DOI: 10.5061/dryad.73n5tb2vv
title: Light and temperature measurements and untargeted proteomic measurements

description: The right timing of animal physiology and behavior ensures the stability
 of populations and ecosystems. In order to predict anthropogenic impacts
 on these timings, more insight is needed into the interplay between
 environment and molecular timing mechanisms. This is particularly true in
 marine environments. Using high-resolution, long-term daylight
 measurements from a habitat of the marine annelid Platynereis dumerilii,
 we find that temporal changes in UVA/deep violet intensities, more than
 longer wavelengths, can provide annual time information, which differs
 from annual changes in photoperiod. We developed experimental setups that
 resemble natural daylight illumination conditions, and automated,
 quantifiable behavioral tracking. Experimental reduction of UVA/deep
 violet light (app. 370-430nm) under long photoperiod (LD16:8)
 significantly decreases locomotor activities, comparable to the decrease
 caused by short photoperiod (8:16). In contrast, altering UVA/deep violet
 light intensities does not cause differences in locomotor levels under
 short photoperiod. This modulation of locomotion by UVA/deep violet light
 under long photoperiod requires c-opsin1, an UVA/deep violet-sensor
 employing Gi-signalling. C-opsin1 also regulates the levels of
 rate-limiting enzymes for monogenic amine synthesis and of several
 neurohormones, including PDF, Vasotocin (Vasopressin/Oxytocin) and NPY-1.
 Our analyses indicate a complex inteplay between UVA intensities and
 photoperiod as indicators of annual time.
description: Light and temperature measurements: Measurements were recorded every
 15min between July, 27th 2010 and July, 10th 2011 at 10m depth and
 between May, 31st 2011 - July, 10th 2011
 at 4m depth. Natural light measurements were acquired by using two
 RAMSES-ACC-VIS hyperspectral radiometers (TriOS GmbH) for UV to IR
 spectral range. Both instruments were tied to a cement plate and deployed
 on a sand flat on the east coast of the island of Ischia, in the Gulf of
 Naples (Italy) at about 10m (40°43'50.6"N -
 13°58'02.9"E) and 4m (40°43'56"N 13°57'44"E)
 depth, respectively. The locations were near <i>Posidonia
 oceanica</i> meadows, a natural habitat of
 <i>Platynereis</i>. Data were retrieved and instrument sensors
 cleaned every 2-3 months. The radiometers provided individual values of
 light intensity over 255 individual wavelengths between 305 nm and 1150
 nm, the wavelength range for which they had been calibrated directly
 before usage by the company Trios. As sensor sensitivity drops
 significantly below 310nm and above 710nm, we concentrated our analyses on
 the wavelengths range between 310-710nm. Following the manufacturer’s
 instructions and associated software (msda_xe v8.9), the background data
 and electronic offset of the instrument were subtracted from the raw data,
 and the spectrum’s values were calibrated for underwater conditions. The
 final calibrated raw data for light and temperature are deposited under
 the tab labels: "Natural light data" and "Temperature
 data". Next, the acquired raw measurements in
 mW/m<sup>2</sup>/nm/sec were converted to corresponding number
 of photons/cm<sup>2</sup>/nm/sec or, to enable comparisons
 with previous published works, also converted to
 uW/cm<sup>2</sup>/nm/sec,
 uMolphotons/m<sup>2</sup>/nm/sec,
 photons/m<sup>2</sup>/um/sec and log
 photons/m<sup>2</sup>/nm/sec (Examples for such a conversion
 are deposited under the tab labels "24.08.2010" and
 "04.07.2011").   Untargeted
 Mass-Spectrometry (Proteomics) <i>Platynereis
 c-opsin1<sup>+/+</sup></i><sup> </sup>and
 <i>c-opsin1<sup>Δ8/Δ8</sup></i> immature worms
 were grown at long day (LD 16:8) under monochromatic UVA-light (Extended
 Data Fig.10a,b) for three consecutive lunar months. After entrainment, the
 heads (n=3BR; 20heads/BR) were sampled on new moon phase at ZT4, ZT19 and
 ZT22 by anesthetizing the worm with 1:1 7.5%
 MgCl<sub>2</sub>:Natural Seawater (NSW) solution. The sampled
 heads were collected in 2ml tubes containing metal beads on ice,
 snap-frozen in liquid nitrogen and stored at -80℃ until further
 use. For tissue
 lysis, worm heads were resuspended in sample buffer (7.5 M urea, 1.5 M
 thiourea, 4 % CHAPS. 0.05 % SDS, 100 mM DTT) and sonicated. After
