Developmental behavioural plasticity and DNA methylation patterns in response to predation stress in Trinidadian guppies

Early-life experiences can predict the environments experienced later in life, giving individuals an opportunity to develop adaptive behaviour appropriate to a likely future environment. Epigenetic mechanisms such as DNA methylation (DNAm) have been implicated in developmental behavioural plasticity, however, studies investigating this possibility are limited in taxonomic breadth and ecological relevance. We investigated the impact of early-life exposure to predation stress on behaviour and DNAm in the brains of Trinidadian guppies (Poecilia reticulata). We exposed guppies throughout development to either alarm cue (conspecific skin extract), inducing predation stress, or a control cue (water) for eight weeks and then raised them to adulthood under identical conditions. We then conducted two behavioural assays, an open-field and a grouping test, before performing whole-genome bisulfite sequencing on whole brains. Guppies exposed to alarm cue during development exhibited increased grouping (shoaling) in adulthood compared to those exposed to the control treatment, but there were no detectable impacts on activity, boldness, or exploratory behaviour. We also identified stable shifts in brain DNAm in response to developmental alarm cue exposure in genes involved in behavioural regulation. Some differentially methylated sites were significantly associated with shoaling propensity in both males and females. Additionally, males and females differed in the magnitude of DNAm responses and the genes impacted, suggesting distinct roles for DNAm between the sexes. This study shows how early-life predation stress can induce behavioural changes in adulthood and that shifts in neural DNAm could be an underlying mechanism responsible for these changes.

Methods
Juvenile guppies were exposed to either alarm cue or control cue throughout development three times a week for eight weeks. After a 22-week period without exposure to cue guppies went through behavioural assays.

Behavioural assays were carried out between 9:00 and 17:00 from May to September 2021. Fish were not fed on the day of behavioural assays. Arena tanks were 20 L rectangular glass tanks with the sides covered in white corrugated plastic sheets to prevent reflections. We filled tanks with fresh conditioned and heated (25 +/- 1oC) water to 6 cm of depth and loosely scattered light-coloured gravel along the bottom. For each assay, fish were allowed to habituate in the arena tank for three minutes in a transparent cylinder (diameter = 6 cm) placed at the center of the tank. We then slowly lifted the cylinder to release the fish and begin the assay. The experimenter hid behind a barrier for the duration of the assay. The assays lasted for five minutes and were recorded using a 1080P HD Model N5 webcam (HDZIYU, Shenzhen, China) positioned 60 cm above the tank. In the modified open field test, a 10 cm x 10 cm artificial lawn aquarium plant that fish could hide in was placed in one corner of the tank (Supplemental Figure 1). For five minutes, the fish was allowed to explore the tank or hide in the plant shelter. We used EthoVision XT v11.5 to quantify distance travelled (cm), time spent in the shelter (s), time spent in outer edge of tank (within the outer squares) (s), time spent moving (s), and time spent frozen (s). The last two measurements were only recorded while the fish was not in the shelter. Additionally, a virtual 4 x 8 grid was overlayed onto the arena video and we extracted the amount of time a fish spent within each unique square. A fish had to spend at least three seconds within a square for it to count as “explored”. ”. Immediately afterwards, we ran the shoaling test. Two identical glass cylinders with a 9 cm diameter were placed on each side of the tank - one empty, and the other containing a shoal of four, unfamiliar adult females from the Paria population. The fish was then allowed to move around the tank monitored for five minutes. The side of the tank that contained the shoal container was alternated between every assay to control for any effect of tank side. One observer blind to cue treatment used BORIS v7.12.2 to record time spent within four body lengths with each container (s). Data was cleaned to ensure labels match across datasheets and to add on data regarding time of behavioural assay.

Immediately following behavioural assays, we euthanized fish by immersion in ice water. Within three minutes, we measured fish mass and length and dissected out brains for whole genome bisulfite sequencing (data stored on SRA).