrichtius robustus):
Eschrichtius%20robustus.jpg
Do whales sleep? You may have heard that dolphins do – one hemisphere at the time, while swimming, and not for very long periods at a time. A combined Russian/US team of researchers published a study in 2000 – to my knowledge the best to date – on sleep-wake and activity patterns of the grey whale: Rest and activity states in a gray whale (pdf) by Lyamin, Manger, Mukhametov, Siegel and Shpak.

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Cortisol necessary for circadian rhythm of cell division

A new paper just came out today on PLoS-Biology: Glucocorticoids Play a Key Role in Circadian Cell Cycle Rhythms. The paper is long and complicated, with many control experiments, etc, so I will just give you a very brief summary of the main finding.
One of the three major hypotheses for the origin of circadian clocks is the need to shield sensitive cellular processes – including cell division – from the effects of UV radiation by the sun, thus relegating it to night-time only:

The cyclic nature of energetic availability and cycles of potentially degrading effects of the sun’s ultraviolet rays on particular pigmented enzymes, provided the selective environment. A cell with a timer can predict the changes and adjust its metabolic activities to minimize energetic and material loss. This cell will outcompete the other cells in the Archeozoic sea (Pittendrigh 1967).

Biological clocks in various organisms regulate timing of many different biochemical, physiological and behavioral events, but the circadian control of cell-cycle is really ubiqutous – it has been found in everything from bacteria to humans.
In many large organisms, the distinction between pacemakers and peripheral clocks is mainly in the ability of the pacemaker to synchronize itself to the outside environment and to send daily signals that act to synchronize all the peripheral clocks in all the cells in the body. The local clocks, then, regulate timing of local events.
In some organisms, peripheral clocks are also capable of direct sampling of the environment. Zebrafish is one such animal – its peripheral clocks (in every cell of the body) are photosensitive and entrainable directly by light-dark cycles. Only in constant light conditions does the brain pacemaker assume the role of the “conductor of the orchestra”, synchronizing all the clocks in the body.
In vertebrates, it has been thought for a long time now that the central pacemaker (the SCN in the hypothalamus of the brain) uses, among else, cortisol as a signal for synchronizing the peripheral clocks. It times the release of corticotropins from the pituitary which in turn releases cortisol from the adrenal gland into the circulation at particular time of day. Various tissues are sensitive to cortisol and will use its surge as a timing/entraining signal.
In this paper, circadian rhythms of cell-division were shown to get attenuated in mutants that do not produce corticotropins (and thus do not produce cortisol). However, the clock genes still cycle normally in the periphery. Placing the fish in continous bath of cortisol agonist reinstates the circadian rhythms of cell division.
This suggests that cortisol is not a timing signal from the center to the periphery as the peripheral clocks keep cycling in its absence (and entrain directly – no need for any input from the eyes).
This also suggests that cortisol is neccessary for the coupling of the peripheral clock mechanism and its own output – the cell cycle. The presence of cortisol need not be rhythmic – it just needs to be there if the clock is to time the daily rhythms of cell division.

Me and the Copperheads

Last week I had lunch with a good old friend of mine, Jim Green. He got his degree in Zoology, then a law degree (patent law) and is now coming back for yet another degree in biological and chemical engineering. He did his research on snakes, so we reminisced and laughed about the time several years ago (that was before Kevin joined the lab, which is why I was recruited for this study in the first place) when we were taking blood samples from copperheads.
What we wanted to do is see if snakes have melatonin and if so, if it shows a diurnal rhythm in concentration like it does in other Vertebrates (believe it or not, nobody’s done that yet) and the copperheads were the only snakes he had, about ten of them, each in its own terrarium in a tiny shed outside of campus.
So, we needed to take blood samples at noon and, after a few days of recovery, again at midnight. So, we went in at noon one day. Jim would pick up a snake and hold it by its head. My lab budy Christ Steele was holding the body of the snake. Jim’s advisor Hal Heatwole was taking the blood samples straight from the heart, and I was the “nurse assistant” taking care of needles, syringes, anticoagulant, test-tubes, etc. The whole thing, ten snakes, took perhaps an hour or so and worked out perfectly without any glitches.
copperhead.jpg
About a week later, when we came for a repeat session at midnight, we were starkly reminded that copperheads are nocturnal animals. They were active. And I mean ACTIVE! Due to acute effects of light on depressing melatonin release, we had to take samples in very dim red light, with some highly uncooperative snakes. The process took hours!
At one point one of the snakes got lose in the room and, since the room was practically completely dark, I could not see where it was underneath the cages. So I said “OK, you snake guys figure out where it is and call me back once you have it under control” and I slid out of the door. I got teased for this act of cowardice for years afterwards.
Unfortunately, the melatonin essay repeatedly did not work and we did not have enough blood volume to try with a new kit, so the study was never completed. The snakes got used in other experiments, Jim finished and defended his Thesis and left town and nobody else wanted to try to do a repeat. I hope one day someone will. Perhaps with a non-venomous snake species for a change – makes midnight sampling much safer and easier!
[image]

