Analysis of the heritability of epigenetic traces in clonality context – methylclonome

The technical approaches were broad. Briefly we proceed to RNAseq analysis: Full transcriptome for both the aphid and its endosymbiont. Pull down of methylated DNA fragments and high through put sequencing (NGS). Full sequencing of genome for both the aphid and endosymbiont after bisulfite treatment (WGBS) was performed. A full mapping of methyl group was realized with bio informatic tools allowing the browsing of decorated scaffolds from aphid base web site. The examination by GO enrichment analysis allowed to determine the p value regarding the fold changes for each category of genes related to biological process of molecular function. Bio informatics tools were used in order to map correlatively gene expression changes and density of methyl groups on genome. All these tools were used at wide scale genome for both the host and its endosymbiont. On the other side we have investigated similar events in Drosophila model for which powerful genetic tools are available. Using transgenic reporter genes that acts as calcium sensor, we have shown the functionality of gustatory receptors in wing. Those are partially under control of environmental factors. On the other side, the viral charges in plant infected by aphid vector was measured by Dombrovsky group.

The WGBS analysis leads to the conclusion that about 200.000 methylated CpGs in each sample were found and this would correspond to a CpG methylation level of 1.5%, which appears low compared to mammals and plants. To investigate the reality of aphid DNA methylation, we merged the WGBS data with that of another technical approach, the NGS sequencing of a pull down of methylated DNA fragments. Therefore the two methods were applied in parallel on the two aphid clonal variants derived from a unique parthenogenetic mother in order to provide a comprehensive view of genome-wide DNA methylation patterns in aphid host and its obligatory endosymbiont genomes. This approach allowed us to generate high-resolution maps for cytosine methylation. Only genes that were found in these two lists (WGBS and NGS reads from the pull down) were kept for GO enrichment analysis. The methylation in the endosymbiont genome appears robust in accordance with the levels found in E coli suggesting a 3D chromosome architecture guided by methylase action. Regarding the host, the transcriptome analysis show that most of the top genes appearing in the heat map are presently unknown. Hot spots of methylation were consistently observed indicating a probable regulatory mechanism for distant genes in the chromosomal 3D context. The global increase of methylation in the green aphid host seems solid arguing for a role to adapt to environmental stress. To verify the results obtained with aphid, we investigate the Drosophila model in order to establish similar epigenetic process. We used genetic tools to demonstrate the functionality of gustatory receptors in wing tissue. Although there is no measurable DNA methylation in Drosophila we observed epigenetic regulation of molecular set up in wing tissue. The same way the spreading of viral charges in plants by the aphid dispersion/invasion likely depends at least partially on aphid epigenetic status.

A lot of data obtained during the funding period and few ongoing publications writting encourage us to continue the topic of non mendellian regulatory processes in adaptation of organisms to environment. This work is still at basic level of sciences without timeable follow ups in application. The diffusion of plant virus by aphid vectors whose adaptation rely strongly on epigenetic regulation, reenforce this paradigm framework to envisage a pest control strategy to protect plants and crops.

The chapter opened by this project has shed light on a new topic which is non medellian heredity in insects that appears tremendously important to achieve refined adaptation to fluctuating environment. This means that beside the genetic background selection by environment pressure, the epigenetic regulation by marks like methyl constitutes a refined response for appropriate level of gene expression dictated by external factors. The data obtained in “methylclonome” likely are valid in the overall insect word whatever the specificity of epigenetic regulations that vary depending on species and speciation. The observation related to this program has to be amplified to investigate new scenario of epigenetics like those mediated by non coding RNA in order to unravel the diversity of epigenetic modes.

Adenine methylation may contribute to endosymbiont selection in a clonal aphid population.Victoria Reingold, Neta Luria, Alain Robichon, Aviv Dombrovsky BMC Genomics 2014, 15:999

Environmentally Selected Aphid Variants in Clonality Context Display Differential Patterns of Methylation in the Genome. Claude Pasquier, Mathilde Clément, Aviv Dombrovsky, Stéphanie Penaud, Martine Da Rocha, Corinne Rancurel, Neil Ledger, Maria Capovilla, Alain Robichon. PLoS ONE 2014 9(12):e115022


Functional Gustatory Role of Chemoreceptors in Drosophila Wings. Hussein Raad, Jean-François Ferveur, Neil Ledger, Maria Capovilla, Alain Robichon. Cell Reports, Vol. 15, Issue 7, p1442–1454 2016

Free-flight odor tracking in Drosophila: Effect of natural variation, sex, wing chemosensors and pheromonal gene regulation. Benjamin Houot, Steven Fry, Vincent Gigot, Alain Robichon, Jean-François Ferveur. Scientific reports 9 january 2017

