All data are presented as mean ± SEM (standard error of the
mean). Statistical data analysis was carried out using Statistica 8
(StatSoft, Inc. 2008) and R 3.1.1 [49]. Data were tested for normality
and homogeneity of variances. Daily mortality was calculated and
was analysed in R using KaplaneMeier estimates of survival [50].
Multivariate analysis (MANOVA) were performed to test for differences
in the entire gene expression pattern of all 30 genes but
also divided into functional gene classes (Groups see Table 1). 2-
way nested ANOVAs (tank nested in treatment) were performed
to test for significant effects of tank, treatment, time and time*-
treatment interactions. For data sets that displayed significant
time*treatment interactions nested ANOVAs were performed for
the individual time points. Significance was defined as p 0.05 and
gene expression results were Bonferroni corrected for multiple
testing. For gene expression studies statistical analysis was performed
on Dct values, whilst graphs represent x-fold gene
expression relative to the control group. Graphs were constructed
with GraphPad Prism 5 and data are plotted as independent data
points (n ¼ 18), whilst statistical analysis controlled for potential
tank effects through nested ANOVA.