DOI: 10.5061/dryad.79cnp5j07
title: Personality traits and Pan I locus data of Atlantic cod juveniles

description: Animals show among-individual variation in behaviours, including migration
 behaviours, which are often repeatable across time periods and contexts,
 commonly termed “personality”. These behaviours can be correlated, forming
 a behavioural syndrome. In this study, we assessed the repeatability and
 correlation of different behavioural traits i.e., boldness, exploration,
 and sociality and the link to feeding migration patterns in Atlantic cod
 juveniles. To do so, we collected repeated measurements within two
 short-term (three days) and two long-term (two months) intervals of these
 personality traits and genotypes of the Pan I locus, which is correlated
 to feeding migration patterns in this species. We found high
 repeatabilities for exploration behaviour in the short- and long-term
 intervals, and a trend for the relationship between exploration and the
 Pan I locus. Boldness and sociality were only repeatable in the second
 short-term interval indicating a possible development of stability over
 time and did not show a relation with the Pan I locus. We found no
 indication of behavioural syndromes among the studied traits. We were
 unable to identify the existence of a migration syndrome for the frontal
 genotype which is the reason that the link between personality and
 migration remains inconclusive, but we demonstrated a possible link
 between exploration and the Pan I genotype. This supports the need for
 further research that should focus on the effect of exploration tendency
 and other personality traits on cod movement, including the migratory
 (frontal) ecotype to develop management strategies based on behavioural
 units, rather than treating the population as a single homogeneous stock.
description: The methods followed the same protocol as
 described in Beukeboom et al. 2022; 102 age 0+ cod juveniles (weight range
 = 0.75-4.39 g and mean = 1.87 g; standard length range = 3.83-7.55 cm and
 mean = 5.72 cm) were beach seined from the 3<sup>rd</sup> to
 12<sup>th</sup> of October 2019 in three different fjords
 around the Westfjords of Iceland to obtain genetical variation. They were
 transported to a laboratory in Bolungarvik, Iceland and housed
 individually in 9.5-litre tanks (~29x21x19cm, water level 16 cm,
 Aquaneering Inc.). The recirculating system contained freshwater mixed
 with marine salt to achieve a natural salinity of 30 ± 2‰, a temperature
 of 11 ± 1°C (November) and 10 ± 1°C (December-June), ammonia levels of
 &lt;0.5 ppm, oxygen levels of 10.4 ± 0.1 mg/L and a constant
 photoperiod of 12 hr:12 hr (7 AM-7 PM GMT). The water circulated through
 the Aquaneering system, passing through all the tanks, a biofilter, sieves
 (mesh size 25 µm), and a UV light for sterilization. Every tank had a grey
 PVC pipe to provide shelter to the fish. Fish were fed twice a day
 alternating defrosted shrimp and bloodworms daily ad libitum. On the
 experimental days, feeding took place after the experiment to avoid any
 differences in feeding motivation. After the first trial of the open field
 experiments every month, the fish were measured for weight and standard
 length. In November, a set of trial experiments was performed which are
 presented in Beukeboom et al., 2022. <em>Behavioural
 tests</em> Of the initial 102 caught individuals, 43 were
 included in the analysis (see Results). Each individual underwent a cycle
 of a shelter test (ST, boldness), open field test (OFT, exploration),
 novel object test (NOT, boldness), and mirror test (MT, sociality) during
 four trials: January 19-21 (Trial A), and January 22-24 (Trial B), March
 15-17 (Trial C) and March 18-20 (Trial D) (figure 1). All fish started
 with the ST, followed by the OFT. Consecutively, 20 fish were subjected to
 first the NOT and then the MT, while the rest (N=23) received the MT first
 and then the NOT to take any influence of the NOT or MT test order into
 account (figure 1). In addition, all fish were tested in the same overall
 order to standardize the intervals between the tests for each fish. Data
 in the ST was collected manually, while OFT, NOT, and MT videos were
 analyzed with video-tracking software (Ethovision, Noldus, v. 15.0). A
 smoothing parameter of 0.1 was set in the video-tracking software, which
 set the sample points to the previous location until the distance moved
 was more than 0.1 cm which removed any noise of moving pixels due to
 imperfect light conditions, as this was found to be appropriate for the
 setup we used (Beukeboom
 et al., 2022). The water
 temperature of the experimental tank ranged from
 9.2-11.5°C. <em>Shelter test
 (ST)</em> The shelter test is commonly used as a measure
 of boldness (see for an overview Toms et al. (2010)), where bolder fish have a shorter exit time.
