DOI: 10.5061/dryad.x0k6djhw6
title: Rapid neural DNA methylation responses to predation stress in Trinidadian guppies

description: 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.
description: 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.
description: # Rapid neural DNA methylation responses to predation stress in
 Trinidadian guppies
 [https://doi.org/10.5061/dryad.x0k6djhw6](https://doi.org/10.5061/dryad.x0k6djhw6) ## Description of the data and file structure **Rapid neural DNA methylation responses to predation stress in Trinidadian guppies** ### DOI: [http://doi.org/10.1111/mec.17774](http://doi.org/10.1111/mec.17774). ### Experiment Summary: Adult guppies were exposed to either alarm cue or control cue one time. Behaviour was measured for 5 minute before and after cue exposure. At set time points following cue exposure (0.5h, 1h, 4h, 24h, 72h), brains were dissected out for whole genome bisulfite sequencing. ### File structure: Raw and clean behavioural and tank info in data files (raw and clean folders) are provided. Sequencing data are stored on SRA. Analysis scripts are provided in separate folders for each analysis type (01_behavioural_analysis and 02_methylation_analysis). Within each folder, each script should be run in order of numbering.  All variables and abbreviations defined in metadata. Missing data = NA.  ### Files and variables #### File: data.zip **Description:** zip file containing all data #### File: all\_cue\_data.csv **Description:** Cleaned full behavioural responses dataset (10 minutes of behaviour before and after cue exposure) ##### Variables * obs_id: observation ID * sex: sex (female/male) * length_obs_s: length of time of observation  * sig_s: length of time engaging in sigmoidal displays (seconds) - males only * sig_nb: number of sigmoidal displays - males only * chasing_s: length of time spent chasing the female (seconds) - males only * feeding_s: length of time spent feeding (seconds) * frozen_s: length of time spent frozen (seconds) * Q1_s: length of time in quadrant 1 (seconds) * Q2_s: length of time in quadrant 2 (seconds) * Q3_s: length of time in quadrant 3 (seconds) * Q4_s: length of time in quadrant 4 (seconds) * Q5_s: length of time in quadrant 5 (seconds) * Q6_s: length of time in quadrant 6 (seconds) * dash_nb: Number of dashes  * bin: which time bin observation is a part of  * tank: tank ID number * time: before or after cue exposre * fish_ID: fish ID  * cue: which cue tank was exposed to (alarm or control) #### File: clean\_cue\_fivemin.csv **Description:** Cleaned five minute behavioural responses dataset (5 minutes of behaviour before and after cue exposure) ##### Variables * fish_ID: fish ID * sex: male/female * time: before/after cue exposure * cue: alarm cue/control cue * tank: tank ID * length_obs_s: length of time of observation (seconds) * sig_s:  length of time spent engaging in sigmoidal display (seconds) - males only * sig_nb: number of sigmoidal displays - males only * chasing_s: length of time spent chasing female (seconds) - males only * feeding_s: length of time spent feeding (seconds) * frozen_s: length of time spent frozen (seconds * Q1_s: length of time in quadrant 1 (seconds) * Q2_s: length of time in quadrant 2 (seconds) * Q3_s: length of time in quadrant 3 (seconds) * Q4_s: length of time in quadrant 4 (seconds) * Q5_s: length of time in quadrant 5 (seconds) * Q6_s: length of time in quadrant 6 (seconds) * dash_nb: number of dashes #### File: cueresponse.csv **Description:** raw cue response data ##### Variables * obs_id: Observation ID * length_obs_s: length of obervation (seconds) * sex: male/female * sig_s: length of time spent engaging in sigmoidal displays - male only (seconds) * sig_nb: number of sigmoidal displays - male only  * chasing_s: length of time spent chasing the female - male only (seconds) * feeding_s: length of time spent feeding (seconds) * frozen_s: length of time spent frozen (seconds) * Q1_s: length of time in quadrant 1 (seconds) * Q2_s: length of time in quadrant 2 (seconds) * Q3_s: length of time in quadrant 3 (seconds) * Q4_s: length of time in quadrant 4 (seconds) * Q5_s: length of time in quadrant 5 (seconds) * Q6_s: length of time in quadrant 6 (seconds) * dash_nb: Number of dashes #### File: cuetanks.csv **Description:** raw tank and cue exposure data ##### Variables * obs_id: Observation ID * tank: tank ID * time: before/after cue exposure * sex: male/female * fish_ID: fish ID * cue: alarm cue/control ## Code/software All required packages and functions are listed at the top of each script.  **Setup** 1\. Download project repository from Github or Dryad 2\. Download data folders: * Behavioural data: from Github or Dryad * Sequencing data: from SRA (Short-term: SubmissionID: SUB15053745, BioProject ID: PRJNA1220975) **Code Running** All code for analysis is provided in the github repository or in scripts.zip file on Dryad. Each folder contains all scripts for a specific type of analysis (01_behavioural_analysis and 02_methylation_analysis) and the files within the folder should be run in order of numbering. Files with a and b lettering represent code paired with its slurm submission file. Note that any code with this pairing was written to be run on Compute Canada HPC system and may need to be adapted to your needs if you do not have access. All required software is commented at top of each script. Uncomment and run install lines if required. Analysis was originally run on R v4.3.2. The nf-core/methylseq pipeline v1.6.1 was used to process sequencing reads. Other details on versions of software used can be found in manuscript. ## Access information Other publicly accessible locations of the data: * SRA: SUB15053745, BioProject ID: PRJNA1220975