Environmentally selected aphid variants in clonality context display differential patterns of methylation in the genome.

Heritability of acquired phenotypic traits is an adaptive evolutionary process that appears more complex than the basic allele selection guided by environmental pressure. In insects, the trans-generational transmission of epigenetic marks in clonal and/or sexual species is poorly documented. Aphids were used as a model to explore this feature because their asexual phase generates a stochastic and/or environment-oriented repertoire of variants. The a priori unchanged genome in clonal individuals prompts us to hypothesize whether covalent methyl DNA marks might be associated to the phenotypic variability and fitness selection. The full differential transcriptome between two environmentally selected clonal variants that originated from the same founder mother was mapped on the entire genomic scaffolds, in parallel with the methyl cytosine distribution. Data suggest that the assortments of heavily methylated DNA sites are distinct in these two clonal phenotypes. This might constitute an epigenetic mechanism that confers the robust adaptation of insect species to various environments involving clonal reproduction

Representative scaffolds decorated with the mapping of the exact matches corresponding to the methyl reads and the transcriptomic contigs.

<p>The methylome reads were directly mapped to the genome scaffolds of <i>A. pisum</i> strain LSR1 version Acyr_1.0 with the program Razers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115022#pone.0115022-Weese1" target="_blank">[43]</a>. The contigs were searched for sequence similarity using BLAST <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115022#pone.0115022-Altschul1" target="_blank">[46]</a>. Vertical bars are the number of reads corresponding to the contigated fragments obtained after subtractive enrichment of transcripts (pink for <i>orange</i> aphids and green for <i>green</i> aphids). Each pink and green horizontal trait represents a methyl read that is at least represented twice in pyrosequencing in the <i>orange</i> and <i>green</i> aphids and the thick traits are overlapping or contiguous reads. The methyl reads were mapped on scaffolds disregarding the fact that some of them match the genome elsewhere. (A) One scaffold shows a strong gene expression of five genes in the <i>green</i> aphids, whereas these transcripts are absent in the <i>orange</i>. (B) Expression and methylation of one specific gene encoding <i>Argonaute-2</i>.</p

Selection of the green variant.

<p>(A) The scheme summarizes the results of experiments using injection of aphid extracts adapted to 8°C or to 22°C in aphid mothers adapted to 8°C (green squares) or to 22°C (red squares). The empty box represents injection of buffer (Ringer solution) only. Arrows show the passage <i>orange</i> to <i>green</i> or <i>vice versa</i>. The cross indicates the death of progenies. (B) Ten orange adult aphids reared at 22°C from a single founder were placed at 8°C each day (X-axis) before the emergence of a <i>green</i> phenotype (black circles) after 5 months. <i>Orange</i> aphids injected with <i>green</i> aphid extract gave immediately <i>green</i> progenies in cold (black triangles). <i>Green</i> aphids placed at 22°C lost their green pigments after few days and their progenies were immediately orange (open circles). (C) <i>Green</i> aphids were placed at 22°C for 8 generations and for each one, 20 newly <i>orange</i> adults were placed back at 8°C. A preliminary experiment allowed us to determine the time required to get 20 newly <i>green</i> adult aphids from the 20 original orange at the first generation. This time scale was used to count the newly <i>green</i> adult aphids at each generation and data are represented as the ratio newly <i>green</i> adults versus initial <i>orange</i> aphids. Dots are the mean+/− SEM, n = 4.</p

Comparative analysis of the methylome hits matching to genome, genes and transposons between the <i>orange</i> and <i>green</i> variants.

<p>(A) Several hundreds of distinct reads found in both variants map on a large number of locations in scaffolds. Two graphs represent the number of unique reads (X axis) corresponding to the number of genomic locations retrieved by exact match (Y axis) (A1 and A2). The 50 reads the most represented in either variant totalize 107,258 locations for the <i>orange</i> and 118,966 for the <i>green</i>, which provides an internal control for further comparative analysis. On the other hand, the number of distinct reads that fits from 1 to 10 locations in the genome by exact match is plotted for both variants. About 65,615 and 103,529 unique reads match one location; 6,094 and 10,078 match two locations; 361 and 603, 5 locations for the <i>orange</i> and the <i>green</i>, respectively. (A3 and 4) (B) Distribution of methyl reads represented twice only in <i>green</i> (7,735), <i>orange</i> (5,339) and one in either variants (2,639). The same analysis was performed with methyl reads represented more than 5 times in both variants (917) exclusively in the <i>green</i> (181) or the <i>orange</i> (115). (C) The methyl fragments reads obtained with the <i>orange</i> and the <i>green</i> samples were mapped on scaffolds. X and Y-axis correspond to the number of hits per scaffold for the <i>orange</i> and the <i>green</i> aphids, respectively. Coefficient of correlation r = 0.995. (D) Comparative number of hits in introns, in promoters (2 and 3 kb upstream), border of transposons and arbitrary at 50 kb (49–51 kb) upstream of transposons.</p

Statistical analysis of the correlation GO term enrichment and methylation.

