Animal care and maintenance

Stock populations of P. formosa (Amazon molly, obtained from Manfred Schartl, University of Würzburg) are maintained in large (100 l) stock aquariums. The all-female Amazon molly originates from a single natural hybridization event between the sailfin molly Poecilia latipinna and the Atlantic molly Poecilia mexicana44,45. It reproduces gynogenetically and females require sperm from one of the parental species to stimulate egg production45,46,47. Therefore, several (two–four) males of P. mexicana were kept with each stock population aquarium. Stock populations experience ambient light conditions similar to the local light cycle (∼14:10 L:D). Fish were fed ab libitum three times daily on standard flake fish food. We performed weekly water changes to replace ∼30% of the total water volume of each tank. To generate the experimental individuals, we isolated gravid females from a single isogenic line (strain 1304, lab code Manfred Schartl) in separate 35 l tanks containing a gravel bottom and plastic plants. To further limit the potential for differences among mothers (for example, in maternal effects), these gravid females were all sisters from the same brood ensuring all individuals were the same age, from the same mother and had experienced similar conditions for their entire lives. Intermittent genetic samplings of this strain confirm that all individuals are clones. We checked females’ tanks twice daily for evidence of offspring. In case a female gave birth, we took 12 juveniles from the brood and randomly assigned four siblings to each of our three treatments (see more details below; Fig. 1). This split-brood design helped to ensure that any differences in maternal effects were at least split among all our treatments. Additionally, we only used broods of similar size (12–24 offspring) to reduce the potential for differences in maternal provisioning. In total, seven different mothers (one mother contributed two clutches) contributed to the experimental individuals.

Experimental set-up

Newly born offspring were randomly assigned to one of three treatments directly after birth (Fig. 1).

In the 0-day treatment, directly after birth, four individuals from a single brood were transferred into a single 30 cm × 30 cm × 30 cm square tank, which was covered on all sides with black foil to limit outside disturbances. The tank was divided into four equally sized quadrants using a blue filter sponge. Each compartment housed a single individual and the sponge divider allowed water exchange between the four compartments but no visual or direct interactions of the fish. Such a design allows olfactory communication among physically isolated fish which we know is important in this species37,38 and should circumvent the development of behavioural abnormalities reported in other studies using teleost fishes that were entirely socially deprived48,49. An air-driven filter was integrated into the sponge to maintain water quality. We exchanged 50% of the water on a weekly routine. Water temperature was maintained constantly at 24 °C through room temperature, and as all tanks were placed on a large table at the same height no temperature variation greater than 1 °C was observed in our weekly measurements. The tank contained no gravel or any other substrate. To (i) avoid size (growth) differences between individuals due to competition for food (we observed no differences in body size between individuals, see results) and (ii) maintain good health of all fish throughout the experiment (only 2 out of 96 experimental individuals died before phenotyping), fish were fed ad libitum with live Artemia-nauplii three times a day as well as with commercially available dusted flake food (TetraMin) twice a day. Such a feeding regime follows standard protocols for common garden experiments in mollies50,51. After 7 weeks of isolation, each fish was individually phenotyped for its exploration and activity (see ref. 52 for a similar protocol). We chose exploration and activity patterns as our target trait since locomotion is of central importance for all non-sessile animals and individual differences in this trait are thought to have substantial ecological consequences53. Furthermore, activity is influenced by the social environment in the Atlantic molly (Poecilia mexicana), one of the parental species of the Amazon molly32.

In the 7-day treatment, four individuals from a single brood were transferred to a similar square tank as described for the 0-day treatment. However, fish were reared as a group without a sponge divider for 7 days, allowing unlimited direct social interactions (for example, social hierarchies31). Afterwards, fish were isolated via a sponge divider and reared separately for another 6 weeks before being phenotyped. Maintenance was as described for 0-day treatment.

Finally, in the 28-day treatment, four individuals from a single brood were transferred to a similar square tank as described for the 0-day treatment. Fish were then reared as a group without a sponge divider for 28 days, allowing unlimited direct social interactions. Afterwards, fish were isolated via a sponge divider and reared separately for another 3 weeks before being phenotyped. Maintenance was as described for 0-day treatment.

