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Circadian clock without DNA–History and the power of metaphor 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.


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.


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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

Sigma Xi pizza lunch lecture – Science in the current media environment

Next Tuesday at Sigma Xi:

Hi all. Normally we aim to hold pizza lunch on the 3rd Tuesday of each month. In November, that date conflicts with the ship date of the January-February 2011 issue of American Scientist. So we’ll convene a week later. Still, I think you’ll find the session—something different this time—worth the wait.

Join us on Tuesday, Nov. 23 to hear one of our own, veteran science blogger Bora Zivkovic, talk about the shifting ecosystems within his craft. Zivkovic has had a front seat to much of that change, as author of the influential A Blog Around The Clock, as co-founder (with Anton Zuiker) of the international conference ScienceOnline in RTP, as the former online community manager at Public Library of Science and, now, as the new blog and community editor for Scientific American magazine. For a long time, people spoke of the day when print and online media would converge. In a growing share of the publishing world, that convergence has occurred. And Bora, when it comes to science journalism, has been a catalyst in that change.

Thanks to a grant from the N.C. Biotechnology Center, American Scientist Pizza Lunch is free and open to science journalists and science communicators of all stripes. Feel free to forward this message to anyone who might want to attend. RSVPs are required (for the slice count) to

Directions to Sigma Xi, the Scientific Research Society in RTP, are here:

ScienceWriters2010 – NASW/CASW meeting this week

ScienceWriters2010 is starting on Friday afternoon at Yale University in New Haven, CT. This is a joint meeting of the National Association of Science Writers (NASW) and the Council for the Advancement of Science Writing (CASW).

I am not exactly sure, but I think most sessions will be recorded in some fashion and made available online later.

It is much better, if you want to follow live, to bookmarks the official ScienceWriters2010 blog where recipients of the travel grants – mostly science journalism students or young freelancers – will cover all the sessions in as close to real-time as is possible.

The fellows will also tweet from the meeting, and you can follow them by subscribing to this Twitter list. Or you can follow everyone from the conference by saving a Twitter search for the #sciwri10 hashtag.

I will be on a panel Rebooting science journalism: Adapting to the new media landscape, put together by David Dobbs. My co-panelists are Emily Bell and Betsy Mason. That should be fun!

Definitely check out the rest of the schedule – it is awesome. Everyone’s biggest problem is that all those great sessions are happening simultaneously, so we’ll all also have to wait for recordings of our colleagues’ sessions afterward.

And if you are there and you see me, please come and say Hi!

Quick Links

Do Open Networks Threaten Brands? (Pt. 2)

Blogging: The skill that begets all others

5 things journalists should learn from bloggers

Another example of the power of blogging

Blaschko’s Lines

An Ancient Sea Monster’s Fearsome Fins

The heart of an octopus is a fickle thing…

Social Cognition in Polar Bears

Expedition records show severe orangutan decline

Why Russians Don’t Get Depressed

Bleached to Death

Megafauna meltdown and Step-dads from Hell

The Zen of Presentations, Part 34: Lessons from the blind

Argument from Authority vs. Trusting Experts

Scientific spectating

Non-traditional alternatives to grant funding

Ecology in Prison

Yay! J. Neuroscience Agrees with Me that ‘Supplementary Materials’ is BS and Ruining Science! and Supplemental materials or no? and More questions about supplemental materials and Disrupting with data.

The shroud of retraction: Virology Journal withdraws paper about whether Christ cured a woman with flu

It’s Going Too Fast — Can Embargoes Manage the Real-time Web?

You Are the Person You Are Now

What I know about Marc Hauser, the recently ‘investigated’ Harvard primatologist

It’s that time again: ‘Broken’ peer review and Let us not skip so lightly past censorship effects of bad peer review, Orac.

XMRV: Not in spooge

Smells From the Past: The Fulton Fish Market

Pro gamers head to Raleigh for showdown

Toward a better agriculture… for everyone

Stop wasting food, save the world’s energy

Millions Of Barrels Of Oil Safely Reach Port In Major Environmental Catastrophe

New and Exciting in PLoS this week

There are new articles in four PLoS journals today. As always, you should rate the articles, post notes and comments and send trackbacks when you blog about the papers. You can now also easily place articles on various social services (CiteULike, Mendeley, Connotea, Stumbleupon, Facebook and Digg) with just one click. Here are my own picks for the week – you go and look for your own favourites:

Do Humans Optimally Exploit Redundancy to Control Step Variability in Walking?:

Existing principles used to explain how locomotion is controlled predict average, long-term behavior. However, neuromuscular noise continuously disrupts these movements, presenting a significant challenge for the nervous system. One possibility is that the nervous system must overcome all neuromuscular variability as a constraint limiting performance. Conversely, we show that humans walking on a treadmill exploit redundancy to adjust stepping movements at each stride and maintain performance. This strategy is not required by the task itself, but is predicted by appropriate stochastic control models. Thus, the nervous system simplifies control by strongly regulating goal-relevant fluctuations, while largely ignoring non-essential variations. Properly determining how stochasticity affects control is critical to developing biological models, since neuro-motor fluctuations are intrinsic to these systems. Our work unifies the perspectives of time series analysis researchers, motor coordination researchers, and motor control theorists by providing a single dynamical framework for studying variability in the context of goal-directedness.

Morphometric Relationship, Phylogenetic Correlation, and Character Evolution in the Species-Rich Genus Aphis (Hemiptera: Aphididae):

The species-rich genus Aphis consists of more than 500 species, many of them host-specific on a wide range of plants, yet very similar in general appearance due to convergence toward particular morphological types. Most species have been historically clustered into four main phenotypic groups (gossypii, craccivora, fabae, and spiraecola groups). To confirm the morphological hypotheses between these groups and to examine the characteristics that determine them, multivariate morphometric analyses were performed using 28 characters measured/counted from 40 species. To infer whether the morphological relationships are correlated with the genetic relationships, we compared the morphometric dataset with a phylogeny reconstructed from the combined dataset of three mtDNA and one nuclear DNA regions. Based on a comparison of morphological and molecular datasets, we confirmed morphological reduction or regression in the gossypii group unlike in related groups. Most morphological characteristics of the gossypii group were less variable than for the other groups. Due to these, the gossypii group could be morphologically well separated from the craccivora, fabae, and spiraecola groups. In addition, the correlation of the rates of evolution between morphological and DNA datasets was highly significant in their diversification. The morphological separation between the gossypii group and the other species-groups are congruent with their phylogenetic relationships. Analysis of trait evolution revealed that the morphological traits found to be significant based on the morphometric analyses were confidently correlated with the phylogeny. The dominant patterns of trait evolution resulting in increased rates of short branches and temporally later evolution are likely suitable for the modality of Aphis speciation because they have adapted species-specifically, rapidly, and more recently on many different host plants.

Widespread Presence of Human BOULE Homologs among Animals and Conservation of Their Ancient Reproductive Function:

While sexual reproduction is widespread among animals, it remains enigmatic to what extent sexual reproduction is conserved and when sex-specific gametogenesis (spermatogenesis and oogenesis) originated in animals. Here we demonstrate the presence of the reproductive-specific protein Boule throughout bilaterally-symmetric animals (Bilateria) and the conservation of its male reproductive function in mice. Examination of Boule evolution in insect and mammalian lineages, representing the Protostome and Deuterostome clades of bilateral animals, failed to detect any evidence for accelerated evolution. Instead, purifying selection is the major force behind Boule evolution. Further investigation of Boule homologs among Deuterostome species revealed reproduction-specific expression, with a strong prevalence of testis-biased expression. We further determined the function of a deuterostomian Boule homolog by inactivating Boule in mice (a representative mammal, a class of Deuterostomes). Like its counterpart in Drosophila (a representative of the opposing Protostome clade), mouse Boule is also required only for male reproduction. Loss of mouse Boule prevents sperm production, resulting in a global arrest of spermatogenesis in remarkable similarity to that of Drosophila boule mutants. Our findings are consistent with a common origin for male gametogenesis among metazoans and reveal the high conservation of a reproduction-specific protein among bilaterian animals.

Dynamics of Person-to-Person Interactions from Distributed RFID Sensor Networks:

Digital networks, mobile devices, and the possibility of mining the ever-increasing amount of digital traces that we leave behind in our daily activities are changing the way we can approach the study of human and social interactions. Large-scale datasets, however, are mostly available for collective and statistical behaviors, at coarse granularities, while high-resolution data on person-to-person interactions are generally limited to relatively small groups of individuals. Here we present a scalable experimental framework for gathering real-time data resolving face-to-face social interactions with tunable spatial and temporal granularities. We use active Radio Frequency Identification (RFID) devices that assess mutual proximity in a distributed fashion by exchanging low-power radio packets. We analyze the dynamics of person-to-person interaction networks obtained in three high-resolution experiments carried out at different orders of magnitude in community size. The data sets exhibit common statistical properties and lack of a characteristic time scale from 20 seconds to several hours. The association between the number of connections and their duration shows an interesting super-linear behavior, which indicates the possibility of defining super-connectors both in the number and intensity of connections. Taking advantage of scalability and resolution, this experimental framework allows the monitoring of social interactions, uncovering similarities in the way individuals interact in different contexts, and identifying patterns of super-connector behavior in the community. These results could impact our understanding of all phenomena driven by face-to-face interactions, such as the spreading of transmissible infectious diseases and information.