The Mapping of Predicted Triplex DNA:RNA in the Drosophila Genome Reveals a Prominent Location in Development- and Morphogenesis-Related Genes. Claude Pasquier, Sandra Agnel, Alain Robichon . G3 (Bethesda). 2017;7:2295-2304

A new paramutation-like example at the Delta gene of Drosophila. Maria Capovilla, Alain Robichon, Minoo Rassoulzadegan. PLoS One. 2017;12(3):e0172780

Cucumber green mottle mosaic virus: Rapidly Increasing Global Distribution, Etiology, Epidemiology and Management. Aviv Dombrovsky et al. Annual Review of Phytopathology Vol 55, 2017

A New Israeli Tobamovirus Isolate Infects Tomato Plants Harboring Tm-22 Resistance Genes.Luria, Neta., Smith, E., Reingold, V., Bekelman, I., Lapidot, M., Levin, I., Elad, N., Tam, Y., Sela, N., Abu-Ras, A., Ezra, N., Haberman, A., Yitzhak, L. and Dombrovsky, A. PLoS One 2017. 12:e0170429.

The honey bee Apis mellifera contributes to Cucumber green mottle mosaic virus spread via pollination.Darzi, E., Smith, E., Shargil, D., Lachman, O., Ganot, L., Dombrovsky, A. (2017). Plant Pathology (under press)

Epigenetic marks on genomic DNA over a limited number of generations are considered non-Mendelian heritability. This phenomenon is not related to transmissible mutations or changes in DNA sequence. Partly, they are produced by environmentally induced covalent binding of a methyl group to cytosine, which initiates chromatin remodeling and regulation of gene expression. The question at the basis of this proposal is as follows: how can adaptive traits emerge in organisms harboring an unchanged genome and be heritable over a limited number of generations depending on the nature of the environmental pressure?
We have benefited of an ANR funding (acronym “EXDISUM”) over a period of 4 years (2006-2009) for which A.R. was coordinator. This proposal is in the prolongation of what has been achieved during this period. The aim of EXDISUM was about “frequency and density dependent genes” for which the mode of expression seems heritable over a limited number of generations depending on the population density and/or the environmental conditions. This ANR program was the logical next step coming from our work on Drosophila and honey bee (Ben-Shahar et al., Science 2002; Osborne et al., Science 1997) and allowed us to achieve the analysis of these genes in clonality context using aphids as an insect model (Consortium, Plos Biology 2010; Dombrovsky et al., Genome Research 2009) and in sexuality context using Drosophila (Ben Rokia-Mille et al., Plos One 2008 and Arthaud et al., Plos One 2011). Of considerable interest during EXDISUM, is the observation that asexuality of aphids leads to an extensive repertoire of variants stochastically produced and/or oriented by external factors so that the environment in fine selects the profiles presenting the best fitness. We have also reported that the aphid genome is extensively methylated and the repertoire of variants is under the control of these methylations (Dombrovsky et al., Genome Research 2009). Moreover a substantial effort during the funding period of EXDISUM has been devoted to the annotation of gene families following the aphid genome sequencing process (Consortium, Plos Biology 2010).
The present proposal consists in examining whether a clonal species of insect is capable to transmit across generations epigenetic marks as a signature of a transient and singular environmental episode. We expect transmissible epigenetic marks as the main cue for adaptation to these fluctuating environments. The aphid model used in this proposal (Acyrtosiphon pisum) has major advantages for epigenetic studies: parthenogenetic reproduction in spring and summer and sexual reproduction in fall leading to a new cycle of clonal reproduction the next spring. The aim of this proposal is to investigate the range of phenotypic plasticity of this clonal organism and the heredity of the induced adaptive traits that exclude the classic Darwinian genetics of allele selection.
Our analysis will be focused on the methylation of aphid DNA as a putative molecular mechanism to orchestrate these phenomena. The range of phenotypic plasticity of environmentally selected variants in clonality context will be examined and the large panel of “methylated” genes will be analyzed to assess their role in this phenomenon along with their heritable status. A large number of methylated loci on the genome (500 to 1.000 fragments) will be analyzed through the successive generations of ancestors having experienced extreme climatic episodes (extreme temperatures). The associated epigenetic marks on the genome will be checked across clonal generations and the marks kept or preserved by the sexual barrier will be comparatively assessed. The extensive analysis of the genes which are “hot spot” of methylation will be performed to assess the heredity of their mode of expression in clonality and in sexuality contexts.