Exposure to Yeast Shapes the Intestinal Bacterial Community Assembly in Zebrafish Larvae

Abstract

Establishment of the early-life gut microbiota has a large influence on host development and succession of microbial composition in later life stages. The effect of commensal yeasts - which are known to create a conducive environment for beneficial bacteria - on the structure and diversity of fish gut microbiota still remains unexplored. The present study examined the intestinal bacterial community of zebrafish (Danio rerio) larvae exposed to two fish-derived yeasts by sequencing the V4 hypervariable region of bacterial 16S rRNA. The first stage of the experiment (until 7 days post-fertilization) was performed in cell culture flasks under sterile and conventional conditions for germ-free (GF) and conventionally raised (CR) larvae, respectively. The second phase was carried out under standard rearing conditions, for both groups. Exposure of GF and CR zebrafish larvae to one of the yeast species Debaryomyces or Pseudozyma affected the bacterial composition. Exposure to Debaryomyces resulted in a significantly higher abundance of core bacteria. The difference was mainly due to shifts in relative abundance of taxa belonging to the phylum Proteobacteria. In Debaryomyces-exposed CR larvae, the significantly enriched taxa included beneficial bacteria such as Pediococcus and Lactococcus (Firmicutes). Furthermore, most diversity indices of bacterial communities in yeast-exposed CR zebrafish were significantly altered compared to the control group. Such alterations were not evident in GF zebrafish. The water bacterial community was distinct from the intestinal microbiota of zebrafish larvae. Our findings indicate that early exposure to commensal yeast could cause differential bacterial assemblage, including the establishment of potentially beneficial bacteria.

Keywords: 16S rRNA; Debaryomyces; Pseudozyma; amplicon sequencing; germ-free; microbiota; yeast; zebrafish.

FIGURE 1

Design of the yeast exposure study. The alterations in the intestinal bacterial communities of conventionally raised (CR) and germ-free (GF) zebrafish larvae were examined after exposing them to the yeasts Debaryomyces sp. or Pseudozyma sp. for 24 h. The larvae in the control group were exposed to PBS. The intestine samples were collected on day 14 and water samples from their respective flasks/tanks were collected at days 2, 3, 7, and 14.

FIGURE 2

Relative abundance of the intestinal bacterial communities of zebrafish larvae, at different taxonomic levels. Phyla (A) and the top abundant genera (B) in CR and GF zebrafish. Each bar segment representing the average relative abundance of a particular bacterial taxon within a group is color coded: Proteobacteria – shades of purple and pink, and Bacteroidetes – green. CRC, conventionally raised control; CRD, conventionally raised Debaryomyces-exposed; CRP, conventionally raised Pseudozyma-exposed; GFC, germ-free control; GFD, germ-free Debaryomyces-exposed; GFP, germ-free Pseudozyma-exposed.

FIGURE 3

Principal coordinate analysis plots based on weighted UniFrac distance metric show the distinct intestinal bacterial communities in CR (A), and GF (B) of zebrafish larvae. Ellipses include 95% of samples from normally distributed data. CRC, conventionally raised control; CRD, conventionally raised Debaryomyces-exposed; CRP, conventionally raised Pseudozyma-exposed; GFC, germ-free control; GFD, germ-free Debaryomyces-exposed; GFP, germ-free Pseudozyma-exposed.

FIGURE 4

Alpha diversity indices of the intestinal bacterial communities of CR zebrafish larvae. Species richness (A), Shannon diversity (B), Simpson diversity (C), phylogenetic diversity (D), core abundance (E), and rare low abundance (F). Different letters above the bars indicate significant differences as determined by Dunn’s tests. CRC, conventionally raised control; CRD, conventionally raised Debaryomyces-exposed; CRP, conventionally raised Pseudozyma-exposed.

FIGURE 5

Alpha diversity indices of the intestinal bacterial communities of GF zebrafish larvae. Species richness (A), Shannon diversity (B), Simpson diversity (C), phylogenetic diversity (D), core abundance (E), and rare low abundance (F). Different letters above the bars indicate significant differences as determined by Dunn’s tests. GFC, Germ-free control; GFD, germ-free Debaryomyces-exposed; GFP, germ-free Pseudozyma-exposed.

FIGURE 6

Differentially abundant bacterial taxa in CR (A) and GF (B) zebrafish larvae intestine. LEfSe was employed to find the differential abundance using a cut-off of 3.5 and a significance threshold of P < 0.05. Y-axis labels are color coded for different bacterial taxa: Proteobacteria – purple, Bacteroidetes – light green, Firmicutes – dark green, Chlamydiae – coral, Chloroflexi– cyan, Planctomycetes – dark red, and Actinobacteria – light orange. CRC, conventionally raised control; CRD, conventionally raised Debaryomyces-exposed; CRP, conventionally raised Pseudozyma-exposed; GFC, germ-free control; GFD, germ-free Debaryomyces-exposed; GFP, germ-free Pseudozyma-exposed.

FIGURE 7

Relative abundance of bacterial phyla in the water samples (A) and principal coordinate analysis plot (B). The PCoA plot based on weighted UniFrac distance metric shows the distinction between the bacterial communities in water and zebrafish intestine. Ellipses include 95% of samples from normally distributed data. CRCW, CRDW, and CRPW represent the water samples collected from their respective flasks/tanks on day 2, 3, 7, and 14. GFCW, GFDW, and GFPW represent the water samples collected only on day 14. ZSW represents the water samples collected from the zebrafish facility on days 7 and 14.