Developmental and light-entrained expression of melatonin and its relationship to the circadian clock in the sea anemone...

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Title:
Developmental and light-entrained expression of melatonin and its relationship to the circadian clock in the sea anemone Nematostella vectensis
Physical Description:
Mixed Material
Language:
English
Creator:
Peres, Rafael
Reitzel, Adam M.
Passamaneck, Yale
Afeche, Solange Castro
Cipolla-Neto, Jose
Marques, Antonio Carlos
Martindale, Mark Q.
Publisher:
Bio-Med Central (Evo Devo)
Publication Date:

Notes

Abstract:
Background: The primary hormone of the vertebrate pineal gland, melatonin, has been identified broadly throughout the eukaryotes. While the role for melatonin in cyclic behavior via interactions with the circadian clock has only been reported in vertebrates, comparative research has shown that the transcription-translation loops of the animal circadian clock likely date to the cnidarian-bilaterian ancestor, leaving open significant questions about the evolutionary origin of melatonin signaling in circadian behavior by interacting with the molecular clock. Results: Expression of melatonin in adult anemones showed peak expression at the end of light period (zeitgeber time (ZT) = 12) when cultured under diel conditions, coinciding with expression of genes and enzyme activity for members of the melatonin synthesis pathway (tryptophan hydroxylase and hydroxyindol-O-methyltransferase), which also showed rhythmic expression. During embryogenesis and juvenile stages, melatonin showed cyclic oscillations in concentration, peaking in midday. Spatial (in situ hybridization) and quantitative (real-time PCR) transcription of clock genes during development of N. vectensis showed these ‘clock’ genes are expressed early in the development, prior to rhythmic oscillations, suggesting functions independent of a function in the circadian clock. Finally, time-course studies revealed that animals transferred from diel conditions to constant darkness lose circadian expression for most of the clock genes within 4 days, which can be reset by melatonin supplementation. Conclusions: Our results support an ancient role for melatonin in the circadian behavior of animals by showing cyclic expression of this hormone under diel conditions, light-dependent oscillations in genes in the melatonin synthesis pathway, and the function of melatonin in initiating expression of circadian clock genes in the cnidarian N. vectensis. The differences in expression melatonin and the circadian clock gene network in the adult stage when compared with developmental stages of N. vectensis suggests new research directions to characterize stage-specific mechanisms of circadian clock function in animals. Keywords: Circadian clock, Embryogenesis, In situ hybridization, Melatonin
General Note:
Peres et al. EvoDevo 2014, 5:26 http://www.evodevojournal.com/content/5/1/26; Pages 1-18
General Note:
doi:10.1186/2041-9139-5-26 Cite this article as: Peres et al.: Developmental and light-entrained expression of melatonin and its relationship to the circadian clock in the sea anemone Nematostella vectensis. EvoDevo 2014 5:26.

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Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
© 2014 Peres et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
System ID:
AA00026998:00001

Full Text

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Supplementary Information 1: Primers information: Clock, 5'TCGGCTCCCCGAGTCTAGCG-3'(sense) and 5'GCATCGCGGCGAACCCAGTA-3' (antisense), 59.8 C, 110bp, accesion number XP_001639742 Cycle, 5'TGTCTCGTGGCCGTCGGAAG-3'(sense) and 5'GGCGACATCCGGTCGGGAAAA-3' (antisense), 59 C, 72bp, accesion number XP_001624731 Timeout, 5'GTGCCTTCGCAGGTGGGACG-3'(sense) and 5'GGCTTTCGTTGTCCGACTCGCT-3' (antisense), 60 C, 96bp, accesion number XP_001641000 Cry1a, 5'GCGGCATGGCAAGACAGGCT-3'(sense) and 5'CACCCAATGGACTGCCGGGC-3' (antisense), 60.3 C, 99bp, accesion number XP_001631029 Cry1b, 5'TCGGACCGGACCATGCTGACA-3'(sense) and 5'TCGTCGCAAAGCTTCGGGCT-3' (antisense), 59.6 C, 122bp, accesion number XP_001632849 Cry2, 5'AGCTCGCCGTAAAAGGGCGG-3'(sense) and 5'ATATAAGCGCCTGCACCCGCG-3' (antisense), 59.8 C, 75bp, accesion number XP_001623146 TPH: 5'AAAAACTCGCCACGCTCTACTGG-3'(sense) and 5'CGGTCAAGCAATACTGTAACTCCCC-3' (antisense), 56.4 C, 123bp, accesion number XM_001623795.1 HIOMT: 5'CGAGATTTGATTGCGTTCACCG-3'(sense) and 5'AAAACAGCCAGTTCCTCCTCCAAG-3' (antisense), 59 C, 107bp, accesion number XM_001627179.1 Ribosomal protein P0: 5'GGCTTCGTCTTCACCAAGGAGGAG-3'(sense) and 5'CCAGCAGGGACAAACACATCAATA-3' (antisense), 60.5 C, 117bp, accesion number

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XM_001626244.1



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Formatted Alignments hiomt_nematostella hiomt_qpcr hiomt_cloned 10 20 30 C T T T C T C A T T G T G A A A T G G C A G A A T T G A C A T C A T T G T G A A A T G G C A G A A T T G A C A C T T T C T C A T T G T G A A A T G G C A G A A T T G A C A hiomt_nematostella hiomt_qpcr hiomt_cloned 40 50 60 A G T A C T C T C G G T A C A A T C A T A C C T A C A G C C A G T A C T C T C G G T A C A A T C A T A C C T A C A G C C A G T A C T C T C G G T A C A A T C A T A C C T A C A G C C hiomt_nematostella hiomt_qpcr hiomt_cloned 70 80 90 C T T T C A G A T A T T T T T C A T G G A T T T G T T A A A C T T T C A G A T A T T T T T C A T G G A T T T G T T A A A C T T T C A G A T A T T T T T C A T G G A T T T G T T A A A hiomt_nematostella hiomt_qpcr hiomt_cloned 100 110 120 T C A A A A G T A T T G T T C A C C G C C T G T G A A T T A T C A A A A G T A T T G T T C A C C G C C T G T G A A T T A T C A A A A G T A T T G T T C A C C G C C T G T G A A T T A hiomt_nematostella hiomt_qpcr hiomt_cloned 130 140 150 G G T A T A T T T G A C G T A C T G T G C G A T A A G C A T G G T A T A T T T G A C G T A C T G T G C G A T A A G C A T G G T A T A T T T G A C G T A C T G T G C G A T A A G C A T hiomt_nematostella hiomt_qpcr hiomt_cloned 160 170 180 T C G G C A G A A C A T G T G G C A G A A A A T G T T A A A T C G G C A G A A C A T G T G G C A G A A A A T G T T A A A T C G G C A G A A C A T G T G G C A G A A A A T G T T A A A hiomt_nematostella hiomt_qpcr hiomt_cloned 190 200 210 C C A A G C T G C A A A A C T G G C T C A A C T C G T C G T C C A A G C T G C A A A A C T G G C T C A A C T C G T C G T C C A A G C T G C A A A A C T G G C T C A A C T C G T C G T hiomt_nematostella hiomt_qpcr hiomt_cloned 220 230 240 C T T C T T G A C A C A C T T G T A G C T A T G C A A T T A C T T C T T G A C A C A C T T G T A G C T A T G C A A T T A C T T C T T G A C A C A C T T G T A G C T A T G C A A T T A hiomt_nematostella hiomt_qpcr hiomt_cloned 250 260 270 C T T G T C A A A G A A A T G G A C A G C G A C C C G C C T C T T G T C A A A G A A A T G G A C A G C G A C C C G C C T C T T G T C A A A G A A A T G G A C A G C G A C C C G C C T hiomt_nematostella hiomt_qpcr hiomt_cloned 280 290 300 G T C T A C T T C A A T A G C C A A A C T G C A G A G G C A G T C T A C T T C A A T A G C C A A A C T G C A G A G G C A G T C T A C T T C A A T A G C C A A A C T G C A G A G G C A hiomt_nematostella hiomt_qpcr hiomt_cloned 310 320 330 T T T C T T A C T A G G A A A A G T C C C A A G T C T C T C T T T C T T A C T A G G A A A A G T C C C A A G T C T C T C T T T C T T A C T A G G A A A A G T C C C A A G T C T C T C hiomt_nematostella hiomt_qpcr hiomt_cloned 340 350 360 A T A A G C T A C T T T G A A T T T C T T C A C G C C A C C A T A A G C T A C T T T G A A T T T C T T C A C G C C A C C A T A A G C T A C T T T G A A T T T C T T C A C G C C A C C hiomt_nematostella hiomt_qpcr hiomt_cloned 370 380 390 G A T T A C A A G C T A T T T G A C A A C C T G A A G C A T G A T T A C A A G C T A T T T G A C A A C C T G A A C A T G A T T A C A A G C T A T T T G A C A A C C T G A A G C A T hiomt_nematostella hiomt_qpcr hiomt_cloned 400 410 420 G C G G T T G T C G A G G A G C A G C C A C A A T G G C A A G C G G T T G T C G A G G A G C A G C C A C A A T G G C A A G C G G T T G T C G A G G A G C A G C C A C A A T G G C A A hiomt_nematostella hiomt_qpcr hiomt_cloned 430 440 450 C G A G C C T T C G A T C A G C C G G C T T C A G A A A T T C G A G C C T T C G A T C A G C C G G C T T C A G A A A T T C G A G C C T T C G A T C A G C C G G C T T C A G A A A T T hiomt_nematostella hiomt_qpcr hiomt_cloned 460 470 480 T A C A A A A T A G T G C A T T C C A A T A A A G A A G A G T A C A A A A T A G T G C A T T C C A A T A A A G A A G A G T A C A A A A T A G T G C A T T C C A A T A A A G A A G A G hiomt_nematostella hiomt_qpcr hiomt_cloned 490 500 510 C T G G T G C G T A T C T A C T C G A G A T T T G A T T G C A G A T T T G A T T G C C T G G T G C G T A T C T A C T C G A G A T T T G A T T G C C T G G T G C G T A T C T A C T C G A G A T T T G A T T G C hiomt_nematostella hiomt_qpcr hiomt_cloned 520 530 540 G T T C A C C G A T T G G A A G C A C C T G G G G T G A T G G T T C A C C G A T T G G A A G C A C C T G G G G T G A T G G T T C A C C G A T T G G A A G C A C C T G G G G T G A T G G T T C A C C G A T T G G A A G C A C C T G G G G T G A T G hiomt_nematostella hiomt_qpcr hiomt_cloned 550 560 570 T C T G C G T T T G A C T T G A A C C C T T T T A A A C A C T C T G C G T T T G A C T T G A A C C C T T T T A A A C A C T C T G C G T T T G A C T T G A A C C C T T T T A A A C A C T C T G C G T T T G A C T T G A A C C C T T T T A A A C A C hiomt_nematostella hiomt_qpcr hiomt_cloned 580 590 600 A T G T G T G A T C T T G G A G G A G G A A C T G G C T G T A T G T G T G A T C T T G G A G G A G G A A C T G G C T G T A T G T G T G A T C T T G G A G G A G G A A C T G G C T G T A T G T G T G A T C T T G G A G G A G G A A C T G G C T G T hiomt_nematostella hiomt_qpcr hiomt_cloned 610 620 630 T T T T C A T A C G A G G C G T G C A A G C A G T A T C C T T T T T T C A T A C G A G G C G T G C A A G C A G T A T C C T T T T T C A T A C G A G G C G T G C A A G C A G T A T C C T hiomt_nematostella hiomt_qpcr hiomt_cloned 640 650 660 G C A T T A A A A A T C A C A A T T T A C G A A A T G C A G G C A T T A A A A A T C A C A A T T T A C G A A A T G C A G G C A T T A A A A A T C A C A A T T T A C G A A A T G C A G hiomt_nematostella hiomt_qpcr hiomt_cloned 670 680 690 C C A A T C T T A G A T G T C G C G C C T G C T T T C A A G C C A A T C T T A G A T G T C G C G C C T G C T T T C A A G C C A A T C T T A G A T G T C G C G C C T G C T T T C A A G hiomt_nematostella hiomt_qpcr hiomt_cloned 700 710 720 C C A A C C A T C G C T G A T T G C C C T A A C C A A T C A C C A A C C A T C G C T G A T T G C C C T A A C C A A T C A C C A A C C A T C G C T G A T T G C C C T A A C C A A T C A hiomt_nematostella hiomt_qpcr hiomt_cloned 730 740 750 A A C G T C A C C T A C G T A G C G G G A G A C T T T T T T A A C G T C A C C T A C G T A G C G G G A G A C T T T T T T A A C G T C A C C T A C G T A G C G G G A G A C T T T T T T hiomt_nematostella hiomt_qpcr hiomt_cloned 760 770 780 A A A G A C C C A C T T C C G G T T G C T G A C C T G T A T A A A G A C C C A C T T C C G G T T G C T G A C C T T A T A A A G A C C C A C T T C C G G T T G C T G A C C T G T A T hiomt_nematostella hiomt_qpcr hiomt_cloned 790 800 810 T T T C T G G C C C A T G T T C T C C A C A A T T G G G C A T T T C T G G C C C A T G T T C T C C A C A A T T G G G C A T T T C T G G C C C A T G T T C T C C A C A A T T G G G C A hiomt_nematostella hiomt_qpcr hiomt_cloned 820 830 840 G A A G A A A A G G T T G A C T T G C T A C T T A G C A A A G A A G A A A A G G T T G A C T T G C T A C T T A G C A A A G A A G A A A A G G T T G A C T T G C T A C T T A G C A A A hiomt_nematostella hiomt_qpcr hiomt_cloned 850 860 870 G T T T T T G C A G T G C T G C C A C C A G G T G G A G G T G T T T T T G C A G T G C T G C C A C C A G G T G G A G G T G T T T T T G C A G T G C T G C C A C C A G G T G G A G G T hiomt_nematostella hiomt_qpcr hiomt_cloned 880 890 900 A T A C T T C T T G G G G A A G T T C T T C T T C C T G T T A T A C T T C T T G G G G A A G T T C T T C T T C C T G T T A T A C T T C T T G G G G A A G T T C T T C T T C C T G T T hiomt_nematostella hiomt_qpcr hiomt_cloned 910 920 930 G A C G A G C C G A A T C C A C A G C T C T C T G C A C C C G A C G A G C C G A A T C C A C A G C T C T C T G C A C C C G A C G A G C C G A A T C C A C A G C T C T C T G C A C C C hiomt_nematostella hiomt_qpcr hiomt_cloned 940 950 960 T T C C T G G A C C T C A C C A T G C T A G T G A C C T G C T T C C T G G A C C T C A C C A T G C T A G T G A C C T G C T T C C T G G A C C T C A C C A T G C T A G T G A C C T G C hiomt_nematostella hiomt_qpcr hiomt_cloned 970 980 990 G A G T C A G G G G C A C G T G A C C G T T C G G G C C C C G A G T C A G G G G C A C G T G A C C G T T C G G G C C C C G A G T C A G G G G C A C G T G A C C G T T C G G G C C C C hiomt_nematostella hiomt_qpcr hiomt_cloned 1000 1010 1020 G A G T A C A A A C G A T T A C T G G A A A G A C A T G G A G A G T A C A A A C G A T T A C T G G A A A G A C A T G G A G A G T A C A A A C G A T T A C T G G A A A G A C A T G G A hiomt_nematostella hiomt_qpcr hiomt_cloned 1030 1040 1050 T T T C A A G A C G T G C G G A T G A A A T C C T T G C C A T T T C A A G A C G T G C G G A T G A A A T C C T T G C C A T T T C A A G A C G T G C G G A T G A A A T C C T T G C C A hiomt_nematostella hiomt_qpcr hiomt_cloned 1060 1070 1080 G G C G C C A A A A C G A C C G A T G C T G T T T T C G C C G G C G C C A A A A C G A C C G A T G C T G T T T T C G C C G G C G C C A A A A C G A C C G A T G C T G T T T T C G C C hiomt_nematostella hiomt_qpcr hiomt_cloned 1090 1100 1110 C G G A A A C C T T A A A A T C G C C A C G G C G C G A C G C G G A A A C C T T A A A A T C G C C A C G G C G C G A C G C G G A A A C C T T A A A A T C G C C A C G G C G C G A C G hiomt_nematostella hiomt_qpcr hiomt_cloned 1120 1130 1140 G C A C T T A T A C T T T A C C A T A C G T A A T T T T C T G C A C T T A T A C T T T A C C A T A C G T A A T T T T C T G C A C T T A T A C T T T A C C A T A C G T A A T T T T C T hiomt_nematostella hiomt_qpcr hiomt_cloned 1150 1160 1170 A C G A C A G C T T G T G A A C A A A A T T A C A A A A T A A C G A C A G C T T G T G A A C A A A A T T A C A A A A T A A C G A C A G C T T G T G A A C A A A A T T A C A A A A T A hiomt_nematostella hiomt_qpcr hiomt_cloned 1180 1190 1200 T C C G C T T T G G C A T T A A A C C T C C G C T T T G T C C G C T T T G G C A T T A A A C C