 The fish was gently captured with a dipnet out of its home tank and placed
 in a shelter (26L; 40x40x40 cm, water level 16 cm) and after five minutes
 of acclimation, the door was lifted. The fish was given five minutes to
 leave the shelter, and latency to exit was recorded. If it did not leave
 voluntarily, it was gently forced out using a dip net into the arena (51L;
 80x40x40cm, water level 16 cm) and given a maximum score of 300 seconds.
 Unfortunately, in 95 out of 158 ST trials (60.1%), fish did not leave the
 shelter voluntarily. This
 was most likely due to the test duration being too short, which resulted
 in insufficient variation in this behaviour. This original boldness
 measurement was therefore excluded from further analysis, but a binary
 factor (left shelter y/n) was included in our models to account for any
 influence of the fish being pushed out of the shelter rather than leaving
 voluntarily. <em>Open field test
 (OFT)</em> In absence of a predator, juvenile cod have been
 shown to move freely across an open space (Nordeide
 & Svåsand, 1990),
 which most likely serves the function of information gathering (Hughes,
 1997). Therefore, the OFT was used as a proxy for exploration, where it is
 expected that explorative fish swim greater distances and cover a bigger
 area than non-explorative individuals. The test was carried out as
 follows: as soon as the fish entered the arena during the ST (51L;
 80x40x40cm, water level 16 cm, figure 1), the shelter was closed and the
 fish was video recorded for five minutes. The total distance travelled and
 the total area covered (i.e. traversed, calculated by using the total
 unique x/y coordinates rounded to the nearest integer) were extracted.
 Area covered and distance travelled (both log-transformed) were highly
 correlated (PEARSON: r = 0.97; CI 95% [0.96 to 0.98]; P &lt; 0.001).
 Because the area covered is statistically ceiled (i.e., there is a maximum
 number of unique X/Y coordinates available), the total distance travelled
 was used in further analysis, to catch the maximum variation
 possible. <em>Novel object test
 (NOT)</em> The novel object test was used as a measure of
 boldness, where shy individuals are expected to flee, retreat, or are
 inactive, while bold individuals are expected to become more active and
 approach the novel object (Toms et al., 2010). The NOT was carried out
 after the OFT (N=20) or after the MT (N=23). When present, the mirror was
 removed, and a novel object was dropped in the middle of the arena (Figure
 1). In January, this was a red, tin can (⌀ 6 cm,
 113 cm<sup>2</sup>) and in March this was a blue plastic
 pipette tip rack (13x10 cm; 117 cm<sup>2</sup>) to reduce
 habituation to the object. Five minutes were video recorded and the mean
 distance to the object was extracted as a proxy for
 boldness. <em>Mirror test
 (MT)</em> Cod juveniles can show plastic social behaviour,
 from shoaling to aggression (Meager et al., 2018). The mirror test can be
 used to measure both sociality and aggression, depending on the species
 and developmental phase. While Villegas-Ríos et al.