Phylogenomic Analysis of Marine Roseobacters:

Members of the Roseobacter clade which play a key role in the biogeochemical cycles of the ocean are diverse and abundant, comprising 10-25% of the bacterioplankton in most marine surface waters. The rapid accumulation of whole-genome sequence data for the Roseobacter clade allows us to obtain a clearer picture of its evolution. In this study about 1,200 likely orthologous protein families were identified from 17 Roseobacter bacteria genomes. Functional annotations for these genes are provided by iProClass. Phylogenetic trees were constructed for each gene using maximum likelihood (ML) and neighbor joining (NJ). Putative organismal phylogenetic trees were built with phylogenomic methods. These trees were compared and analyzed using principal coordinates analysis (PCoA), approximately unbiased (AU) and Shimodaira-Hasegawa (SH) tests. A core set of 694 genes with vertical descent signal that are resistant to horizontal gene transfer (HGT) is used to reconstruct a robust organismal phylogeny. In addition, we also discovered the most likely 109 HGT genes. The core set contains genes that encode ribosomal apparatus, ABC transporters and chaperones often found in the environmental metagenomic and metatranscriptomic data. These genes in the core set are spread out uniformly among the various functional classes and biological processes. Here we report a new multigene-derived phylogenetic tree of the Roseobacter clade. Of particular interest is the HGT of eleven genes involved in vitamin B12 synthesis as well as key enzynmes for dimethylsulfoniopropionate (DMSP) degradation. These aquired genes are essential for the growth of Roseobacters and their eukaryotic partners.

Risk-Sensitive Optimal Feedback Control Accounts for Sensorimotor Behavior under Uncertainty:

In economic decision-making it is well-known that when decision-makers have several options, each associated with uncertain outcomes, their decision is not purely determined by the average payoff, but also takes into account the risk (that is, variability of the payoff) associated with each option. Some actions have a highly variable payoff, such as betting money on a horse, whereas others are much less variable, such as the return from a savings account. Whether an individual favors one action over the other depends on their risk-attitude. In contrast to economic decision-making, models of human motor control have exclusively focussed on models that maximize average rewards (minimize average cost). Here, we consider a computational model (an optimal feedback controller) that takes the variance of the cost into account when calculating the best movement strategy. We compare the model with the performance of human subjects in a sensorimotor task and find that the subjects’ behavior is consistent with the predictions of a risk-sensitive optimal feedback controller with most subjects being risk-averse.

New and Exciting in PLoS ONE

There are 29 new articles in PLoS ONE today. As always, you should rate the articles, post notes and comments and send trackbacks when you blog about the papers. You can now also easily place articles on various social services (CiteULike, Mendeley, Connotea, Stumbleupon, Facebook and Digg) with just one click. Here are my own picks for the week – you go and look for your own favourites:

Chimpanzees Extract Social Information from Agonistic Screams:

Chimpanzee (Pan troglodytes) agonistic screams are graded vocal signals that are produced in a context-specific manner. Screams given by aggressors and victims can be discriminated based on their acoustic structure but the mechanisms of listener comprehension of these calls are currently unknown. In this study, we show that chimpanzees extract social information from these vocal signals that, combined with their more general social knowledge, enables them to understand the nature of out-of-sight social interactions. In playback experiments, we broadcast congruent and incongruent sequences of agonistic calls and monitored the response of bystanders. Congruent sequences were in accordance with existing social dominance relations; incongruent ones violated them. Subjects looked significantly longer at incongruent sequences, despite them being acoustically less salient (fewer call types from fewer individuals) than congruent ones. We concluded that chimpanzees categorised an apparently simple acoustic signal into victim and aggressor screams and used pragmatics to form inferences about third-party interactions they could not see.

