DOI: 10.5061/dryad.wm37pvmq8
title: Raw data for the article: A Cryptochrome adopts distinct moon- and sunlight states and functions as sun- versus moonlight interpreter in monthly oscillator entrainment

description: The moon’s monthly cycle synchronizes reproduction in countless marine
 organisms. The mass-spawning bristle worm Platynereis dumerilii 
 uses an endogenous monthly oscillator to phase reproduction to specific
 days. Classical work showed that this oscillator is set by full moon. But
 how do organisms recognize such a specific moon phase? We uncover that the
 light receptor L-Cryptochrome (L-Cry) is able to discriminate between
 different moonlight durations, as well as between sun- and moonlight.
 Consistent with L-Cry’s function as light valence interpreter, its genetic
 loss leads to a faster re-entrainment under artificially strong nocturnal
 light. This suggests that L-Cry blocks “wrong” light from impacting on the
 monthly oscillator. A biochemical characterization of purified L-Cry
 protein, exposed to naturalistic sun- or moonlight, reveals the formation
 of distinct sun- and moonlight states characterized by different
 photoreduction- and recovery kinetics of L-Cry’s co-factor Flavin Adenine
 Dinucleotide. In vivo, L-Cry’s sun- versus moonlight states correlate with
 distinct sub-cellular localizations, indicating different signalling. In
 contrast, r-Opsin1, the most abundant ocular opsin, is not required for
 monthly oscillator entrainment. Our work reveals a new concept for correct
 moonlight interpretation involving a “valence interpreter” that provides
 entraining photoreceptor(s) with light source and moon phase information.
 These findings advance our mechanistic understanding of a fundamental
 biological phenomenon: moon-controlled monthly timing. Such level of
 understanding is also an essential prerequisite to tackle anthropogenic
 threats on marine ecology.
description: This dataset consists of 1. Western blot
 data<br>2. Records of spawning behaviour<br>3.
 Immunohistochemistry data<br>4. Light spectra
 measurement<br>5. SEC/MALS data<br>6. UV/VIS spectroscopy
 data 1. Western blot<br>Worm head protein lysates
 were analysed using SDS-PAGE (10% acrylamide) and subsequent transfer to a
 0.45µm nitrocellulose membrane. The membrane was probed with two primary
 antibodies against the <em>Platynereis dumerilii</em> L-Cry
 protein (raised in mouse) and a primary antibody against beta-actin
 (raised in rabbit). The primary antibodies were specifically bound with
 the corresponding secondary antibodies: anti-mouse IgG-peroxidase-linked
 and anti-rabbit IgG-peroxidase-linked or fluorescently labelled antibody.
 Detection was performed accordingly, either using a peroxidase substrate
 or detection of fluorescence. This dataset comprises all membranes for
 L-Cry and/or beta-actin detection for every biological replicate (if BRs
 are applicable). 2. Records of spawning
 behaviour<br>Mature male and female worms were recorded on a daily
 basis, taking into consideration days of the lunar month.
 3. Immunohistochemistry data<br>Immunohistochemistry
 samples were imaged using Zeiss LSM 700 confocal microscope with a 40x oil
 objective. In order to determine fluorescence in the nuclei, images of the
 405 nm channel (Hoechst staining) were segmented using the deep
 learning-based algorithm Cellpose. The resulting regions were overlayed as
 Regions Of Interest (ROI) on images of the 555nm channel (L-Cry antibody
 staining) in Fiji/ImageJ. In addition, the entire image was selected as a
 ROI as well. As background regions, nuclei of the cells that do not
 express L-Cry and/or some non-nuclear regions of the image were used (at
 least 3 background ROIs per image).Fiji/ImageJ was used to determine the
 fluorescence signal intensity (by measuring the Area and Mean Gray Value
 of each ROI). Corrected Total Cell Fluorescence (CTCF) was calculated the
 following way: CTCF =Area (ROI_1 )*Mean (ROI_1 )-Area (ROI_1
 )*Mean(ROI_(background ROIs) ). Total nuclear CTCF was calculated by
 adding all nuclear ROI together. To obtain the cytoplasmic fluorescence
 intensity, the summed nuclear values were subtracted from the CTCF of the
 ROI for the entire image. In order to compare results across experiments,
 total nuclear CTCF and total cytoplasmic CTCF values were normalized to
 the highest value within each experiment. The ratio between nuclear and
 cytoplasmic signal intensity was calculated as well. In some experiments,
 the analysis was performed in a "blinded" manner, i.e. the names
 of the images were enciphered. Experiment 1 and 3 contained all three
 conditions, while the experiments 2 and 4 contained only two conditions.
 Experiments 3 and 4 contained the <em>l-cry</em> -/- mutant
 samples that were used as threshold control. 4. Light
 spectra measurement<br>Under water measurements of natural sun- and
 moonlight at the habitat of Platynereis were acquired using a
 RAMSES-ACC-VIS hyperspectral radiometer (TriOS GmbH) for UV to IR spectral
 range (see V. B. Veedin Rajan et al., Nat. Ecol. Evol. 5, 204–218 (2021)
 for details). Radiometers were placed at 4m and 5m water depth close to
 <em>Posidonia oceanica</em> meadows, which are a natural
 habitat for <em>P. dumerilii</em>. Measurements were recorded
 automatically every 15min across several weeks in the winter 2011/2012 (at
 5m depth) and during spring 2011 (at a 4m depth). <br>To obtain an
 exemplary sunlight spectrum, the sunlight measurements taken at 5m depth
 between 10 am-4 pm on 25.11.2011 were averaged.  To obtain a fullmoon
 spectrum (FM) for the 5m depth location measurements taken from 10pm to
 1am on a clear fullmoon night (10-11.11.2011) were averaged. To subtract
 baseline noise from this measurement, a new moon (NM) spectrum was
 obtained by averaging measurements between 7:15pm to 5am on a new moon
 night on 24.11.2011, and subtracted from the fullmoon spectrum. To
 validate that this spectrum is representative of a typical full moon
 spectrum at the habitat of <em>Platynereis</em>, we averaged
 moonlight measured at 4m depth between 10:15 pm to 2am during a full moon
 night in April (17.-18.04.2012) and subtracted a new moon spectrum
 measured two weeks earlier. To benchmark these moonlight spectra measured
 under water with moonlight measured on land, we compared the underwater
 spectra to a publicly available full moon spectrum measured on land on
 14.04.2014 in the Netherlands (http://www.olino.org/blog/us/articles/2015/10/05/spectrum-of-moon-light). The resulting light spectra are plotted in the Supplementary Figure 1. <br>For primary data, see Zurl et al. 2022, Data accompanying article: Two light sensors decode moonlight versus sunlight to adjust a plastic circadian/circalunidian clock to moon phase, Dryad, Dataset, https://doi.org/10.5061/dryad.2v6wwpzkr 5. SEC/MALS data<br>Purified L-Cry protein was analyzed for its oligomeric state by SEC and SEC-MALS. 6. UV/VIS spectroscopy data<br>The L-Cry light responses to blue light, sunlight and moonlight and recovery kinetics were analyzed by UV/VIS spectroscopy, observing the light-induced photoreduction and reoxidation (recovery) of L-Cry’s cofactor flavin adenine dinucleotide (FAD).
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