To detect the presence of behavioral oscillations in N. vectensis, locomotor activity was evaluated using an automated system in an environmentally controlled chamber. Animals exposed to a 24 hr photoperiod (12 hr light: 12 hr dark) exhibited locomotor behavior that was both rhythmic and predominantly nocturnal. The activity peak occurred in the early half of the night with a 2-fold increase in locomotion. Upon transfer to constant lighting conditions (constant light or constant dark), an approximately 24 hr rhythm persisted in most animals, suggesting that the rhythm is controlled by an endogenous circadian mechanism. Fourier analysis revealed the presence of multiple peaks in some animals suggesting additional rhythmic components could be present. In particular, an approximately 12 hr oscillation was often observed. The nocturnal increase in generalized locomotion corresponded to a 24 hr oscillation in animal elongation.

Although much is known about how circadian systems control daily cycles in the physiology and behavior of Drosophila and several vertebrate models, marine invertebrates have often been overlooked in circadian rhythms research. This study focuses on the starlet sea anemone, Nematostella vectensis, a species that has received increasing attention within the scientific community for its potential as a model research organism. The recently sequenced genome of N. vectensis makes it an especially attractive model for exploring the molecular evolution of circadian behavior. Critical behavioral data needed to correlate gene expression patterns to specific behaviors are currently lacking in N. vectensis.

Introduction

Throughout the natural world, rhythms in behavior and physiology have been observed in most organisms, including eukaryotes, prokaryotes, and even in members of the Archaea. The range of frequencies in which these biological oscillations are expressed within an individual can be quite diverse and is likely to depend on the organism’s specific needs for survival. The spectrum of rhythms includes ultradian (minutes), circatidal (12.4 hrs), circadian (24 hrs), circalunar-day (24.8 hrs), semilunar (15 days), lunar (28 days), or circannual (yearly) oscillations [1], [2], [3], [4]. Among these, the most widely studied is the circadian rhythm, a cyclic pattern that has been documented in most species studied to date. Undoubtedly, the ubiquitous presence of the circadian rhythm is an indication of the evolutionary significance of this particular temporal oscillation.

Although regulation of circadian cycles has been thoroughly investigated in some derived metazoan taxa, much remains to be learned about circadian control in pleisiomorphic groups such as the Cnidaria. In this phylum, which includes organisms such as sea anemones, corals, jellyfish, and Hydra, diel patterns of behavior have been documented in species of the subphyla Anthozoa [5], [6], [7], [8], [9], [10], [11], [12], [13] and Medusozoa [14], [15], [16], [17], [18], [19], [20], but only a few investigators have attempted to discern whether these behaviors are direct responses to changes in light availability or whether the behavior is endogenously generated by a circadian clock. Depending on the species tested and the environmental conditions, studies have reported the presence of behaviors dependent on an internal clock, as well as behaviors that cycle merely in response to the exogenous photoperiod. Specifically, in some of the earliest studies, Bohn [5] found that retraction and expansion of the body column in the sea anemone Actinia equina followed a daily pattern that persisted in constant darkness for 3 to 8 days in the laboratory. Similar observations were made in the sea anemone Metridium senile [6] and the sea pen Cavernularia obesa [7], [8]. In contrast, an early study by Parker [5] reported that the sea anemone Metridium marginatum exhibited daily oscillations in expansion and retraction that failed to persist under constant dim lighting. These inconsistencies have also been observed in some coral species. The comparative work of Sweeney [9] looked at circadian behaviors in 21 coral species. In most corals studied, tentacle expansion fluctuated daily. Typically, individuals were nocturnal, extending their tentacles at night and retracting them during the day. When exposed to continuous darkness, only the fungids continued to exhibit rhythmic patterns of contraction and expansion. The non-persistent behaviors reported in some of the corals and sea anemones may indicate that these measured behaviors are not under the control of a circadian clock, but this does not exclude the possibility of a circadian mechanism governing oscillations of other behaviors in these organisms. Moreover, these early studies typically utilized equipment that may not have permitted full characterization of the observed behaviors. Thus, the presence and function of an internal circadian clock within the phylum Cnidaria has not been completely resolved.

We have chosen to study circadian cycles in N. vectensis because this easily manipulated species has the potential to reveal the molecular basis of circadian cycles both in basal taxonomic groups and in economically desirable species such as corals. This species has become a critical model for studies in developmental biology and molecular evolution because it is quite hardy, can be induced to spawn throughout the year, and has a genome that is publicly accessible [21], [22], [23]. Also, as a cnidarian, it occupies a basal taxonomic position among metazoan phyla and, as a member of the class Anthozoa, it is pleisiomorphic within that phylum. These features make N. vectensis especially useful for detecting traits that may have existed among ancestral cnidarians or metazoans and, cumulatively, these characteristics make N. vectensis an ideal model to probe the evolution of the circadian clock.

Molecular clocks in insects and mammals have been fairly well described [24], however, only in the last few years have there been investigations into the molecular underpinnings of rhythmic behavior in cnidarians [25], [26], [27], [28]. A recent study [26] investigating circadian genes in N. vectensis strongly suggests that critical molecular components of the circadian clock mechanism have been conserved in these animals. Unfortunately, without clear characterization of behavior, it will be impossible to fully understand how these circadian genes function. The present study complements the molecular studies by defining locomotor behavior of N. vectensis across the circadian cycle. We have developed a protocol to assess locomotor behavior, setting the stage for future studies aimed at understanding the cellular and molecular control of circadian behavior in this species. In the present study, the locomotor activity of N. vectensis was monitored under photoperiodic and continuous light cycles. Animals exposed to a 12 hr light: 12 hr dark photoperiod were most active in the dark phase. In order to determine whether the rhythmic behavior was generated by an endogenous circadian clock or was merely a direct response to the changes in lighting, animals were exposed to conditions of continuous darkness (DD) or continuous light (LL). Cyclic patterns of locomotor activity persisted in many of these individuals, indicating endogenous circadian regulation. Also, under constant conditions, some individuals exhibited a secondary activity component. These observations suggest that one or more endogenous pathways function to regulate patterns of locomotor activity in the sea anemone N. vectensis.