 (2018) used the mirror test as a measure of aggression in adult cod, the
 life stage where the risk of being predated is minimal,
 we assume that the mirror
 test in this study on juveniles elicited social behaviour instead for two
 reasons. Firstly, the arena was open with no shelter to hide, which has
 been shown to elicit shoaling behaviour in juvenile cod (Laurel et al.,
 2004). Secondly, aggressive/submissive behaviour often occurs when
 opponents differ in size (McCormick & Weaver, 2012; Sverdrup et
 al., 2011), and as the mirror just reflects the fish itself, these size
 differences are non-existent. The MT was carried out after the OFT (N=20)
 or after the NOT (N=23). When present, the object was removed, and a
 mirror was placed opposite the shelter door (Figure 1). Five minutes were
 video recorded, and the total time spent in a 10 cm zone in front of the
 mirror was extracted as a proxy of sociality. After the experiment, visual
 inspection of the videos for aggressive and/or social behaviours confirmed
 our decision; social behaviours, such as repeated approaches to the mirror
 at cruising speed and “hanging around” the mirror were highly represented,
 and aggressive behaviours such as accelerations towards the mirror, biting
 or c-shaping were absent (Sverdrup et al., 2011). 
 <em>Genotyping</em>
 In June 2020, at the end of the overall project,
 the fish were euthanized with phenoxyethanol (1.6mg/L) and fin clips were
 taken to assign the fish to either coastal (<em>Pan
 </em>I<sup>AA</sup>), frontal (<em>Pan
 </em>I<sup>BB</sup>), or heterozygote (<em>Pan
 </em>I<sup>AB</sup>) using PCR analyses as described in
 Pampoulie et al. (2006). <em>Statistics</em>
 All data were analyzed using the same method as
 in Beukeboom et al.
 (2022) using R (v. 4.1.2;
 R Core Team, 2021). We fit multivariate linear mixed models to estimate
 the repeatabilities of and the correlations between the total distance
 travelled (OFT), mean distance to object (NOT), and time spent in mirror
 zone (MT). The models were fit using the Bayesian software Stan (Carpenter
 et al., 2017) run via the ‘<em>brms</em>’ package
 (Bürkner,
 2017). We ran four
 separate models containing four different subsets of the data: short-term
 (∆ three days) for January (trials 1 and 2; SJ) and March (trials 3 and 4;
 SM) and long-term (∆ two months) for the first trials (trial 1 and 3; LA)
 and the second trials (trial 2 and 4; LB) of each month. The models
 simultaneously regressed each dependent variable (i.e., OFT, NOT and MT
 estimates) against a set of fixed and random effects while also
 quantifying the covariance between the dependent variables. The fixed
 effects of weight (g), standard length (cm), Fulton’s condition factor
 (<em>K</em>= <em>Weight </em>/<em>Length</em><sup>3</sup> × 100), and specific growth rate (<em>SGR=</em> ∆ ln(weight) *100 / ∆ day) that could influence the personality estimates were evaluated for collinearity. Pearson correlations revealed that all measurements were substantially correlated (Figure A1) and therefore only SGR was included in the analysis. The three personality estimates and SGR were scaled using z-scoring (subtracted the mean and divided by the standard deviation) separately for the subset of data used in each model. The full version of each model was fit with the scaled personality measurement as response variables (i.e. total distance travelled, mean distance to object, and total time in mirror zone), the four fixed effects of genotype, SGR (since the previous month for short-term, January-March for long-term), trial and shelter leave (y/n). We include a binary covariate to indicate the order in which fish were tested for the NOT and MT. The random-effects structure included individual fish identity (ID) as a grouping variable, allowing us to calculate the repeatability of the personality estimates as the ratio of the among-individual variance and the sum of the among-individual and residual-level variances (Johnson & Koch, 2011). Moreover, the model estimated covariances between the personality estimates at both the ID and residual levels. The among-individual covariance quantified the degree to which the personality estimates were correlated among individuals across multiple trials (i.e. behavioural syndrome), while the residual level covariance quantified the degree to which the personality estimates were correlated among observations independently of the identity of individuals. The model was run for 4000 iterations (2000 for warmup and 2000 for sampling), four chains, an adapted delta of 0.9 and all other parameters set to their defaults. Convergence was assessed using the standard diagnostics provided by Stan (Bürkner, 2017; Carpenter et al., 2017), including the potential scale reduction factor ( ), effective sample size, and visual inspection of trace plots and histograms for each model parameter. We used medians for point estimates and quantiles with 95% coverage for uncertainty intervals (UI<sub>95%</sub>).