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.

发育环境对动物表型产生显著影响。近期，表观遗传修饰作为一种调节发育可塑性以应对环境变异性响应的机制已逐渐凸显。例如，激素受体基因启动子区域的DNA甲基化模式可以影响其表达和激素释放模式。在分类群中，表观遗传的改变已被与糖皮质激素（GC）生理学的变化联系起来。GC是影响生长、发育、生命史阶段之间的转变以及因此适应性的一种代谢激素。迄今为止，关于表观遗传对野生动物表型性状影响的研究相对较少，尤其是在两栖动物中。在本研究中，我们考察了捕食威胁暴露和实验性DNA甲基化操纵对入侵型甘蔗蟾蜍（Rhinella marina）蝌蚪和变态幼虫的皮质酮（CORT）水平的影响。我们包括了来自该物种澳大利亚分布范围内不同群体的蟾蜍后代。在这些动物中，暴露于受伤同种生物的化学线索会引发发育轨迹的改变，这可能是作为一种减少捕食脆弱性的适应性响应。我们将蝌蚪暴露于这些警报信号中，并测量了DNA甲基化和CORT水平的变化，这两者都是与蝌蚪表型可塑性响应控制有关的机制。为了检验DNA甲基化驱动GC生理学转变的观点，我们还使用药物zebularine实验性地操纵了甲基化水平。我们发现，与对照组蝌蚪及其暴露于警报信号、zebularine或两种处理的同胞相比，存在不同的甲基化区域。然而，这些操纵对甲基化模式的影响弱于窝次（例如遗传的、母性的等）的影响。暴露于警报信号和zebularine的幼虫蟾蜍的CORT水平较高。我们发现，在响应警报信号或zebularine暴露时，糖皮质激素受体基因（NR3C1）启动子区域中的DNA甲基化没有显著变化。在暴露于警报信号和zebularine的个体中，我们发现了抑制细胞因子信号传导3基因（SOCS3）中的差异甲基化DNA，这可能涉及捕食者回避行为。总的来说，我们的数据揭示了警报信号对蝌蚪生理学有显著影响，但仅显示出DNA甲基化和CORT水平之间的弱联系。我们还确定了在暴露于警报信号和zebularine的蝌蚪中存在差异甲基化区域的基因，尤其是在分布边缘的种群中，这些基因值得进一步研究。