Small Arctic Mammals Entrain to Something during the Long Summer Day

There are several journals dedicated to biological rhythms or sleep. Of those I regularly check only two or three of the best, so I often miss interesting papers that occur in lower-tier journals. Here is one from December 2006 that caught my eye the other day:
Mammalian activity – rest rhythms in Arctic continuous daylight:

Activity – rest (circadian) rhythms were studied in two species of Arctic mammals living in Arctic continuous daylight with all human-induced regular environmental cues (zeitgebers) removed. The two Arctic species (porcupine and ground squirrel) lived outdoors in large enclosures while the Arctic summer sun circled overhead for 82 days. Would local animals maintained under natural continuous daylight demonstrate the Aschoff effect described in previously published laboratory experiments using continuous light, in which rats’ circadian activity patterns changed systematically to a longer period, expressing a 26-hour day of activity and rest? The outdoor experiments reported here, however, showed that under natural continuous daylight, both species (porcupine and ground squirrel) had specific times of activity and rest on a nearly 24-hour scale, and their activity peaks did not come later each day. The daily rhythms of the two species were recorded using implanted physiological radio capsules, and from direct observation.

You may recall that I wrote about a similar study in a much larger Arctic mammal – the reindeer, which loses the overt behavioral rhythmicity during the long summer. Apparently, these two small mammals, the porcupine and ground squirrel are different.
In the press release, they explain:

It seemed that although the scientists were very careful not to provide time cues of any sort, the animals had managed to latch onto something that gave them regularity.
“I have written for years that experimental animals seem to be hungry for cues, or time signals, to keep on a regular cycle,” Folk said. “So we tried to figure out what cue the wild animals were using, and we could find only one thing that kept a 24 hour periodicity. At Barrow, the sun travels in a circle overhead for 82 days, but at midnight the circle is tipped to the north.
“We postulate that the animals are conscious of where the sun is in the sky and that the nearness of the sun to the horizon could be a clue to animals, and even plants, to keep on a 24-hour schedule.”

This is an interesting hypothesis: not just using the clock to orient by Sun, but also using the Sun posiiton to entrain the clock. I hope this gets tested and that this was not just a case of investigators missing an alternative environmental cue. Changes in the Earth’s magnetic field show daily oscillations and are potentially one of such alternative cues that animals could use. Just like Dr.Folk states in the article, I’d also like to see this study replicated in Arctic birds, as they are known to be sensitive to the magnetic field which they can use for migratory orientation.

