DOI: 10.5061/dryad.ncjsxkt5x
title: Parental predator exposure affects offspring boldness and laterality in the stickleback

description: Parental influences on offspring phenotype occurring through pathways
 other than via inherited DNA sequences are known as parental effects.
 Parental effects profoundly influence offspring behaviour, including
 behaviour laterality and personality, two traits that are widespread and
 of fundamental importance in the animal kingdom with clear fitness
 consequences. However, the impact of parental effects on the interaction
 between behavioural laterality and personality within the same species has
 not been previously explored. If such a link exists, it would deepen our
 understanding of personality traits, extending them to brain laterality
 and its underlying neurobiology. In addition, if both traits are causally
 linked, it may constrain evolution as changing one of these traits would
 affect the other.  The aim of this study was to examine whether
 offspring personality traits and behavioural laterality are related and
 can simultaneously be influenced by parental effects, suggesting a common
 underlying mechanism. Here we exposed parents of an egg laying species,
 the stickleback (Gasterosteus aculeatus), to predator cue or not and
 examined the impact of this exposure on the two behavioural traits of
 their offspring. Shortly after laying, clutches were split: half were
 reared without predator cues for 12 weeks before behavioural testing, and
 the other half were used for cortisol analysis. We found that
 both parents and offspring from predator-exposed parents were bolder, with
 the offspring more likely to show lateralized behaviour, and were smaller
 than offspring from parents that were not exposed to predation. The egg
 cortisol levels were too low to be detected by LC/MS-MS. To our knowledge,
 these results are the first to indicate that parental effects under
 varying predation conditions can influence laterality, personality, and
 growth of offspring within the same individuals, though further evaluation
 and experiments are needed to determine the role of maternal cortisol.
description: We observed the response of the adult fish  to a predator model
 under controlled lab conditions. We tested 26 fish from the predation
 treatment and 24 from the control. These fish were moved from their
 home-pond a day before the test and placed into one of four aquaria
 (80x35x40 cm) to allow them to acclimatize.  A focal
 fish was selected from the housing aquarium and gently placed in a novel
 experimental tank (40x30x40 cm). The experimental tank was furnished with
 a tube as shelter (5x10 cm), gravel on the floor and a tethered perch
 model of which the movement was remotely controlled by hand (Fig.2). To
 further simulate the presence of predators in the environment, we added
 Perch odour (5ml) to the test aquarium. The experimental tank was
 positioned in an illuminated wooden box to avoid external disturbance. The
 duration of the experiment was 10 mins and the behaviour of the subjects
 was recorded using an overhead camera (Raspberry Pi NoIR Camera Board V2 –
 8MP, Raspberry Pi Foundation, UK). We replaced the water in the
 experimental tank with freshwater and new predator odour each time after 4
 fish were tested. Behaviour was scored using the
 software BORIS 7.10.2 (Friard & Gamba, 2016). We recorded the time
 spent in the safe zone (a third of the arena nearest to the shelter) and
 the predator zone (the third of the tank nearest the predator
 model). <em>Offspring
 boldness</em> Offspring were tested at 4 months
 old with a predator inspection test twice, three weeks apart to assess
 consistency in boldness following (Ramesh et al., 2023). The sample size
 tested was 100 fish from the control group and 94 from the treatment
 group. Two tanks placed side-by-side, one (40x20x30 cm) housed a live
 perch (<em>Perca fluviatilis</em>) and the other (25x20x20 cm)
 the focal fish and a shelter . The tanks were positioned in an illuminated
 wooden box to avoid external disturbance. The duration of the experiment
 was 10 mins. We scored the time spent inside the shelter  and outside the
 shelter as proxies for shyness and boldness respectively.
 <em>Offspring Shoaling (anti-predator
 behaviour)</em> To measure shoaling behaviour, 5
 individuals from the same home tank were placed in a novel arena (33x18x33
 cm) which was surrounded with black plastic. The test lasted 10 minutes.
 Every 10s a snapshot of the video was taken and the xy coordinates of each
 fish’s head was identified. The area of the polygon generated by
 coordinates was calculated using ImageJ 13.1 (Schneider et al., 2012) as a
 measure of shoal cohesion. We averaged the area generated by the shoal at
 each snapshot over the total observation time (Bibost & Brown,
 2013; Bisazza & Dadda, 2005).
