Animals and housing conditions
The zebra finch (Taeniopygia guttata castanotis) is a well-studied model organism [60] which can be bred and kept in captivity readily and has a short generation time, as juveniles are independent after 35 days and sexually mature after 65 days.
We conducted experiments on animal personality in zebra finches in a project on divergent bi-directional selection lines. The monitoring of the birds included five different personality tests, three of which served as selection parameters for the differing selection lines. The long-term design of the selection line study allowed us to assess repeatability and the development of behavioural syndromes over a longer time scale than is possible in most studies on long-lived organisms.
We chose 52 wild-type zebra finches from the F1-generation, 22 males and 30 females, to study long-term consistency of personality traits. They were bred from 31 different pairs, with between 5 and 14 individuals coming from the same selection line and maximum 3 full-sibs per family. They showed no behavioural trait values at the extreme end of the personality axis in the direction their parents were selected on.
The zebra finches were reared in outdoor aviaries (6 × 2 × 3 m) at Bielefeld University in the different selection line groups, each consisting of five breeding pairs plus their offspring.
After independence from their parents at ca. 40 days of age (median 43 d, min 30 d, max 62 d, IQR 8 d), subadult birds were translocated from their natal aviary groups into indoor mixed-sex tutor groups (cages: 81 × 60 × 50 cm). Tutor groups consisted of seven to ten juveniles with a pair of unrelated, unfamiliar adults. Before sexual maturation, at about 60 days of age (median 60 d, min 47 d, max 81 d, IQR 9 d), birds were then transferred to double cages (82 × 40 × 30 cm) in groups of three to four same-sex individuals. Tutor groups and same-sex cage groups were both arranged so they comprised animals originating from several different selection lines.
All animals were maintained in the same room with auditory and visual contact between cages. At all times, birds had ad libitum access to commercial zebra finch seed mix (Elles, Mischfutter für Exoten, L. Stroetmann Saat, 48163 Münster, Germany) and fresh water. Additionally, a mixture of germinated seeds and egg food (Cédé N.V., 9940 Evergem, Belgium) was provided daily in outdoor aviaries and tutor groups and thrice weekly after the transfer to double cages. The diet was weekly supplemented with fresh greens. In the housing room, birds were kept on a 14:10 light-dark-cycle, additional to the natural light conditions.
Test schedule and description of behavioural tests
Behavioural tests started while birds were still kept in the tutor groups. All individuals were tested in five behavioural tests
(a) twice at the 'subadult' stage
(b) twice shortly after reaching adulthood ('young adult')
(c) twice at approximately one year of age ('mature adult')
leading to a total of six test rounds, with approximately 14 days apart within a given life stage. The interval between life stages was about one month for the transition to adulthood, and seven to ten months for the transition between young adult and mature adult. For all rounds, mean age and intervals between tests are given in table 1.
Experiments were conducted always in the same order: (TI - AG - GA - NO - NE) between 8:30 and 20:00 hours, and we took care that between tests, birds had at least one day break.
Tonic immobility (TI): An empty wire cage (40 × 31.5 × 21 cm) located on a table in a sound proof chamber (to exclude noise disturbances during the test) was used to conduct the tonic immobility test. The bird was placed on its back with wings pressed to its body on a metal holding cradle padded with foam rubber. An experimenter locked the bird in this position for five seconds by gently pressing index and middle fingers on its chest and then retracting the hands. Movements by the observer were minimized to prevent disturbances. Birds were considered successfully immobilized if they stayed in this position for at least five seconds. If a bird was not immobilized at once the procedure was repeated up to a maximum of 10 inductions. The test was terminated after a maximum of 20 minutes immobility (ceiling value of latency = 1200 s). Tests were performed by YW and student assistants Simon Tiersch and Nele Heitland (rounds 1-4) and Ivonne Kienast (rounds 5 and 6). Tonic immobility latencies and number of inductions are considered a measure of fearfulness [61] or boldness in predator contexts [62].
Aggression (AG): The aggression test cage (after [42]) was a small wire cage (40 × 31.5 × 21 cm) with one perch (39 cm). At the far end of the perch, a mirror (15 × 15 cm) was fixed and a food dispenser placed underneath.
