In neo-Darwinian theory, adaptation results from a response to selection on relatively slowly accumulating genetic variation. However, more rapid adaptive responses are possible if selectable or plastic phenotypic variation is produced by epigenetic differences in gene expression. This rapid path to adaptation may prove particularly important when genetic variation is lacking, such as in small, bottlenecked, or asexual populations. To examine the potential for an epigenetic contribution to adaptive variation, we examined morphological divergence and epigenetic variation in genetically impoverished asexual populations of a freshwater snail, Potamopyrgus antipodarum, from distinct habitats (two lakes versus two rivers). These populations exhibit habitat specific differences in shell shape, and these differences are consistent with adaptation to water current speed. Between these same habitats, we also found significant genome wide DNA methylation differences. The differences between habitats were an order of magnitude greater than the differences between replicate sites of the same habitat. These observations suggest one possible mechanism for the expression of adaptive shell shape differences between habitats involves environmentally induced epigenetic differences. This provides a potential explanation for the capacity of this asexual snail to spread by adaptive evolution or plasticity to different environments.

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Epigenetic variation has the potential to influence environmentally dependent development and contribute to phenotypic responses to local environments. Environmental epigenetic studies of sexual organisms confirm the capacity to respond through epigenetic variation. An epigenetic response could be even more important in a population when genetic variation is lacking. A previous study of an asexual snail, Potamopyrgus antipodarum, demonstrated that different populations derived from a single clonal lineage differed in both shell phenotype and methylation signature when comparing lake versus river populations. Here, we examine methylation variation among lakes that differ in environmental disturbance and pollution histories. Snails were collected from a more pristine rural Lake 1 (Lake Lytle), and two urban lakes, Lake 2 (Capitol Lake) and Lake 3 (Lake Washington) on the Northwest Pacific coast. DNA methylation was assessed for each sample population using methylated DNA immunoprecipitation, MeDIP, followed by next-generation sequencing. The differential DNA methylation regions (DMRs) identified among the different lake comparisons suggested a higher number of DMRs and variation between rural Lake 1 and one urban Lake 2, and between the two urban Lakes 2 and 3, but limited variation between the rural Lake 1 and urban Lake 3. DMR genomic characteristics and gene associations were investigated. The presence of site-specific differences between each of these lake populations suggest an epigenetic response to varied environmental factors. The results do not support an effect of geographic distance in these populations. The role of dispersal distance among lakes, population history, environmental pollution and stably inherited methylation versus environmentally triggered methylation in producing the observed epigenetic variation are discussed. Observations support the proposal that epigenetic alterations may associate with phenotypic variation and environmental factors and history of the different lakes.

Background Permethrin and N,N-diethyl-meta-toluamide (DEET) are the pesticides and insect repellent most commonly used by humans. These pesticides have been shown to promote the epigenetic transgenerational inheritance of disease in rats. The current study was designed as an epigenome-wide association study (EWAS) to identify potential sperm DNA methylation epimutation biomarkers for specific transgenerational disease. Methods Outbred Sprague Dawley gestating female rats (F0) were transiently exposed during fetal gonadal sex determination to the pesticide combination including Permethrin and DEET. The F3 generation great-grand offspring within the pesticide lineage were aged to 1 year. The transgenerational adult male rat sperm were collected from individuals with single and multiple diseases and compared to non-diseased animals to identify differential DNA methylation regions (DMRs) as biomarkers for specific transgenerational disease. Results The exposure of gestating female rats to a permethrin and DEET pesticide combination promoted transgenerational testis disease, prostate disease, kidney disease, and the presence of multiple disease in the subsequent F3 generation great-grand offspring. The disease DMRs were found to be disease specific with negligible overlap between different diseases. The genomic features of CpG density, DMR length, and chromosomal locations of the disease specific DMRs were investigated. Interestingly, the majority of the disease specific sperm DMR associated genes have been previously found to be linked to relevant disease specific genes. Conclusions Observations demonstrate the EWAS approach identified disease specific biomarkers that can be potentially used to assess transgenerational disease susceptibility and facilitate the clinical management of environmentally induced pathology.