VIP synchronizes mammalian circadian pacemaker neurons

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No other aspect of behavioral biology is as well understood at the molecular level as the mechanism that generates and sustains circadian rhythms. If you are following science in general, or this blog in particular, you are probably familiar with the names of circadian clock genes like per, tim, clk, frq, wc, cry, Bmal, kai, toc, doubletime, rev-erb etc.
The deep and detailed knowledge of the genes involved in circadian clock function has one unintended side-effect, especially for people outside the field. If one does not stop and think for a second, it is easy to fall under the impression that various aspects of the circadian oscillations, e.g., period, phase and amplitude, are determined by the clock genes. After all, most of these genes were discovered by the study of serendipitously occuring mutations, usually period-mutations.
If the circadian properties are really deteremined by clock genes, then the predictions from this hypothesis are that: 1) every cell in the body shows the same period (phase, amplitude, etc.), 2) every cell in the body has the same period throughout its life, 3) every cell removed from the body and placed in the dish continues to oscillate with the same period as it had inside the body, and 4) the properties of the circadian rhythms are not alterable by environmental influences.
Stated this bluntly, one has to recoil in horror: of course it is not determined by genes! But without such an exercise in thinking, much work and writing (especially by the press) tacitly assumes the strict genetic determination. However, the experimental data show this not to be true. Period (and other properties) of the whole organism’s rhythms are readily modified by environmental influences, e.g., light intensity (Aschoff Rule), heavy water, lithium, sex steroids and melatonin. They change with age and reproductive state. There is individual variation even in clonal species (or highly-inbred laboratory strains). The period of the rhythms measured in cells or cell-types in a dish is not always the same as exhibited by the same cells inside the organism. Finally, the occurrence of splitting (of one unified circadian output into two semi-independent components differing in period)suggests that two or more groups of circadian pacemakers simultaneously exhibit quite different periods within the same animal.
Several years ago, Dr. Eric Herzog (disclosure – a good friend) has shown that even the individual pacemaker cells within the same SCN (suprachiasmatic nucleus – the site of the main mammalian circadian clock in the brain) exhibit different periods. When dispersed in culture, pacemaker neurons (originally taken from a single animal) tend to show a broad variation in periods (amplitudes, etc…).
As they grow cellular processes, two neurons in a dish may touch and form connections. As soon as they do, their periods change and from then on the two cells show the SAME period, i.e., they are synchronized. As more and more cells connect, they build an entire network of neurons, all cycling in sync with each other – same period, same phase, same amplitude. This is assumed to happen inside the whole animal as well – the unconnected SCN neurons of the fetus start making connections just before (or immediately after, depending on the species) birth and as a result, an overt, whole-organism rhythm emerges out of arrhytmic background.
But, what molecules are involved in cell-cell communication that allows the pacemaker cells to synchronize their rhythms? For several years, the most likely candidate was thought to be GABA, an inhibitory neurotransmitter produced by all SCN cells. Sara Aton, a graduate student in Eric Herzog’s lab (now postdoc at UPenn), set out to test this hypothesis. Over a few years and several papers, a different story emerged, culminating in this months paper in PNAS:
GABA and Gi/o differentially control circadian rhythms and synchrony in clock neurons. (by Aton SJ, Huettner JE, Straume M and Herzog ED).
What the paper shows – and there is a lot of detail there, so you can read the paper for yourself if interested, or at least the media coverage (here, here and here) – is that various perturbations of the GABA system, either at the synthesis end or the reception end, have, at best, some mild effects on amplitude and phase. There was no effect of GABA on period of individual pacemaker neurons. Yet, effect on period is neccessary for mutual synchronization of cells into a network. Instead, VIP (vasoactive intestinal polypeptide) was shown to be the agent that, by modulating period, allowed spatially coupled cells to also temporally couple – to synchronize their circadian oscillations.
This is a much more important finding than you may think at the first glance. The naive idea of a single clock driving all the overt rhythms has been abandoned for more than half a century. Every important problem in chronobiology – coherence of rhythms, temperature compensation, communication between pacemakers and peripheral clocks, entrainment to environmental cycles, etc. – hinges on the properties of the multi-clock networks. Understanding the biochemical mechanism by which pacemaker cells syncronize with each other is thus a key finding that will allow us to study those phenomena at a cellular and molecular level. Right now, and due to Sara Aton’s work, VIP is the “handle” we will use to revisit those old problems and test our pet hypotheses about the coupling of circadian systems in various animals (the reasonable assumption being that mouse is not unique in using VIP and that this molecule is probably used for the same function in all vertebrates). We can now study exactly how two cells communicate by VIP to synchronize their clocks – is the pattern of VIP release, for instance, used as a kind of temporal “code”?
Probably the most important such phenomenon to study is splitting. Different kinds of spliting have been observed in lizards, starlings, tree-shrews (Tupaia), mice, rats, hamsters, marmosets and many other animals under various experimental conditions, e.g., constant light, constant darkness, removal of the pineal, infusion with testosterone, or exposure to skeleton photocycles. Splitting can be induced by highly artificial experimental protocols, e.g., alternate eye-patching, or they may appear spontaneusly in animals out in the wild, something that can be replicated in the laboratory.
In some cases, it has been shown that the splitting is lateral, i.e., left SCN drives one component and the right SCN drives the other. In the case of alternate eye-patching, it is reasonable to hypothesize that the same thing is happening. But in other cases, it is more likely that each SCN splits into two subsets of neurons, synchronized within but not between the two groups. Is VIP the synchronizer in both groups? In all cases? In all animals?
If, for instance, rhythms split into two componenets under the influence of testosterone, is VIP used for coupling within each of those two semi-independent “clocks”? Does one group of neurons, insensitive to testosterone, use VIP to synchronize its output, while the other group of neurons, under the influence of testosterone, changes its period and also uses VIP to synchronize its output?
Now that we know to use VIP and not GABA as an entry into the system, all of these questions will be much more amenable to future research – an exciting prospect for me and many others in the field.