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Formatted Alignments tph_nematostella tph_qpcr tph_cloned 10 20 30 G T G T G T T T C T T T G A G T G A G G A A A A T G C A C G C T T T G A G T G A G G A A A A T G C A C G G T G T G T T T C T T T G A G T G A G G A A A A T G C A C G tph_nematostella tph_qpcr tph_cloned 40 50 60 C T G T A A G G C G C A A A A G A A C C T A C G A A A T C G C T G T A A G G C G C A A A A G A A C C T A C G A A A T C G C T G T A A G G C G C A A A A G A A C C T A C G A A A T C G tph_nematostella tph_qpcr tph_cloned 70 80 90 A G C C G G A C C A T G G C C C T G C T A A G A A G C A T G A G C C G G A C C A T G G C C C T G C T A A G A A G C A T G A G C C G G A C C A T G G C C C T G C T A A G A A G C A T G tph_nematostella tph_qpcr tph_cloned 100 110 120 A G G G C G A A T C A G A A A A G A A A G C T A C C A C G G A G G G C G A A T C A G A A A A G A A A G C T A C C A C G G A G G G C G A A T C A G A A A A G A A A G C T A C C A C G G tph_nematostella tph_qpcr tph_cloned 130 140 150 T A G T G T T T T C C C T G A A T G A G G A G G T T G G C G T A G T G T T T T C C C T G A A T G A G G A G G T T G G C G T A G T G T T T T C C C T G A A T G A G G A G G T T G G C G tph_nematostella tph_qpcr tph_cloned 160 170 180 C G T T G G C C A G A G C T T T G A A A T T A T T T G A G G C G T T G G C C A G A G C T T T G A A A T T A T T T G A G G C G T T G G C C A G A G C T T T G A A A T T A T T T G A G G tph_nematostella tph_qpcr tph_cloned 190 200 210 A T C A C C A T G T G A A T A T G A C G C A C A T T G A A T A T C A C C A T G T G A A T A T G A C G C A C A T T G A A T A T C A C C A T G T G A A T A T G A C G C A C A T T G A A T tph_nematostella tph_qpcr tph_cloned 220 230 240 C C C G C C C G T C C A A G G C A T G C A A G A C C C G C T C C C G C C C G T C C A A G G C A T G C A A G A C C C G C T C C C G C C C G T C C A A G G C A T G C A A G A C C C G C T tph_nematostella tph_qpcr tph_cloned 250 260 270 A T G A T T T C T T C G C A G A C T G T G A G G G G C T G C A T G A T T T C T T C G C A G A C T G T G A G G G G C T G C A T G A T T T C T T C G C A G A C T G T G A G G G G C T G C tph_nematostella tph_qpcr tph_cloned 280 290 300 A T G G T G C T A G T C T G A A T C A C T T T G T T G A T G A T G G T G C T A G T C T G A A T C A C T T T G T T G A T G A T G G T G C T A G T C T G A A T C A C T T T G T T G A T G tph_nematostella tph_qpcr tph_cloned 310 320 330 C A C T A A A G A A G A G A G C C G T C A A T A T T A C T G C A C T A A A G A A G A G A G C C G T C A A T A T T A C T G C A C T A A A G A A G A G A G C C G T C A A T A T T A C T G tph_nematostella tph_qpcr tph_cloned 340 350 360 T T C T A T C T G A T G A T A A G G G C T C A G T G C C A T T T C T A T C T G A T G A T A A G G G C T C A G T G C C A T T T C T A T C T G A T G A T A A G G G C T C A G T G C C A T tph_nematostella tph_qpcr tph_cloned 370 380 390 G G T T C C C C C G C A A G A T T T C T G A T C T G G A C A G G T T C C C C C G C A A G A T T T C T G A T C T G G A C A G G T T C C C C C G C A A G A T T T C T G A T C T G G A C A tph_nematostella tph_qpcr tph_cloned 400 410 420 A G T T T G C T G A C A G A G T G C T G A G C T A T G G A G A G T T T G C T G A C A G A G T G C T G A G C T A T G G A G A G T T T G C T G A C A G A G T G C T G A G C T A T G G A G tph_nematostella tph_qpcr tph_cloned 430 440 450 C T G A G C T A G A C T C A G A C C A T C C T G G C T T T A C T G A G C T A G A C T C A G A C C A T C C T G G C T T T A C T G A G C T A G A C T C A G A C C A T C C T G G C T T T A tph_nematostella tph_qpcr tph_cloned 460 470 480 C A G A C C A A G T A T A C A G A G C C A G A C G A A A G G C A G A C C A A G T A T A C A G A G C C A G A C G A A A G G C A G A C C A A G T A T A C A G A G C C A G A C G A A A G G tph_nematostella tph_qpcr tph_cloned 490 500 510 A G T T T G C T G A C A T T G C C T T C A A G C A C A A A C A G T T T G C T G A C A T T G C C T T C A A G C A C A A A C A G T T T G C T G A C A T T G C C T T C A A G C A C A A A C tph_nematostella tph_qpcr tph_cloned 520 530 540 A C G G T G A A A T T A T T C C A C A G G T C A C T T A T A A C G G T G A A T T A T T C C A C A G G T C A C T T A T A A C G G T G A A A T T A T T C C A C A G G T C A C T T A T A tph_nematostella tph_qpcr tph_cloned 550 560 570 C A G A T A T C G A G A T T G C A A C C T G G G A T A A A G C A G A T A T C G A G A T T G C A A C C T G G G A T A A A G C A G A T A T C G A G A T T G C A A C C T G G G A T A A A G tph_nematostella tph_qpcr tph_cloned 580 590 600 T G T T C A C A G A G C T G A C C A A G T T G T A T C C T A T G T T C A C A G A G C T G A C C A A G T T G T A T C C T A T G T T C A C A G A G C T G A C C A A G T T G T A T C C T A tph_nematostella tph_qpcr tph_cloned 610 620 630 C C C A T G C T T G C C G T G A A C A T A A C T T T G T C T C C C A T G C T T G C C G T G A A C A T A A C T T T G T C T C C C A T G C T T G C C G T G A A C A T A A C T T T G T C T tph_nematostella tph_qpcr tph_cloned 640 650 660 G G C C A T T G C T A G T G C A A A A T T G T G G T T A C A G G C C A T T G C T A G T G C A A A A T T G T G G T T A C A G G C C A T T G C T A G T G C A A A A T T G T G G T T A C A tph_nematostella tph_qpcr tph_cloned 670 680 690 G A C T T G G G A A T A T T C C T C A G C T T C A G G A T G G A C T T G G G A A T A T T C C T C A G C T T C A G G A T G G A C T T G G G A A T A T T C C T C A G C T T C A G G A T G tph_nematostella tph_qpcr tph_cloned 700 710 720 T C T C A A T G T T T C T G A A A G A A T G C A C T G G G T T C T C A A T G T T T C T G A A A G A A T G C A C T G G G T T C T C A A T G T T T C T G A A A G A A T G C A C T G G G T tph_nematostella tph_qpcr tph_cloned 730 740 750 T C A C T T T A C G G C C T G T G G C T G G A C T T C T G T T C A C T T T A C G G C C T G T G G C T G G A C T T C T G T T C A C T T T A C G G C C T G T G G C T G G A C T T C T G T tph_nematostella tph_qpcr tph_cloned 760 770 780 C A T C C C G G G A C T T C C T G G C G G G C C T G G C A T C A T C C C G G G A C T T C C T G G C G G G C C T G G C A T C A T C C C G G G A C T T C C T G G C G G G C C T G G C A T tph_nematostella tph_qpcr tph_cloned 790 800 810 T C C G T G T G T T C C A C A G T A C T C A G T A C A T C C T C C G T G T G T T C C A C A G T A C T C A G T A C A T C C T C C G T G T G T T C C A C A G T A C T C A G T A C A T C C tph_nematostella tph_qpcr tph_cloned 820 830 840 G T C A C C C A A C A A A A C C C A T G T A C A C C C C A G G T C A C C C A A C A A A A C C C A T G T A C A C C C C A G G T C A C C C A A C A A A A C C C A T G T A C A C C C C A G tph_nematostella tph_qpcr tph_cloned 850 860 870 A G C C T G A C G T T G T G C A T G A G C T A A T C G G T C A G C C T G A C G T T G T G C A T G A G C T A A T C G G T C A G C C T G A C G T T G T G C A T G A G C T A A T C G G T C tph_nematostella tph_qpcr tph_cloned 880 890 900 A T G T G C C T C T G T T T G C T G A T C C A G A T T T C G A T G T G C C T C T G T T T G C T G A T C C A G A T T T C G A T G T G C C T C T G T T T G C T G A T C C A G A T T T C G tph_nematostella tph_qpcr tph_cloned 910 920 930 C A C A A T T T A G T C A G G A A A T T G G T C T T G C C T C A C A A T T T A G T C A G G A A A T T G G T C T T G C C T C A C A A T T T A G T C A G G A A A T T G G T C T T G C C T tph_nematostella tph_qpcr tph_cloned 940 950 960 C C C T G G G A G C A C C A G A C G A G T G G G T G G A A A A C C C T G G G A G C A C C A G A C G A G T G G G T G G A A A C C C T G G G A G C A C C A G A C G A G T G G G T G G A A A tph_nematostella tph_qpcr tph_cloned 970 980 990 A A C T C G C C A C G C T C T A C T G G T T C A C T G T T G A A C T C G C C A C G C T C T A C T G G T T C A C T G T T G A A C T C G C C A C G C T C T A C T G G T T C A C T G T T G A A C T C G C C A C G C T C T A C T G G T T C A C T G T T G tph_nematostella tph_qpcr tph_cloned 1000 1010 1020 A G T T T G G G C T G T G T C G G C A G G A G G G C G A G G A G T T T G G G C T G T G T C G G C A G G A G G G C G A G G A G T T T G G G C T G T G T C G G C A G G A G G G C G A G G A G T T T G G G C T G T G T C G G C A G G A G G G C G A G G tph_nematostella tph_qpcr tph_cloned 1030 1040 1050 T G A A A G C G T A C G G C G C T G G T C T A T T G T C C T T G A A A G C G T A C G G C G C T G G T C T A T T G T C C T T G A A A G C G T A C G G C G C T G G T C T A T T G T C C T T G A A A G C G T A C G G C G C T G G T C T A T T G T C C T tph_nematostella tph_qpcr tph_cloned 1060 1070 1080 C C T T T G G G G A G T T A C A G T A T T G C T T G A C C G C C T T T G G G G A G T T A C A G T A T T G C C T T T G G G G A G T T A C A G T A T G C T T G A C C G C C T T T G G G G A G T T A C A G T A T T G C T T G A C C G tph_nematostella tph_qpcr tph_cloned 1090 1100 1110 A T G A G C C C A A G A A A T A C C C T C T G G A G C C T G A T G A G C C C A A G A A A T A C C C T C T G G A G C C T G A T G A G C C C A A G A A A T A C C C T C T G G A G C C T G tph_nematostella tph_qpcr tph_cloned 1120 1130 1140 A A A A G A C C A G T G T A C A G A A G T A C C C C A T C A A A A A G A C C A G T G T A C A G A A G T A C C C C A T C A A A A A G A C C A G T G T A C A G A A G T A C C C C A T C A tph_nematostella tph_qpcr tph_cloned 1150 1160 1170 C G C A G T A C C A G C C A G T C T A C T T C A T T G C C G C G C A G T A C C A G C C A G T C T A C T T C A T T G C C G C G C A G T A C C A G C C A G T C T A C T T C A T T G C C G tph_nematostella tph_qpcr tph_cloned 1180 1190 1200 A C A G T T T C T T G T C G G C C A G A A A C A A A G T T A A C A G T T T C T T G T C G G C C A G A A A C A A A G T T A A C A G T T T C T T G T C G G C C A G A A A C A A A G T T A tph_nematostella tph_qpcr tph_cloned 1210 1220 1230 G G A A A C C C T G T T T T A T T A C C A A G G A A T A G A G G A A A C C C T G T T T T A T T A C C A A G G A A T A G A G G A A A C C C T G T T T T A T T A C C A A G G A A T A G A tph_nematostella tph_qpcr tph_cloned 1240 1250 1260 A C T T A A G C T T A T A A G C A A T G T A T T G C A A G G A C T T A A G C T T A T A A G C A A T G T A T T G C A A G G A C T T A A G C T T A T A A G C A A T G T A T T G C A A G G tph_nematostella tph_qpcr tph_cloned 1270 1280 1290 C C T T G T A G T T T T A T A A C A G C A A G A A T T G T A C C T T G T A G T T T T A T A A C A G C A A G A A T T G T A C C T T G T A G T T T T A T A A C A G C A A G A A T T G T A tph_nematostella tph_qpcr tph_cloned 1300 1310 1320 G G A A G A A T T T T G T G C A A T A T A T G T A A A G G T G G A A G A A T T T T G T G C A A T A T A T G T A A A G G T G G A A G A A T T T T G T G C A A T A T A T G T A A A G G T tph_nematostella tph_qpcr tph_cloned 1330 1340 1350 A T T C T T A G G C A A G G G A A T A T G T G T T A A G G A A T T C T T A G G C A A G G G A A T A T G T G T T A A G G A A T T C T T A G G C A A G G G A A T A T G T G T T A A G G A tph_nematostella tph_qpcr tph_cloned 1360 1370 1380 C T G T T T T G T T G T G A T T T T A G A A A A C T A T C C C T G T T T T G T T G T G A T T T T A G A A A A C T A T C C C T G T T T T G T T G T G A T T T T A G A A A A C T A T C C tph_nematostella tph_qpcr tph_cloned 1390 1400 1410 C C C A T C C C T T T C C C T C C A T T G G T A A T G T A G C C C A T C C C T T T C C C T C C A T T G G T A A T G T A G C C C A T C C C T T T C C C T C C A T T G G T A A T G T A G tph_nematostella tph_qpcr tph_cloned 1420 1430 1440 T G A T A T C A A G A A A A A A T T C A T T A G G T A T T T T G A T A T C A A G A A A A A A T T C A T T A G G T A T T T T G A T A T C A A G A A A A A A T T C A T T A G G T A T T T tph_nematostella tph_qpcr tph_cloned 1450 1460 1470 A A G C T C T G T G T T A T T T A T A G A C T G G A T T T T A A G C T C T G T G T T A T T T A T A G A C T G G A T T T T A A G C T C T G T G T T A T T T A T A G A C T G G A T T T T tph_nematostella tph_qpcr tph_cloned 1480 1490 1500 T C C C G A C C T G T A G T A T C T T A A A G A C C G G T A T C C C G A C C T G T A G T A T C T T A A A G A C C G G T A T C C C G A C C T G T A G T A T C T T A A A G A C C G G T A tph_nematostella tph_qpcr tph_cloned 1510 1520 1530 G T A T C T T A A G A T C T G T G G A T T A A A C T T T T T G T A T C T T A A G A T C T G T G G A T T A A A C T T T T T G T A T C T T A A G A T C T G T G G A T T A A A C T T T T T tph_nematostella tph_qpcr tph_cloned 1540 1550 1560 A T T A A A A G A A C C A T T A T C A C C C A C A C C G T T A T T A A A A G A A C C A T T A T C A C C C A C A C C G T T A T T A A A A G A A C C A T T A T C A C C C A C A C C G T T tph_nematostella tph_qpcr tph_cloned 1570 1580 1590 G T T A T T G T T T C A C A A T A T T A A C C A T A A A C A G T T A T T G T T T C A C A A T A T T A A C C A T A A A C A G T T A T T G T T T C A C A A T A T T A A C C A T A A A C A tph_nematostella tph_qpcr tph_cloned 1600 1610 1620 C A A G A A C A A T A T C C A C A G C T T A C A G T G T C A C A A G A A C A A T A T C C A C A G C T T A C A G T G T C A C A A G A A C A A T A T C C A C A G C T T A C A G T G T C A tph_nematostella tph_qpcr tph_cloned 1630 1640 1650 C A A T G T T T T A A G G A G T G C A C T G T C T G A T T A C A A T G T T T T A A G G A G T G C A C T G T C T G A T T A C A A T G T T T T A A G G A G T G C A C T G T C T G A T T A tph_nematostella tph_qpcr tph_cloned 1660 1670 1680 A A A T G A A C T T A A A T A A C T G G T G T G G C A A A G A A A T G A A C T T A A A T A A C T G G T G T G G C A A A G A A A T G A A C T T A A A T A A C T G G T G T G G C A A A G tph_nematostella tph_qpcr tph_cloned 1690 1700 1710 G A T G A T T C A A T G C A C A T C A G A A C A G G T G T G G A T G A T T C A A T G C A C A T C A G A A C A G G T G T G G A T G A T T C A A T G C A C A T C A G A A C A G G T G T G tph_nematostella tph_qpcr tph_cloned 1720 1730 1740 T G C C C G G G A T T G G C T G A T G G G G G T G C A C A G T G C C C G G G A T T G G C T G A T G G G G G T G C A C A G T G C C C G G G A T T G G C T G A T G G G G G T G C A C A G tph_nematostella tph_qpcr tph_cloned 1750 1760 1770 T C A T A G G T A T A T G G A A A G A G C A T T G T A T T T T C A T A G G T A T A T G G A A A G A G C A T T G T A T T T T C A T A G G T A T A T G G A A A G A G C A T T G T A T T T tph_nematostella tph_qpcr tph_cloned 1780 1790 1800 G A A G A C C A A C T T T T T T T T T T T G G G G G G G G G G A A G A C C A A C T T T T T T T T T T T G G G G G G G G G G A A G A C C A A C T T T T T T T T T T T G G G G G G G G G tph_nematostella tph_qpcr tph_cloned 1810 1820 1830 G G G G G A G A G G G T G A T C G T G C A C C C T G T G C A G G G G G A G A G G G T G A T C G T G C A C C C T G T G C A G G G G G A G A G G G T G A T C G T G C A C C C T G T G C A tph_nematostella tph_qpcr tph_cloned 1840 1850 1860 C C C C C C T G C G T C T G C A C C T G C A G A A T A T G A C C C C C C T G C G T C T G C A C C T G C A G A A T A T G A C C C C C C T G C G T C T G C A C C T G C A G A A T A T G A tph_nematostella tph_qpcr tph_cloned 1870 1880 1890 T G G A C A T T T C C A A G T T A T C A A C T T A A T C C A T G G A C A T T T C C A A G T T A T C A A C T T A A T C C A T G G A C A T T T C C A A G T T A T C A A C T T A A T C C A tph_nematostella tph_qpcr tph_cloned 1900 1910 1920 G T G A T T C T T G C G A C C T A A T T A G C T A A C T T C G T G A T T C T T G C G A C C T A A T T A G C T A A C T T C G T G A T T C T T G C G A C C T A A T T A G C T A A C T T C tph_nematostella tph_qpcr tph_cloned 1930 1940 1950 C A C T T T G A C A A T G G T G G C A A C G G C A T T A T T C A C T T T G A C A A T G G T G G C A A C G G C A T T A T T C A C T T T G A C A A T G G T G G C A A C G G C A T T A T T tph_nematostella tph_qpcr tph_cloned 1960 1970 1980 T G A T A A C C G T C T T A T C A T C T G A A A T T G A A A T G A T A A C C G T C T T A T C A T C T G A A A T T G A A A T G A T A A C C G T C T T A T C A T C T G A A A T T G A A A tph_nematostella tph_qpcr tph_cloned 1990 2000 2010 G A G C T G C T C A T G A A A C A T T A A A T T A T A T A T G A G C T G C T C A T G A A A C A T T A A A T T A T A T G A G C T G C T C A T G A A A C A T T A A A T T A T A T A T tph_nematostella tph_qpcr tph_cloned 2020 2030 2040 G C C T A T T T C A A A T G C C T A T T T C A A A T