Conserving the Stage: Climate Change and the Geophysical Underpinnings of Species Diversity:

Conservationists have proposed methods for adapting to climate change that assume species distributions are primarily explained by climate variables. The key idea is to use the understanding of species-climate relationships to map corridors and to identify regions of faunal stability or high species turnover. An alternative approach is to adopt an evolutionary timescale and ask ultimately what factors control total diversity, so that over the long run the major drivers of total species richness can be protected. Within a single climatic region, the temperate area encompassing all of the Northeastern U.S. and Maritime Canada, we hypothesized that geologic factors may take precedence over climate in explaining diversity patterns. If geophysical diversity does drive regional diversity, then conserving geophysical settings may offer an approach to conservation that protects diversity under both current and future climates. Here we tested how well geology predicts the species diversity of 14 US states and three Canadian provinces, using a comprehensive new spatial dataset. Results of linear regressions of species diversity on all possible combinations of 23 geophysical and climatic variables indicated that four geophysical factors; the number of geological classes, latitude, elevation range and the amount of calcareous bedrock, predicted species diversity with certainty (adj. R2 = 0.94). To confirm the species-geology relationships we ran an independent test using 18,700 location points for 885 rare species and found that 40% of the species were restricted to a single geology. Moreover, each geology class supported 5-95 endemic species and chi-square tests confirmed that calcareous bedrock and extreme elevations had significantly more rare species than expected by chance (P<0.0001), strongly corroborating the regression model. Our results suggest that protecting geophysical settings will conserve the stage for current and future biodiversity and may be a robust alternative to species-level predictions.

The Influence of Perceptual Training on Working Memory in Older Adults:

Normal aging is associated with a degradation of perceptual abilities and a decline in higher-level cognitive functions, notably working memory. To remediate age-related deficits, cognitive training programs are increasingly being developed. However, it is not yet definitively established if, and by what mechanisms, training ameliorates effects of cognitive aging. Furthermore, a major factor impeding the success of training programs is a frequent failure of training to transfer benefits to untrained abilities. Here, we offer the first evidence of direct transfer-of-benefits from perceptual discrimination training to working memory performance in older adults. Moreover, using electroencephalography to evaluate participants before and after training, we reveal neural evidence of functional plasticity in older adult brains, such that training-induced modifications in early visual processing during stimulus encoding predict working memory accuracy improvements. These findings demonstrate the strength of the perceptual discrimination training approach by offering clear psychophysical evidence of transfer-of-benefit and a neural mechanism underlying cognitive improvement.

Very Bright Green Fluorescent Proteins from the Pontellid Copepod Pontella mimocerami:

Fluorescent proteins (FP) homologous to the green fluorescent protein (GFP) from the jellyfish Aequorea victoria have revolutionized biomedical research due to their usefulness as genetically encoded fluorescent labels. Fluorescent proteins from copepods are particularly promising due to their high brightness and rapid fluorescence development. Here we report two novel FPs from Pontella mimocerami (Copepoda, Calanoida, Pontellidae), which were identified via fluorescence screening of a bacterial cDNA expression library prepared from the whole-body total RNA of the animal. The proteins are very similar in sequence and spectroscopic properties. They possess high molar extinction coefficients (79,000 M−1 cm−) and quantum yields (0.92), which make them more than two-fold brighter than the most common FP marker, EGFP. Both proteins form oligomers, which we were able to counteract to some extent by mutagenesis of the N-terminal region; however, this particular modification resulted in substantial drop in brightness. The spectroscopic characteristics of the two P. mimocerami proteins place them among the brightest green FPs ever described. These proteins may therefore become valuable additions to the in vivo imaging toolkit.

Effectiveness of Biodiversity Surrogates for Conservation Planning: Different Measures of Effectiveness Generate a Kaleidoscope of Variation:

Conservation planners represent many aspects of biodiversity by using surrogates with spatial distributions readily observed or quantified, but tests of their effectiveness have produced varied and conflicting results. We identified four factors likely to have a strong influence on the apparent effectiveness of surrogates: (1) the choice of surrogate; (2) differences among study regions, which might be large and unquantified (3) the test method, that is, how effectiveness is quantified, and (4) the test features that the surrogates are intended to represent. Analysis of an unusually rich dataset enabled us, for the first time, to disentangle these factors and to compare their individual and interacting influences. Using two data-rich regions, we estimated effectiveness using five alternative methods: two forms of incidental representation, two forms of species accumulation index and irreplaceability correlation, to assess the performance of ‘forest ecosystems’ and ‘environmental units’ as surrogates for six groups of threatened species–the test features–mammals, birds, reptiles, frogs, plants and all of these combined. Four methods tested the effectiveness of the surrogates by selecting areas for conservation of the surrogates then estimating how effective those areas were at representing test features. One method measured the spatial match between conservation priorities for surrogates and test features. For methods that selected conservation areas, we measured effectiveness using two analytical approaches: (1) when representation targets for the surrogates were achieved (incidental representation), or (2) progressively as areas were selected (species accumulation index). We estimated the spatial correlation of conservation priorities using an index known as summed irreplaceability. In general, the effectiveness of surrogates for our taxa (mostly threatened species) was low, although environmental units tended to be more effective than forest ecosystems. The surrogates were most effective for plants and mammals and least effective for frogs and reptiles. The five testing methods differed in their rankings of effectiveness of the two surrogates in relation to different groups of test features. There were differences between study areas in terms of the effectiveness of surrogates for different test feature groups. Overall, the effectiveness of the surrogates was sensitive to all four factors. This indicates the need for caution in generalizing surrogacy tests.

