Final Report Summary - EPIWORM (Finding the Ghost in the Genome: Assessing the contribution of epigenetics to environmental plasticity in the soil sentinel Lumbricus rubellus)
It has been established that the epigenetic landscape (cytosine methylation, histone modification and non-coding RNAs) impacts on the underlying genomic architect. Animals inhabiting extreme terrestrial environments provide a unique opportunity to better understand how organisms’ cope with challenges posed by multiple geogenic stressors. High abundance of an invasive pantropical earthworm, in the Furnas geothermal field (Sao Miguel Island, Azores) indicates its outstanding tolerance to high soil temperature, exceptionally high carbon dioxide and low oxygen levels, and elevated metal bioavailability. These conditions would be lethal for the majority of terrestrial metazoans. In the present work, we assessed how this metazoan tolerates such stressful environment determining whether epigenetic control is an additional (and previously unrecognised) mechanism that is used as a means of regulating adaptive genome responses by earthworm populations. The project aimed to pursue three specific objectives (see below) to test the overarching hypothesis that: "Alterations in the epigenetic landscape caused by exposure to environmental contaminants directly modulate the expression of specific genes, increasing phenotypic plasticity and providing the mechanistic basis underpinning the organism's responses to the stress inducing causative stimuli."
Two sites in São Miguel, differing in their contemporary volcanic activity (thermal and degassing outputs), were selected for mesocosms exposures. Furnas, which displays the most conspicuous degassing and geothermal activity in the entire Azores archipelago and Macela, which does not presently display any thermal and degassing phenomena. A group of adult earthworms from Furnas and another group from Macela were assigned to a factorial-design (with earthworm source and exposure site as factors) and exposed for 28 days.
The first objective included the identification and description of the alterations in the global epigenetic landscape as well as geochemist characterization and measure of metal body burden. We studied the soil geochemistry thoroughly. Metal concentrations in actively volcanic (Furnas) and volcanically inactive (Macela) soils were found to be similar; however, Furnas soil was characterised by elevated temperature (15 °C differential), relative hypoxia, extremely high CO2 tension, and accompanying acidity. We also measured the body burden for an array of metals, analysed from the powdered tissue of the earthworms. Elemental concentrations in actively volcanic (Furnas) and volcanically inactive (Macela) soils were found to be similar although increased metal mobility was influenced by the environmental conditions in the volcanically active site, in particular heavy metals such as lead, copper, nickel, cadmium and arsenic. A draft genome of the focus species was assembled, and at the moment over a hundred thousand contigs have been very valuable for mapping the epigenetic changes. We have performed MeDIP-seq for powdered earthworms under the four conditions. This constitutes the first description of methylation patterns of an earthworm. This data has also led to the development of software which is capable of rapidly performing investigative analysis of large scale epigenetic data sets.We prepared libraries for small RNAs sequencing and identified novel miRNAs for A. gracilis for the first time. We performed differential expression analyses and identified the miRNAs that seemed more important for the adaptation to the volcanic conditions as well as those having population-specific regulatory functions. In situ hybridizations in histological sections were performed, showing that the in silico predicted miRNAs were real and that they show specific tissue expression.
The second objective aimed to determine the spatial association of epigenetic modification with loci displaying altered expression. For that, we firstly generated RNA-seq data that revealed an array of mechanistic pathways associated with survival in a multi-stressor environment including genes involved in the response to hypoxia, hypercapnia, heat stress and metals. Furthermore, preliminary ontological analysis of those loci with displaying differential methylation revealed a similar association. Interestingly, another general term which was highly overrepresented in the genes which changed methylation was alternative splicing. In general, higher gene expression correlates positively with increases in methylation. However there is also a tendency for a group of the ~10% most highly methylated genes to exhibit an inverse relationship of expression level with methylation. Targets of miRNAs were much more likely to occur in the promotors of lowly expressed genes than in highly expressed ones and there was a pretty clear linear inverse relationship between promotor miRNA targeting and gene expression. However, these analyses are still ongoing for more definitive results.
The third objective was directed towards establishing the functional relationships between epigenetic changes and phenotypic responses. The epidermis of earthworms transplanted to the non-volcanic soil (Macela) was approximately twice the thickness of the epidermis of conspecifics exposed to volcanic soil (Furnas), regardless the origin source of the earthworm. Some of the miRNAs that were differentially expressed showed to be regulating epidermis. We studied as well the microbiome of the exposed earthworms by means of NGS sequencing of the region 16S. It seems that the worms mainly integrate the bacteria that are in the soil where they are exposed indicating the potential role of bacteria for aiding the adaptation of these earthworms to the new environments. We explored the selection among those populations, first comparing the transcriptomic data and calculating the nonsynonymous versus the synonymous substitutions and then analysing the populations with RAD-seq. The first analysis indicated that genes showing positive selection were involved in pathways related to response to hypoxia and oxygen levels, ion binding and channels, cell differentiation, sensory perception of chemical stimuli. This means that the earthworms are not only responding to the changes from the epigenetic point of view, by regulating changes in gene expression, but also the basal sequence information of the genome has been modified as a result for the adaptation. RAD-seq data is currently being analysed..
