Melatonin Distribution Reveals Clues to Its Biological Significance in Basal Metazoans

Citation: Roopin M, Levy O ( Melatonin Distribution Reveals Clues to Its Biological Significance in Basal Metazoans Modi Roopin 0 Oren Levy 0 Nicholas S. Foulkes, Karlsruhe Institute of Technology, Germany 0 The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University , Ramat-Gan , Israel Although nearly ubiquitous in nature, the precise biological significance of endogenous melatonin is poorly understood in phylogenetically basal taxa. In the present work, we describe insights into the functional role of melatonin at the most ''basal'' level of metazoan evolution. Hitherto unknown morphological determinants of melatonin distribution were evaluated in Nematostella vectensis by detecting melatonin immunoreactivity and examining the spatial gene expression patterns of putative melatonin biosynthetic and receptor elements that are located at opposing ends of the melatonin signaling pathway. Immuno-melatonin profiling indicated an elaborate interaction with reproductive tissues, reinforcing previous conjectures of a melatonin-responsive component in anthozoan reproduction. In situ hybridization (ISH) to putative melatonin receptor elements highlighted the possibility that the bioregulatory effects of melatonin in anthozoan reproduction may be mediated by interactions with membrane receptors, as in higher vertebrates. Another intriguing finding of the present study pertains to the prevalence of melatonin in centralized nervous structures. This pattern may be of great significance given that it 1) identifies an ancestral association between melatonin and key neuronal components and 2) potentially implies that certain effects of melatonin in basal species may be spread widely by regionalized nerve centers. - Melatonin is one of the phylogenetically oldest biological molecules in nature. An exceptional spectrum of action for this pleiotropic agent has been uncovered in a nearly ubiquitous range of organisms (e.g., [16]); however, the principle significance of melatonin in many non-vertebrate species remains uncertain. This apparent knowledge gap is largely because insufficient effort has been exerted in the investigation and characterization of the specific role of melatonin in many of the numerous non-vertebrate organisms that have been demonstrated to possess endogenous melatonin activity. Investigations in such species have primarily focused on either the classical vertebrate function of melatonin as a transducer of photoperiodic information, or more recently on its putative role as an antioxidative protectant (e.g., [24,79]). Of the well-established functions in vertebrates, the involvement of melatonin in the detoxification of destructive radicals, is neither species- nor cell-specific (e.g., [10,11]) and has therefore been hypothesized to represent the primary role of melatonin in living organisms [9]. Nonetheless, the benefits of melatonin as an antioxidant have only been demonstrated in a small number of non-vertebrate species to date, primarily dinoflagellates (e.g., [12,13]). The extent to which antioxidant activity represents the principal biological significance of melatonin in multicellular heterotrophic non-vertebrates remains largely theoretical. Another fundamental function of melatonin in vertebrates relates to its key role as a photoperiodic synchronizer of biological processes (reviewed in [14,15]). However, while hypotheses concerning an early evolutionary photoperiodic role of melatonin in basal species have been historically appealing, the combined findings from various non-vertebrate species suggest potentially divergent patterns among different organisms and even different organs [25]. Anthozoa represent the most basal class within the Cnidaria [1618], which is the simplest animal phylum at the level of tissue organization. Thus, the role of melatonin in anthozoans may represent an informative and restricted subset of early-evolved functionality at the base of metazoan evolution. Nonetheless, only isolated studies of melatonin exist for this phylogeny level. The findings from previous studies conflict regarding the daily pattern of melatonin changes. For example, the sea pen R. koellikeri exhibits 24-hour arhythmicity in melatonin levels [19], whereas the sea anemone A. equina exhibits significant day-night rhythms [20]. However, all studies generally agree on the potential involvement of this indoleamine in reproductive processes. In the sea pen R. koellikeri, the involvement of melatonin in reproduction was strongly implied by three parameters: 1) the correspondence of seasonal alterations in melatonin levels with the first stages of sexual maturation, 2) substantially higher melatonin levels in the gamete-bearing colonial mass, and 3) a potentially neuronal distribution of melatonin immunoreactivity in the endodermal filaments that are wrapped around gametes [19]. Given that anthozoans are the lowest animal group with a nervous system [2124], any melatonin activity that involves neuronal interactions in these species is intriguing. Neuronal melatonin may represent a primordial association between melatonin and the nervous system and may also provide a novel understanding of the function of melatonin during early metazoan evolution. However, comparative evaluations of melatonins neural interactions across additional anthozoans and other basal species are required to fully assess the significance of this potential relationship at the base of metazoan evolution. In vertebrates, the neuro-regenerative and neuro-protective effects of melatonin are known to involve both receptor-coupled and receptor-independent processes (reviewed in [25]). Although receptor-independent neuro-protective effects of melatonin can be hypothesized to occur in basal metazoan species as part of the general antioxidant role of this molecule, even the most basic mechanistic understanding of the relationship between melatonin and the ancient neural network in anthozoans is lacking. The present study reveals for the first time the distribution of melatonin in whole specimens of the starlet sea anemone Nematostella vectensis, which is a key anthozoan model organism. Although the distribution patterns of melatonin in Nematostella support previous conjectures concerning its role in reproduction [19,20], elevated melatonin was predominantly observed in distinct neural locations within Nematostella, suggesting a dominant neuronal role in its mechanism of action. Moreover, spatial expression profiles of putative genes for melatonin biosynthetic and receptor elements [26], which were hitherto unstudied, supported the neural distribution of this molecule and suggested that the action of melatonin in anthozoans may be modulated through receptor interactions, as in vertebrates. Materials and Methods Nematostella Culture Nematostella vectensis were cultured as previously described [27]. Adult polyps were fed at least four times a week with Artemia nauplii, and water ch (...truncated)