Sporadic feeding regulates robust food entrainable circadian clocks in blind cavefish

The circadian clock represents a key timing system that enables organisms to anticipate the day-night cycle. To ensure synchronization with the environment it is entrained by various periodic signals of which light is the best studied. Many investigations have pointed to the existence of two distinct circadian oscillators: one regulated by the light-dark cycle (light-entrainable oscillator) and the other set specifically by feeding time (food-entrainable oscillator) independently of light, and which regulates various aspects of feeding behaviour. Blind cavefish have evolved under extreme conditions where they completely lack light exposure and also experience food deprivation. Furthermore, they are under particular pressure to optimize the timing of their feeding behaviour. Here, we have investigated feeding regulated clocks in two well-studied cavefish species, the Somalian cavefish Phreatichthys andruzzii and the Mexican cavefish Astyanax mexicanus, in comparison with the surface-dwelling zebrafish Danio rerio. Our results reveal that feeding represents an extremely strong synchronizer for circadian locomotor rhythmicity in subterranean cavefish. Indeed, we showed that consuming just one meal every 4 days is sufficient to entrain circadian rhythmicity in both cavefish species, but not in zebrafish. These profound adaptations to an extreme environment provide unique insight into the connections between feeding and circadian clocks.

Methods
Locomotor activity was measured throughout the experimental period by means of infrared photocells (Omron, mod E3S-AD62, Japan) placed against the aquarium wall. The photocells were connected to a computer, and every time a fish interrupted the infrared light beam it induced an output signal that was recorded and stored in 10-min bins using specialized software (DIO98USB; University of Murcia, Spain).