Atrazine is a common agricultural herbicide previously shown to promote epigenetic transgenerational inheritance of disease to subsequent generations. The current study was designed as an epigenome-wide association study (EWAS) to identify transgenerational sperm disease associated differential DNA methylation regions (DMRs) and differential histone retention regions (DHRs). Gestating female F0 generation rats were transiently exposed to atrazine during the period of embryonic gonadal sex determination, and then subsequent F1, F2, and F3 generations obtained in the absence of any continued exposure. The transgenerational F3 generation males were assessed for disease and sperm collected for epigenetic analysis. Pathology was observed in pubertal onset and for testis disease, prostate disease, kidney disease, lean pathology, and multiple disease. For these pathologies, sufficient numbers of individual males with only a single specific disease were identified. The sperm DNA and chromatin were isolated from adult one-year animals with the specific diseases and analyzed for DMRs with methylated DNA immunoprecipitation (MeDIP) sequencing and DHRs with histone chromatin immunoprecipitation (ChIP) sequencing. Transgenerational F3 generation males with or without disease were compared to identify the disease specific epimutation biomarkers. All pathologies were found to have disease specific DMRs and DHRs which were found to predominantly be distinct for each disease. No common DMRs or DHRs were found among all the pathologies. Epimutation gene associations were identified and found to correlate to previously known disease linked genes. This is one of the first observations of potential sperm disease biomarkers for histone retention sites. Although further studies with expanded animal numbers are required, the current study provides evidence the EWAS analysis is effective for the identification of potential pathology epimutation biomarkers for disease susceptibility.

Environmental exposures such as chemical toxicants can alter gene expression and disease susceptibility through epigenetic processes. Epigenetic changes can be passed to future generations through germ cells through epigenetic transgenerational inheritance of increased disease susceptibility. The current study used an epigenome-wide association study (EWAS) to investigate whether specific transgenerational epigenetic signatures of differential DNA methylation regions (DMRs) exist that are associated with particular disease states in the F3 generation great-grand offspring of F0 generation rats exposed during gestation to the agricultural pesticide methoxychlor. The transgenerational epigenetic profiles of sperm from F3 generation methoxychlor lineage rats that have only one disease state were compared to those that have no disease. Observations identify disease specific patterns of DMRs for these transgenerational rats that can potentially serve as epigenetic biomarkers for prostate disease, kidney disease, obesity, and the presence of multiple diseases. The chromosomal locations, genomic features, and gene associations of the DMRs are characterized. Disease specific DMR sets contained DMR-associated genes that have previously been shown to be associated with that specific disease. Future epigenetic biomarkers could potentially be developed and validated for humans as a disease susceptibility diagnostic tool to facilitate preventative medicine and management of disease.

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Plastic-derived compounds are one of the most frequent daily worldwide exposures. Previously a mixture of plastic-derived toxicants composed of bisphenol A, bis(2-ethylhexyl) phthalate, and dibutyl phthalate at low-dose exposures of a gestating female rats was found to promote the epigenetic transgenerational inheritance of disease to the offspring (F1 generation), grand-offspring (F2 generation), and great-grand-offspring (F3 generation). Epigenetic analysis of the male sperm was found to result in differential DNA methylation regions (DMRs) in the transgenerational F3 generation male sperm. The current study is distinct and was designed to use an epigenome-wide association study to identify potential sperm DNA methylation biomarkers for specific transgenerational diseases. Observations indicate disease-specific DMRs called epimutations in the transgenerational F3 generation great-grand-offspring of rats ancestrally exposed to plastics. The epigenetic DMR biomarkers were identified for testis disease, kidney disease, and multiple (≥2) diseases. These disease sperm epimutation biomarkers were found to be predominantly disease-specific. The genomic locations and features of these DMRs were identified. Interestingly, the disease-specific DMR-associated genes were previously shown to be linked with each of the specific diseases. Therefore, the germline has ancestrally derived epimutations that potentially transmit transgenerational disease susceptibilities. Epigenetic biomarkers for specific diseases could be used as diagnostics to facilitate clinical management of disease and preventative medicine.