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The fish used in the experiment were hatchery-reared juvenile catfish. Two shoals of pigmented and one shoal of albino catfish unfamiliar with each other were obtained from local fish suppliers (Czech Fishery Ltd., Rybářství Třeboň and Rybářství Nové Hrady, Czech Republic) to ensure that the individuals belonging to the distinct shoals had never been in contact. A total of 600 equally sized fish (200 from each shoal) were transported from the hatchery to the laboratory at the age of 4 months. The fish were transported under stable conditions in oxygenated tanks in an air-conditioned loading space, and the transport lasted approximately 2 hours. There was no observable effect of the transport on the health or mortality of the fish. The fish were subsequently kept in 3 separate holding tanks (1000 L each, initial density 1.8 kg m−3; one shoal, i.e., 200 individuals per tank) for 6 weeks prior to the start of the experiment. The fish were fed food pellets ad libitum (Biomar Group, Denmark, www.biomar.com) distributed across the entire tank, providing free access to food to all individuals twice a day. The fish were kept under a natural photoperiod, maintaining the same regime to which they had become accustomed in the hatchery. The water was purified using biological filters with an integrated UV sterilizer (Pressure-Flo 5000, Rolf C. Hagen Inc., www.lagunaponds.com). The water temperature, dissolved oxygen and pH were controlled automatically (HOBO data logger; Onset Computer Corporation, Bourne, Massachusetts, USA). The fish were tagged 10 days prior to the start of the experiment. The fish were anaesthetized with 2-phenoxyethanol (0.2 ml L−1; Merck KGaA, Germany) and then measured (standard length LS; mean 99 mm, range 81–117 mm) and weighed (mean body mass 9.1 g, range 4–16 g); no size differences between the shoals of fish were detected (standard length P > 0.82, n = 600; body mass P > 0.68, n = 600). Passive integrated transponders (PIT; Trovan ID 100, 0.1 g in air, 12 mm 2.1 mm; EID Aalten B.V., Aalten, Netherlands) were inserted into the abdominal cavity using a syringe. This method has been successfully used in behavioural experiments [17]. No adverse effects of PIT implantation or anaesthesia were observed. All experimental fish (600 individuals) survived; after the experiment, the fish were released under the control of Fish Management Authorities into fish ponds with extensive production management.
The laboratory experiment was conducted between November 15 and December 17, 2010, in an oval artificial stream (see [18] for details). For the purpose of this experiment, only the straight part of the stream was used (beige colour, 3.75 m long, 0.49 m wide and 0.32 m deep). This segment was divided into 5 subunits using 7 equidistant PIT antennae (Fig 1), and mesh was placed over the outer antennae to prevent the fish from escaping from the observed stream segment. The antennae (inner area 0.49 m × 0.25 m) were designed to serve as frames for the detection of fish swimming through them and were connected to a recorder that stored the detection information (PIT tag code, date, time and antenna number) in its internal memory. The handling conditions were comparable to those in the holding tanks, and the water quality and flow were controlled by 2 Pressure-Flo 5000 units (60 L/min each). This arrangement generated a visible current (0.01 m s−1) circulating through the stream; however, the fish did not have to swim continuously to maintain their positions.
Figure data removed from full text. Figure identifier and caption: 10.1371/journal.pone.0128279.g001 Illustrative figure of the experimental design and the artificial stream. A group of 8 familiar pigmented catfish was tested for its ability to co-opt an albino (first treatment) or pigmented (second treatment) unfamiliar conspecific in an artificial stream (Fig 1). The treatments were rotated regularly (one day an albino; the other day a pigmented treatment), and every treatment was repeated ten times, resulting in twenty individual trial repetitions. Every experimental trial lasted for 24 h (beginning at 8:00 a.m.). As individual fish were not used repeatedly in the experiment, 180 catfish were used during the entire experiment.
