# Sleeping upside-down: Knockdown of a sleep-associated gene induces daytime sleep in the jellyfish Cassiopea

## TL;DR

This study shows that a specific gene in jellyfish is linked to sleep regulation, suggesting that sleep mechanisms are evolutionarily conserved.

## Contribution

The study identifies a cholinergic-like receptor gene involved in sleep regulation in Cassiopea, revealing deep evolutionary conservation.

## Key findings

- Knockdown of chrnal-E in Cassiopea promotes wakefulness, indicating its role in sleep regulation.
- Chrnal-E expression expands in the ganglia after sleep deprivation, linking it to wakefulness.
- Cholinergic-like receptors are conserved in sleep regulation even in organisms with distributed nervous systems.

## Abstract

Sleep is a fundamental behavioral and physiological process conserved across diverse animals, yet its regulatory mechanisms remain unclear. We investigated the regulation of sleep in an early branching animal lineage with a relatively simple nervous system to gain insight into the evolution of sleep. The upside-down jellyfish Cassiopea xamachana exhibits sleep, behaviorally controlled by marginal ganglia. We focused on how sleep deprivation strongly altered ganglionic expression of a nicotinic acetylcholine receptor alpha-like subunit, chrnal-E. RNAi-mediated knockdown determined that Chrnal-E promotes wakefulness. Our findings suggest deep evolutionary conservation of cholinergic-like receptors in sleep regulation. Understanding how sleep is controlled in relatively simple organisms provides insight into its fundamental biological importance and may inform broader studies of neurobiology across species.

The conservation of sleep among diverse animals provides clear evidence for its physiological importance, but the extent of its regulatory conservation is unknown. The upside-down jellyfish Cassiopea xamachana sleeps, and this behavior is controlled by radially spaced marginal ganglia. After defining a sleep–wake threshold, we compared gene expression profiles of ganglia from animals deprived of sleep and found differential expression in many sleep-related genes including GABAergic, melatonergic, and cholinergic receptors. In particular, a nicotinic acetylcholine receptor alpha subunit-like (Chrnal-E) was strongly differentially expressed in sleep deprived animals. Animals deprived of sleep under both mechanical and light perturbations suggested chrnal-E as a key gene regulating sleep. We found that chemical cholinergic neuromodulators regulate pacemaker activity. Visualization of chrnal-E mRNA revealed it is expressed primarily within the ganglia, and that the area of expression expands after sleep deprivation. In order to confirm the functional role of chrnal-E, we developed RNAi for use in Cassiopea and determined that Chrnal-E promotes wakefulness. Finally, we field-sampled control and sleep deprived animals and found chrnal-E has lowest expression late at night in controls, while in sleep deprived animals, chrnal-E peaks at this time, supporting a link to wakefulness. Our finding that Cassiopea sleep is regulated by the cholinergic-like system underscores that some components of sleep regulation are deeply conserved even in an animal with a distributed nervous system.