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.