The focal individual was transferred to the test cage with the mirror being covered with a piece of cardboard. After an habituation phase of five minutes, the mirror was uncovered and the recording started for five minutes. Birds were tested with the mirror fixed on either the right (= R) or left (= L) side of the cage in a given sequence (R-L-L-R-R-L). During the test, another cage of birds was present in the test room for auditory contact (always the same birds).
The first five seconds after removal of the division were discarded. Then, the frequency of the following interactions was determined within five minutes of video recording:
- Pecking/beak contact: each contact of the bill with the mirror.
- Flying against the mirror: body contact with the mirror while feet are not touching the perch.
- Breast contact: touching mirror with breast, usually after straightening up to full body height.
- Head contact: Usually occurring when sitting quite close to the mirror, it is touched with the head while the feet still remain on the perch.
We used the sum of aggressive interactions with the mirror as measure for aggressive behaviour.
General activity (GA): The general activity test was conducted in the birds' home cage. All birds were removed from the cage prior to testing and relocated to a waiting cage (without visual contact to the focal individual). The food dispenser remained in the home cage during testing. After replacing the focal individual to its home cage and a two minute recovery time, the activities of the bird were video-recorded for ten minutes.
In the video analysis, the first 20 s were discarded (settling period). Then, the number of flights in the cage was determined. A flight was defined as a movement between perches or between perch and cage floor. We also determined the events of visits to all seven possible positions in the cage (four perches, left, middle and right bottom part of the cage) and calculated a position diversity index (PDI, equivalent to the calculation of the Exploration Diversity Index (EDI) as described in [53]). A higher position diversity index means that an animal visited more different places in the cage and thus indicates a higher activity.
Novel object (NO): Directly following the activity test, the divider was placed in the cage and a novel object inserted in the empty cage compartment. The position of the novel object (far left or far right perch) was randomly assigned beforehand. The novel object was fixed to a perch and placed inside the cage, while the focal individual was in the other compartment. The divider was then removed after the recording started, and the experimenter left the room. Behaviour was video-recorded for ten minutes.
This procedure was repeated with the remaining cage mates, while the already tested birds were transferred to the waiting cage.
As novel objects we used wooden blocks, styrofoam balls and chipboard discs of different colours. Details regarding the type of novel object used in each test round are given in part 6 in additional file 1.
Data of the activity test as well as the novel object data were analysed using CowLog [63]. The events and durations of visits to each of the seven positions in the cage were determined, as well as the latency to land on the novel object perch. We used the novel object latency, the frequency to visit the NO perch and the percentage of time spent there as variables for boldness. GA and NO tests were both conducted in the animal housing room.
Novel environment (NE): As a novel environment, we used standard double cages whose interiors were covered with adhesive foil. Otherwise, they were equipped as the usual home cages (sand on floor, four wooden perches, food dish with seed mixture, water bowl). Descriptions of the cage interiors used as novel environments are given in part 6 in additional file 1. Up to four individuals were tested simultaneously (in separate cages); in general all cage mates at a time. The focal individuals were transferred from their home cage into separated deprivation cages (30 × 40 × 40 cm) without food or water about 3.5 h (207 ± 10 min) prior to testing. Each focal individual was then put into a start box (11 × 17 × 11 cm) which was attached on either the left or right side of the experimental cage. A cardboard division between cages ensured that there was no visual contact between focal individuals in the start boxes. In the cages a food dish was placed on the same side as the start box, a water tray in the other compartment. The division between start box and novel environment was lifted and the behaviour video-recorded for one hour. A cage with flock mates was present in the experimental room during testing for auditory contact with the focal individuals.
As explorative behaviour, we determined how many of the seven possible positions in the cage were visited within one hour and we measured the latency to enter the novel environment cage and the latency to visit all seven positions if applicable. If the start box was not left or not all positions were explored, birds received a maximum value of 3601 seconds.
Statistical analysis
All statistical analyses were run in statistical software R [64] within the RStudio environment [65], using the packages lme4, rptR, lattice, psych and psychometric [66–70].
Principal components analysis
For each round per test we ran a principal component analysis (PCA) calculated with correlation matrix to extract the first axis for each personality trait. PCA were performed using the maximum available dataset (max. N = 52, for sample sizes per test and round see part 2, additional file 1). The included behavioural variables and resulting PC loadings are given in part 2 in additional file 1. Because seven data points from six individuals were missing, the final dataset for subsequent analyses was reduced to 46 individuals (n = 276). For general activity (GA) round 1, the derived scores were inverted by multiplication with (-1), so that higher scores represent bolder behaviours in all tests and rounds (GA = more active; NO = bolder towards object; NE = faster exploration; TI = lower latency and more inductions; AG = more aggressive). As the loadings differed between test rounds for aggression (AG) and the derived PC scores were not normally distributed, we instead used the decimal logarithm of the sum of aggressive interactions with the mirror as a score for later analysis, which showed a distribution closer to normality.
