Circadian Clock Gene Expression in the Coral Favia fragum over Diel and Lunar Reproductive Cycles

Pyott SJ (2011) Circadian Clock Gene Expression in the Coral Favia fragum over Diel and Lunar Reproductive Cycles. PLoS ONE 6(5): e19755. doi:10.1371/journal.pone.0019755 Circadian Clock Gene Expression in the Coral Favia fragum over Diel and Lunar Reproductive Cycles Kenneth D. Hoadley 0 Alina M. Szmant 0 Sonja J. Pyott 0 Christian R. Voolstra, King Abdullah University of Science and Technology, Saudi Arabia 0 1 Department of Biology and Marine Biology, University of North Carolina Wilmington, Wilmington, North Carolina, United States of America, 2 Department of Biology and Marine Biology and the Center for Marine Science, University of North Carolina Wilmington , Wilmington, North Carolina , United States of America Natural light cycles synchronize behavioral and physiological cycles over varying time periods in both plants and animals. Many scleractinian corals exhibit diel cycles of polyp expansion and contraction entrained by diel sunlight patterns, and monthly cycles of spawning or planulation that correspond to lunar moonlight cycles. The molecular mechanisms for regulating such cycles are poorly understood. In this study, we identified four molecular clock genes (cry1, cry2, clock and cycle) in the scleractinian coral, Favia fragum, and investigated patterns of gene expression hypothesized to be involved in the corals' diel polyp behavior and lunar reproductive cycles. Using quantitative PCR, we measured fluctuations in expression of these clock genes over both diel and monthly spawning timeframes. Additionally, we assayed gene expression and polyp expansion-contraction behavior in experimental corals in normal light:dark (control) or constant dark treatments. Well-defined and reproducible diel patterns in cry1, cry2, and clock expression were observed in both fieldcollected and the experimental colonies maintained under control light:dark conditions, but no pattern was observed for cycle. Colonies in the control light:dark treatment also displayed diel rhythms of tentacle expansion and contraction. Experimental colonies in the constant dark treatment lost diel patterns in cry1, cry2, and clock expression and displayed a diminished and less synchronous pattern of tentacle expansion and contraction. We observed no pattern in cry1, cry2, clock, or cycle expression correlated with monthly spawning events suggesting these genes are not involved in the entrainment of reproductive cycles to lunar light cycles in F. fragum. Our results suggest a molecular clock mechanism, potentially similar to that in described in fruit flies, exists within F. fragum. - Funding: The authors acknowledge the following funding sources: UNC Wilmington Center for Marine Science Pilot Project to SJP and AMS; UNC Wilmington Academic Affairs funding to AMS for support of coral reef research; Friends of UNC Wilmington to SJP; Project AWARE Foundation grant to KDH; and funding from UNC Wilmington to SJP. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Predictable and cyclic diel patterns of sunlight and monthly cycles of moonlight occur in most geographic locations around the globe. Accordingly, many species have evolved mechanisms to entrain behaviors to these environmental light patterns [1,2,3,4,5]. Scleractinian corals display behavioral and reproductive changes corresponding to both diel solar and monthly lunar light cycles. Many reef corals retract their tentacles during the day and extend them at night to feed [6,7,8,9]. Diel light cycles may also be involved with determining the time of day of coral spawning [10]. Over longer periods of time, the lunar cycle provides a light cue that is thought to play a role in synchronization of reproductive events such as gametogenesis, spawning or larval release in some scleractinian coral species [11,12]. Within a given geographic region, spawning time occurs simultaneously for all corals of a particular species. This precise and simultaneous release of gametes is thought to be an adaptation for increasing the probability of successful fertilization [13]. Although rhythmic coral behaviors such as diel tentacle expansion-contraction and synchronous spawning have been well characterized, little is known about the molecular signaling pathways responsible for these behaviors. In model systems such as fruit flies and mice, circadian behaviors are maintained by a well-studied core molecular clock composed of the transcriptional activators CLOCK and CYCLE (orthologus to BMAL1 in vertebrates) and other positive and negative regulatory components including PERIOD, TIMELESS and CRYPTOCHROME [14,15,16]. Molecular clock components, including the cryptochrome genes, are also thought to play fundamental roles in the timing of reproductive processes in these taxa [17]. A recent meta-analysis has shown that orthologs of many of these genes are present in the basal metazoan phylum Cnidaria, specifically, in the coral Acropora millepora and the sea anemone Nematostella vectensis [18]. Further, correlative evidence suggests that upregulation of one of the molecular clock genes, cryptochrome 2 (cry2), may play a role in the timing of spawning of the scleractinian coral A. millepora [19]. Whether these genes are involved in entraining cnidarian behaviors remains unclear. Based on the established roles these genes play in maintaining both shorter circadian and longer timeframe reproductive rhythms within insect and mammalian species [16,17,20,21,22], these genes may also be important in synchronizing both diel and monthly behaviors within scleractinian corals. In this study, we investigated whether the brooding coral, Favia fragum, had diel or lunar cycles of cry1, cry2, clock, and cycle transcript abundance that correlated with diel sunlight cycle and/ or key events in the monthly reproductive cycle of F. fragum. F. fragum is a small Caribbean reef coral that reproduces monthly throughout the year in a predictable lunar pattern [23], in contrast to A. millepora and many other broadcast spawning corals that reproduce annually [13,24]. Understanding the patterns of expression of these genes will help elucidate the circadian molecular clock mechanism in corals and the evolution of clock mechanisms within the metazoan lineage. Phylogenetic analyses of sequenced rtPCR gene products was used to confirm the presence of orthologous clock gene products within the F. fragum transcriptome. Quantitative PCR (qPCR) methods were then used to measure fluctuations in clock gene expression over both short term (diel) and longer term (reproductive) cycles. Behavioral outputs, specifically polyp expansion and contraction over the diel cycle, and gametogenesis over the lunar cycle were also monitored and compared to fluctuations in clock gene expression. Changes in behavior and gene expression under constant darkness (...truncated)