The energetic rhythm of Mexican tetra

Studies of both mammals and fish have shown that the metabolism follows an autonomous circadian (daily) rhythm independent of the metabolic costs related to the daily variation in movement and feeding. Varying the metabolism throughout the day probably helps animals tune their physiology to daily rhythms in feeding, digestion and movement (e.g. hunting or evading predators).

But what about animals living in habitats with a constant food supply and no predators - do they need a circadian rhythm in metabolism? Caves are such habitats, and while caves are often food-poor places, they are usually predator free (due to the low abundance of potential prey) and many different kinds of animals have taken advantage of this and evolved cave-dwelling populations from surface ancestors. If we studied the daily metabolic rhythm of fully cave-adapted animals would they still retain the circadian rhythm of their surface ancestors?

The Mexican tetra Astyanax mexicanus is the perfect animal to investigate this question. This species has two distinct morphs: a fully-eyed surface-dwelling form and multiple cave-dwelling forms (with partial to total visual system degeneration) descended from surface ancestors.

Eyed surface and eyeless cave form of Astyanax mexicanus. The two forms can be bred together to produce hybrids.

Eyed surface and eyeless cave form of Astyanax mexicanus. The two forms can be bred together to produce hybrids.

This species is very useful for investigating evolutionary process as the different forms are still inter-breedable, allowing researchers to create hybrids and study the genetic traits that lead to different behaviours and appearances from similar genetic architecture. The surface form is assumed to be a good representative of the cave form ancestor based on the high degree of genetic similarity between the different living forms. This allows researchers to test interesting evolutionary hypotheses, such as whether the circadian rhythm of cave forms changed while evolving in a habitat virtually devoid of daily queues.

We investigated the daily variation in metabolic rate of surface and cave (Pachón) forms of A. mexicanus. To do this we used a standard piece of fish physiology equipment, a swim flume respirometer. This is a device that can measure the oxygen consumption rate of a single fish at a set swimming speed. After a few hours inside this flume, the fish acclimate to their new environment and swim steadily into the water current. Freshly aerated water is intermittently and automatically flushed through the respirometer, and the rate of oxygen decrease measured by opt odes (the oxygen consumption rate is strongly correlated with energy expenditure). Below is a schematic of the respirometer we used. 

A schematic of the 1.6L swim flume respirometer used to measure oxygen consumption over 7 days.

A schematic of the 1.6L swim flume respirometer used to measure oxygen consumption over 7 days.

Single surface or cave fish were placed in the respirometers and their oxygen consumption measured over 7 days in either a 12:12 hour light:dark photoperiod (12:12LD), or 24 hour constant darkness (24D).

Under a 12:12LD photoperiod, the surface exhibited a daily rhythm in oxygen consumption characteristic of many animals, with an increase during the day light hours (showed in the graphs below). Under constant dark conditions the surface fish also exhibited increased energy expenditure during the times of day that previously represented day light (called the subjective daytime). The increase in oxygen consumption during the subjective daytime shows that the metabolism of surface fish is under the control of an internal molecular circadian clock. Conversely, the cave form lacked a measurable circadian variation in oxygen consumption (see below).

Daily variation in oxygen consumption of A. mexicanus at a set swimming speed

Daily variation in oxygen consumption of A. mexicanus at a set swimming speed

Around the time we were preparing our findings for publication, Beale et al published a paper describing interesting alterations in the circadian behaviour and molecular clock of cave forms of Mexican tetra. As yet it is unclear whether the metabolism has been decoupled from an altered molecular clock in cave forms, or whether the altered molecular clock has caused a loss in metabolic rhythm. In any case, something very interesting has happened to the circadian biology during the evolution of these cave fish.

Would the loss of the metabolic circadian rhythm be useful to a cave fish? If food was limited in the caves, and the body didn't need to prepare for regular daily activities like feeding and predator avoidance, then removing the day time increase in energy use would certainly be advantageous in a cave. We calculated the predicted daily energy use for cave and surface forms under the different photoperiods (using the area under the oxygen consumption curve and an oxygen-energy conversion factor). 

Comparison the daily energy expenditure of surface and cave Mexican tetra at a low swimming speed

Comparison the daily energy expenditure of surface and cave Mexican tetra at a low swimming speed

If we assume the surface ancestor of cave Mexican tetra was the same as modern surface fish, and compare the daily energy expenditure of cave and surface forms under their natural photoperiod (see above), we can estimate that cave forms have saved something like 27% of their daily energy needs by eliminating the day time increase in metabolism. That's quite a big saving for the loss of a trait that is probably not very relevant in  a cave. We suspect that this is a widespread feature of cave animals.

This research has been published.

Moran, D, Softley, R, Warrant, EJ (2014). Eyeless Mexican cavefish save energy by eliminating circadian rhythm in metabolism. PLoS ONE September 24, 2014.

Eric J Warrant and Rowan Softley worked with me on this project.

This work was made possible thanks to a Marie Curie Fellowship from the European Research Council.

This worked has been written about in the following places...