Rapid neural DNA methylation responses to predation stress in Trinidadian guppies

DNA methylation (DNAm) is a well-studied epigenetic mechanism implicated in environmentally induced phenotypes and phenotypic plasticity. However, few studies investigate the time scale of DNAm shifts. Thus, it is uncertain whether DNAm can change on timescales relevant for rapid phenotypic shifts, such as during the expression of short-term behavioural plasticity. DNAm could be especially reactive in the brain, potentially increasing its relevance for behavioural plasticity. Most research investigating neural changes in methylation has been conducted in mammalian systems, on isolated individuals, and using stressors that are less ecologically relevant, reducing their generalizability to other natural systems. We exposed pairs of male and female Trinidadian guppies (Poecilia reticulata) to alarm cue, conspecific skin extract that reliably induces anti-predator behaviour, or a control cue. Whole genome bisulfite sequencing on whole brains at various time points following cue exposure (0.5h, 1h, 4h, 24h, and 72h) allowed us to uncover the timescale of neural DNAm responses. Males and females both showed rapid shifts in DNAm in as little as 0.5 hours. However, males and females differed in the time-course of their responses: both sexes showed a peak in the number of loci showing significant responses at 4 hours but males showed an additional peak at 72 hours. We suggest that this finding could be due to differing longer-term plastic responses between the sexes. This study shows that DNAm can be rapidly induced by an ecologically relevant stressor in fish and suggests that DNA methylation could be involved in short-term behavioural plasticity.

Methods
We exposed guppies to alarm cue or control cue and then measured behavioural responses for 10 minutes (only 5 minutes of data used in paper) before and after cue exposure. Tanks had a back board that visually divided the tank into three equal horizontal sections so that fish position could be recorded as upper, middle, and bottom of tank. All behavioural responses to cue exposure were scored by a single observer that was blind to the treatment using BORIS v7.12.2. Behavioural videos were binned into 60 second segments for scoring. The observer recorded the time spent in each section of the tank, time spent frozen, and time spent foraging. Foraging was defined as active pecking at substrate and ended when the fish was no longer oriented towards the substrate and had not pecked for two seconds. We also recorded the time males spent pursuing females and the time males engaged in sigmoidal mating displays, a courtship display where males curve their bodies into an S-shape. After the assigned time point (0.5h, 1h, 4h, 24h, or 72h), we euthanized fish and removed brains for whole genome bisulfite sequencing.