A Model for Transgenerational Imprinting Variation in Complex Traits:

Despite the fact that genetic imprinting, i.e., differential expression of the same allele due to its different parental origins, plays a pivotal role in controlling complex traits or diseases, the origin, action and transmission mode of imprinted genes have still remained largely unexplored. We present a new strategy for studying these properties of genetic imprinting with a two-stage reciprocal F mating design, initiated with two contrasting inbred lines. This strategy maps quantitative trait loci that are imprinted (i.e., iQTLs) based on their segregation and transmission across different generations. By incorporating the allelic configuration of an iQTL genotype into a mixture model framework, this strategy provides a path to trace the parental origin of alleles from previous generations. The imprinting effects of iQTLs and their interactions with other traditionally defined genetic effects, expressed in different generations, are estimated and tested by implementing the EM algorithm. The strategy was used to map iQTLs responsible for survival time with four reciprocal F populations and test whether and how the detected iQTLs inherit their imprinting effects into the next generation. The new strategy will provide a tool for quantifying the role of imprinting effects in the creation and maintenance of phenotypic diversity and elucidating a comprehensive picture of the genetic architecture of complex traits and diseases.

The Genetic, Morphological, and Physiological Characterization of a Dark Larval Cuticle Mutation in the Butterfly, Bicyclus anynana:

Studies on insect melanism have greatly contributed to our understanding of natural selection and the ultimate factors influencing the evolution of darkly pigmented phenotypes. Research on several species of melanic lepidopteran larvae have found that low levels of circulating juvenile hormone (JH) titers are associated with a melanic phenotype, suggesting that genetic changes in the JH biosynthetic pathway give rise to increased deposition of melanin granules in the cuticle in this group. But does melanism arise through different molecular mechanisms in different species? The present study reports on a Bicyclus anynana (Lepidoptera: Nymphalidae) dark larvae single locus mutation, in which larvae exhibit a darker cuticle relative to wild type. Unlike other lepidopteran melanic larvae mutations, this one is autosomal recessive and does not appear to involve a deficiency in JH titers. Unlike JH deficiency mutants, dark larvae mutants display similar growth rates and sexual behaviors as wild type, and topical application of a JH analogue failed to rescue the wild type cuticular coloration. Finally, transmission electron microscopy showed that sclerotization or deposition of diffuse melanin, rather than deposition of melanin granules, produces the dark coloration found in the cuticle of this species. We conclude that different molecular mechanisms underlie larval melanism in different species of Lepidoptera.

Seasonal Changes in Colour: A Comparison of Structural, Melanin- and Carotenoid-Based Plumage Colours:

Plumage coloration is important for bird communication, most notably in sexual signalling. Colour is often considered a good quality indicator, and the expression of exaggerated colours may depend on individual condition during moult. After moult, plumage coloration has been deemed fixed due to the fact that feathers are dead structures. Still, many plumage colours change after moult, although whether this affects signalling has not been sufficiently assessed. We studied changes in coloration after moult in four passerine birds (robin, Erithacus rubecula; blackbird, Turdus merula; blue tit, Cyanistes caeruleus; and great tit, Parus major) displaying various coloration types (melanin-, carotenoid-based and structural). Birds were caught regularly during three years to measure plumage reflectance. We used models of avian colour vision to derive two variables, one describing chromatic and the other achromatic variation over the year that can be compared in magnitude among different colour types. All studied plumage patches but one (yellow breast of the blue tit) showed significant chromatic changes over the year, although these were smaller than for a typical dynamic trait (bill colour). Overall, structural colours showed a reduction in relative reflectance at shorter wavelengths, carotenoid-based colours the opposite pattern, while no general pattern was found for melanin-based colours. Achromatic changes were also common, but there were no consistent patterns of change for the different types of colours. Changes of plumage coloration independent of moult are probably widespread; they should be perceivable by birds and have the potential to affect colour signalling.

