What can a blind cavefish tell us about circadian clocks?

Circadian clocks and a revolving planet go hand-in-hand. But why so many plants and animals have a circadian clock from an evolutionary perspective is relatively unknown. One way to find out is to study animals that live in non-rhythmic environments. And at the end of 2013, my team published a study on exactly that: the circadian clock of the Mexican blind cavefish, Astyanax mexicanus, from data collected in the laboratory and in the fishes’ natural habitat. We showed that these cavefish fish shows wonderful daily patterns of behaviour and gene expression, confirming that it has a functional circadian clock.

The cavefish, Astyanax mexicanus, is a particularly special animal in that within the same species there are two very different body forms: a blind cave-dwelling fish and an eyed river-dwelling fish, which is very much like the ancestor of the cavefish. By comparing the surface fish and the cavefish you are able to see a snapshot in evolution, knowing that the main difference between the two forms is the environment of the cave, an extremely useful feature when studying something as connected to the environment as the circadian clock. The fish can also survive outside of caves and actually make popular pet fish, making it an excellent study animal.

Fishing in the dark
Fishing in the dark
CC-BY-SA 4.0 Andrew Beale

In this study, surface fish, cavefish from the Pachón cave and cavefish from the Chica cave were kept in an aquarium and given daily cycles of light and dark, simulating day and night. In this simulated environment, both cavefish and surface fish were highly rhythmic: circadian gene expression oscillated with peaks and troughs every 24 hours and both forms were more active in the light period than the dark. The oscillation of gene expression continued even when the lights were turned off, a strong indicator that an internal timer is in control of the expression pattern of these genes.

Paper Fig1 - behaviour traces greys
Cavefish have a working circadian clock. (a) A key core clock component, per1, is expressed in an oscillatory pattern over the 4 days of sampling – two days of 12 hours light and 12 hours dark, shown by white and grey backgrounds, and two days of constant dark. Per1 expression oscillates strongly in surface fish (red) as expected for fish, but it also oscillates in two populations of cavefish, Pachón and Chica, though the rhythm is a little weaker. (b) The fish are also behaviourally rhythmic, more active in the day than the night. However, the behaviour rhythm does not persist in constant darkness. Zeitgeber or circadian time refers to the number of hours after the start of the period of light, and is equivalent to clock time.
From: Figure 1 – Beale et al. (2013)

However, in the natural habitat a slightly different observation was made. Though surface fish in the rivers of Mexico show gene expression rhythms just like in the lab, the cavefish don’t show any rhythm at all – it is as if the clock is silent in the cave.

A closer look at the difference between the gene rhythms in the surface fish and the cavefish revealed something intriguing: the cavefish clock seemed to be actively repressed, as the expression level of one of the key components of the molecular clock, period1, was very low and not oscillating.

Paper Figure 4
Rhythms in the wild. Chica cave fish (b) show a very low level of per1 expression, and no circadian rhythm is detectable.
From: Figure 4 – Beale et al. (2013)

In fish, light is signalled to the molecular mechanism of the circadian clock through two genes in particular, per2 and cry1a. During the light of the day these two genes are highly expressed, in the night they are off. In zebrafish, when the expression of either of these two genes is increased artificially, the circadian clock is repressed – and the result looks something like what is seen in cavefish.

Could it be that this was the way by which the cavefish clock is repressed in caves? Quite extraordinarily, yes. Cavefish showed a significantly raised expression of per2 relative to surface fish as if the cavefish was experiencing a constant light input, even in the pitch blackness of the caves.

Paper Figure 2
Per2 is upregulated in cavefish – it’s expressed at a higher level in Pachón and Chica cavefish than surface fish during the night, when it should only be expressed at a very low level.
From: Figure 2 – Beale et al. (2013)

Why is it like this?

To answer this question, we need to consider the wider impact of evolving in the dark. Cavefish have adapted to life in the dark with a suite of characteristics – no eyes, lower metabolism, loss of pigmentation, larger jaws and a more sensitive touch or vibration sense. But, especially for fish, light signals also have a strong impact on cell biology, such as the activation of DNA repair processes and the regulation of the cell cycle. 

DNA repair shares a close relationship with the circadian clock, especially with genes in the pathway that signals light. Indeed, one class of DNA repair proteins, the photolyases, are within the same gene family as cryptochrome (cry) which, like per, is a key element of the molecular circadian clock. Especially in fish, many DNA repair genes and proteins are light-dependent.

Could there be a link in cavefish between changes in the light-signalling pathway of the circadian clock and DNA repair activity? It seems so – cavefish in the laboratory and in the field show higher expression of DNA repair genes and a greater ability to repair DNA damage in the dark, a really quite interesting result.

Paper Figure 5
DNA repair are upregulated in cavefish in the dark (compare light blue points in d and e with the grey points). In the lab, cavefish show significantly improved DNA repair in the dark, with fewer damaged DNA bases remaining 24 hours after the damage event
From: Figure 5 – Beale et al. (2013)

This increased activity in DNA repair is likely to be beneficial in an extreme environment like caves. The cave environment, especially that of the trapped water in the pools where the fish live, is particularly harsh. An animal that has ‘lost’ the ability to activate DNA repair events due to the lack of light would be at risk of significantly increased DNA damage. By increasing the activity of DNA repair genes in the dark through turning on the light-signalling pathway in the absence of light (by an as yet unknown mechanism), the cavefish would be at an advantage in the struggle to survive and reproduce.

That a functional clock is retained at all in cavefish, even in its damped form, is interesting. It cannot be due to coordinating with the external environment: this and two previous studies noted how there is no circadian rhythm in behaviour and a further study showed that another outputs of the circadian clock, the daily oscillation in basal metabolism, has been abolished in cavefish. Moran et al. suggested that the loss of the circadian rhythm in metabolism was an energy saving adaptation in an environment that did not require a rhythmic increase in energy metabolism to prepare for a day of foraging and predator evasion – another example of an external synchronisation mechanism that is not needed in the darkness of the caves.

All the results point to timing internal process. A molecular clock can still time aspects of internal physiology even when obvious signs of its action, like behaviour patterns and metabolic rhythms, are absent. How it does this, and what it is timing, is currently unknown. We’ve still got a great deal to discover about Astyanax and the secrets of its life underground.

IMG_9359 - no eye cavefish copy
An Astyanax mexicanus cavefish, just caught from its cave pool. After this picture, it went back into the water.
CC-BY-SA 4.0 Andrew Beale

Featured research

Beale et al., (2013), Circadian rhythms in Mexican blind cavefish Astyanax mexicanus in the lab and in the field, Nature Communications

Moran et al., (2014), Eyeless Mexican Cavefish Save Energy by Eliminating the Circadian Rhythm in Metabolism, PLoS ONE


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