Fig 1.
Housing and behavioural testing.
A) Zebrafish (N = 100) were housed in pairs (1 female:1 male) and individually tested over a 21 week period to assess locomotor and anxiety-like behaviour at several adult stages (90, 120, and 150 dpf). B) The multiple-arena modified novel tank dive arena dimensions allowed for a deeper water level when compared to traditional arenas, as well as a narrower tank-depth to reduce 3D exploratory behaviour. Swimming behaviour was tracked and recorded to identify the potential trait-like characteristics of anxiety-like and locomotor behaviour. C) Virtual zones created within EthoVision: the upper zone, the transition zone, and the lower zone.
Fig 2.
Zone preference and locomotion.
A) Zebrafish spent significantly more time in the upper zone at 150 dpf when compared to 90 and 120 dpf and B) less time in the lower zone at 150 dpf when compared to 90 and 120 dpf. C) Distance from bottom also differed with age, with fish at 150 dpf positioned higher in the water column compared to 90 and 120 dpf. D) Zebrafish swimming velocity significantly increased at 150 dpf when compared to 90 and 120 dpf. E) Zebrafish also spent less time immobile at 150 dpf when compared to 120 dpf. F) Single-exposure cohort (S) compared to longitudinal cohort (L). Naïve zebrafish tested only once at 90, 120, or 150 dpf did not differ from the longitudinal zebrafish used in this study repeatedly tested across time in time spent in the lower zone, indicating repeated exposure did not alter behaviour. All data are presented as mean ± S.E.M. Significant differences are indicated by * (p < 0.05), *** (p < 0.001), and **** (p < 0.0001).
Fig 3.
A) There were no significant sex differences in time spent in the upper zone. However, females spent more time in the upper zone at 150 dpf compared to 90 dpf, and males spent more time in the upper zone at 150 dpf compared to both 90 and 120 dpf. B) There were no significant sex differences in time spent in the lower zone. However, females spent less time in the lower zone at 150 dpf compared to 90 dpf, and males spent less time in the lower zone at 150 dpf compared to both 90 and 120 dpf. C) There were significant sex differences in swimming velocity. Males had higher swimming speed at 90, 120, and 150 dpf compared to females. Interestingly, females’ swimming velocity did not change over time, whereas males’ swimming velocity increased over time. D) There were no sex differences in immobility. Both males’ and females’ time spent immobile did not change over time. All data are presented as mean ± S.E.M. Significant differences are indicated by * (p < 0.05), ** (p < 0.01), *** (p < 0.001), and **** (p < 0.0001).
Table 1.
Sources of variation in time spent in the lower zone of the tank (in seconds) over a 10-minute observation.
Table 2.
Sources of variation in velocity (cm/s) of zebrafish over a 10-minute observation.
Table 3.
Sources of variation in time spent immobile (seconds) in zebrafish over a 10-minute observation.
Fig 4.
Sex- and age-related changes in individual variance and behavioural repeatability.
Model-derived estimates (± 95% credible intervals) of among-individual variance, within-individual (residual) variance, and repeatability for each behavioural trait, separated by sex and age group (Early = 90–120 dpf; Late = 120–150 dpf). Note, y-axis labels provide the description (and units) of the response variable to which the variance component, though it should be noted that variance components themselves, including repeatability, are unitless. A) Among-individual variance in time spent in the lower zone was higher in females than males and increased with age in both sexes. B) Within-individual variance in lower zone time was higher in males and decreased with age in both sexes. C) Repeatability for lower zone time increased with age and was higher in females. D) Among-individual variance in velocity was higher in males and increased with age in both sexes. E) Within-individual variance in velocity was higher in males and decreased with age in both sexes. F) Velocity repeatability increased with age and was higher in females in the early period and higher in males in the later period. G) Among-individual variance in immobility was higher in females and increased with age in both sexes. H) Within-individual variance in immobility was higher in males and decreased with age in both sexes. I) Immobility repeatability increased with age and was higher in females than males. All data are presented as point estimates with error bars representing 95% confidence intervals.
Fig 5.
A) There were no differences in time spent in the upper zone in the high (HAZ) or medium (MAZ) anxiety groups across time. Fish in the low (LAZ) anxiety group spent more time in the upper zone at 120 and 150 days old compared to 90 days old. B) There were also no differences in time spent in the lower zone in the HAZ or MAZ groups across time. Fish in the LAZ group spent less time in the lower zone at 120 and 150 days old compared to 90 days old. C) Fish in the HAZ group spent less time in the upper zone compared to the medium and low anxiety groups at 90, 120, and 150 days old. Fish in the LAZ group spent the same amount of time in the upper zone as the MAZ group at 90 days old, then more time at 120 and 150 days old. D) Similarly, fish in the HAZ group consistently spent more time in the lower zone than the MAZ and LAZ groups at 90, 120, and 150 days old. Fish in the LAZ group spent the same amount of time in the lower zone as the MAZ group at 90 days old, but less time in the lower zone at 120 and 150 days old. All data are presented as mean ± S.E.M. Anxiety-level groups were assigned based on lower zone ranking at 150 dpf. Significant differences between groups are indicated by ** (p < 0.01), *** (p < 0.001), and **** (p < 0.0001), as well as different letters above bars (groups not sharing a letter differ significantly, p < 0.05).