Within this project, we have generated for the first time genomic, transcriptomic and miRNAs data for the species Amynthas gracilis as well as the first information for DNA methylation for annelids, as far as we are concerned.
Two sites in São Miguel, differing in their contemporary volcanic activity (thermal and degassing outputs), were selected for mesocosms exposures. Furnas, which displays the most conspicuous degassing and geothermal activity in the entire Azores archipelago and Macela, which does not presently display any thermal and degassing phenomena. A group of adult earthworms from Furnas and another group from Macela were assigned to a factorial-design (with earthworm source and exposure site as factors) and exposed for 28 days.
The first objective included the identification and description of the alterations in the global epigenetic landscape as well as geochemist characterization and measure of metal body burden. We studied the soil geochemistry thoroughly. Metal concentrations in actively volcanic (Furnas) and volcanically inactive (Macela) soils were found to be similar; however, Furnas soil was characterised by elevated temperature (15 °C differential), relative hypoxia, extremely high CO2 tension, and accompanying acidity. We also measured the body burden for an array of metals, analysed from the powdered tissue of the earthworms. Elemental concentrations in actively volcanic (Furnas) and volcanically inactive (Macela) soils were found to be similar although increased metal mobility was influenced by the environmental conditions in the volcanically active site, in particular heavy metals such as lead, copper, nickel, cadmium and arsenic. A draft genome of the focus species was assembled, and at the moment over a hundred thousand contigs have been very valuable for mapping the epigenetic changes. We have performed MeDIP-seq for powdered earthworms under the four conditions. This constitutes the first description of methylation patterns of an earthworm. This data has also led to the development of software which is capable of rapidly performing investigative analysis of large scale epigenetic data sets.We prepared libraries for small RNAs sequencing and identified novel miRNAs for A. gracilis for the first time. We performed differential expression analyses and identified the miRNAs that seemed more important for the adaptation to the volcanic conditions as well as those having population-specific regulatory functions. In situ hybridizations in histological sections were performed, showing that the in silico predicted miRNAs were real and that they show specific tissue expression.
The second objective aimed to determine the spatial association of epigenetic modification with loci displaying altered expression. For that, we firstly generated RNA-seq data that revealed an array of mechanistic pathways associated with survival in a multi-stressor environment including genes involved in the response to hypoxia, hypercapnia, heat stress and metals. Furthermore, preliminary ontological analysis of those loci with displaying differential methylation revealed a similar association. Interestingly, another general term which was highly overrepresented in the genes which changed methylation was alternative splicing. In general, higher gene expression correlates positively with increases in methylation. However there is also a tendency for a group of the ~10% most highly methylated genes to exhibit an inverse relationship of expression level with methylation. Targets of miRNAs were much more likely to occur in the promotors of lowly expressed genes than in highly expressed ones and there was a pretty clear linear inverse relationship between promotor miRNA targeting and gene expression. However, these analyses are still ongoing for more definitive results.
The third objective was directed towards establishing the functional relationships between epigenetic changes and phenotypic responses. The epidermis of earthworms transplanted to the non-volcanic soil (Macela) was approximately twice the thickness of the epidermis of conspecifics exposed to volcanic soil (Furnas), regardless the origin source of the earthworm. Some of the miRNAs that were differentially expressed showed to be regulating epidermis. We studied as well the microbiome of the exposed earthworms by means of NGS sequencing of the region 16S. It seems that the worms mainly integrate the bacteria that are in the soil where they are exposed indicating the potential role of bacteria for aiding the adaptation of these earthworms to the new environments. We explored the selection among those populations, first comparing the transcriptomic data and calculating the nonsynonymous versus the synonymous substitutions and then analysing the populations with RAD-seq. The first analysis indicated that genes showing positive selection were involved in pathways related to response to hypoxia and oxygen levels, ion binding and channels, cell differentiation, sensory perception of chemical stimuli. This means that the earthworms are not only responding to the changes from the epigenetic point of view, by regulating changes in gene expression, but also the basal sequence information of the genome has been modified as a result for the adaptation. RAD-seq data is currently being analysed..
Within this project, we have generated for the first time genomic, transcriptomic and miRNAs data for the species Amynthas gracilis as well as the first information for DNA methylation for annelids, as far as we are concerned.