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Chromatograms of a melatonin standard (black line) and of a Nematostella vectensis sample (blue line). The chromatograms show the same pattern, with the same retention time for melatonin, validating the assay. quinta-feira, 30 de agosto de 12







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RESEARCHOpenAccessDevelopmentalandlight-entrainedexpressionof melatoninanditsrelationshiptothecircadian clockintheseaanemone NematostellavectensisRafaelPeres1,4,AdamMReitzel2,YalePassamaneck1,SolangeCastroAfeche3,JosCipolla-Neto4, AntonioCarlosMarques5,6andMarkQMartindale1,7*AbstractBackground: Theprimaryhormoneofthevertebratepinealgland,melatonin,hasbeenidentifiedbroadly throughouttheeukaryotes.Whiletheroleformelatoninincyclicbehaviorviainteractionswiththecircadianclock hasonlybeenreportedinvertebrates,comparativeresearchhasshownthatthetranscription-translationloopsof theanimalcircadianclocklikelydatetothecnidarian-bilaterianancestor,leavingopensignificantquestionsabout theevolutionaryoriginofmelatoninsignalingincircadianbehaviorbyinteractingwiththemolecularclock. Results: Expressionofmelatonininadultanemonesshowedpeakexpressionattheendoflightperiod(zeitgeber time(ZT)=12)whenculturedunderdielconditions,coincidingwithexpressionofgenesandenzymeactivityfor membersofthemelatoninsynthesispathway(tryptophanhydroxylaseandhydroxyindol-O-methyltransferase), whichalsoshowedrhythmicexpression.Duringembryogenesisandjuvenilestages,melatoninshowedcyclic oscillationsinconcentration,peakinginmidday.Spatial(insituhybridization)andquantitative(real-timePCR) transcriptionofclockgenesduringdevelopmentof N.vectensis showedthese ‘ clock ’ genesareexpressedearlyin thedevelopment,priortorhythmicoscillations,suggestingfunctionsindependentofafunctioninthecircadian clock.Finally,time-coursestudiesrevealedthatanimalstransferredfromdielconditionstoconstantdarknesslose circadianexpressionformostoftheclockgeneswithin4days,whichcanberesetbymelatoninsupplementation. Conclusions: Ourresultssupportanancientroleformelatonininthecircadianbehaviorofanimalsbyshowing cyclicexpressionofthishormoneunderdielconditions,light-dependentoscillationsingenesinthemelatonin synthesispathway,andthefunctionofmelatoninininitiatingexpressionofcircadianclockgenesinthecnidarian N.vectensis .Thedifferencesinexpressionmelatoninandthecircadianclockgenenetworkintheadultstagewhen comparedwithdevelopmentalstagesof N.vectensis suggestsnewresearchdirectionstocharacterizestage-specific mechanismsofcircadianclockfunctioninanimals. Keywords: Circadianclock,Embryogenesis,Insituhybridization,MelatoninBackgroundMostorganismsdisplaydailyresponsestotheperiodic changesintheenvironment,su chaslight,thatincludeanticipationoftheseenvironmentalshiftsandsynchronization ofmolecularandcellularprocesses.Thedieloscillationsinbehaviorandphysiologyofanimalsinresponse toenvironmentalstimuliareregulatedbyacombinationofhormoneexpressionandmolecularnetworks composingacircadianclock.Thecontributionsofand interactionsbetweeneachofthesetwotypesofmechanismsincircadianbehaviorandphysiologyrepresentsa nexusforunderstandinghowdifferentprocessesregulateorganism-environmentinteractionsandhowthese componentsmaychangeoverevolutionarytime. Theprimaryhormoneofthevertebratepinealgland, theindolemelatonin,wasfirstdescribedasacircadian andseasonaloutputsignalofthepacemakerthatallows *Correspondence: mqmartin@whitney.ufl.edu1KewaloMarineLaboratory,UniversityofHawaii,41AhuiStreet,96813 Honolulu,HI,USA7WhitneyLaboratoryforMarineBioscience,UniversityofFlorida,9505Ocean ShoreBlvd,32080St.Augustine,FL,USA Fulllistofauthorinformationisavailableattheendofthearticle 2014Peresetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/4.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycredited.TheCreativeCommonsPublicDomain Dedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle, unlessotherwisestated.Peres etal.EvoDevo 2014, 5 :26 http://www.evodevojournal.com/content/5/1/26

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theanimaltopredictandprepareforchangesintheir environment[1].However,manystudieshaverecently beenpublishedthatexpandtheimportanceofmelatonin inmammalianandnon-mammalianspecies,including newsitesofproduction,suchastheretina[2]and gastrointestinalsystem[3]. Thepresenceofmelatoninininvertebrateswasfirst reportedininsects[4],thensubsequentlyinmanyother invertebrates(seebelow),andlaterstudiesshowedthe presenceofmelatonininadiversearrayofeukaryotic organisms,includingdinoflagellates[5],inwhichitmay regulatecystformation[6];plants,inwhichitregulates flowering[7];andfungi[8],wherenofunctionsfor melatoninhavebeendescribedsofar. Invertebrates,thepeakofmelatoninproductioncharacteristicallyoccursduringthenight[9],anditsproductionisactivelyregulatedbychangesindaylightlength [1].However,differentpatternsofpeakproductionwith respecttotimeofdayhavebeenreportedforinvertebrates.Someprotostomespecies,likeflatworms[10] andthefruitfly Drosophilamelanogaster [11,12] show nocturnalpeaksofmelatonin,similartovertebrates. However,inotherspecies,suchasthecricket Gryllus bimaculatus, melatoninpeaksvarydependingonthetissueexamined,withanocturnalpeakinthebrainandin theeyes,butadiurnalpeakintheantennae,locomotor appendicesandreproductiveorgans[13].Asimilar patternofdifferentialexpressionwasobservedinthe gastropod Aplysiacalifornica ,withanocturnalmelatoninpeakinthebrainanddiurnalpeakintheeye[14]. Thevariationinpeakmelatoninexpressionindifferent organsmaybeexplainedinpartbymelatonin ’ srolein photo-protection[6]. Themolecularpathwaythatleadstothesynthesisof melatoninhasbeenwell-characterizedinvertebrates. Thefirststepinmelatoninsynthesisinvolvestheconversionoftryptophaninto5-hydroxytryptophan(5HTP) bytheenzymetryptophanhydroxylase(TPH).Thenext stepinvolves5HTPdecarboxylationbyanonspecificdecarboxylasethattransformsitintoserotonin.Serotonin isN-acetylatedbyarylalkylamineN-acetyltransferase (AANAT)producingN-acetylserotonin(NAS)[15].In rats,AANATistherate-limitingenzymeduringthedaytime,showinga100%increaseinactivityduringthenight. AANATactivityispreciselycontrolledanddecreases sharply,withahalf-lifeof3minutes,whensympathetic stimulationisceased.Thisresponseisdeterminedby eithertheadministrationofadrenergicantagonistsorasa resultofnocturnalphotostimulation[16-19].While AANAT-likesequenceshavebeenidentifiedinvarious animalandnon-animallineages[20],thevertebratetype AANATthatfunctionsinmelatoninsynthesisisrestricted tovertebratesfollowingneofunctionalizationafterduplicationofthenon-vertebrateAANATgene[21].The finalstepinmelatoninproductionistransformationof NAStomelatoninbyhydroxyindol-O-methyltransferase (HIOMT)[22]. Intheadultstageofvariousvertebratespecies,oneof themainfunctionsofmelatoninisasahormonaloutput ofmolecularsignalingfromaninternalclocklocatedin thesuprachiasmaticnucleus(SCN)inthebrain.The SCNclockismaintainedbytheoscillationsofasetof clockgenes( clock,bmal,cry1 cry2, and period ,among others)thatcontrolsmelatoninproductionthroughsignalingtothepinealgland[23,24].Circulatingmelatonin releasedbythepinealglandthensignalsshiftsinphotoperiodtoperipheralclocksintherestofthebody.Also, melatoninactsasaclockinput,regulatingcircadian rhythmicitythroughthesuprachiasmaticnucleus,resettingtheclockgenesexpression[25].Thus,invertebrates,melatoninandthecircadianclockaretightly associatedwithmaintainingperiodicbehaviorover24hourperiods. Althoughdieloscillationsinmelatoninproductionare knownfrommanyprotostomes,particularlyinsects(see above),apotentialconnectionbetweenmelatoninand themolecularmechanismofthecircadianclockisunknown.Comparativeresearchwithinsectshasshown thatthecircadianclockiscomposedofsimilar,andin manycasesorthologous,genesthatareorganizedin transcription-translationfeedbackloopsasseeninvertebrates[26-28].Thesimilarityofthecircadianclock mechanismandmelatoninsynthesispeaksininsects suggestsaplausiblehypothesiswhere,asinvertebrates, melatoninandcircadianclocksarecloselyassociated. Theontogenyofmelatoninproductionandcircadian clock-relatedgenesandtheirrelationshiptocircadian rhythmsinpre-adultdevelopmentalstagesremainslittle studied.Melatoninwasrecentlyidentifiedintheeggsof rainbowtroutwithclearcircadianrhythmundera12 light(L):12dark(D)photocycle[29],andinbirdegg yolk[30].Thepresenceandpotentialfunctionsofmelatoninduringdevelopmentofinvertebrateshasnotbeen characterized.Moststudiesofthedevelopmentalexpressionofgenescentraltothecircadianclockhavebeen conductedinvertebratespecies.Forexample,previous researchshowedalight-independentinitiationofzygotic per1 transcriptiononthefirstdayofdevelopmentof zebrafish[31].Thisstudyalsodemonstratedthatduring thefirst3daysofdevelopment, clock1 and bmal1 transcriptionwasnotrhythmic,incontrasttolaterdevelopmentalstageswherepronouncedrhythmicexpressionin responsetolightcueswasobserved.However,direct manipulationsshowthatCLOCKisalreadyfunctionalbyregulatingtranscriptionof per1 24-hourspost-fertilization. Thedifferencesinembryosandadultssuggestakeyregulationofthecircadianclockinzebrafishdevelopmentisa productofpost-transcriptionalprocesses.PreimplantationPeres etal.EvoDevo 2014, 5 :26 Page2of18 http://www.evodevojournal.com/content/5/1/26