Data analyses were performed using R software (R Development Core Team, 2010, version 2.11.1). The ‘distance’ between the additional unfamiliar catfish and each of eight individuals from the familiar group was computed as the number of artificial channel sectors between them. A distance equalling zero means that both the evaluated individuals are in the same sector; while a distance equalling four means that the evaluated individuals are separated in the most distant sectors (Fig 1). The ‘average distance’ is the mean of all ‘distance’ values between unfamiliar catfish and members of the familiar group and can be real values from 0 (all fish in one artificial stream sector together) up to 4 (all members from the familiar group in one sector at one end of the channel, with the additional unfamiliar individual at the opposite end of the channel). ‘Isolation’ was defined as a binary variable; 1 means that the additional unfamiliar catfish is isolated in one artificial stream sector; 0 means that there is at least one conspecific from a familiar group present. Another binary variable ‘cohesion’ was used to describe whether the group of familiar pigmented catfish is cohesive (i.e., distributed in three or less artificial stream sectors; value 1) or not (i.e., distributed in more than three artificial stream sectors; value 0). To avoid the dependence of two consecutive points in raw continuous data (a type of M-dependence structure), a regular 10-minute grid approach was applied, resulting in 2880 lines for the final dataset.
Statistical analyses were performed using the SAS software package (version 9.2). Linear mixed effect modelling was applied to analyse the average distance between the unfamiliar catfish and members of the familiar group. The MIXED procedure with a random intercept term to account for variability among the trials was used for this purpose [19]. The final model was built on the basis of the stepwise forward procedure (all two way interactions were tested), always according to a better Akaike’s Information Criterion (AIC) value and considering that the model with the lower AIC fit the data better [20]. The parameter estimates together with the standard error estimates and the appropriate significance tests of the final model are given in Table 1.
Table data removed from full text. Table identifier and caption: 10.1371/journal.pone.0128279.t001 Parameter estimates with corresponding standard errors and p-values for the proposed mixed model. Time covariate enters the model as a third order polynom (Time, Time squared and Time cubed).
Additional binomial distributional data were subjected to a χ2 test to evaluate i) the effect of albinism on the ‘isolation’ of unfamiliar catfish in an artificial stream sector and ii) the ‘cohesion’ (i.e., distribution in three or less artificial stream sectors) of the group of familiar pigmented catfish in the presence of an albino or pigmented unfamiliar conspecific. The FREQ procedure was used for this purpose, designing it to compare the frequency of ‘isolation’ and ‘cohesion’ occurrence under different treatments. Relative deviations from the hypothesized values (equal proportions, i.e. 50% occurrence in this case) were used to express the character of the relationships. The relative deviation for a level is the difference between the observed and hypothesized/expected test percentage occurrence divided by the test percentage occurrence. The GENMOD procedure with binomial distributions was designed to estimate the probability of ‘cohesion’ occurrence. The explanatory variable tested was the average distance between unfamiliar catfish and members of the familiar group across different treatments (albino or pigmented unfamiliar individual). We applied an analysis of repeated measurements based on the generalised estimating equation (GEE) approach [21], which is an extension of a generalised linear model and provides a semi-parametric approach to longitudinal data analysis. To account for repeated measures during the same trials, we used a REPEATED statement with the trial defined in the SUBJECT option. Data entering the statistical analyses are in the supporting information file S1 Table.
All of the laboratory experimental procedures complied with valid legislative regulations (law no. 246/1992, §19, art. 1, letter c); the permit was subjected to O. Slavík, qualified according to Law no. 246/1992, §17, art. 1; permit no. CZ00167. All laboratory sampling including PIT implantation was carried out with the relevant permission from the Departmental Expert Committee for authorization experimental project of the Ministry of Education, Youth and Sports of the Czech Republic (permit no. MSMT-31220/2014-6, registered by the Ministry of Education, Youth and Sports of the Czech Republic). The study did not involve endangered or protected species.
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