Fish were tested once per day in two blocks of trials block 1 trials 13

Fish were tested once per day in two blocks of trials

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Fish were tested once per day in two blocks of trials: block 1, trials 1–3, followed by a 2day break, and then block 2, trials 4–6. All trials were filmed using a camera positioned over the top of the test tanks. The videos were screened after the maze trials were 45 cm 11 cm 18 cm 20 cm Marker Fig.1. Plan view (not drawn to scale) of the maze with test fish inside the isolation tube. The maze was raised up on a platform, creating a water depth of 15cm inside the maze. Each exit (arm) was enclosed but the central arena was open. Solid lines indicate opaque walls that were used to minimize external cues; dashed lines are transparent walls. THE JOURNAL OF EXPERIMENTAL BIOLOGY
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3152 The Journal of Experimental Biology 216 (16) completed and the total number of arms visited during each trial was recorded using Etholog v2.2.5 (Ottoni, 2000). All videos were analyzed with the observer blind to whether a fish was a control or Acid-Al treated fish. Statistical analysis Data were tested for equality of variance and were transformed when there was heterogeneity of variance across the groups being compared. If data did not meet the assumptions of normality and homoscedasticity, non-parametric tests were applied. Mass and fork length, neuroD1 mRNA expression and NKA activity were compared between Acid-Al and control groups using unpaired Student’s t -tests. Gill Al levels were log 10 -transformed before these data were compared using a Student’s t -test. Reciprocal transformation was used for the plasma glucose levels before these data were compared using a Student’s t -test. Plasma cortisol levels were compared between Acid-Al and control groups using a Mann–Whitney U -test. For the behavioral data, there was no difference in the performance of fish in the maze from replicate tanks ( F 1,455 =3.33, P =0.07), thus tanks were pooled for behavioral analysis. The number of arms entered was square-root transformed before being compared using a repeated-measures ANOVA with treatment as a factor. The data were compared across two blocks of three trials, the first block covering trials 1–3 and the second block trials 4–6. Fish were excluded if they never learned to leave the maze and just froze during a trial without visiting any exits (excluded: 11 control, 0 Acid-Al). Freezing is not an uncommon response for salmon, and we believe that the zero value for the Acid- Al fish arose because these fish were more active. The increased activity levels exhibited by the Acid-Al fish may be due to increased levels of stress, resulting in all the Acid-Al fish visiting at least one exit. For the behavior data, variation around the mean is represented as s.e.m. For all other data, variation around the mean is represented as the s.d. All analyses were conducted in StatView (version 5.0.1) and significance was tested at α =0.05.
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  • Winter '20
  • Atlantic salmon

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