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mouseembryosexpressmanyofthecorecircadianclock (forexample, bmal,clock,period, and cryptochromes ),but expressionofthesegenesdidnotshow24-houroscillationsinone-tofour-cellandblastocyst-stageembryos [32].Theauthorsofthestudyhypothesizedthatcircadian genesarenotinvolvedincircadianclockregulationin mousepreimplantationembryosbutareinsteadinvolved inregulationofthecellcycle,particularlymeiosis.In Xenopus embryogenesis,likemouse,transcriptsofthe corecircadianclock( bmal1,per1and2, and clock), have expressedinearlydevelopment,withnoevidenceofa24hourexpressioncycle,butmaybeinvolvedinvariousdevelopmentalpatterningprocessesincludingtheformation andtimingofsomites,heart,andnervoussystem[33]. Theseresultsareconsistentwithresultsfrom Xenopus thatshowed Clock expressioninthemesodermalcellsof thedorsalblastoporelipandtheoverlyingectodermal cells[34].Atpresent,therehavenotbeenanystudiesof theontogenyofmelatoninproductionorclockgene expressionininvertebrates. Together,researchinbilaterianshassuggestedthat melatoniniswidelypresentandthemolecularmechanismscomposingthecircadianclockarebroadlyconserved,butatleastinthevertebrateswhereithasbeen studied,theinteractionofmelatoninandthecircadian clockinregulatingdailybehaviorislikelyrestrictedto theadultstage.Recentresearchincnidarianshasbegun toidentifysimilarconservationofmelatoninproduction andthecircadianclockmechanism.Thecnidariansarea phylumofpredominatelymarinespeciesthatarethe likelysistergrouptothesuperphylumBilateria,which comprisesdeuterostomes(forexample,vertebratesand echinoderms)andprotostomes(forexample,arthropods andmolluscs).Studiesutilizingcnidariansasexperimentalmodelsarethereforeusefulfordeterminingthe antiquityofmolecularfunctionsforsharedproteinsand hormones,aswellasforidentifyingpotentiallynovel featuresforhomologousmoleculeswhencomparedto bilaterians.Studiesofseveralanthozoancnidarianshas revealedthatmostofthecorecircadianclockgenesare conservedincnidarians,andtheyaredifferentially expressedindiellightingconditions[35,36],Despite thesesimilarities,noresearchtodatehasaddressed whetherothermechanismsofthecircadianclockmay beconservedincnidarians,particularlytheroleof hormonesinsignalingandresettingoftheclock.This informationisofimportanceintheunderstandingofthe originandevolutionofmelatonin/circadiancycles. Melatoninhaspreviouslybeenidentifiedinthecolonial anthozoan Renillakllikeri [37] whereitwasconcentratedinneuronalcellsneargametogenictissues.Tissue concentrationfollowedaseasonalbutnotdiurnalpattern, suggestingitcouldserveasapacemakerinvolvedinseasonalreproductionbutnotcircadianrhythms[8,14].Data intwootheranthozoanspecies( Actiniaequina and Nematostellavectensis )[38,39]havesimilarlyshownthat melatoninislocalizednearoringametogenictissues. ExpressionpatternsofHIOMTandputativemelatonin receptorstranscriptsalsoshowedlocalizedexpressionin gametogenictissueaswellassurroundingendodermaltissue.Inaddition,RoopinandLevy[38]showedthatmelatoninlevelsin A.equina varyonadielcyclewithpeak concentrationinearlysubjectivenight,buttheoscillating expressiondissipatesuponexposuretoconstantdarkness. Theselaterresultssuggestthatmelatoninmayservearole inthecircadianclockbecausecnidariansloseoscillations intheexpressionofcircadianclockgeneswhenremoved fromanentraininglightcue[35,36].Together,thesepreviousdatafromcnidarianssuggestanunresolvedpicture forpotentialrole(s)ofmelatoninincnidarians,whichmay includebothseasonalanddailyfunctions.Melatoninin cnidarianshasnotbeenstudiedinpre-adultstagesto determineifitisproducedinstageslackinggametogenic tissueand,ifexpressed,whetherconcentrationvariesina patternconsistentwitharoleincircadianbehavior. Thepresentstudyanalyzesthepresenceofmelatonin anditsrhythmicoscillationinadultsculturedunderdifferentlightingregimesaswellasdevelopmentalstages ofthestarletseaanemone Nematostellavectensis .We alsoreporttheexpressionofgenesencodingtheenzymesofthemelatoninpathwaydescribedfromvertebratespecies,extendingpreviousresultsfor N.vectensis and A.equina [38,39] Wealsostudiedthespatial(in situhybridization)andquantitative(real-timePCR) transcriptionofclockgenesinthedevelopmentof N. vectensis ,showingthattheclockgenesbegintobe expressedearlyinthedevelopment,priortorhythmic oscillations,suggestingfunctionsindependentofafunctioninthecircadianclock.Finally,weexperimentally determinetheimpactofmelatoninsupplementationon thetranscriptionofcircadianclockgenesinthiscnidarian,whichstronglysuggeststhatthefunctionofmelatonininresettingofthecircadianclockisconservedby resettingtranscriptionaloscillationsthatdissipateaftera fewdaysofconstantdarkness.Together,ourdata supporthypothesesthatthefunctionofmelatonininthecircadianclockdatesbacktoatleastthecnidarianbilaterianancestor,thegenesthatcomposethecircadian clockhavedevelopmentalfunctionsinembryogenesis, andlarval N.vectensis potentiallyhavedielrhythms evidencedbytemporalexpressionbymelatonin.MethodsAnimalculturingandexperimentaldesignAdult N.vectensis (measuringbetween2and5cmin length)werekeptundera12-hour:12-hourlight – darkcycle (lightsonat7:00h(Hawaiista ndardtime);ZeitgeberTime ZT=0;lightsoffat19:00h;ZeitgeberTimeZT=12-Peres etal.EvoDevo 2014, 5 :26 Page3of18 http://www.evodevojournal.com/content/5/1/26

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underfullspectrumlights,(Corallife50/50bulb, approximately3,600lux),inglassbowlswith13parts perthousand( ‰ )filteredlocalseawater(dilutedwith distilledwater,referredtoas ‘ 1/3Xseawater ’ )ina temperature-controlledincubator(17C).Waterwas changedeveryday(ZT=0),andanimalswerefedtwice weeklywithfreshlyhatchedbrineshrimp.Asecond groupofadultindividualswerekeptinconstantdarknessfor20daysunderthesameconditions,withwater changesalsooccurringatZT=0.Forasecondexperiment,tomeasuredissipationoftranscriptionaloscillationsinanimalsremovedfromlightcycling,groupsof animalswerekeptinconstantdarknessfor24h,48h and96handotherwisewiththesameconditions. Forthemelatonintreatments,adultanimalswerekept inconstantdarknessfor5daysandthenexposedto 0.1 Mmelatonin(SigmaChemicalCo.,St.Louis,MO, USA)forthesubsequent15consecutivedaysinconstant darkness.Exogenousmelatoninadditionwasadministeredatthetimeofthetransitiontothesubjectivenight (ZT=12),correspondingtoatimeofpeakmelatonin production(seeResults).After12hoursofincubation, theanimalswererinsedinfresh1/3Xseawaterand placedinnewdishesfortheremainderofthe experiment. Todeterminethetemporalproductionofmelatoninin adults,embryos(48-hourpost-fertilization),andjuveniles(1-weekand2-weekpost-fertilization),animals wereindividuallycollectedfromtheirrespectivetreatmentevery3hoursandplacedin1.5mlmicrocentrifuge tubesandflashfrozenwithliquidnitrogen.Embryos andjuvenileswerealsokeptfromeggstageunderthe sameconditionoftheadultsin12-hour:12-hourlight – darkcycle.EggswerefertilizedintheZT=6.Individuals forRNAextractionswereindividuallyplacedintoa 1.5mltubewithTriPureReagent(RocheInc,USA), thenflashfrozen.InsituhybridizationsInsituhybridizationswereperformedaspreviouslydescribed[40].Embryoswerecollectedfromspecifictime pointspost-fertilization(24,48,96,144and168hourspolypswerenotfedduringthestages).Eggswerefertilized intheZT=6.TheanimalcollectionwasalsoatZT=6, occurring24,48,96,144or168hoursafterfertilization andcorrespondingwiththedevelopmentalstages-blastula,lateblastula,gastrula,planula,andlateplanula, respectively.Allstageswerefixedinfreshice-cold3.7% formaldehydewith0.2%glutaraldehydein1/3Xseawater for60secondsandthenpostfixedin3.7%formaldehyde in1/3Xseawaterat4Cfor1hour.Fixedembryoswere rinsedfivetimesinPBSbufferplus0.1%Tween20(PTw) andonceindeionizedwater,andtransferredto100% methanolforstorageat-20C.Earlyembryoswere removedfromthejellyoftheeggmassbytreatingwith freshlymade2%cysteinein1/3Xseawater(pH7.4to7.6) for10minutes.Planulaandlateplanulawererelaxedin 7%MgCl2in1/3Xseawaterfor10minutespriorto fixation.Insituhybridizationusing1to2kbdigoxigeninlabeledriboprobesfor Clock Timeout ,and Cryptochromes wereperformedtodeterminethespatialandtemporaldistributionoftranscriptsineachstage.Probeconcentration rangedfrom0.5to1.0ngml 1,andhybridizationswere performedat70Cfor40hoursin50%formamide.Probe detectionwasachievedbyincubationwithanantidigoxigeninantibodyconjugatedtoalkalinephosphatase(Roche, Inc).Subsequently,thepresenceofalkalinephosphatase wasdetectedbyacolorimetricdetectionreactionusing thesubstrateNBT-BCIP.Specimenswerephotographed onaAxioskopIIwithaZeissZxiocamHRc.MelatoninassaysLevelsofmelatonin(nanograms)wereassayedin N.vectensis (adultsandembryos)byamodifiedandhighlysensitiveenzyme-linkedimmunosorbentassay(ELISA)method (IBLInternational,Hamburg,Germany).Melatoninwas extractedfromtissuewith0.6mlof0.6%perchloricacid. Tonormalizetheassays,2 lofeachsamplewereused forproteinmeasurements.Theamountoftotalprotein (inmilligrams)wasdeterminedspectrophotometrically at280nmwithaNanoDrop1000(ThermoScientific, Inc.).SampleswerethenpassedthroughaC18reversedphasecolumn,extractedwithmethanol,evaporatedto dryness,andreconstitutedwithwater.Eachsamplewas addedtoamicroplatewellcoatedwiththegoat-antirabbitanti-melatoninantibody.Anunknownamount ofantigenpresentinthesampleandafixedamountof enzyme-labeledantigencompetedforthebindingsitesof theantibodiescoatedontothewells.Afterincubationfor twohours,thewellswerewashedtostopthecompetition reaction.Thesubstratep-nitrophenylphosphate(PNPP) wasaddedandtheconcentrationofantigendetermined astheinverseproportionoftheopticaldensitymeasured inaphotometer.Melatoninstandardswereusedtoconstructastandardcurveagainstwhichtheunknownsampleswerecalculated.Specificityofthemethodiscloseto 100%wheremelatoninconcentrationsexceed3pgml-1, thelimitofsensitivityforthisassay.(IBLELISAkitmanual).Aprevioustestaddingknownconcentrationsof melatonintothe N.vectensis samplesshowedtherecovery fromourisolationprotocolwasapproximately85%(data notshown). Foradditionalvalidation,thepresenceof bonafide melatoninwasconfirmedbyhighperformanceliquid chromatography(HPLC)withelectrochemicaldetection (Chromeleon ™ 7.1,DionexSystem,Sunnyvale,CA,USA). MelatoninwasseparatedonAcclaimC18column(2,2 M 2.1100mm,DionexSystem,Sunnyvale,CA,USA).ThePeres etal.EvoDevo 2014, 5 :26 Page4of18 http://www.evodevojournal.com/content/5/1/26

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chromatographicsystemwasisocraticallyoperated(that is,systemsettonotchangecompositionofthesolution duringtherun)withthefollowingmobilephase:0.1M sodiumacetate,0.1Mcitricacid,0.15mMEDTA,32% methanol,pH3.7,ata0.120mlmin 1flowrate.Theelectrochemicaldetectorpotentialwasadjustedto+750mV. Theelutiontimeformelatoninwasapproximately10minutes.Eachtissuesamplewassonicated(MicrosonXL 2005,HeatSystemInc.,Farmingdale,NY,USA)inasolutionof0.1Mperchloricacid(200 l)containing0.02% EDTAand0.02%sodiumbisulfate.Aftercentrifugation (2min,13,000g Eppendorf5415CCentrifuge,Brinkman InstrumentsInc.,Westbury,NY,USA),40 lofthesupernatantwasinjectedintothechromatographicsystemwith anautomaticinjector.Amelatoninstocksolutionwas preparedin0.1MHCLwith0.02%EDTAand0.02%sodiummetabisulfite,andthendilutionstomakeastandard curve(0.145to4.64ng20 l 1)werepreparedwithperchloricacidjustbeforetheassayswereperformed.Blank assayscontainingnomelatoninwereusedasnegative controls.SerotoninassaysSerotonin(5HT)contentwasquantifiedusingasensitive enzyme-linkedimmunosorbentassaymethod(IBLInternational,Hamburg,Germany).Theembryossamples (approximately60embryospersample)werecentrifuged (3,000gfor2min)andtheseawaterremoved.Sampleswerereconstitutedin120 loftheassaybufferof thekit.Tonormalizetheassays,2 lofeachsamplewas usedtoproteinmeasures.Briefly,theassaywasperformedasfollows:20 lofsamplesand5HTstandard dilutionswereappliedto96-wellmicrotiterplatespreviouslycoatedwithgoatanti-rabbitantibodyfollowedby 50 lbiotin-labeled5HTand50 lrabbitantibody against5HT.Afterincubationovernightat4Candwashingwithwashbuffer,150 loffreshpreparedenzyme conjugatedwasadded.Samplesfromthedevelopmental stages,standards,positiveandnegativecontrolswereincubatedfor1houratroomtemperaturewithgentlemixing,washedagain,then200 lp-nitrophenylphosphate substratewasadded,andtheenzymereactionwas terminatedafter60minutesbyadditionof50 lofpnitrophenylphosphatestopsolution.Absorptionwas measuredat405nminaspectrophotometerandtheconcentrationof5HTwascalculatedfromthereference curve.Aprevioustestaddingknownconcentrationsof serotonintothe N.vectensis samplesshowedusthatthe recoverywasapproximately90%(datanotshown).TotalRNAextraction,reverse-transcriptionreaction, andPCRTotalRNAwasextractedfromsamplesusingTriPure Reagent(RocheInc,USA)accordingtothemanufacturer's specifications.Briefly,adultanimals/embryoswerelysed in0.5mlTriPurereagentandincubatedfor5minatroom temperature.Two-hundred lof1-bromo-3-chloropropane(BCP)wasaddedtothetubesandcentrifugedat 12,000gfor15minutes.Theaqueousphasewastransferredtoafreshtube,treatedwithDNAse(Ambion,Life Technologies,GrandIsland,NY,USA)andtotalRNAwas pelletedbyprecipitationwithisopropylalcoholandcentrifugation(12,000gfor10min).TheRNApelletwas washedwithethanol(75%)andpelletedat7,500gfor 5minandair-driedatroomtemperature.RNApellets werereconstitutedinRNase-freewater.RNAwasquantifiedspectrophotometricallyat260nmwith260/280ratios between1.8and2.0.RNAqualitywasalsocheckedby 1.4%agarosegelelectrophoresisstainedwith5 gml 1ethidiumbromide.ComplementaryDNA(cDNA)was synthesizedwiththeAdvantageRT-for-PCRKitprotocol (ClontechLaboratories,MountainView,CA,USA)followingthesupplier'sinstructions.cDNAwasstoredin waterat-20C. PrimersforquantitativePCRweredesignedfor genesofinteresttoamplifygenefragmentsoflength 75to150bp[seeAdditionalfile1]usingMacVector (MacVector,Inc,NorthCarolinaUSA).Thegenes studiedincludepresumptivecircadianclockgenespreviouslyreportedbyReitzeletal.[35],aswellasidentifiedgeneslikelyinvolvedinthemelatoninsynthesis pathway[39].RibosomalproteinP0[XM_001626244.1] wasusedasanormalizationgeneforallexperiments. PreviousqPCRassaysshowedthatthisgenedoesnothave variationintranscription,eitherbetweendifferenttime pointswithinatreatmentorbetweendifferenttreatments. Thecyclingparametersusedwere10secondsat95C, 20secondsatspecificannealingtemperatureforeachprimer[seeAdditionalfile1],and20secondsat72Cduring 45cycles.qPCRwasconductedonaLightCycler ™ 480 Real-TimePCRSystem(Roche,Inc.)usingSYBRGreenI Mastermix(Roche,Inc.).EnzymeactivityassaysTryptophanhydroxylase(TPH)activitywasquantified aspreviouslydescribed[41].Briefly,eachtissuesample wassonicatedinsodiumphosphatebuffer(2mM,pH7, 100 L),andthefollowingmixturewasaddedtoeach sample:HEPES(50mM,pH7),catalase(100 gml 1), tryptophan(50 M),dithiothreitol(5mM),Fe(NH4)2(SO4)2(10 M),6-MPH4(500 M)and1 Lof[3H] tryptophan(1mCiml 1-previouslydriedundernitrogen).Thematerialwasincubatedat37Cfor10minutes. Anactivatedcharcoalsolutionwasadded(7.5%in1M HCl)toterminatethereaction.Two-hundred Lofthe supernatantwastransferredtoscintillationvials,and radioactivitywasdeterminedbyaBeckmanLS6500 counter(BeckmanCoulterInc.,CA,USA).Positive(ratPeres etal.EvoDevo 2014, 5 :26 Page5of18 http://www.evodevojournal.com/content/5/1/26