Eight Hours a Circadian Rhythm Do Not Make

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Eight Hours a Circadian Rhythm Do Not MakeThis post is a relatively recent (May 24, 2006) critique of a PLoS paper.

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Serotonin, Melatonin, Immunity and Cancer

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Serotonin, Melatonin, Immunity and CancerMaking connections (from January 22, 2006)…

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Waking Experience Affects Sleep Need in Drosophila

Blogging on Peer-Reviewed Research

There is nothing easier than taking a bad paper – or a worse press release – and fisking it with gusto on a blog. If you happen also to know the author and keep him in contempt, the pleasure of destroying the article is even greater.
It is much, much harder to write (and to excite readers with) a blog post about an excellent paper published by your dear friends. But I’ll try to do this now anyway (after the cut).

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Ah, Zugunruhe!

Ah, Zugunruhe! How birds know when and where to migrate (from April 03, 2006)

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Estrogen, Aggression and Photoperiod

Randy Nelson is a wonderful person, an engaging speaker and the author of the best textbook on Behavioral Endocrinology. I heard that he is also a great teacher which does not surprise me and he has a talent for attracting some of the best students and postdocs to work in his lab. Oh, by the way, he also does some great research.
For decades, the study of seasonality and photoperiodism was a hustling bustling field, until everyone jumped on the clock-gene bandwagon. Randy Nelson is one of the rare birds to remain in the photoperiodism field, coming out every year with more and more exciting papers. Here is just the latest one – supercool! [excerpt under the fold]

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Circadian Rhythms, or Not, in Arctic Reindeer

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Circadian Rhythms, or Not, in Arctic ReindeerA January 20, 2006 post placing a cool physiological/behavioral study into an evolutionary context.

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Another Clock Gene

Considering that circadian clocks were first discovered in plants, and studied almost exclusively in plants for almost a century before people started looking at animals in the early 20th century, it is somewhat surprising that the molecular aspects of the circadian rhythm generation mechanisms have lagged behind those in insects, vertebrates, fungi and bacteria. It is always nice to see a paper reporting a discovery of a new plant clock gene:
New function for protein links plant s circadian rhythm to its light-detection mechanism:

Plants set their clocks by detecting the light cycle, and Chua’s lab found that an accessory protein, called SPA1, is important for keeping the internal clock set. When they bred Arabidopsis plants with a mutated SPA1 protein, the plants flowered early, producing shoots and flowers weeks ahead of wild-type plants.
“The regulation of flowering initiation in response to the length of the day is mediated by the interaction of light with the plant s circadian clock system,” says Chua. Plants detect light with proteins called phytochromes and cryptochromes. SPA1 regulates one of these phytochromes, called PhyA.
The PhyA protein links light detection with the circadian clock system and directly influences when a plant flowers. But Chua’s finding suggests that SPA1 normally represses PhyA function, holding the plant back from flowering until the right time. “We knew that SPA1 negatively regulated PhyA immediately after germination, but didn t know if it played a role in the adult,” says Chua. “Our results show that SPA1 is important in the adult for regulating PhyA and the circadian period. When SPA1 is mutated, the plants precociously flower, affecting their entire reproductive cycle.”

Diversity of insect circadian clocks – the story of the Monarch butterfly

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Diversity of insect circadian clocks - the story of the Monarch butterflyFrom January 20, 2006, on the need to check the model-derived findings in non-model organisms.

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Influence of Light Cycle on Dominance Status and Aggression in Crayfish

Influence of Light Cycle on Dominance Status and Aggression in CrayfishIn this post from April 06, 2006, I present some unpublished data that you may find interesting.

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Clocks in Bacteria V: How about E.coli?

Clocks in Bacteria V: How about E.coli?Fifth in the five-part series on clocks in bacteria, covering more politics than biology (from May 17, 2006):

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Clocks in Bacteria IV: Clocks in other bacteria

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Clocks in Bacteria IV: Clocks in other bacteriaFourth in the five-part series on clocks in bacteria (from April 30, 2006):

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Clocks in Bacteria III: Evolution of Clocks in Cyanobacteria

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Clocks in Bacteria III: Evolution of Clocks in CyanobacteriaThe third installment in the five-part series on clocks in bacteria (from April 19, 2006):

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Clocks in Bacteria II: Adaptive Function of Clocks in Cyanobacteria

