Computational protocol: Low levels of Cd induce persisting epigenetic modifications and acclimation mechanisms in the earthworm Lumbricus terrestris

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Protocol publication

[…] Comet assay was performed using a previously published protocol [] with slight modifications (see ).Tissue samples for measuring oxidative stress parameters were homogenized using glass beads on a Precellys®24 Homogenizer (Bertin Technologies, USA) at 8.400 x g for 60 s in 1 ml of 50 mM potassium phosphate buffer (1 M KH2PO4, 1 M K2HPO4, pH 7.0 with 0.1 mM EDTA). The homogenates were centrifuged at 10.000 x g at 4°C for 12 min. The supernatant was collected and the protein content was determined using a NanoDrop 2000c (Thermo Fisher Scientific, USA). Supernatants were used for catalase (CAT) and malondialdehyde (MDA) measurements on a spectrophotometer (EnSpire® Multimode Plate Reader, Perkin Elmer, USA).CAT activity was measured at 240 nm as previously published [] with modifications to measure in UV transparent 96 well plates []. The level of lipid peroxidation was indirectly determined as the formation of MDA (absorbance measured at 600 nm), a by-product of lipid peroxidation that reacts with thiobarbituric acid (TBA) []. A detailed procedure on CAT and MDA measurements is available in .All results are presented as mean±SEM. Statistical analyses were performed using Mann-Whitney Rank Sum Test (p≤0.05) in SigmaPlot 13.0 [...] Genomic DNA was extracted using the GenElute Mammalian DNA miniprep kit (Sigma-Aldrich, Germany) according to the manufacturer’s instructions. MSAP was performed following a standard protocol [,] with slight modifications (see ).10 samples were replicated for all primer combinations to ensure consistency between runs. Error rates and fragment filtering was calculated and performed as previously recommended []. Prior to fragment filtering, the mean error rate across all primer combinations was 12.21%, and the median was 10% (N = 1236). A conservative filtering strategy was performed to retain only fragments with error rates equal or lower than the median of the error distribution. The mean error rate across all primers after fragment filtering was 4.33% (N = 728). Using this data set, each replicate was analysed independently (see ). MSAP profiles were analysed using the R package msap [] and classified as either “methylation-susceptible loci” (MSL) or “non-methylation loci” (NML). NML fragments were utilized to test for genomic diversity of earthworms (Fst values) using Arlequin software V 3.5.2.2. Samples from time point 0 from the control and Cd treatment group in both replicas were considered as 4 “cohorts”. The percentage of methylation for each individual across all MSL was calculated for each time point and treatment group. Groups were statistically compared using a Student’s t-test (level of significance p≤0.05) and the results are presented as mean±SEM. Differentially methylated MSL were defined as those showing a difference of more than 10% of the average methylation of Cd_4 and/or Cd_12 groups compared to the Cd_0 group. GenAlEx 6.5 software was applied to perform a Principal Coordinates Analysis (PCoA) using differentially methylated MSL. PCoA graphs were generated from the mean binary epigenetic distance between treatment groups. Differentially methylated MSL were further analysed to determine whether methylation after 4 and 12 weeks was either increased, decreased or the same as in the Cd_0 group. In order to define persistent DNA methylation modifications, differentially methylated MSL were analysed in replica I after a 7 month recovery period. Fragments that had the same methylation pattern (increased or decreased) after the recovery as during the exposure period were defined as persistent differentially methylated MSL. Results are presented as percentages of fragments that remained altered after the recovery period. […]

Pipeline specifications

Software tools SigmaPlot, Arlequin, GenAlEx
Applications Miscellaneous, Population genetic analysis
Organisms Lumbricus terrestris
Chemicals Cadmium, Phytochelatins