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pinealglands)andnegative(water)controlswereincluded ineachassay. Hydroxyindol-O-methyltransferase(HIOMT)activity wasassayedaspreviouslydescribed[22].Sampleswere sonicatedinphosphatebuffer(0.05M,pH7.9,50 L). Onehundredfifty Lofasolutioncontaining14C-Sadenosyl-L-methionine(specificactivity43.8mCi-Sigma ChemicalCo.,St.Louis,MO,USA)andN-acetylserotonin (1mM)wasthenadded.Thehomogenateswereincubatedfor30minutesat37C.Thereactionwasterminated byadding200 Lofsodiumboratebuffer(12.5mM, pH10)and1mLofbuffersaturatedchloroform.The tubeswerecentrifugedat13,000xgfor5minutesat4C. The14[C]melatoninproductwasextractedin800 Lof chloroform,air-dried,andtheradioactivitywasdeterminedbyaBeckmanLS6500 counter(BeckmanCoulter Inc.,CA,USA).Positive(ratpinealglands)andnegative (water)wereincludedineachassay.Tonormalizethe TPHandHIOMTassays,2 lofeachsamplewereused quantifyproteinconcentrationtotheTPHandtothe HIOMTexperiments.Theresultsarepresentedasthe ratioofactivitypermilligramofprotein.StatisticalanalysisDataarepresentedasthemeanoffourindependent replicatesSEM.Melatoninandserotonincontentwas expressedasngmg-1ofprotein.Statisticalanalyses (GraphPadPrism5.0,GraphPadSoftwareInc.,San Diego,CA,USA)wereperformedusinganANOVA (one-ortwo-way,asrequired)followedbyBonferroni post-hoctest. One-wayANOVAwasusedtoevaluatetheinfluence ofthevariable ‘ timeofday ’ oneachtemporalseries. Givenanoverallsignificantone-wayANOVA,the cosinormethodwasutilizedtoevaluatethepresenceof adaily24-hourrhythm.Thetheoreticalcosinecurvefittingwasappliedineachtemporalseriesusingtheleastsquarecalculation.Thebestfittingcurveforeachday wasdeterminedwithF-statistics.Thenullhypothesis testedwaszeroamplitude,thatis,norhythmicityata 24-hourfrequency.Foreachtemporalseries,three parametersoftheadjustedcurvewerecalculated:acrophase(timeofthemaximumvalueoftheadjusted curve),mesor(valueofthemeanleveloftheadjusted curve)andamplitude(distancebetweenthemesorand themaximumorminimumvalueoftheadjustedcurve). Theserhythmicparameterswerecomparedbetween groupsusingtheStudent ’ s t -test[42].Allstatistical analyseswereconsideredsignificantfor P 0.05.ResultsMelatoninconcentrationinadultsMelatoninwassuccessfullydetectedinadultsand embryosusingtheELISAkit.Highperformanceliquid chromatography(HPLC)withelectrochemicaldetection assayconfirmedthepresenceofmelatonin,withthe sameretentiontimefortheindolinthestandardsand inallsamples[seeAdditionalfile2].Fortheadultsin thelight – darktreatment,weobservedpeakexpression at4hoursbeforethetransitiontothedarkphase(that is,ZT=8,Figure1a,light-grayline)thatwassustained untiltheZT=20(4hoursbeforethetransitiontosubjectivedayphase).Inordertoevaluateiftheseoscillationsinmelatoninproductionwerearesponsetothe presenceorabsenceoflight,werepeatedtheexperiment usinganimalsthatwerekeptinconstantdarknessfor 20days.After20daysofconstantdarkness,melatonin stillshowedsignificantlyhigherconcentrationinthe transitionfromthesubjectivedaytothesubjective night(ZT=12,Figure1a,blackline,one-wayANOVA, P =0.0124).However,thenightpeakofmelatoninwas notsustainedduringthesubjectivenightwherewe measuredsignificantlylowerconcentrationsofmelatonininZTs=16and20.Thisobservationisconfirmed byrhythmicanalysisusingthecosinorprocedures, whichshowedthatmelatoninlevelscycledinthelight – darkcycle,butnotinconstantdarkness(Figure1b).Melatoninandserotoninconcentrationduring developmentFor N.vectensis embryos,wemeasuredapeakexpressionofmelatonin6hoursafterlightson(ZT=6)in48hourembryos(Figure1c)aswellasjuvenilepolypstages (1-weekpost-fertilization,Figure1d;2-weekspostfertilization,Figure1e).Thepeakamountofmelatonin wassimilarinthe48-hour(14.111.04pgmg 1fprotein)and1-weekjuveniles(14.040.88pgmg 1ofprotein)butwasalmostthehalfoftheamountobservedin the2-weekjuveniles(25.410.54pgmg 1ofprotein). Thelowestmelatoninconcentrationwasobservedinthe middleofthedarkperiod(ZT=18)forallthegroups (4.340.86pgmg 1ofproteinforthe48-hour;5.81 0.62pgmg 1ofproteinfor1-weekand8.64 0.701pgmg 1ofproteinfor2-weekjuveniles). Tobetterunderstandthedynamicsofthemelatonin productionintheembryosof N.vectensis ,wealsomade serotoninmeasuresonthesamedevelopmentaltime points.Serotoninwassuccessfullydetectedinallsamples.Thepatternofproductionreplicatestheone showedinthemelatoninassays.Again,therewasapeak expression6hoursafterlightson(ZT=6)inthe48-hour embryos(Figure2a),1-week(Figure2b)and2-week-old polyps(Figure2c).Thelowestserotoninpeakwasobservedinthe48-hourembryos(14.730.91pgmg 1of protein).Inthe1-weekjuveniles,thepeakwas61.43 1.02pgmg 1ofprotein,whichwashigherthanthe2weekjuveniles(53.251.30pgmg 1ofprotein).ThisdifferencebetweenthejuvenilestagespotentiallyindicatesaPeres etal.EvoDevo 2014, 5 :26 Page6of18 http://www.evodevojournal.com/content/5/1/26

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higheramountofserotoninisbeingconvertedin melatoninintheseolderjuveniles.DieloscillationsinmelatoninproductionpathwayInordertocharacterizethe melatoninproductionin N.vectensis ,weidentifiedandmeasuredtheexpression patternoftranscriptscodingforenzymesintheclassic pathwayofmelatoninproduction Bysearchingthe N. vectensis genemodelsandESTlibrary(JointGenome Institute Nematostella genomebrowser,http://genome. jgi-psf.org/Nemve1/Nemve1.home.html),weidentified candidatesforthefirstandthelastenzymesinthe classicalmelatoninpathwa y,tryptophanhydroxylase [XM_001623795.1]andhydroxyindol-O-methyltransferase [XM_001627179.1].HIOMThasbeenpreviouslydescribed in N.vectensis [39] Bothgeneswereamplified,clonedand sequenced,withthesamenucleotidesequencepresent intheESTlibrary[seeAdditionalfile3andAdditional file4].Thesequenceshadhighsimilaritytosequences previouslydescribedinotherspecies.Phylogenetic analysesshowthatthecandidate N.vectensis TPH genegroupswithPaH(phenylalaninehydroxylases), thesistergrouptoTPH,withnoclearTPHortholog inthegenome[Additionalfile5].Becausethe N.vectensis TPH-likegenegroupswithPaH,werefertoitas TPH/PaH,indicatingitsco-orthologousrelationship tobothTPHandPaHfrombilaterians. N.vectensis HIOMTgroupswithpreviouslyreportedHIOMTproteinsamongvariousanimalspecies.Thephylogenetic analysisofthe N.vectensis HIOMTgeneclearlysupportsorthologywithotheranimalHIOMTgenestothe exclusionofnon-animalgenes(o-methyltransferases). Figure1 Melatonincontentin Nematostellavectensis. Resultsin (a) Nematostellavectensis adultsculturedinalight – dark(lightgrayline) (12:12h)orconstantdarknesscycle(blackline);andin48-h (c) ,1-week(d) and2-week-old (e) embryosinlightdarkcycle.* P <0.05.Two-way analysisofvariance(ANOVA)followedbyBonferronimultiplecomparison(n=5individuals/samplespertimepoint,pergroup). (b) Curveobtained withthecosinortestwiththeadultanimalsculturedinthelight – darkcycle.Thehorizontalline,themesor,representsthemeanoscillation,andthe verticalarrow,theacrophase,thetimeofoccurrenceofthedaily-peakofmelatonin. Peres etal.EvoDevo 2014, 5 :26 Page7of18 http://www.evodevojournal.com/content/5/1/26

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HumanandzebrafishHIOMT-likegenesaresupportedas morerecentduplicationeventsinthedeuterostomeor chordatelineage[seeAdditionalfile6]. NvTPH/PaHshowedlittlechangeinexpressioninadult animalsexposedtolight – darkconditions.Therewasa moderate,butmeasurable,increaseintranscriptionbefore thetransitionfromthelighttothedarkperiod(Figure3a, light-grayline),whichcorrelateswiththepatternobserved inthemelatoninproduction.Therewassignificant variationinexpressionofTPH/PaHintheanimalsinconstantdarknessinapatternwithtwopeaksthatdoesnot resembletheoneobservedformelatonin(Figure3a,black line).ThePearsoncorrelationtestbetweentheTPH/PaH expressionandmelatonincontentwaspositivefortheanimalsinthelight – darkcycle(r=0.871, P =0.0053).There wasnocorrelationfortheanimalsinconstantdarkness (rvalue=0.286, P =0.267).ExpressionofTPH/PaHin the2-weekembryosshowedapatternthatagaincorrelatedwiththeserotoninandmelatoninproductionprofile(Figure3c).ThePearsoncorrelationtestbetween theTPH/PaHexpressionandserotonincontentwas positivefortheembryos(r=0.926, P =0.0044).Asimilar resultwithlowerexpressionwasobservedforthe48-hour embryosand1-weekjuveniles(datanotshown). NvHIOMThadincreasedexpressionatthebeginningof thelightperiodandhadsignificantlylowerexpression duringthedarkperiodforadultanimals(Figure3b, light-grayline).Thispatterncorrelatedwiththepatternof melatoninproductionobserved,witha4-houradvanceof theHIOMTgeneexpressionrelativetothemelatonin peakdeterminedwithELISA(Pearsoncorrelationtestr value=0.696, P =0.042).Foranimalsinconstantdarkness, HIOMTexpressionwasnearconstantduringthe24-hour period(Figure3b,blackline).ExpressionoftheHIOMT geneinthe2-week-oldjuvenilesalsowassimilartothat patternofserotoninandmelatoninproduction(Figure3d), withapeak6hoursafterlightson(ZT=6).ThePearson correlationtestbetweentheHIOMTexpressionand melatonincontentwaspositivefortheembryos(r=0.931, P =0.0108).Asimilarresultwasobservedforthe48-hour embryosand1-weekjuvenile(datanotshown). Tofurthervalidatetheserotoninandmelatoninassays, weperformedenzymaticassaysfortryptophanhydroxylaseandhydroxyindol-O-methyltransferaseforadults fromZT=8andZT=20.Thesetimeperiodsshowedactivityforeachenzymeatbothtimepoints(Table1).HighestactivitywasmeasuredattheZT=8,thesamepointof themelatoninpeak.Enzymaticassayswerealsoperformed withsamplesfromembryosfromZT=6.Themeasured activityoftryptophanhydroxylasewas38.947.32picomolesper200mgofproteinperhour(n=4)and61.51 0.74picomolesper200mgofproteinperhour(n=4)for hydroxyindol-O-methyltransferase.Developmentalexpressionofthecircadianclockgenes andgenerationofrhythmicityTocharacterizethepotentialroleof N.vectensis clock genesinembryonicdevelopment,weexaminedthe spatialandtemporalexpressiondynamicsofthosegenes bywholemountinsituhybridization.Figure4showsthe Figure2 Serotonincontentin Nematostellavectensis embryos culturedinalight – dark(12:12h)cycle. Samplesfrom48-h (a) 1-week (b) and2-weeks (c) post-fertilization. Peres etal.EvoDevo 2014, 5 :26 Page8of18 http://www.evodevojournal.com/content/5/1/26