 centrifugation, protein concentrations were assessed by applying a
 Bradford assay (Bio-Rad-Laboratories, Vienna, Austria). Protein samples
 were subjected to a filter-assisted proteolytic digestion with a modified
 version of the FASP protocol (Wisniewski et al. 2009, PMID: 19377485). In
 short, 20 µg protein were loaded onto a pre-wetted MWCO filter (Pall
 Austria Filter GmbH, Vienna, Austria) with a pore size of 3 kD, followed
 by reduction of disulfide bonds with dithiothreitol (DTT), alkylation with
 iodoacetamide (IAA) and washing steps with 50 mM ammonium bicarbonate
 buffer. Digestion of proteins was achieved by applying Trypsin/Lys-C with
 Mass Spec Grade quality (Promega, Mannheim, Germany) for an overnight
 digest followed by the further addition of fresh enzyme and an additional
 incubation for four hours. Resulting peptides were eluted through the
 filter by centrifugation, and a clean-up step was performed using C-18
 spin columns (Pierce, Thermo Fisher Scientific, Austria).   LC-MS/MS analysis:
 For LC-MS/MS analyses,
 samples were reconstituted in 5 µl 30 % formic acid (FA), supplemented
 with four synthetic peptide standards for internal quality control, and
 diluted with 40 µl mobile phase A (97.9 % H2O, 2 % ACN, 0.1 % FA). Of this
 solution, 5 µl were injected into a Dionex Ultimate 3000 nano LC-system
 coupled to a Q Exactive orbitrap mass spectrometer equipped with a
 nanospray ion source (Thermo Fisher Scientific, Austria). All samples were
 analyzed as technical replicates. As a pre-concentration step, peptides
 were loaded on a 2 cm x 75 µm C18 Pepmap100 pre-column (Thermo Fisher
 Scientific, Austria) at a flow rate of 10 µl/min using mobile phase A.
 Elution from the pre-column to a 50 cm x 75 µm Pepmap100 analytical column
 (Thermo Fisher Scientific, Austria) and subsequent separation was achieved
 at a flow rate of 300 nl/min using a gradient of 8 % to 40 % mobile phase
 B (79.9 % ACN, 2 % H2O, 0.1 % FA) over 235 min with a total
 chromatographic run time of 280 min. For mass spectrometric detection, MS
 scans were performed in the range from m/z 400-1400 at a resolution of
 70000 (at m/z =200). MS/MS scans of the eight most abundant ions were
 achieved through HCD fragmentation at 30 % normalized collision energy and
 analyzed in the orbitrap at a resolution of 17500 (at m/z =200).
   Proteome data analysis:
 The MaxQuant software
 (version 1.6.0.1), including the Andromeda search engine, was used for
 data analysis (Cox and Mann, 2008, PMID: 19029910). For positive protein
 identification, as a minimum two peptides, at least one of them being
 unique, had to be detected. Trypsin/P was specified in the digestion mode.
 Peptide mass tolerance was set to 50 and 25 ppm for the first and the main
 search, respectively. The false discovery rate (FDR) was set to 0.01 both
 on peptide and protein level. Peptides were mapped against a reference
 proteome set established before<sup>82</sup>.
 Carbamidomethylation was set as fixed modification, methionine oxidation
 and N-terminal acetylation as variable modifications. Each peptide was
 allowed to have a maximum of two missed cleavages and two modifications.
 “Match between runs” was enabled and the alignment and match time window
 set to 10 and 1 min, respectively. The analysis of the quantitative
 protein abundance has been performed using the Perseus software platform
 (Tyanova et al. 2016, PMID: 27348712), and the FDR (according to the
 Benjamini-Hochberg procedure) has been set at 0.2. Only proteins detected
 in at least four biological replicates have been considered in the
 analysis. Data
 analyzed using the Perseus computational platform<sup>
 </sup>(Tyranova, S et al,
 doi:10.1038/nmeth.3901)<sup> </sup> were processed
 additionally and independently. Arithmetic means were calculated from
 technical duplicates and then filtered with all treatments (combinations
 of genotype and diel time point) being excluded where no protein was
 detected in &gt;1 of the 3 replicates. Diel time points were then
 pooled and genotypes were compared via 2-sided t-tests. p-values were
 corrected for multiple testing using the “Permutation-based FDR” option
 (250 randomizations). Significant hits were annotated via BLAST against a
 recently published <i>Platynereis</i> proteome (Schenk,
 S.<i> et al.</i> doi:10.7554/eLife.41556).