Do epigenetic changes drive corticosterone responses to alarm cues in larvae of an invasive amphibian?

The developmental environment can exert powerful effects on animal phenotype. Recently epigenetic modifications have emerged as one mechanism that can modulate developmentally plastic responses to environmental variability. For example, the DNA methylation profile at promoters of hormone receptor genes can affect their expression and patterns of hormone release. Across taxonomic groups, epigenetic alterations have been linked to changes in glucocorticoid (GC) physiology. GCs are metabolic hormones that influence growth, development, transitions between life-history stages, and thus fitness. To date, relatively few studies have examined epigenetic effects on phenotypic traits in wild animals, especially in amphibians. Here, we examined the effects of exposure to predation threat and experimentally manipulated DNA methylation on corticosterone (CORT) levels in tadpoles and metamorphs of the invasive cane toad (Rhinella marina). We included offspring of toads sampled from populations across the species’ Australian range. In these animals, exposure to chemical cues from injured conspecifics induces shifts in developmental trajectories, putatively as an adaptive response that lessens vulnerability to predation. We exposed tadpoles to these alarm cues, and measured changes in DNA methylation and CORT levels, both of which are mechanisms that have been implicated in the control of phenotypically plastic responses in tadpoles. To test the idea that DNA methylation drives shifts in GC physiology, we also experimentally manipulated methylation levels with the drug zebularine. We found differentially methylated regions between control tadpoles and their full-siblings exposed to alarm cues, zebularine or both treatments. However, the effects of these manipulations on methylation patterns were weaker than clutch (e.g. genetic, maternal, etc.) effects. CORT levels were higher in larval cane toads exposed to alarm cues and zebularine. We found little evidence of changes in DNA methylation across the glucocorticoid receptor gene (NR3C1) promoter region in response to alarm cue or zebularine exposure. In both alarm cue and zebularine-exposed individuals, we found differentially methylated DNA in the suppressor of cytokine signaling 3 gene (SOCS3), which may be involved in predator avoidance behavior. In total, our data reveal that alarm cues have significant impacts on tadpole physiology, but show only weak links between DNA methylation and CORT levels. We also identify genes containing differentially methylated regions in tadpoles exposed to alarm cues and zebularine, particularly in range-edge populations, that warrant further investigation.

Methods
Please see associated manuscript for full methods. Briefly, cane toad tadpoles were exposed to zebularine, alarm cues, both or neither (controls). 18-day old tadpoles were euthanased and DNA was extracted from whole tadpoles. Reduced-representation bisulfite sequencing was conducted and analysed following the methods in the main manuscript. We removed zero data from CpG reports to reduce file size and uploaded these data here.

Usage Notes
This dataset is comprised of CpG reports (zero data removed) for the associated manuscript. Columns represent: sequence name, position of cytosine, strand, cytosine count, methylated cytosine count, type of methylation, methylation context.

发育环境可对动物表型产生强烈调控作用。近年来，表观遗传修饰（epigenetic modifications）已被证实是介导动物对环境变化产生发育可塑性响应的关键机制之一。例如，激素受体基因启动子区域的DNA甲基化谱式，可影响其基因表达与激素释放模式。在多个生物分类群中，表观遗传改变均与糖皮质激素（glucocorticoid, GC）生理功能的变化密切相关。糖皮质激素是一类代谢激素，可调控生长发育、生活史阶段转换，进而影响个体适合度。迄今为止，针对野生动物表型性状的表观遗传调控研究仍相对匮乏，在两栖动物类群中更是如此。

本研究以入侵物种蔗蟾（Rhinella marina）的蝌蚪及变态个体为研究对象，探究了捕食威胁暴露与实验性调控DNA甲基化对皮质酮（corticosterone, CORT）水平的影响。我们采集了该物种在澳大利亚分布范围内多个种群的蔗蟾后代。在该物种中，暴露于同种个体损伤释放的化学警报信号，可诱导其发育轨迹发生改变，这被认为是一种降低捕食风险的适应性响应。我们将蝌蚪暴露于此类警报信号，并检测其DNA甲基化与皮质酮水平的变化——这两种机制均被证实参与调控蝌蚪的表型可塑性响应。为验证“DNA甲基化驱动糖皮质激素生理变化”这一假说，我们还使用药物zebularine（西布他林）实验性调控甲基化水平。

我们在对照组蝌蚪与暴露于警报信号、zebularine或二者联合处理的全同胞蝌蚪之间，发现了差异甲基化区域（differentially methylated regions）。然而，这些处理对甲基化谱式的影响弱于同窝效应（如遗传效应、母体效应等）。暴露于警报信号与zebularine处理的幼体蔗蟾，其皮质酮水平均有所升高。我们未发现警报信号或zebularine暴露可诱导糖皮质激素受体基因（NR3C1）启动子区域的DNA甲基化发生显著变化。在暴露于警报信号与zebularine的个体中，我们均在细胞因子信号转导抑制因子3基因（SOCS3）中发现了差异甲基化DNA区域，该基因可能参与捕食回避行为调控。

综上，本研究数据表明警报信号可对蝌蚪生理产生显著影响，但DNA甲基化与皮质酮水平之间仅存在较弱的关联。我们还鉴定出了在警报信号与zebularine暴露的蝌蚪中携带差异甲基化区域的基因，尤其是在分布边缘种群中，这些基因值得进一步深入研究。

## 方法
详见关联研究手稿以获取完整实验方法。简要而言，将蔗蟾蝌蚪分为四组：对照组、zebularine处理组、警报信号暴露组、以及二者联合处理组。对18日龄蝌蚪实施安乐死后，提取全蝌蚪组织的DNA，开展简化代表性亚硫酸氢盐测序，分析流程遵循主手稿中的方法。为减小文件体积，我们移除了CpG报告中的零值数据，并将处理后的数据上传至本数据集。

## 使用说明
本数据集包含经零值数据移除处理的CpG报告文件，对应本关联研究手稿。各列含义依次为：序列名称、胞嘧啶位点位置、DNA链方向、胞嘧啶总计数、甲基化胞嘧啶计数、甲基化类型、甲基化序列上下文。