Computational protocol: Herbivore-induced chemical and molecular responses of the kelps Laminaria digitata and Lessonia spicata

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[…] Metabolic extracts were obtained from 100 mg of frozen algal powder of L. digitata (n = 4 except for grazed-48h n = 2) or L. spicata (n = 3 except for control-48h n = 2) samples collected after 6h, 12h, 24h and 48h, with 1 mL MeOH:H2O (8:2), as already described []. For untargeted metabolomic analyses, 1.25 μg of 12-OH-lauric acid was added in each sample, as internal standard. After extraction, aliquots of 50 μL were separated by ultrahigh-pressure liquid chromatography (UPLC) and analyzed by mass spectrometry (MS) on a Thermo Scientific LTQ-Orbitrap Discovery™ mass spectrometer (Thermo Scientific) equipped with an Electro Spray Ionization (ESI) source running on the negative mode, as described []. Samples were separated using an Acclaim RSLC 120 C18 1.9 μm (2.1 x 100 mm) column 545 (Dionex; Thermo Fisher Scientific, Courtaboeuf, France) maintained at 20°C using 5 μL injection volume and a flow-rate of 250 μL.min-1 and the mobile phase (0.2% acetic acid in water–acetonitrile) was programmed from 95: 5 to 5: 95 acetonitrile–water (v: v).Mass spectra data were processed by XCMS using the online version of Galaxy-Workflow4metabolomics [], after conversion of raw spectra to mzXML format. Data processing was performed using centWave method for the peak picking, with a maximum deviation of 4 ppm. The signal/noise threshold was fixed at 10, the prefilter at 3,100 and the noise filter at 5000. For the first group step, density method was used, with the band width set at 30 and the minimum fraction of samples necessary at 0.7. For correction of retention time, the obiwarp method was used and a step size of 0.1 m/z. The second group step was performed using density method and a band width of 10. Fillpeaks step was used with the chrom filling method. Finally, annotation by CAMERA was set using a max ion charge of 2, a general ppm error of 5 and a precision of 4 decimals of m/z values. Areas for all peaks were normalized using that of the internal standard in the same sample, and normalized areas were used as ion relative abundances. The multivariate data analysis of relative ions abundances using Partial Least Squares—Discriminant Analysis (PLS-DA) was performed to test for differences in metabolite profiles among grazed and control samples. Further statistical multiple testing analyses were performed to identify the different ions, based on Student T-test and FDR-adjusted p-values (P). The analysis was carried out on Pareto scaling and log10-transformed data using the software SIMCA (13.0, Umetrics, Umeå, Sweden).For amino acid, free fatty acid and oxylipin targeted profiling in L. spicata samples (n = 3 except for control-48h n = 2), the same extraction and quantification process was applied as for the untargeted analysis (see above), adding 1.25 μg of 12-OH-lauric acid and 10 μg of atropine in each sample, as internal standards for negative or positive ionization mode analyses, respectively. Free fatty acid and oxylipin profiles were obtained through negative mode LCMS analyses, and amino acid measurements in positive mode. The Hierarchical Clustering analysis of relative abundances changes was carried out using Pearson Correlation values in Multi Experiment Viewer 4.9 []. Statistical Kruskal-Wallis tests were then performed for each metabolite with grazing treatment and time as factors, using the free software R version 3.2.5. [...] Total RNA was extracted from 100 mg of frozen algal materials according to Apt et al. [], treated with RNAse-free DNAse I (Stratagene, La Jolla, CA, USA) to eliminate genomic DNA contamination, and quantified using a Nanodrop ND 1000 spectrophotometer (Labtech International Ltd, Lewes, UK). For each species, total RNA was extracted from three independent biological replicates of control or grazed algal samples, collected after 6, 12, 24 and 48 hours for L. digitata (Ld) and after 6, 12 and 24 hours for L. spicata (Ls). For cDNA library sequencing, two pools of RNA, corresponding to Control or Grazed treatments for each species, were prepared by pooling the same amount of RNA samples extracted at each time point from independent biological triplicates, i.e. 12 RNA samples for Ld and 9 RNA samples from Ls. The four non-normalized shot-gun cDNA libraries, namely Ld-Control, Ld-Grazed, Ls-Control and Ls-Grazed, were constructed using the SMART cDNA library kit from Clontech (CA, USA), differentially- tagged with a specific adaptor and sequenced using 454 sequencing technology (Roche, Branford, CT, USA) at the Max Planck Institute of Berlin-Dahlem.Raw sequencing data were deposited in the SRA database at NCBI (accessions SRR4149283 for Ld-Control, SRR4159455 for Ld-Grazed, SRR4240918 for Ls-Control, SRR4240919 for Ls-Grazed sequences). Raw reads were cleaned by removing adaptor sequences, empty reads and filtering reads with poor quality using SeqClean ( The cleaned 454-generated reads constituted the Expressed Sequence Tags (ESTs). For each species, all ESTs derived from Control and Grazed libraries were assembled together into non-redundant unigenes using Newbler v2.0.01.14 (, by including cDNA sequences of the two species available in public databases, to increase the quality of clustering. The unigenes sequences are available upon request. Each EST, belonging to Control or Grazed libraries, was associated to one unigene sequence (contigs or singletons). In order to compare the relative abundance of EST per unigene between the two libraries, we calculated their relative abundance with the following formula: Relative EST abundance of Unigene U in Library Lib = number of EST mapped to Unigene U in Library Lib * 1/(Total number of ESTs in Library Lib * Length of Unigene U (bp)).Putative differentially-expressed unigenes were further identified according to their relative EST abundance into the two libraries, using IDEG6 web tool [], based on concordant results from four different statistical tests, namely Pairwise Audic & Claverie, Pairwise Fisher Exact, Pairwise Chi-squared and Stekel & Falciani R tests []. The putative function of these differentially-expressed sequences was analyzed using Blastx and Blastn against the nr and nt databases (March 2016). [...] To validate the putative molecular markers of grazing pressure, total RNA was extracted from 100 mg of frozen algal material of L. digitata or L. spicata sampled during laboratory-controlled grazing kinetics or in natural populations. For L. digitata grazing laboratory kinetics, the RNA samples (n = 3 for each time point) were those later pooled for EST analyses, whereas for L. spicata, a new experimental grazing experiment was conducted up to 48 hours, corresponding to a new set of RNA samples for both control and grazed algae (n = 3 for each time point). For in situ survey, grazed and un-grazed fronds from adult sporophytes of L. digitata and L. spicata (n = 7–8) were sampled at the same locations at Roscoff and Las Cruces (see above and ). Field samples were immediately flash frozen in liquid nitrogen and kept at -80°C before RNA extraction. Gene expression was then analyzed by qPCR as described by Cosse et al. [], using the primers sequences provided in . Gene transcript level was normalized to the geometrical mean of two reference gene transcript levels in the same sample (tubulin and EF1a genes for L. digitata; EF1a and RPL36 genes for L. spicata). The Hierarchical Clustering analyses of gene expression changes was carried out on Pearson Correlation values in Multi Experiment Viewer 4.9 []. A two-way analysis of variance (ANOVA) with grazing treatment and time as categorical predictors was applied to qPCR expression data. All tests were performed using Statistica 7 (Statsoft, Tulsa, OK, USA). […]

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