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expressionpatternsof NvClock,NvTimeout,NvCry1a and NvCry1b ( NvCycle reportedin[43]).Diffuseexpression of NvClock beginsatblastulastage(Figure4a).Expressionisconcentratedintheoralpoleduringlateblastula (Figure4b)andthenremainsrestrictedtotheendodermduringthegastrula(Figure4c)andlateplanula (Figure4d-e)stagesofdevelopment.Expressionappearedtobeuniforminallcellsoftheendodermand didnotshowthe ‘ saltandpepper ’ patterncharacteristic ofcellsintheendodermalnervoussystem[44]. NvTimeout showedapatternsimilarto NvClock ,weak expressionattheblastulastage(Figure4f)withexpressionintensifyingattheoralpoleduringinvagination (Figure4g).Afterthisstage,expressionisthenrestricted totheendoderm,duringearly(Figure4h)andlateplanula (Figure4i-j). Thecryptochromegenes NvCry1a and NvCry1b show similarpatterns.Theexpressionbeginsattheblastula stagediffuselyinfutureectoderm(Figure4k,p).During invaginationofprospectiveendodermatgastrulation, expressionbeginstobeexpandtotheendodermfor NvCry1a (Figure4l),andovertimerestrictsitsexpressiontobecomemorerestrictedtotheendoderminlater stages(Figure4m-o).Asimilarpatternofexpressionis observedfor NvCry1b ;predominantlyectodermalduring gastrulation(Figure4q)butswitchingtoendodermduringlatergastrulastages(Figure4r).Intheplanula stages,theexpressionismoreconcentratedintheendoderm,butfaintexpressionwasevidentinectodermal tissues(Figure4s-t). WeperformedqPCRassaysontheclockgenesto investigatethegenerationoftherhythmicityduring N. vectensis embryogenesis. NvClock NvTimeout NvCry1a and NvCry1b ,atsixhourtimepointsovera24-hourperiod (thatis,6,12,18,and24-hourspost-fertilization(blastula)), 48-hours(lateblastula),1-week(lateplanula)and2-weeks (earlypolypwithtentaclebuds)post-fertilization. Uptothe48-hourspost-fertilization,therewasno clearrhythmofexpressionof NvClock (Figure5a).OnewayANOVAtestshowednodifferenceinexpression levelsbetweenanyofthetimepoints( P =0.4179).At1weekpost-fertilization,therewasevidenceofarhythm (Figure5b),withapeakinthetransitionlight – dark (ZT=12),thatwasconfirmedbytheone-wayANOVA Figure3 RelativemRNAexpressionofmelatoninpathwayproductionin Nematostella vectensis. Resultsof a) tryptophanhydroxylase (TPH)/phenylalaninehydroxylases(PaH)and b) hydroxyindol-O-methyltransferase(HIOMT)inadultindividualsof Nematostellavectensis keptina light – dark(12:12h)orconstantdarknesscycle;and c) TPH/PaHand d) HIOMTexpressionin2-week-oldembryos.Datawerenormalizedbythe genetotheribosomalproteinP0.Eachpointrepresentsfiveindividuals/samples.*Statisticallydifferentfromlight – darkwith P <0.05. Table1Measuresofactivityoftryptophanhydroxylase (TPH)andhydroxyindol-O-methyltransferase(HIOMT) withsamplesoftheanimalsatZeitgebertime(ZT)=8 (thatis,8hoursafterlightson)andZT=20(thatis, 8hoursafterlightsoff)Enzymaticassay/TimepointZT=8ZT=20 TPH78.947.3258.964.73 HIOMT91.710.8043.510.99ValuesaremeanSEMforfoursamplesperassay.Activityexpressedin picomolesper200mgofproteinperhour.ZT20valuesarestatistically differentfromZT=8; P <0.05.Peres etal.EvoDevo 2014, 5 :26 Page9of18 http://www.evodevojournal.com/content/5/1/26

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( P <0.0001).However,thecosinortestwasnotvalid, indicatingthateventhattherewasvariationbetween timepointsthatdidnotfollowa24-hourperiod.Inthe 2-weekpolyps,however,therewasaclearcircadianpatternofexpression,withvariationconfirmedbythe one-wayANOVAtest( P <0.0001),andthecosinortest wasvalid,( P =0.048),indicatingthatthisgenehad acquiredacircadianpatternofexpression(Figure5c). Forthe NvTimeout ,againtherewasnorhythmicityin expressionin48-hourembryos(Figure5d),confirmed bytheone-wayANOVA( P =0.7247).Inthe1-week polyps,however,itwasevidenceofrhythmicexpression (Figure5e).Theone-wayANOVAshowedthatexpression wassignificantlydifferent( P =0.0008)andthecosinor testwasvalid( P =0.045),revealingthatthegene alreadyhadacircadianpatternofexpression(graph notshown).Rhythmicexpressionwasmaintainedin the2-weekpolyps(Figure5f),wherethecosinormodel wasalsovalid( P =0.025). Thegene NvCry1a hadapatternsimilarto NvClock :no rhythmofexpressioninthe48-hourembryos(Figure5g, P =0.3745),butwithadifferencebetweenthetimepoints inthe1-weekembryos( P <0.0001),withoutvalidation fromthecosinor(Figure5h).At2weeks,thepolypshada circadianpatternofexpressionofthegene(Figure5i), withvalidationoftheone-wayANOVA( P <0.0001)and fromthecosinor( P =0.003).Thepatternfor NvCry1b was similarto NvTimeout .In48-hourembryos,therewasno rhythmicityinexpression(Figure5j),whichwasconfirmedbytheone-wayANOVA,whichshowednodifferenceinexpressionbetweentimepoints( P =0.928). Inthe1-weekpolyps,however,wedetectedarhythm NvClock NvTimeout NvCry1a NvCry1bab c d e fgh i j k l mn o p q r s t24h48h96h144h168h 0.1mm 0.1mm 0.1mm 0.1mm 0.1mm Figure4 Temporalexpressiondynamicsof NvClock,NvTimeout,NvCry1a and NvCry1b genesbywholemountinsituhybridization. The stagesforeachgenerepresentembryos24(a,f,k,p) ,48(b,g,l,q) 96(c,h,m,r) ,144(d,i,n,s) and168(e,j,o,t) hourspost-fertilization. Peres etal.EvoDevo 2014, 5 :26 Page10of18 http://www.evodevojournal.com/content/5/1/26

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(Figure5k)withsignificantdifferencesinexpression overtime( P =0.0004)andasignificantcosinorresult ( P =0.0072,graphnotshown).Thiscircadianpatternof expressionwascontinuedinthe2-week-oldpolyps (Figure5l),whereagaintheone-wayANOVAshowed differencebetweenthepoints( P <0.0001)andthecosinor modelwasvalid( P =0.0052).Clockattenuationandmelatonin-initiatedclockgene activationWeanalyzedtranscriptionofclockgenesinadultstodeterminehowtheirexpressionwasalteredbyremovalof alightcycleandinvestigateiftheadditionofexogenous melatoninalterstheexpressionofthesegenesinthe absenceofalightcue.Previousworkshowedthat 30daysofconstantdarknessappearstoresultintheloss ofrhythmicityinthesameclockgeneswhenassayed withqPCR[35]. First,inordertoinvestigatehowrapidlythe N.vectensis clockgeneslosttheirrhythmicityinadultsremoved fromtheentraininglightcue,wemovedanimals entrainedinthenormal12:12light – darkcycletoconstantdarknessandsampledindividualsat24,48and 96-hours. NvClock ,lostrhythminthefirst24hoursof Figure5 RelativemRNAexpressionof NvClock NvTimeout NvCry1a and NvCry1b inembryosof Nematostellavectensis normalizedby thegenetotheribosomalproteinP0. Samplesfrom48-h,1-week,and2-weekspost-fertilization.Eachpointrepresentsfiveindividuals/samples. Peres etal.EvoDevo 2014, 5 :26 Page11of18 http://www.evodevojournal.com/content/5/1/26

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constantdarkness,withnodifferenceinthepointsalong theperiod(Figure6a,one-wayANOVA, P =0.1880). NvTimeout,NvCry1a and NvCry2 maintainedrhythmic expressionforlongerthan NvClock ,butby96hoursall showedexpressionprofileslackingoscillations(one-way ANOVA, P =0.2532for NvTimeout ,0.1081for NvCry1a and0.4186for NvCry2 ,Figure6b,candd). NvCry1b lost evidenceofrhythmicexpressionat48hours(Figure6e, one-wayANOVA, P =0.6111). Next,wemeasuredexpressionofthesegenesin N. vectensis adultsculturedindarknessfor20days(approximately16daysafterlossofrhythmicgeneexpression)and supplementedwithmelatoninatZT=12.Melatonintreatmentdidnotaltertheexpressionpatternof NvClock ;gene expressionlackedoscillationsinexpressionsimilartoanimalsinconstantdarkness,whichcontrastswithincreased expressionof NvClock duringlightperiodswhenanimals areculturedonadielcycle(Figure7a)[38].Thecosinor testshowedthatthevariationintheexpressionof NvClock hada24-hourperiodonlyinanimalsinanormal light – darkcycle(Figure8a). Theresultobservedforthegene NvCycle wasinterestingbecausealteringthelightcycledidnotaltersubstantiallythepatternofexpressionofthegene(Figure7b). Thetimepointsweresignificantlydifferentbothbetween andwithintreatments(one-wayANOVA, P <0.005), whichenabledustodoacosinortestthatwasvalidforall groups(Figure8b). NvTimeout showedasignificantlydifferentpatternof expressionintheanimalsthroughoutthelight – dark cycle,withapeak3hoursafterthetransitiontothedark period(Figure7c).Theone-wayANOVAtestshowed thattheexpressionlevelsweredifferentduringthe24hourperiod( P =0.0006)forthisgroupandforthe melatonin-treatedanimals( P <0.0001)butnotsignificantlydifferentfortheanimalsinconstantdarkness ( P =0.7985).Thecosinortestshowedthatthegene hadacircadianpatternofexpression,butwithadifference Figure6 RelativemRNAexpressionofClockGenesin Nematostellavectensis. Dataof a) Clock b) Timeout c) Cry1a d) Cry1band e) Cry2 in individualsof Nematostellavectensis inlight – darkcycleand24h,48hor96haftertheanimalsweremovedtotheconstantdarkness.*indicates themomentwheretheone-wayANOVAtestindicatesthatthereisnomoredifferencebetweenthetimepointswithinthe24-hcycle( P >0.05.) Eachpointrepresentsfiveindividuals/samples. Peres etal.EvoDevo 2014, 5 :26 Page12of18 http://www.evodevojournal.com/content/5/1/26

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intheacrophaseofalmost13hoursbetweenthelight – dark andthemelatonin-treatedanimals(Figure8c,Table2). ThetwoTypeIandsingleTypeIIcryptochromes from N.vectensis showeddifferentialresponses NvCry1a showedasimilarexpressionpatternas NvTimeout (Figure7d)withthecosinormodelshowingthatthe light – darkandthemelatonin-treatedanimalsshowed significant24-hrhythmsth atwerelostintheconstant darknessgroup(Figure8d). NvCry1b showedcircadian expressionforthelight – darkandmelatonin-treatedanimals,butinphaseoppositiontooneanother(Figure7e). Thecosinoranalysisshowedrhythmicexpressionforboth thosegroups,showingagaintheabilityofmelatoninto alterthepatternofexpressionofgeneslikelyassociated withtheanemonecircadianclock(Figure8e).Thepattern ofthegene NvCry2 wasverysimilartotheoneof NvCry1b ,withphaseoppositionbetweenthelight – dark andthemelatonin-treatedanimalsandanexpression patternwithoutoscillationsforthoseinconstantdarkness (Figure7f,8f).DiscussionWeprovidemultiplelinesofempiricalevidence(HPLC andELISA)showingthepresenceofmelatonininthe anthozoan Nematostellavectensis indifferentlifestages, aswellasenzymaticandtranscriptiondatasuggesting conservationofportionsofthemelatoninsynthesis pathwaydescribedinvertebrates.Thesedataareconsistentwithpreviouspapersthathavedescribedmelatonin inanthozoancnidariansaswellasthedieloscillationsin somespecieswhenexposedtolight:darkcycles.Weextendthesepreviousobservationstoshowthatmelatonin exposurecouldbeimportantasaclockinputregulating theexpressionofthegenesthatlikelydrivethecnidariancircadianclockintheabsenceoflightcues.PatternsofmelatoninsynthesisincnidariansPreviousresearchhasshownvariationintheseasonal anddielsynthesisofmelatonininanthozoancnidarians. Melatoninwasfirstreportedinthecolonialanthozoan Renillakllikeri [37] wheremelatoninamountsfluctuatedbyseasonbutnotdielcycles.Melatoninwasalsoat highestconcentrationingametogenicregions.Thiswork supportedaroleformelatoninincnidarianseasonal reproductionbutnotcircadianprocesses,whichdiffers frombilaterians[8,14].Subsequentstudiesintwoother anthozoanspecies( Actiniaequina and Nematostellavectensis )[38,39]showedthatmelatoninwasalsolocalized Figure7 RelativemRNAexpressionofClockGenesin Nematostellavectensis. Dataof a) Clock b) Cycle c) Timeout d) Cry1a, e) Cry1b and f) Cry2 inN ematostellavectensis keptinalight – dark(12:12h),constantdarkness,orconstantdarknesswithmelatoninsupplementation(0.1 M). DatanormalizedbytheribosomalproteinP0.Eachpointrepresentsfiveindividuals.*Statisticallydifferentfromlight – darkwith P <0.05. Peres etal.EvoDevo 2014, 5 :26 Page13of18 http://www.evodevojournal.com/content/5/1/26

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nearoringametogenictissues.Inaddition,Roopinand Levy[38]showedthatmelatoninlevelsin A.equina vary onadielcyclewithpeakconcentrationinearlysubjectivenight,buttheoscillatingexpressiondissipatesupon exposuretoconstantdarkness.Theselaterresultssuggestthatmelatoninmayservearoleinthecircadian clockbecausecnidariansloseoscillationsingeneexpressionwhenlightcuesareremoved[35,43,45].Weshow thatmelatoninconcentration,aswellasthegenesinthe melatoninsynthesispathway,haveoscillatingpatternsin N.vectensis .Thepresenceofnocturnalpeaksof melatoninproductionin N.vectensis issimilartopeak productionofmelatoninindiversespecies.Nocturnal peaksarepresentinallthevertebratesinvestigatedthus far[46],aswellasspeciesofalgae[8,47],flatworms[10] andinsects,including Drosophila [48] Combined,these resultssuggestthatmelatoninhasdielpatternsinanthozoans,whicharelikelyrelatedtothecircadianclock(see below). WedeterminedthatTPH/PaHandHIOMT,central componentsofthemelatoninpathway,arepresentand expressedin N.vectensis .Bothenzymeshavealready Figure8 CosinorcurvesfortherelativemRNAexpressionofClockGenesin Nematostellavectensis. Dataof a) Clock b) Cycle c) Timeout d) Cry1a e) Cry1b and f) Cry2 inindividualsof Nematostellavectensis keptinalight – dark(12:12h),constantdarknesscycle,orconstantdarkness withmelatoninsupplementation(0.1 M)normalizedbytheribosomalproteinP0,whereasignificantcurvefitwasobtained.Thehorizontalline, themesor,representsthemeanoscillation,andtheverticalarrow,theacrophase,thetimeofoccurrenceofthedaily-peakofmelatonin.Each pointrepresentsfiveindividuals/samples. Table2CosinorparametersoftheclockgeneswhereasignificantcurvefitwasobtainedParameterAcrophaseMesorAmplitude GeneLDMelatoninLDMelatoninLDMelatonin NvTimeout 13.27 0.32 0.041.19 0.0079 0.0008 0.00630.0011 0.0049 0.0010.0042 0.0015 NvCry1a 10.04 0.174 6.242.17 0.0066 0.0003 0.00410.0005 0.003 0.0003 0.0010.0007 NvCry1b 13.18 0.17 1.21.18 0.011 0.02 0.01440.0023 0.007 0.00080.0086 0.0031 NvCry2 12.32 1.13 1.111.04 0.28 0.0330.30 0.0680.14 0.044 0.3430.091‘ LD ’ referstoanimalskeptinalight – darkcycle. ‘ Melatonin ’ referstoanimalskeptinaconstantdarknesscyclewithmelatonin(0.1 M)supplementation.Values aremeanSEMforfivesamplesperassay.Boldindicatessignificantdifference.Peres etal.EvoDevo 2014, 5 :26 Page14of18 http://www.evodevojournal.com/content/5/1/26

