Computational protocol: Cadmium bioaccumulates after acute exposure but has no effect on locomotion or shelter-seeking behaviour in the invasive green shore crab (Carcinus maenas)

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Protocol publication

[…] The exposure chamber containing the crabs was moved to the testing facility 10 min prior to the first trial to minimize any potential stress due to transportation. Crabs were individually moved by hand from the exposure container to the open field testing arena, which was a white plastic circular arena (69 cm diameter, 25 cm walls). Seawater of composition identical to that of the exposure, but without Cd2+, was filled to a depth of 8 cm prior to the testing of each exposure group. The order of testing of Cd2+ groups was reversed for each replicate. Individual crabs were placed in the centre of the arena and were recorded with motion-tracking software (Ethovision XT, v.10; Noldus Information Technology, Leesburg, VA, USA). All open field trials were 5 min in duration and began immediately when crabs were placed into the arena. At the end of the trial the novel object/shelter was placed into the arena in the same location against the wall of the arena and the crab was gently moved to the opposite side of the arena with a plastic metre stick. The shelter was a PVC pipe (10 cm diameter, 8.5 cm long) that was weighted down to prevent movement. This was used as a shelter because all crabs were previously exposed to these PVC pipes when habituated upon arrival to the field station and PVC pipes were also present in the exposure containers.All testing trials were 5 min in duration, and data were recorded and analyzed with Ethovision XT. Distance moved (cm), immobility (s) and time in zones (s) were quantified in Ethovision XT. In the open field test we quantified time in the inner zone (a circle of 48 cm diameter centred in the middle of the arena) as this is a measure of exploration and a lack of time in the outer zone (thigmotaxis zone) is a common proxy for decreased anxiety-like behaviour. In the shelter test we quantified the time spent in the shelter zone (a circle of 34.5 cm centred on top of the shelter) and average distance to the shelter. [...] Normality was tested via the D’Agostino and Pearson omnibus test. One-way ANOVAs were used for parametric data, and Kruskal–Wallis tests with Dunn’s multiple comparison post hoc tests were used for data that were not normally distributed. All accumulation and haemolymph data were analyzed by a one-way ANOVA using a Tukey post hoc analysis. An α-level of 0.05 and 95% confidence intervals were used for assessing statistical significance in all tests. We used t-tests and Mann–Whitney tests to compare the two replicate groups per treatment condition. Non-significant differences allowed us to combine groups. There were significant differences in the open field test between the replicates in the Medium (3.3 μmol/L) group for distance moved, (P = 0.0211, t= 2.526, df = 18, unpaired t-test) and immobility, (P = 0.045, t = 2.154, df = 18, unpaired t-test). Similarly, in the novel object/shelter test a significant difference was also observed in the low (0.3 μmol/L) replicate groups for distance moved (P = 0.028, t = 2.390, df = 18, unpaired t-test). However, in every case neither replicate was different from the control group so these data were still pooled. Behavioural data were analyzed using GraphPad Prism (v.5, San Diego, CA, USA). Bioaccumulation significance was tested via SigmaPlot 11 (Systat software Inc., San Jose, CA, USA) and Sigma Stat 3.5 (Systat Software Inc., San Jose, CA, USA) using a one-way ANOVA and a Tukey post hoc test. All data are presented as mean ± SEM. […]

Pipeline specifications

Software tools EthoVision, SigmaPlot
Applications Miscellaneous, Macroscope & basic digital camera imaging
Chemicals Cadmium