A Quantitative Analysis of Flight Feather Replacement in the Moustached Tree Swift Hemiprocne mystacea, a Tropical Aerial Forager:

The functional life span of feathers is always much less than the potential life span of birds, so feathers must be renewed regularly. But feather renewal entails important energetic, time and performance costs that must be integrated into the annual cycle. Across species the time required to replace flight feather increases disproportionately with body size, resulting in complex, multiple waves of feather replacement in the primaries of many large birds. We describe the rules of flight feather replacement for Hemiprocne mystacea, a small, 60g tree swift from the New Guinea region. This species breeds and molts in all months of the year, and flight feather molt occurs during breeding in some individuals. H. mystacea is one to be the smallest species for which stepwise replacement of the primaries and secondaries has been documented; yet, primary replacement is extremely slow in this aerial forager, requiring more than 300 days if molt is not interrupted. We used growth bands to show that primaries grow at an average rate of 2.86 mm/d. The 10 primaries are a single molt series, while the 11 secondaries and five rectrices are each broken into two molt series. In large birds stepwise replacement of the primaries serves to increase the rate of primary replacement while minimizing gaps in the wing. But stepwise replacement of the wing quills in H. mystacea proceeds so slowly that it may be a consequence of the ontogeny of stepwise molting, rather than an adaptation, because the average number of growing primaries is probably lower than 1.14 feathers per wing.

Sexual Experience Promotes Adult Neurogenesis in the Hippocampus Despite an Initial Elevation in Stress Hormones:

Aversive stressful experiences are typically associated with increased anxiety and a predisposition to develop mood disorders. Negative stress also suppresses adult neurogenesis and restricts dendritic architecture in the hippocampus, a brain region associated with anxiety regulation. The effects of aversive stress on hippocampal structure and function have been linked to stress-induced elevations in glucocorticoids. Normalizing corticosterone levels prevents some of the deleterious consequences of stress, including increased anxiety and suppressed structural plasticity in the hippocampus. Here we examined whether a rewarding stressor, namely sexual experience, also adversely affects hippocampal structure and function in adult rats. Adult male rats were exposed to a sexually-receptive female once (acute) or once daily for 14 consecutive days (chronic) and levels of circulating glucocorticoids were measured. Separate cohorts of sexually experienced rats were injected with the thymidine analog bromodeoxyuridine in order to measure cell proliferation and neurogenesis in the hippocampus. In addition, brains were processed using Golgi impregnation to assess the effects of sexual experience on dendritic spines and dendritic complexity in the hippocampus. Finally, to evaluate whether sexual experience alters hippocampal function, rats were tested on two tests of anxiety-like behavior: novelty suppressed feeding and the elevated plus maze. We found that acute sexual experience increased circulating corticosterone levels and the number of new neurons in the hippocampus. Chronic sexual experience no longer produced an increase in corticosterone levels but continued to promote adult neurogenesis and stimulate the growth of dendritic spines and dendritic architecture. Chronic sexual experience also reduced anxiety-like behavior. These findings suggest that a rewarding experience not only buffers against the deleterious actions of early elevated glucocorticoids but actually promotes neuronal growth and reduces anxiety.

Dietary Determinants of Changes in Waist Circumference Adjusted for Body Mass Index – a Proxy Measure of Visceral Adiposity:

Given the recognized health effects of visceral fat, the understanding of how diet can modulate changes in the phenotype “waist circumference for a given body mass index (WCBMI)”, a proxy measure of visceral adiposity, is deemed necessary. Hence, the objective of the present study was to assess the association between dietary factors and prospective changes in visceral adiposity as measured by changes in the phenotype WCBMI. We analyzed data from 48,631 men and women from 5 countries participating in the European Prospective Investigation into Cancer and Nutrition (EPIC) study. Anthropometric measurements were obtained at baseline and after a median follow-up time of 5.5 years. WCBMI was defined as the residuals of waist circumference regressed on body mass index, and annual change in WCBMI (ΔWCBMI, cm/y) was defined as the difference between residuals at follow-up and baseline, divided by follow-up time. The association between energy, energy density (ED), macronutrients, alcohol, glycemic index (GI), glycemic load (GL), fibre and ΔWCBMI was modelled using centre-specific adjusted linear regression, and random-effects meta-analyses to obtain pooled estimates. Men and women with higher ED and GI diets showed significant increases in their WCBMI, compared to those with lower ED and GI [1 kcal/g greater ED predicted a ΔWCBMI of 0.09 cm (95% CI 0.05 to 0.13) in men and 0.15 cm (95% CI 0.09 to 0.21) in women; 10 units greater GI predicted a ΔWCBMI of 0.07 cm (95% CI 0.03 to 0.12) in men and 0.06 cm (95% CI 0.03 to 0.10) in women]. Among women, lower fibre intake, higher GL, and higher alcohol consumption also predicted a higher ΔWCBMI. Results of this study suggest that a diet with low GI and ED may prevent visceral adiposity, defined as the prospective changes in WCBMI. Additional effects may be obtained among women of low alcohol, low GL, and high fibre intake.