<p>This table displays significantly enriched GO terms (using a p-value<0.05) with respect to the “Molecular Function” GO category. (A) Molecular function of genes that show at least two-fold increase of expression value in the <i>green</i> compared to the <i>orange</i> sample. (B) Significantly enriched GO terms in the list of over-expressed genes that show a reduction of methylation. (C) Enriched terms found in the list of under-expressed genes (at least two fold) in the <i>green</i>. (D) Significant molecular function associated to the under-expressed genes that have an increase of methylation. No enrichment was found for GO categories that show both an increase of expression level and an increase of methylation and/or for under expressed genes that show a decrease of methylation.</p><p>Statistical analysis of the correlation GO term enrichment and methylation.</p

Quantitative PCR analysis of selected genes to assess the pyrosequencing transcriptomic data.

<p>Bars represent fold expression of gene of interest in the <i>orange</i> aphids (black) and the <i>green</i>-cold-adapted ones (grey). For each gene of interest, the normalized expression was rescaled by minimal sample value. The accumulation of each transcript was measured in three independent biological samples for each aphid variant. Statistically significant differences were determined by Student’s test analyses. The numbers in the bars are the reads obtained by RNA enrichment (SSH) followed by pyrosequencing of the corresponding cDNA.</p

Gene Ontology term enrichment for genes showing a variation (>twofold) of methylation.

<p>Both diagrams display enriched GO terms and their hierarchical relationships with respect to the “Molecular Function” GO category. Boxes represent GO terms. (A) Significantly enriched GO terms that present a strong reduction of reads in the <i>green</i> compared to the <i>orange</i> aphids (using a p-value<0.05, see main text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115022#pone.0115022.s019" target="_blank">S1 Procedures</a> for the cut off threshold). Terms marked pink have a minimum two-fold increase of methylation, whereas terms marked blue do not show methylation differences. (B) Significantly enriched GO terms that present a strong increase of reads in the <i>green</i> compared to the <i>orange</i> aphids using a p-value<0.05 (see main text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115022#pone.0115022.s019" target="_blank">S1 Procedures</a> for the cut off threshold selection). Terms marked green have a minimum two-fold decrease of methylation, whereas terms marked blue do not show methylation differences. The degree of color saturation of each box is positively correlated with the significance of enrichment of the corresponding GO term. Dots represent omitted terms that are not significantly found. Edges (arrows) stand for connections between different GO terms. Red edges stand for relationships between two enriched GO terms, black solid edges for relationships between enriched and non enriched terms and black dashed edges for relationships between two un-enriched GO terms.</p

Effect of chemoprevention by low-dose aspirin of new or recurrent colorectal adenomas in patients with Lynch syndrome (AAS-Lynch): study protocol for a multicenter, double-blind, placebo-controlled randomized controlled trial

Abstract Lynch syndrome (LS) is the most common cause of inherited colorectal cancer (CRC) and confers a high lifetime risk of CRC estimated to be up to 60%. Colonoscopy is recommended every 2 years in LS patients above the 20–25-year-old age bracket, and every year when colonic neoplasia has been detected. Efficient chemoprevention has the potential to represent a cost-effective intervention in these high-risk patients and could allow a delay in colonoscopy surveillance. Several epidemiological studies have shown that regular use of low dose aspirin is associated with a 20 to 30% reduction in the risk of sporadic colonic adenomas and colorectal cancer regardless of family risk. However, in recent large randomized trials in specific populations, aspirin use showed no protection for colorectal cancer. A prospective randomized CAPP-2 trial evaluated the effect of aspirin use in LS patients. The primary analysis of this trial showed no significant decrease in CRC in LS patients under daily aspirin. However, a preplanned secondary analysis after an extended follow-up showed a significant reduced risk of CRC in the aspirin group in the per-protocol analysis. The real effect and clinical benefit of aspirin are still to be consolidated in this population. The AAS-Lynch trial—a prospective, multicentric, double-blind, placebo-controlled, randomized clinical trial—was designed to investigate if daily aspirin therapy, at a dose of 100 or 300 mg, would decrease the occurrence or recurrence of colorectal adenomas in patients under 75 years of age, compared with placebo. Trial registration ClinicalTrials.gov NCT02813824 . Registered on 27 June 2016. The trial was prospectively registered