Treatment effects on BC composition
Aquatic BC composition: To test for the effect of treatment on
the BC composition in the hold tank water, taxonomical compositions of
the BCs were visualized using stacked barplots and Pie charts of the
relative abundance of the bacteria at the phylum and family level using
the online tool MicrobiomeAnalyst (Chong et al., 2020). Moreover,
differences in alpha diversity indices (Chao1 and PD) among the
treatments (antibiotic, probiotic, control) for the tank water bacterial
communities (BCs) were tested using a Kruskal-Wallis (KW) rank test. In
the case of a significant association, a post hoc Dunn tests with
Bonferroni corrected P values was done. To visualize among-treatment
divergence in the tank water BCs, a Principal-coordinate analysis (PCoA)
using the Bray Curtis distance matrix was used. Subsequently, the
significance of the observed clusters was assessed using permutational
multivariate analysis of variance (PERMANOVA) analysis permutations in
Primer 6 (v6.1.15). Pairwise comparisons were performed in cases of
significant PERMANOVA among treatment groups.
Fish gut bacterial community composition: The effect of treatment
on taxonomic composition of the gut sample BCs was visualized using Pie
charts and stacked barplots of the relative abundance of the bacterial
taxa at the family and phylum level (Chong et al., 2020). To identify
treatment and parental effects on gut microbial community, alpha (Chao1
and PD) diversity indices for gut samples were compared using the KW
rank test in SPSS (IBM SPSS Statistics for Windows, Version 27.0.
Armonk, NY: IBM Corp). To visualize treatment effects on BC structure, a
PCoA using the Bray-Curtis distance matrix was used to generate
scatterplot of the first two PCoA axes in R (version 4.1.1). Moreover,
PERMANOVA analyses were performed in R (version 4.1.1) to test for
treatment and parental (dams, sires) effects on BC composition. Pairwise
comparisons were performed when significant differences among the
treatment groups were detected to identify specific treatment effects.
Comparison between fish gut and aquatic bacterial community
composition: Fish gut BC composition was compared against the rearing
water BC at both the alpha and beta diversity level. Alpha diversity
measures (Chao1 and PD) of gut and water samples were compared using
Mann-Whitney U test in SPSS (IBM SPSS Statistics for Windows, Version
27.0. Armonk, NY: IBM Corp). PCoA first and second axes were used to
visualize clustering of the samples based on sample type (gut or water).
Subsequently, PERMANOVA analysis were performed in R (version 4.1.1) to
test sample type effect on BC composition.