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beendescribedindifferentgroupsofinvertebrates, includingflatworms[49],seaurchins[50]andcnidarians [51]andtheactivityoftheenzymesinmollusks[52]and arthropods[53-55].Wefoundacorrelationbetweenthe rhythmofthetranscriptionofTPH/PaHandproduction ofserotonin(productofTPHinvertebrates)andHIOMT andproductionofmelatonin(productofHIOMT),which providesconsistentevidencethattheseenzymesparticipateinthemelatoninpathway.Thefactthatwewerealso abletomeasuretheactivityoftheenzymesgivesaddition supportforthepresenceofavertebrate-likemelatonin pathwayincnidarians. Ourworkpresentedhereisthefirsttoshow melatoninsynthesisinacnidarianduringinvertebrate embryogenesis.Therearefewreferencesintheliterature ofmelatonininembryos,showingitspresenceinfishes [29],birds[30]andmammals[56].Themeasurementof arhythminthemelatoninproductionduringdevelopmentindicatesaregulatedproductionofthehormone inresponsetolightenvironment,insteadofmaternal depositionintomaturingoocytes.Inaddition,because developmentalstageslackgametogenictissue,therole formelatoninincnidariansincludesnon-reproductive functionsinadditiontothehypothesizedrolein gametogenesisreportedinpreviousstudies[37-39].The melatoninpatternofproductionintheembryosresemblestheadults,withpeaksduringthesecondhalfofthe lightphase.Thesedatasuggestthatacircadianpattern isinitiatedin N.vectensis priortotheadultstage.However,themelatonincontenthashighervariationbetween theselifecyclestages,withapeakvaluecloseto26picogramspermilligramofproteininthe2-week-oldembryosandaround6picogramspermilligraminadults. Thisdifferencemayindicateeitherahigherproduction intheembryosorahigherdegradationofmelatoninin theadults.MelatoninasaninputintothecnidariancircadianclockTheanalysisofthegeneslikelyinvolvedinthecircadian clockintheadultsshowedthat4daysofconstantdarknesswereadequatetoabolishthecircadianexpression forvirtuallyallofthem.Ourdataarenearlyidenticalto thepatternpreviouslyobserved,althoughthatstudy kept N.vectensis for30daysinconstantdarkness[35]; however,ourresultsshowthatquenchingofcyclical geneexpressionoccurswithindaysofremovaloflight cues.Datafromcoralshavesimilarlyshownlossofthe rhythmicityofsomeclockgeneswithin24hours( Acroporamillepora [57])or72hours (Faviafragum [45]). Together,thesedatasuggestthatlossofrhythmicgene expressionischaracteristicofcnidarianclocks,inoppositiontotheclassicaldescriptionofthebilaterianclock, whichiscapableofmaintainingrhythmicityevenafter severaldaysinconstantdarkness[58-60]. Wetestedwhethermelatonincouldre-synchronize theclockgenesinindividualsculturedintotaldarkness becauseworkinmammalshasshownthatmelatonin canserveasapotentmoleculeforcircadianclockresetting[61,62].Previousstudiesinmammalsshowedthata shortmelatoninpulse(2hours)isnotenoughtoalter theexpressionofclock-relatedgenes[63].Weobserved thattheadditionofmelatoninin12-hourpulsesresults inoscillationsinexpressionof N.vectensis circadian clockgenes,supportingahypothesisthattheinteraction ofmelatoninandclockgenesappearedearlyinthe evolutionoftheanimals. Thedifferencesinthetimingofpeakmelatonin expressionobservedforclockgenesbetweenanimalsin thelight:darkculturesandconstantdarknesswithmelatoninsupplementationarelikelyduetothetimingof melatoninintroduction.Animalsinalight – darkcycle showedincreasingmelatoninbeginningatZT=8that wassustainedintosubjectivenight.Weaddedmelatonin toculturesofanimalsinconstantdarknessatthesubjectivetransitiontothedarkphase(thatis,ZT=12), whichcouldexplainthetemporalshiftingeneexpression.Invivomelatoninisproducedinaprogressiveway, withlowconcentrationsinthebeginningoftheday, whichincreaseduringsubjectivedayandpeakduring thenightphaseofthelight:darkcycle.Inourtreatment, weaddedmelatoninatthebeginningofsubjectivenight (ZT=12)tocoincidewiththehighestconcentrationof melatonin.Futureexperimentsthatmanipulatethe timingofmelatoninsupplementationwouldprovidean additionalempiricaltestforhowmelatoninadditions mayshifttranscriptionofgenesinvolvedinthecircadian clockandhowthetimingofadditionrelatestothe endogenousmelatoninlevels. Consideringthepotentialinterplaybetweenmelatonin andclockgenes,itisdifficulttopostulatewhetherthe changesweobservedinthemelatoninproductioninthe animalsinconstantdarknessareaproductofthedisruptionoftherhythmicityoftheclockgenes,orviceversa. Manipulationstoblockthemelatoninproductionwith morpholinostoTPH/PaHorHIOMT,ortoknockdown theexpressionoftheclockgenesthemselves,couldbe usefultobetterunderstandtheinteractionbetweenthe twofactors.Thesestudieswouldtestforconservedfunctionsfortheroleofhormonesinthetranscriptiontranslationfeedbackloopofthecnidariancircadianclock.Additionalpotentialfunction(s)ofmelatoninin NematostellavectensisAlthoughmelatoninconcentrationpeaksinthedark periodoflight:darkcycles,thepresenceofmelatoninin thelightperiodcouldalsohavefunctionsthatextend beyonditsroleinacircadianclock.MelatonincanfunctionasanantioxidantcapableofreducingreactivePeres etal.EvoDevo 2014, 5 :26 Page15of18 http://www.evodevojournal.com/content/5/1/26

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species[64].Theseoxidativespeciesarenotonlyproducedbyendogenoussources,likemetabolism,butalso byexogenousones,suchaspollutants(forexample,toxic metals)andultravioletradiation[65,66]. N.vectensis livesintheshallowintertidalzone,withpotentiallyhigh exposuretoultravioletradiation[67],andthusmelatoninmayserveasimilar,additionalfunctionasanantioxidant.Consideringthatduringembryogenesisthereis ahighmetabolismrate,(forexample,highmitoticactivity)[68],melatoninproductioncouldalsohelpmitigatetheproductionoffreeradicalsduringthesestages. Anotherpossiblefunctionforthemelatoninobserved intheearlyembryoscouldbeitsparticipationina ‘ diffuseneuroendocrinesystem ’ [69],actingatearlydevelopmentalstagesbeforedifferentiationofthenervous system.Inthisway,serotonin,whichwedemonstrateto bepresentinthe N.vectensis embryos,couldhavea functionintheregulationofbasicdevelopmentalprocesses(cellmigration,differentiationandproliferation). Aroleforserotoninindevelopmenthasbeenreported forseaurchin,mouse,andothervertebrateembryos [70-73].Melatoninhasalsoalreadybeenreportedtoact tostimulatecellproliferationandacceleratedevelopmentinzebrafishembryos[74].Ontologyofthecnidariancircadianclockandrelationship tomelatoninsynthesisQuantitativeandspatialanal ysisofclockgeneexpression duringdevelopmentindicatesthattheirexpressionbegins earlyinthedevelopmentthatprecedesoscillatingexpressioninresponsetoenvironmentalcues,similartozebrafish. Insituexpressionanalysisofthesecircadiangenesshowed thatallgenesareexpressedbroadlyintheendodermpostgastrulation.ThetwoTypeIcryptochromeparalogs Cry1a and Cry1b wereexpressedpriortogastrulationwithgeneralexpressionintheblastula.Thelackofspecificexpressioninanyregionoftheendodermprecludesanyclear hypothesesregardingpotentialdevelopmentalfunctionsof thesegenes.Previousworkdescribingexpressionofcircadianclockgenesinthedevelopmentofzebrafishhassuggestedthatthesegenesmayfunctionincellcyclecontrol priortoaroleincircadianclo ckfunctioninlaterstages.A similarfunctionmaybepresentin N.vectensis Twogenes( Timeout and Cry1b )aretranscribedina circadianfashioninthe1-week-oldembryos,andattwo weeks,alltheclockgenesweevaluatedwerebeing expressedinacircadianrhythm,withasimilarpatternto thatobservedintheadults.Theseresultssuggestthatthe circadianclockmaynotbeginoscillatinguntilthepolyp stage.Interestingly,weobservedmelatoninconcentrations fluctuatedduringembryogenesis,despitethelackofoscillatingexpressionofthecircadianclock.Thedifferencein timingofoscillatorybehaviorofmelatoninandcircadian clockmayindicateanon-circadianroleformelatoninin development,potentiallyinrespondingtooxidativestress. Futureresearchmeasuringbehavioralentrainmentof larvalstageswillbenecessarytodeterminein N.vectensis larvaehavedielactivitypatternslikethosedescribedin adultpolyps[75].ConclusionsOurresultssupportanancientroleformelatonininthe circadianbehaviorofanimalsbyshowingcyclicexpressionofthishormoneunderdielconditions,bothin adultsandinembryos.Wealsoshowlight-dependent oscillationsingeneexpressioninthemelatoninsynthesispathway,andinactivityofthoseenzymes,indicating thatthemelatoninpathwayofproductionislikelyconservedsincethecnidarian-bilaterianancestor.Ourwork ispioneeringinshowingmelatonininitiatingexpression ofcircadianclockgenesinthecnidarian N.vectensis, an eventonlyshowedbeforeinmammals,supportinga hypothesisthattheinteractionofmelatoninandclock genesappearedearlyintheevolutionoftheanimals. Duringdevelopment,melatoninshowsoscillationsduringembryogenesiswhendevelopmentalstagesexperience diellightingconditions;however,thecircadianclockgenes shownoevidenceofrhythmicity,despitehavingspecific spatialexpressionintheendoderm.Wesuggestthatmelatoninintheseearlystagescanhavefunctionstomitigate theproductionoffreeradical[68]orparticipateinpotentialneuroendocrinesignaling[69].Thedifferences inexpressionofmelatoninandthecircadianclockgene networkintheadultstagewhencomparedwithdevelopmentalstagesof N.vectensis suggestsnewresearch directionstocharacterizestage-specificmechanismsof circadianclockfunctioninanimals.AdditionalfilesAdditionalfile1: PrimersusedintheqPCRreactions. Additionalfile2: Chromatogramsofamelatoninstandard(black line)andofa Nematostellavectensis sample(blueline). The chromatogramsshowthesamepattern,withthesameretentiontimefor melatonin,validatingtheassay. Additionalfile3: FormattedAlignmentsofthesequencesofTPH genein Nematostellavectensis genome,ourcloneandtheqPCR amplifiedfragment. Additionalfile4: FormattedAlignmentsofthesequencesofHIOMT genein Nematostellavectensis genome,ourcloneandtheqPCR amplifiedfragment. Additionalfile5: PhylogeneticanalysesofTPHgene. Additionalfile6: PhylogeneticanalysesofHIOMTgene. Abbreviations AANAT: arylalkylamineN-acetyltransferase;BCP:1-bromo-3-chloropropane; D:dark;HIOMT:hydroxyindol-O-methyltransferase;HPLC:highperformance liquidchromatography;L:light;NAS:N-acetylserotonin;PaH:phenylalanine hydroxylases;SCN:suprachiasmaticnucleus;TPH:tryptophanhydroxylase; ZT:zeitgebertime;5HTP:5-hydroxytryptophan.Peres etal.EvoDevo 2014, 5 :26 Page16of18 http://www.evodevojournal.com/content/5/1/26