Generalized Linear Mixed Models for extraction of best linear unbiased predictors
For each personality axis in each life stage, we ran a univariate Generalized Linear Mixed Model (GLMM) with Gaussian error structure fit with Maximum Likelihood. This was done to extract the best linear unbiased predictors (BLUPs) for behavioural syndrome structure calculation and to check for effects of fixed factors such as sex differences in mean level behaviour.
Models were run in R using the function lmer from the R package lme4 [66]. In the full model we included the three fixed effects sex, test round and selection line and the two-way interaction of sex and test round as well as the random effects batch (to control for possible season effects, as all birds from the F1-generation were transferred from outdoor aviaries to indoor cages in a total of 10 batches between mid-June and mid-December 2012) and individual ID nested within mother ID. We conducted a stepwise deletion of fixed effects if model comparison using a likelihood-ratio analysis between the full and reduced model was non-significant. The random effects as well as test round and selection line as a fixed effect to control for were always retained in the final model.
Because Fligner-Kileen tests indicated heterogeneity of variance in NO scores for the interaction of the fixed factors sex and round, we weighted this effect (by the ratio between the variances of the factor levels) to improve the fit of the model. NO models were calculated with and without weights and the model results for both are presented in parts 4 and 5 in additional file 1. Although likelihood-ratio tests showed that the weights significantly improved the fit, we decided to use the unweighted models for subsequent analyses after visual inspection of the residuals plotted against the fitted values.
Because of ceiling or floor effects of the data distribution, we created subsets of data containing only valid data points (without zero- or maximum-inflated data) as follows: TI was maximum-inflated as individuals that did not enter the immobile state after ten inductions received a latency of one second (leading to high PC scores). The reduced dataset included data points with latencies larger than one. AG, NO, NE and GA all were zero-inflated, as some individuals did not interact with the mirror (interactions = 0), not approach the novel object (events on perch = 0), not explore all seven positions the novel environment (received a maximum latency of 3601 s resulting in lowest PC scores) or not move in the home cage (flights = 0) at all. In the respective reduced datasets these data points were removed. Each model (one per life stage and personality trait) was run for the inflated dataset and reduced dataset separately and the model outputs are given in parts 4 and 5 in additional file 1. Although results of the reduced dataset deviated from results of the inflated dataset in some cases, we continued calculations with the complete (inflated) dataset, as using the reduced dataset would have decreased our overall sample size (see part 5 additional file 1 for reduced dataset sample sizes) immensely. We discuss these cases carefully.
Repeatabilities ('differential consistency')
Repeatability is calculated as the ratio of two variances, the variance within groups and the variance among groups as follows:
We used the R package rptR [67] to calculate repeatabilities within and between life stages. All response variables (PC axes and logarithmized sum of aggressive interactions) were approximately normally distributed and thus analysed with Gaussian fit and REML estimation. We report repeatability estimates with their significance levels and 95% confidence intervals, calculated with 1000 permutations and 1000 bootstrappings. We calculated repeatabilities within each life stage (comprising two subsequent test rounds), for the complete dataset including all six test rounds (referred to as 'across life stages') and also including only test rounds 1 and 6, to get an estimate across life stages using only two data points (referred to as 'long interval'). We compared the overlap of confidence intervals, showing non-significance at the 5% threshold if applicable [71]. As the mixed models had indicated a significant effect of sex in some cases, we calculated repeatabilities for males and females separately as well.
Behavioural syndrome structure ('structural consistency')
We extracted the best linear unbiased predictors (BLUPs) of the GLMMs. BLUPs represent scores that are "controlling for" the effects included in the model. Behavioural syndrome structure at the three life stages was assessed by computing Spearman rank correlation matrices with Holm's correction using the life stage mean of the fitted values of the minimum adequate models of the five personality traits for each life stage (R package psych [69]). The lower and upper bounds of 95% confidence intervals were estimated with the R package psychometric [70]. We also calculated behavioural syndrome structure separately for males and females.