We repeatedly assayed all individual’s behaviour at the age of 7 weeks. To limit the possibility of differences in energy status among individuals affecting our behavioural measures, we did not feed fish the morning before the tests, which were always done between 10:00 hours and 13:00:00 hours. We used an open-field arena that consisted of a circular tank (48.5 cm in diameter, made of white plastic) filled with system water to a depth of 3 cm. Lighting was provided from neon tubes positioned at the room ceiling, which helped to avoid shadows or reflections within the tank. In each trial, a single fish was introduced into an opaque plastic cylinder in the centre of the arena and let to acclimate for 3 min. Then, the cylinder was carefully removed and we videotaped fish exploration and activity with a webcam for the next 6 min. After that, we transferred the fish back into its rearing compartment. We exchanged the water in the open-field arena after every trial to exclude any effect of released chemicals on subsequently tested fish. Measurements were repeated every other day until we completed four measurements per individual. Fish were tested in random order, so that no fish was always tested as first or last in a day. Videos were analysed using the automated video tracking software EthoVision XT Version 10.1 (Noldus Information Technologies, Inc.), thus, experimenters were blinded about individual fish treatment identity. Position scoring started 10 s after the fish was released from the cylinder and we measured the total distance swam within the following 5 min. After completing the four activity measures, standard length of all fish were measured to the nearest 0.1 mm. Two experimental individuals died before phenotyping, resulting in final sample sizes of: 0-day treatment N=31; 7-day treatment N=31; 28-day treatment N=32. Choosing our sample size, we followed the recommendations in ref. 54, and the number of animals as well as the number of repeats per individual is large enough to yield sufficient power in detecting repeatability if present54. The reported experiments comply with current German law approved by LaGeSo Berlin (GO124/14 to D.B.).

Statistical analysis

Our behavioural measure, total distance swam, was square-root transformed before analysis to meet assumptions of normality and homogeneity of variance. We first tested for differences in body size (standard length) among fish from our different treatments using a linear mixed model with standard length as the response variable and treatment as the fixed effect; mother identity was included as a random effect. To investigate whether levels of individual behavioural variation differed across treatments, we used a linear mixed model including the covariates ‘observation (trial 1 to 4)’ to account for behavioural changes over the course of the experiment55 and ‘standard length’ to account for individual body size differences as well as ‘treatment’ as a fixed factor. We included treatment-specific variance estimates for individual and residual variance. Additionally, we included the global (that is, not treatment specific) random effect of mother identity. The resulting variance components were used to estimate the proportion of variance attributable to the individual, that is, behavioural repeatability54,56. We did not include any variation attributed to mother identity in these repeatability estimates as this random effect was not well supported by the model (see Results section) and any variation attributable to mother identity would be split across all treatments (due to our split-brood design). A significant repeatability estimate is interpreted as evidence of consistent individual differences. Significant differences in variance components and repeatability between the treatments can be assumed when the 95% confidence intervals of the estimates do not overlap. We additionally tested whether there was overall support for the treatment-specific variance estimates (that is, whether among-individual variance was different across treatments) by comparing this model to a model where we did not allow the among-individual variance in individual intercepts to vary across treatments (that is, we fit one variance estimate for individuals across all three treatments). We compared the resulting deviance information criterion (DIC) of each model, and if the DIC was reduced by greater than three by including the treatment-specific random effect, we considered this statistical support that the treatment-specific variance estimates better fit our data. Finally, in preliminary analyses, we tested for the possibility that individuals within each treatment exhibited differing plasticity over the repeated testing (that is, random slopes/regression). We did this by running a separate model for each treatment where we included random intercepts and slopes for individuals and mothers. However, there was no evidence that inclusion of the random slopes increased model fit (ΔDIC <1), and actually impaired model convergence, therefore, we did not retain these terms in our final model (see SI data, Table 1). Additionally, there was no evidence for an interaction between treatment and body size, or treatment and observation (data not shown).

For all analyses, we used Markov chain Monte Carlo estimation, assuming a Gaussian error distribution with the MCMCglmm package in R v3.1.3 (ref. 57). We used parameter-expanded proper priors and preliminary analyses indicated that our results were not sensitive to changes in prior specification. To ensure model convergence, we ran five chains for each model with 500,000 iterations, 1,000 burn-in and thinning every 100 iterations. We visually checked the posterior density plots to ensure proper model mixing and convergence.

Data availability

All data are accessible via dryad respository58.