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Clocks in Bacteria II: Adaptive Function of Clocks in CyanobacteriaSecond post in a series of five (from April 05, 2006):

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Clocks in Bacteria I: Synechococcus elongatus

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Clocks in Bacteria I: Synechococcus elongatus
First in a series of five posts on clocks in bacteria (from March 08, 2006)…

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Circadian Clocks in Microorganisms

Circadian Clocks in MicroorganismsThe first in a series of posts on circadian clocks in microorganisms (from February 23, 2006)…

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Biological Effects of the Moon

I rarely write about biological rhythms outside of circadian range (e.g., circannual, circalunar, circatidal rhythms etc.), but if you liked this post on lunar rhythms in antlions, you will probably also like this little review of lunar rhythms in today’s Nature:
Pull of the Moon:

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Studies of fiddler crabs, for example, have shown that even when kept in the lab under constant light and temperature, the animals are still most active at the times that the tide would be out. A similar internal ‘circalunar’ clock is thought to tick inside many animals, running in synchrony with the Moon and tides, and working in conjunction with the animal’s 24-hour circadian clock. This is thought to help animals anticipate tide movements; a skill that might give some creatures an edge. Ecologist Martin Wikelski of Princeton University, New Jersey, has found for example, that Galapagos marine iguanas with the most accurate circalunar clock are more likely to survive tough times, presumably because they are best at reaching feeding spots first.
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Moonlight can also change animal behaviour. Many marine organisms move up and down in the sea depending on the level of moonlight in order to keep their light levels constant. On land, some nocturnal animals come out on a well-lit night to hunt, others stay hidden to avoid predators.
And African dung beetles, oddly, can walk in a straighter line when the Moon is out: Eric Warrant at the University at the University of Lund, Sweden, and his colleagues reported in 2003 that Scarabaeus zambesianus can detect the pattern of polarized moonlight in the night sky and use it to navigate2. This means they can roll their dung balls in a straight line on a moonlit night.
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And yeah, for the anthropocentric readers, the article has a bunch on humans as well….

Chossat’s Effect in humans and other animals

Chossat’s Effect in humans and other animalsChossat's Effect in humans and other animalsThis April 09, 2006 post places another paper of ours (Reference #17) within a broader context of physiology, behavior, ecology and evolution.
The paper was a result of a “communal” experiment in the lab, i.e., it was not included in anyone’s Thesis. My advisor designed it and started the experiment with the first couple of birds. When I joined the lab, I did the experiment in an additional number of animals. When Chris joined the lab, he took over the project and did the rest of the lab work, including bringin in the idea for an additional experiment that was included, and some of the analysis. We all talked about it in our lab meetings for a long time. In the end, the boss did most of the analysis and all of the writing, so the order of authors faithfully reflects the relative contributions to the work.
What is not mentioned in the post below is an additional observation – that return of the food after the fasting period induced a phase-shift of the circadian system, so we also generated a Phase-Response Curve, suggesting that food-entrainable pacemaker in quail is, unlike in mammals, not separate from the light-entrainable system.
Finally, at the end of the post, I show some unpublished data – a rare event in science blogging.

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Phase-Response Curve and T-Cycles: Clocks and Photoperiodism in Quail

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This is a summary of my 1999 paper, following in the footsteps of the work I described here two days ago. The work described in that earlier post was done surprisingly quickly – in about a year – so I decided to do some more for my Masters Thesis.
The obvious next thing to do was to expose the quail to T-cycles, i.e., non-24h cycles. This is some arcane circadiana, so please refer to the series of posts on entrainment from yesterday and the two posts on seasonality and photoperiodism posted this morning so you can follow the discussion below:
There were three big reasons for me to attempt the T-cycle experiment at that time:

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Does circadian clock regulate clutch-size in birds? A question of appropriatness of the model animal.

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 Does circadian clock regulate clutch-size in birds? A question of appropriatness of the model animal.This post from March 27, 2006 starts with some of my old research and poses a new hypothesis.

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Quail: How many clocks?

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One of the assumptions in the study of circadian organization is that, at the level of molecules and cells, all vertebrate (and perhaps all animal) clocks work in roughly the same way. The diversity of circadian properties is understood to be a higher-level property of interacting multicelular and multi-organ circadian systems: how the clocks receive environmental information, how the multiple pacemakers communicate and synchronize with each other, how they convey the temporal information to the peripheral clocks in all the other cells in the body, and how perpheral clocks generate observable rhythms in biochemistry, physiology and behavior.

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