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Light and temperature measurements and untargeted proteomic measurements

Citation

Tessmar-Raible, Kristin (2021), Light and temperature measurements and untargeted proteomic measurements, Dryad, Dataset, https://doi.org/10.5061/dryad.73n5tb2vv

Abstract

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.

Methods

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 Posidonia oceanica meadows, a natural habitat of Platynereis. 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/m2/nm/sec were converted to corresponding number of photons/cm2/nm/sec or, to enable comparisons with previous published works, also converted to uW/cm2/nm/sec, uMolphotons/m2/nm/sec, photons/m2/um/sec and log photons/m2/nm/sec (Examples for such a conversion are deposited under the tab labels "24.08.2010" and "04.07.2011").

 

Untargeted Mass-Spectrometry (Proteomics)

Platynereis c-opsin1+/+ and c-opsin1Δ8/Δ8 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% MgCl2: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 before82. 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 (Tyranova, S et al, doi:10.1038/nmeth.3901)  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 >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 Platynereis proteome (Schenk, S. et al. doi:10.7554/eLife.41556).

Funding

European Research Council, Award: 337011

H2020 European Research Council, Award: 819952

Austrian Science Fund, Award: SFB F78

Austrian Science Fund, Award: AY0041321

Austrian Science Fund, Award: P28970

Human Frontiers Science Foundation, Award: RGY0082/2010

Human Frontiers Science Foundation, Award: RGY0082/2010