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Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Authors ’ contributions RPperformedmostoftheexperimentsanddraftedthearticle.RP,AMRand MQMperformedtheanalysisofthedata.RPandYPperformedthe experimentswithembryos.RPandSCAperformedtheenzymeactivity assays.RP,AMR,MQM,ACMandJCNparticipatedinthedesignofthestudy. AMRandMQMhelpedtodraftthemanuscript.Allauthorsreadand approvedthefinalmanuscript. Acknowledgements ThisworkwassupportedbySoPauloStateResearchFoundation(FAPESP), NationalCouncilforScientificandTechnologicalDevelopment(CNPq),and bytheCoordinationfortheImprovementofHigherLevelPersonnel(CAPES). AMRacknowledgesgenerousfundingfromtheUniversityofNorthCarolina atCharlotteduringthecourseofthisproject. Authordetails1KewaloMarineLaboratory,UniversityofHawaii,41AhuiStreet,96813 Honolulu,HI,USA.2DepartmentofBiologicalSciences,UniversityofNorth CarolinaatCharlotte,9201UniversityCityBlvd,Charlotte,28223-0001 Charlotte,NorthCarolina,USA.3LaboratoryofPharmacology,Butantan Institute,SoPaulo,Brazil.4DepartmentofPhysiologyandBiophysics, InstituteofBiomedicalSciences,UniversityofSoPaulo,Av.VitalBrasil,1500, Butant,SoPauloSP,05503-900,Brazil.5DepartmentofZoology, BiosciencesInstitute,UniversityofSoPaulo.RuaMatao,Trav.14,101, 05508-090SaoPaulo,Brazil.6CenterforMarineBiology,UniversityofSo Paulo.RodoviaManoelHyplitodoRego,km.131,5-PraiadoCabeloGordo, SoSebastioSP,11600-000,CEP,Brazil.7WhitneyLaboratoryforMarine Bioscience,UniversityofFlorida,9505OceanShoreBlvd,32080St.Augustine, FL,USA. Received:4March2014Accepted:15July2014 Published:14August2014 References1.ReiterRJ: Themelatoninrhythm:bothaclockandacalendar. Experientia 1993, 49: 654 – 664. 2.DoCarmoBuonfiglioD,Peliciari-GarciaRA,DoAmaralFG,PeresR,Nogueira TC,AfecheSC,Cipolla-NetoJ: Early-stageretinalmelatoninsynthesis impairmentinstreptozotocin-induceddiabeticwistarrats. InvestOphthalmol VisSci 2011, 52: 7416 – 7422. 3.BubenikGA: Gastrointestinalmelatonin:localization,function,andclinical relevance. DigDisSci 2002, 47: 2336 – 2348. 4.Vivien-RoelsB,PevetP,BeckO,Fevre-MontangeM: Identificationofmelatonin inthecompoundeyesofaninsect,thelocust(Locustamigratoria),by radioimmunoassayandgaschromatography – massspectrometry. NeurosciLett 1984, 49: 153 – 157. 5.PoeggelerB,HardelandR: Detectionandquantificationofmelatoninina dinoflagellate,Gonyaulaxpolyedra:solutionstotheproblemof methoxyindoledestructioninnon-vertebratematerial. JPinealRes 1994, 17: 1 – 10. 6.HardelandR: Newactionsofmelatoninandtheirrelevanceto biometeorology. IntJBiometeorol 1997, 41: 47 – 57. 7.KolarJ,MachackovaI: Melatonininhigherplants:occurrenceand possiblefunctions. JPinealRes 2005, 39: 333 – 341. 8.HardelandR: Melatoninand5-methoxytryptamineinnon-metazoans. ReprodNutrDev 1999, 39: 399 – 408. 9.ArendtJ: Melatonin. BMJ 1996, 312: 1242 – 1243. 10.ItohMT,ShinozawaT,SumiY: Circadianrhythmsofmelatonin-synthesizing enzymeactivitiesandmelatoninlevelsinplanarians. BrainRes 1999, 830: 165 – 173. 11.HintermannE,JenoP,MeyerUA: Isolationandcharacterizationofan arylalkylamineN-acetyltransferasefromDrosophilamelanogaster. FEBSLett 1995, 375: 148 – 150.12.HintermannE,GriederNC,AmherdR,BrodbeckD,MeyerUA: Cloningofan arylalkylamineN-acetyltransferase(aaNAT1)fromDrosophilamelanogaster expressedinthenervoussystemandthegut. ProcNatlAcadSciUSA 1996, 93: 12315 – 12320. 13.ItohMT,HattoriA,SumiY,SuzukiT: Day-nightchangesinmelatonin levelsindifferentorgansofthecricket(Gryllusbimaculatus). JPinealRes 1995, 18: 165 – 169. 14.HardelandR,PoeggelerB: Non-vertebratemelatonin. JPinealRes 2003, 34: 233 – 241. 15.DeguchiT,AxelrodJ: SuperinductionofserotoninN-acetyltransferaseand supersensitivityofadenylcyclasetocatecholaminesindenervated pinealgland. MolPharmacol 1973, 9: 612 – 618. 16.DeguchiT,AxelrodJ: InductionandsuperinductionofserotoninNacetyltransferasebyadrenergicdrugsanddenervationinratpineal organ. ProcNatlAcadSciUSA 1972, 69: 2208 – 2211. 17.DeguchiT,AxelrodJ: SensitiveassayforserotoninN-acetyltransferase activityinratpineal. AnalBiochem 1972, 50: 174 – 179. 18.KleinDC,WellerJL: Rapidlight-induceddecreaseinpinealserotonin N-acetyltransferaseactivity. Science 1972, 177: 532 – 533. 19.ParfittA,WellerJL,KleinDC: Betaadrenergic-blockersdecrease adrenergicallystimulatedN-acetyltransferaseactivityinpinealglandsin organculture. Neuropharmacology 1976, 15: 353 – 358. 20.PavlicekJ,SauzetS,BesseauL,CoonSL,WellerJL,BoeufG,GaildratP, OmelchenkoMV,KooninEV,FalconJ,KleinDC: EvolutionofAANAT: expansionofthegenefamilyinthecephalochordateamphioxus. BMCEvolBiol 2010, 10: 154. 21.FalconJ,CoonSL,BesseauL,Cazamea-CatalanD,FuentesM,MagnanouE, PaulinCH,BoeufG,SauzetS,JorgensenEH,MazanS,WolfYI,KooninEV, SteinbachPJ,HyodoS,KleinDC: Drasticneofunctionalizationassociated withevolutionofthetimezymeAANAT500Mya. ProcNatlAcadSciUSA 2014, 111: 314 – 319. 22.RibelaygaC,GauerF,CalgariC,PevetP,SimonneauxV: Photoneural regulationofratpinealhydroxyindole-O-methyltransferase(HIOMT) messengerribonucleicacidexpression:ananalysisofitscomplex relationshipwithHIOMTactivity. Endocrinology 1999, 140: 1375 – 1384.23.KalsbeekA,Perreau-LenzS,BuijsRM: Anetworkof(autonomic)clock outputs. ChronobiolInt 2006, 23: 521 – 535. 24.BorjiginJ,SamanthaZhangL,CalinescuAA: Circadianregulationofpineal glandrhythmicity. MolCellEndocrinol 2012, 349: 13 – 19. 25.StehleJH,VonGallC,KorfHW: Melatonin:aclock-output,aclock-input. JNeuroendocrinol 2003, 15: 383 – 389. 26.DunlapJC: Molecularbasesforcircadianclocks. Cell 1999, 96: 271 – 290. 27.ReppertSM: Acolorfulmodelofthecircadianclock. Cell 2006, 124: 233 – 236. 28.MerlinC,GegearRJ,ReppertSM: Antennalcircadianclockscoordinatesun compassorientationinmigratorymonarchbutterflies. Science 2009, 325: 1700 – 1704. 29.YamadaH,ChibaH,AmanoM,IigoM,IwataM: Rainbowtrouteyed-stage embryosdemonstratemelatoninrhythmsunderlight – darkconditionsas measuredbyanewlydevelopedtime-resolvedfluoroimmunoassay. GenCompEndocrinol 2002, 125: 41 – 46. 30.OlszanskaB,MajewskiP,LewczukB,StepinskaU: Melatoninandits synthesizingenzymes(arylalkylamineN-acetyltransferase-likeand hydroxyindole-O-methyltransferase)inavianeggsandearlyembryos. JPinealRes 2007, 42: 310 – 318. 31.DekensMP,WhitmoreD: Autonomousonsetofthecircadianclockinthe zebrafishembryo. EMBOJ 2008, 27: 2757 – 2765. 32.AmanoT,MatsushitaA,HatanakaY,WatanabeT,OishiK,IshidaN,AnzaiM, MitaniT,KatoH,KishigamiS,SaekiK,HosoiY,IritaniA,MatsumotoK: Expressionandfunctionalanalysesofcircadiangenesinmouse oocytesandpreimplantationembryos:Cry1isinvolvedinthemeiotic processindependentlyofcircadianclockregulation. BiolReprod 2009, 80: 473 – 483. 33.CurranKL,LaRueS,BronsonB,SolisJ,TrowA,SarverN,ZhuH: Circadian genesareexpressedduringearlydevelopmentinXenopuslaevis. PLoS One 2008, 3: e2749. 34.GreenCB,DurstonAJ,MorganR: ThecircadiangeneClockisrestrictedto theanteriorneuralplateearlyindevelopmentandisregulatedbythe neuralinducernogginandthetranscriptionfactorOtx2.MechDev 2001, 101: 105 – 110. 35.ReitzelAM,BehrendtL,TarrantAM: Lightentrainedrhythmicgene expressionintheseaanemoneNematostellavectensis:theevolutionof theanimalcircadianclock. PLoSOne 2010, 5: e12805. 36.ReitzelAM,TarrantAM,LevyO: Circadianclocksinthecnidaria: environmentalentrainment,molecularregulation,andorganismal outputs. IntegrCompBiol 2013, 53: 118 – 130.Peres etal.EvoDevo 2014, 5 :26 Page17of18 http://www.evodevojournal.com/content/5/1/26

PAGE 18

37.MechawarN,AnctilM: Melatonininaprimitivemetazoan:seasonal changesoflevelsandimmunohistochemicalvisualizationinneurons. JCompNeurol 1997, 387: 243 – 254. 38.RoopinM,LevyO: Temporalandhistologicalevaluationofmelatonin patternsina ‘ basal ’ metazoan. JPinealRes 2012, 53: 259 – 269. 39.RoopinM,LevyO: Melatonindistributionrevealscluestoitsbiological significanceinbasalmetazoans. PLoSOne 2012, 7: e52266. 40.MartindaleMQ,PangK,FinnertyJR: Investigatingtheoriginsof triploblasty: ‘ mesodermal ’ geneexpressioninadiploblasticanimal,the seaanemoneNematostellavectensis(phylum,Cnidaria;class,Anthozoa). Development 2004, 131: 2463 – 2474. 41.BarbosaR,ScialfaJH,TerraIM,Cipolla-NetoJ,SimonneauxV,AfecheSC: TryptophanhydroxylaseismodulatedbyL-typecalciumchannelsinthe ratpinealgland. LifeSci 2008, 82: 529 – 535. 42.CornelissenG,HalbergF,StebbingsJ,HalbergE,CarandenteF,HsiB: Chronobiometrywithpocketcalculatorsandcomputersystems. RicClinLab 1980, 10: 333 – 385. 43.ReitzelAM,PassamaneckYJ,KarchnerSI,FranksDG,MartindaleMQ,Tarrant AM,HahnME: Arylhydrocarbonreceptor(AHR)inthecnidarian Nematostellavectensis:comparativeexpression,proteininteractions, andligandbinding. DevGenesEvol 2014, 224: 13 – 24. 44.MarlowHQ,SrivastavaM,MatusDQ,RokhsarD,MartindaleMQ: Anatomy anddevelopmentofthenervoussystemofNematostellavectensis,an anthozoancnidarian. DevNeurobiol 2009, 69: 235 – 254. 45.HoadleyKD,SzmantAM,PyottSJ: Circadianclockgeneexpressioninthe coralFaviafragumoverdielandlunarreproductivecycles. PLoSOne 2011, 6: e19755. 46.HardelandR: Melatonin,hormoneofdarknessandmore:occurrence, controlmechanisms,actionsandbioactivemetabolites. CellMolLifeSci 2008, 65: 2001 – 2018. 47.BalzerI: Recentprogressinunderstandingthetemporalbehaviorof unicellularorganisms. BrazJMedBiolRes 1996, 29:95 – 99. 48.FinocchiaroL,CallebertJ,LaunayJM,JallonJM: Melatoninbiosynthesisin Drosophila:itsnatureanditseffects. JNeurochem 1988, 50: 382 – 387. 49.NishimuraK,KitamuraY,InoueT,UmesonoY,YoshimotoK,TakeuchiK, TaniguchiT,AgataK: Identificationanddistributionoftryptophan hydroxylase(TPH)-positiveneuronsintheplanarianDugesiajaponica. NeurosciRes 2007, 59: 101 – 106. 50.YaguchiS,KatowH: Expressionoftryptophan5-hydroxylasegeneduring seaurchinneurogenesisandroleofserotonergicnervoussystemin larvalbehavior. JCompNeurol 2003, 466: 219 – 229. 51.PaniAK,AnctilM: Evidenceforbiosynthesisandcatabolismof monoaminesintheseapansyRenillakoellikeri(Cnidaria). NeurochemInt 1994, 25: 465 – 474. 52.BlancA,Vivien-RoelsB,PevetP,AttiaJ,BuissonB: Melatoninand5methoxytryptophol(5-ML)innervousand/orneurosensorystructuresof agastropodmollusc(Helixaspersamaxima):synthesisanddiurnal rhythms. GenCompEndocrinol 2003, 131: 168 – 175. 53.WithyachumnarnkulB,PongtippateeP,AjpruS: N-acetyltransferase, hydroxyindole-O-methyltransferaseandmelatoninintheopticlobesof thegianttigershrimpPenaeusmonodon. JPinealRes 1995, 18: 217 – 221. 54.ItohMT,HattoriA,SumiY: Hydroxyindole-O-methyltransferaseactivity assayusinghigh-performanceliquidchromatographywithfluorometric detection:determinationofmelatoninenzymaticallyformedfromNacetylserotoninandS-adenosyl-L-methionine. JChromatogrBBiomedSciAppl 1997, 692: 217 – 221. 55.BaoX,WangB,ZhangJ,YanT,YangW,JiaoF,LiuJ,WangS: Localization ofserotonin/tryptophan-hydroxylase-immunoreactivecellsinthebrain andsuboesophagealganglionofDrosophilamelanogaster. CellTissueRes 2010, 340: 51 – 59. 56.Seron-FerreM,MendezN,Abarzua-CatalanL,VilchesN,ValenzuelaFJ, ReynoldsHE,LlanosAJ,RojasA,ValenzuelaGJ,Torres-FarfanC: Circadian rhythmsinthefetus. MolCellEndocrinol 2012, 349: 68 – 75. 57.BradyAK,SnyderKA,VizePD: Circadiancyclesofgeneexpressioninthe coral. Acroporamillepora.PLoSOne 2011, 6: e25072. 58.EmeryIF,NoveralJM,JamisonCF,SiwickiKK: RhythmsofDrosophila periodgeneexpressioninculture. ProcNatlAcadSciUSA1997, 94: 4092 – 4096. 59.RubinEB,ShemeshY,CohenM,ElgavishS,RobertsonHM,BlochG: Molecularandphylogeneticanalysesrevealmammalian-likeclockwork inthehoneybee(Apismellifera)andshednewlightonthemolecular evolutionofthecircadianclock. GenomeRes 2006, 16: 1352 – 1365. 60.UryuO,TomiokaK: Circadianoscillationsoutsidetheopticlobeinthe cricketGryllusbimaculatus. JInsectPhysiol 2010, 56: 1284 – 1290. 61.Torres-FarfanC,RoccoV,MonsoC,ValenzuelaFJ,CampinoC,GermainA, TorrealbaF,ValenzuelaGJ,Seron-FerreM: Maternalmelatonineffectson clockgeneexpressioninanonhumanprimatefetus. Endocrinology 2006, 147: 4618 – 4626. 62.Jung-HynesB,ReiterRJ,AhmadN: Sirtuins,melatoninandcircadian rhythms:buildingabridgebetweenagingandcancer. JPinealRes 2010, 48: 9 – 19. 63.Peliciari-GarciaRA,ZanquettaMM,Andrade-SilvaJ,GomesDA,BarretoChavesML,Cipolla-NetoJ: Expressionofcircadianclockandmelatonin receptorswithinculturedratcardiomyocytes. ChronobiolInt 2011, 28: 21 – 30. 64.ReiterRJ,TanDX,AllegraM: Melatonin:reducingmolecularpathology anddysfunctionduetofreeradicalsandassociatedreactants. NeuroEndocrinolLett 2002, 23 (Suppl1):3 – 8. 65.SuzukiT,TakashimaT,IzawaN,WatanabeM,TakedaM: UVradiation elevatesarylalkylamineN-acetyltransferaseactivityandmelatonin contentinthetwo-spottedspidermite,Tetranychusurticae. JInsectPhysiol 2008, 54: 1168 – 1174. 66.VargasMA,GeihsMA,MacielFE,CruzBP,NeryLE,AllodiS: Theeffectsof UVradiationonthevisualsystemofthecrabNeohelicegranulata:a protectiveroleofmelatonin. CompBiochemPhysiolCToxicolPharmacol 2011, 154: 427 – 434. 67.PankowS,BambergerC: Thep53tumorsuppressor-likeproteinnvp63 mediatesselectivegermcelldeathintheseaanemoneNematostella vectensis. PLoSOne 2007, 2: e782. 68.RaddatzE,Eyal-GiladiH,KuceraP: Patternsofoxygenconsumptionduring establishmentofcephalocaudalpolarityintheearlychickembryo. JExpZoolSuppl 1987, 1: 213 – 218. 69.KvetnoyIM: Extrapinealmelatonin:locationandrolewithindiffuse neuroendocrinesystem. HistochemJ 1999, 31:1 – 12. 70.RenaudF,ParisiE,CapassoA,DepriscoP: Ontheroleofserotoninand 5-methoxy-tryptamineintheregulationofcell-divisioninsea-urchin eggs. DevBiol 1983, 98: 37 – 46. 71.LauderJM: Neurotransmittersasgrowthregulatorysignals-roleof receptorsand2ndmessengers. TrendsNeurosci 1993, 16: 233 – 240. 72.WeissER,ManessP,LauderJM: Whydoneurotransmittersactlikegrowth factors? PerspectDevNeurobi 1998, 5: 323 – 335. 73.BuznikovGA,LambertHW,LauderJJ: Serotoninandserotonin-like substancesasregulatorsofearlyembryogenesisandmorphogenesis. CellTissueRes 2001, 305: 177 – 186. 74.DanilovaN,KrupnikVE,SugdenD,ZhdanovaIV: Melatoninstimulatescell proliferationinzebrafishembryoandacceleratesitsdevelopment. FASEBJ 2004, 18: 751 – 753. 75.HendricksWD,ByrumCA,Meyer-BernsteinEL: Characterizationofcircadian behaviorinthestarletseaanemone. Nematostellavectensis.PLoSOne 2012, 7: e46843.doi:10.1186/2041-9139-5-26 Citethisarticleas: Peres etal. : Developmentalandlight-entrained expressionofmelatoninanditsrelationshiptothecircadianclockin theseaanemone Nematostellavectensis EvoDevo 2014 5 :26. Submit your next manuscript to BioMed Central and take full advantage of: € Convenient online submission € Thorough peer review € No space constraints or color “gure charges € Immediate publication on acceptance € Inclusion in PubMed, CAS, Scopus and Google Scholar € Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Peres etal.EvoDevo 2014, 5 :26 Page18of18 http://www.evodevojournal.com/content/5/1/26