New and Exciting in PLoS this week

There are many new articles in four PLoS journals today. As always, you should rate the articles, post notes and comments and send trackbacks when you blog about the papers. You can now also easily place articles on various social services (CiteULike, Mendeley, Connotea, Stumbleupon, Facebook and Digg) with just one click. Here are my own picks for the week – you go and look for your own favourites:

Barcoding Life to Conserve Biological Diversity: Beyond the Taxonomic Imperative:

In the 250 years since the Swedish scientist Carl Linnaeus first started classifying organisms, taxonomists have formally described roughly 1.7 million species. Although seemingly large, this number represents only a small fraction of the estimated tens of millions of species on the planet. Moreover, human activities are causing the extinction of species hundreds of times faster than the natural rate of extinction found in the fossil record. Fully one-third of all species on the planet may be gone by the end of this century–many without ever having been studied or, more importantly, protected [1].

DNA barcoding, developed in 2003 to identify species, has helped to rejuvenate taxonomic research. The science of taxonomy is key to understanding and monitoring biodiversity [2]. The technique is based on a simple but powerful observation: that sequence diversity, in short, standardized gene regions (i.e., DNA barcodes), can serve as a tool to identify known species and potentially discover new ones. Moreover, DNA barcoding allows researchers to develop a system for species identification based on digital characters, eventually allowing for automated identifications, thereby promising to improve the capacity to identify, monitor, and manage biodiversity, with profound societal and economic benefits. It also raises the possibility of identifying the vectors of zoonotic diseases as well as the disease organisms themselves.

Left to Their Own Devices: Breakdowns in United States Medical Device Premarket Review:

Medical devices encompass nearly every medical product that does not achieve its intended purpose through chemical action, from the simple (tongue blades) to the complex (MRI machines), and from the safe (stethoscopes) to the risky (artificial hearts) [1],[2]. Certain drug-device combinations, such as drug-eluting coronary stents, are also regulated as devices.

The number and complexity of medical devices have increased dramatically over the past several decades, often to the betterment of patients’ health. Between 1997 and 2006, the value of device sales roughly doubled to US$123 billion, representing a fairly consistent 6% of the nation’s health care expenditures [3].

The Center for Devices and Radiological Health (CDRH) at the Food and Drug Administration (FDA) is charged with ensuring the safety and effectiveness of medical devices. While a number of serious safety problems with devices have emerged–the Dalkon Shield [4], the Bjork-Shiley heart valve [5], and the Sprint Fidelis defibrillator lead [6], to name a few–problems with effectiveness are not as readily apparent once a device is on the market, in part because postmarket efficacy trials of approved devices are rare. Thus, the burden of ensuring device effectiveness is heavily weighted toward premarket evaluation.

Phylogenetic and Morphologic Analyses of a Coastal Fish Reveals a Marine Biogeographic Break of Terrestrial Origin in the Southern Caribbean:

Marine allopatric speciation involves interplay between intrinsic organismal properties and extrinsic factors. However, the relative contribution of each depends on the taxon under study and its geographic context. Utilizing sea catfishes in the Cathorops mapale species group, this study tests the hypothesis that both reproductive strategies conferring limited dispersal opportunities and an apparent geomorphologic barrier in the Southern Caribbean have promoted speciation in this group from a little studied area of the world. Mitochondrial gene sequences were obtained from representatives of the Cathorops mapale species group across its distributional range from Colombia to Venezuela. Morphometric and meristic analyses were also done to assess morphologic variation. Along a ~2000 km transect, two major lineages, Cathorops sp. and C. mapale, were identified by levels of genetic differentiation, phylogenetic reconstructions, and morphological analyses. The lineages are separated by ~150 km at the Santa Marta Massif (SMM) in Colombia. The northward displacement of the SMM into the Caribbean in the early Pleistocene altered the geomorphology of the continental margin, ultimately disrupting the natural habitat of C. mapale. The estimated ~0.86 my divergence of the lineages from a common ancestor coincides with the timing of the SMM displacement at ~0.78 my. Results presented here support the hypothesis that organismal properties as well as extrinsic factors lead to diversification of the Cathorops mapale group along the northern coast of South America. While a lack of pelagic larval stages and ecological specialization are forces impacting this process, the identification of the SMM as contributing to allopatric speciation in marine organisms adds to the list of recognized barriers in the Caribbean. Comparative examination of additional Southern Caribbean taxa, particularly those with varying life history traits and dispersal capabilities, will determine the extent by which the SMM has influenced marine phylogeography in the region.

Efficient Mitigation Strategies for Epidemics in Rural Regions:

Containing an epidemic at its origin is the most desirable mitigation. Epidemics have often originated in rural areas, with rural communities among the first affected. Disease dynamics in rural regions have received limited attention, and results of general studies cannot be directly applied since population densities and human mobility factors are very different in rural regions from those in cities. We create a network model of a rural community in Kansas, USA, by collecting data on the contact patterns and computing rates of contact among a sampled population. We model the impact of different mitigation strategies detecting closely connected groups of people and frequently visited locations. Within those groups and locations, we compare the effectiveness of random and targeted vaccinations using a Susceptible-Exposed-Infected-Recovered compartmental model on the contact network. Our simulations show that the targeted vaccinations of only 10% of the sampled population reduced the size of the epidemic by 34.5%. Additionally, if 10% of the population visiting one of the most popular locations is randomly vaccinated, the epidemic size is reduced by 19%. Our results suggest a new implementation of a highly effective strategy for targeted vaccinations through the use of popular locations in rural communities.

The Enigma of Soil Animal Species Diversity Revisited: The Role of Small-Scale Heterogeneity:

“The enigma of soil animal species diversity” was the title of a popular article by J. M. Anderson published in 1975. In that paper, Anderson provided insights on the great richness of species found in soils, but emphasized that the mechanisms contributing to the high species richness belowground were largely unknown. Yet, exploration of the mechanisms driving species richness has focused, almost exclusively, on above-ground plant and animal communities, and nearly 35 years later we have several new hypotheses but are not much closer to revealing why soils are so rich in species. One persistent but untested hypothesis is that species richness is promoted by small-scale environmental heterogeneity. To test this hypothesis we manipulated small-scale heterogeneity in soil properties in a one-year field experiment and investigated the impacts on the richness of soil fauna and evenness of the microbial communities. We found that heterogeneity substantially increased the species richness of oribatid mites, collembolans and nematodes, whereas heterogeneity had no direct influence on the evenness of either the fungal, bacterial or archaeal communities or on species richness of the large and mobile mesostigmatid mites. These results suggest that the heterogeneity-species richness relationship is scale dependent. Our results provide direct evidence for the hypothesis that small-scale heterogeneity in soils increase species richness of intermediate-sized soil fauna. The concordance of mechanisms between above and belowground communities suggests that the relationship between environmental heterogeneity and species richness may be a general property of ecological communities.

How Normal Cells Can Win the Battle for Survival Against Cancer Cells:

During the early stages of tumorigenesis, cancerous cells undergo rapid and uncontrolled cell division as they invade the surrounding tissue. How tumors create space around them to accomplish this invasion is not well understood. A recent study showed that cancerous cells in fruit flies manage this feat by inducing neighboring cells to spontaneously destroy themselves and then filling the vacated space left behind in a process known as cell competition. In this issue of PLoS Biology, Yoichiro Tamori et al. provide evidence that this battle also occurs in mammalian tissues and uncover what determines the winners and losers when cells compete.

Limbs Made to Measure:

This year marks the 150th anniversary of the birth of D’Arcy Thompson, the British biologist, classicist, and all round polymath (For more information on D’Arcy Thompson see Like many, he was fascinated by the appearance and structure of living matter, and in his influential book, On Growth and Form [1], he set out to describe and explain the principles of morphogenesis–the way living things grow and acquire their forms. Using a vast range of examples, from the honeycomb in beehives to the spirals in a snail’s shell, he emphasized that form should be studied in the context of growth and that to explain shape it was essential to understand the underlying mechanisms. This led to the central thesis of the book: biological forms are the result of mechanical and physical processes that should be described with mathematical precision.