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Hellbender skin microbial diversity was not affected by experimental vaccination or Bd infection. We used a linear mixed effects model with Faith's Phylogenetic Diversity Index as the response variable, vaccination treatment and infection dose as both independent and interacting fixed effects, and animal as a random effect.

Hellbender skin microbiome between-community diversity was affected by experimental infection, but not by experimental vaccination status. The beta diversity of microbiome communities was evaluated with PERMANOVA of the Bray-Curtis distance between communities.

Pairwise comparisons of gene expression values performed to calculate differential gene expression in hellbender skin throughout experimental vaccination and infection.

Unique biological process functions identified by BLAST2GO in the hellbender skin microbiome following Bd infection.

Pairwise comparisons of gene expression values performed to calculate differential gene expression in Bd throughout experimental infection.

Principal components analysis of global hellbender skin microbiome gene expression during experimental vaccination and challenge with Bd. Gene expression is represented in all skin microbiomes at all experimental time points with Log2 transformed counts per million transcripts. Ellipses indicate 95% CI of expression.

Significantly differentially expressed immune genes identified in each experimental group at each experimental time point. Within each experimental group and time point, immune DEGs are displayed from greatest negative Log fold change to greatest positive Log fold change. All annotated GO terms and the predicted function per putative immune-associated gene were determined using BLAST2GO.

Hellbender skin MHC gene expression was affected by vaccination strategy, Bd infection status, and experimental day. Expression of two isoforms of the MHC Class I gene DN14290 was significantly affected by experimental day and the interaction between experimental day and infection status (isoform 18), and experimental day (isoform 23). Expression of the MHC Class II gene DN4350 was significantly affected by vaccination strategy, infection, experimental day, the interaction between vaccination strategy and infection, and the interaction between infection and experimental day. Changes in gene expression were evaluated with ANOVA-type tests of f2.ld.f1 linear models of gene expression.

Faith's Phylogenetic Diversity Index values (A) and variance (B) were compared between experimental vaccination groups at pre-vaccination and post-vaccination, and between vaccination and infection experimental groups at early infection and late infection with (A) Kruskal-Wallis tests for index values, and (B) Fligner-Killeen tests for homogeneity of variance.

Summary of total and average sequencing read counts used to generate the hellbender skin transcriptome A) by experimental day, and B) by experimental group.

A) Anti-Bd OTU richness was not correlated with Bd load in early infection (Spearman's rank correlation rho = 0.0431, S = 3496, p = 0.828), or (B) in late infection (Spearman's rank correlation rho = 0.272, S = 3271, p = 0.146).

Principal components analysis of global hellbender skin gene expression during experimental vaccination and challenge with Bd. Gene expression is represented in all animals at all experimental time points with Log2 transformed counts per million transcripts. Ellipses indicate 95% CI of expression.

Neither vaccination, infection dosage, nor the interaction of vaccination and infection dosage was a significant predictor of AUDPC in experimental Bd infection of hellbenders. The significance of these effects was tested with Kenward-Roger's F test.