DOI: 10.5061/dryad.6t1g1jwwh
title: Do epigenetic changes drive corticosterone responses to alarm cues in larvae of an invasive amphibian?

description: 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.
description: 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.
description: 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.