 <em>Offspring Laterality</em> We
 employed a rotational mirror test to score laterality in a social context
 (<em>sensu</em> Sovrano et al., 1999). Fish were placed in a
 circular glass tank (diameter 19 cm, water depth 4 cm) where the wall was
 covered with mirror foil to create a continuous mirror for the subject to
 swim alongside their reflection . Subjects were gently placed in the
 arena, and we recorded which eye the individual used to observe their
 mirror image every 5 seconds for 10 mins. When the fish was perpendicular
 to the mirror it was scored as non-lateralized. If the fish was not
 looking at the mirror as indicated with B in figure 5, it was not included
 in the lateralized formula. When the fish was looking at the mirror with
 one eye (&lt;90° to the mirror) we noted if it was using the right or
 left eye and if the fish was &gt; 90° it was excluded from the data
 set (Sovrano et al., 1999).We then calculated the total number of times
 each fish watched the mirror with their left, right or both eyes. The
 sample size tested was 100 fish from the control group and 94 from the
 treatment group.
description: # Parental predator exposure affects offspring boldness and laterality in
 the stickleback
 [https://doi.org/10.5061/dryad.ncjsxkt5x](https://doi.org/10.5061/dryad.ncjsxkt5x) ## Description of the data and file structure Non-parametric tests were performed using SPSS 26.0. We tested the personality and laterality data for normality using the Shapiro–Wilk test and because the data were not normally distributed, we analysed the data using non-parametric procedures. We used the Mann-Whitney U test (MWU) to check for differences in personality traits (shyness- time spent in the safe zone) between the treatments in both the parental and the offspring generations and length in the latter. Boldness Test Parents The Excel file used for these analyses is named “parentaleffects,” and the specific sheet is titled “adults.” The legend is as follows: Files and variables File: lateralitypersonality.csv Description: Variables ID:ID of the fish Group:Control or treatment Tank:Number of the home tank of the fish Tag:The color of the tag a rep1:time in the predator zone b rep1:time in the middle zone c rep1:time in the shelter ab:sum of a plus b total time rep1:total time of the test %a :percentage of time a %b :percentage of time b %c :percentage of time c ab%:percentage of time a plus b leftrightnonlateralized:left score of laterality R:right score of laterality N:non lateralized score LAT INDEX:laterality index ABSolute laterality:absolute laterality n,l,r category:category of laterality if left,right or non lateralized LAT 0,1: category of lateralized or not frequency prop l,n,r:frequency of the right, left or not lateralized in the population frequency prop l.n:frequency of lateralized or not in the population File: parentaleffects.xlsx The Excel file used for these analyses is named “parentaleffects,” and the specific sheet is titled “adults.” The legend is as follows: Id: Identification number of the fish Group: Control or treatment TIME IN A: Time spent in the safe zone TIME IN B: Time spent in the middle zone TIME IN C: Time spent in the predator zone Still in the Excel file “parentaleffects,” the sheet named “offspring length” includes the following legend: * Group: Control or treatment * Tested: Whether it was tested in the other experiments or not * Tagged: Whether it was tagged or not * Tank: The tank it was in * Tag: The colour of the tag, for example, GG, indicates "Green Green" in the same spot underneath the dorsal fin. Alternatively, RPLG indicates "Right Pink" (right side, still underneath the dorsal fin) ,"Left Green" (left side, still underneath the dorsal fin),NT (not tagged), "RG "(Right side pink, still underneath the dorsal fin), "RP" (right side pink,still underneath the dorsal fin), "GG "( left side green, right side green) , "LP" (Left side pink). This pattern continues with different colors, creating an ad hoc system. * M/F: The sex of the offspring * Length: Length in millimeters (mm) Offspring Boldness Test Still in the Excel file “parentaleffects,” the sheet named “offspring boldness” includes the following legend: * Group: Control or treatment * Tank: Tank the fish was in * Tag: Color of the tag * arep1: Time spent near the predator during the first repetition of the test * c rep1: Time spent in the shelter during the first repetition of the test * arep2: Time spent near the predator during the second repetition of the test * c rep2: Time spent in the shelter during the second repetition of the test Offspring Shoaling Behaviour Still in the Excel file “parentaleffects,” the sheet named “offspring schooling” includes the following legend: * Group: Control or treatment * Tank: Number of the home tank of the fish * AVG Rep1: Average area in test 1 * AVG Rep2: Average area in test 2 To analyse the data, we followed the methods used by (Dadda et al., 2015) and categorised right and left lateralised respectively when the index was ≥ 0.75 (right) or ≤ 0.75 (left). Otherwise, the fish were classified as non-lateralized (NL). Because of this categorization, we conducted a two sample Z proportion test to compare the number of lateralised individuals between control and treatment groups. Because the data were highly skewed we analysed the relationship between personality (boldness) and laterality (categorized in three levels: Right Lateralized, Left Lateralized and Non-Lateralized) with a multinomial logistic regression (MLR). For the estimation we used the R package VGAM (Yee, 2010) The csv file named” lateralitypersonality” has the data with categorized individuals, so right, left and non lateralized, then data regarding their personality(boldness). ## Code/software Non-parametric tests were performed using SPSS 26.0. For the estimation of the multinomial logistic regression we used the R package VGAM (Yee, 2010).