Survival and neutrophil counts in conventionally raised (CONV-R) and germ-free Tg(Bacmpx:GFP)ⁱ¹¹⁴ larvae inoculated with yeasts and/or Vibrio anguillarum. (A) Survival rate of germ-free and conventionally raised larvae (CONV-R) (Kaplan–Meier curves). Statistically significant differences were determined with a log-rank test (^∗p < 0.05). (B) Representation of zebrafish larvae with part of the gastrointestinal tract highlighted in red. Neutrophil counts were performed in the green area. (C) Neutrophil counts in germ-free and conventionally raised Tg(Bacmpx:GFP)ⁱ¹¹⁴ zebrafish larvae. Data show box and whisker plot: each box represents the first to third quartiles, center bar the median, and whiskers the maximum and minimum of each dataset. Different letters denote p < 0.05, Kruskal–Wallis followed by Dunn’s test. Control group: non-inoculated larvae. Dh97 and Yl242 groups: larvae inoculated by immersion with 5 × 10⁶ CFU/mL of D. hansenii 97 or Y. lipolytica 242, at 4 dpf. Va group: larvae challenged at 5 dpf by immersion with 10⁷ CFU/mL of V. anguillarum. Dh97 + Va and Yl242 + Va groups: larvae inoculated by immersion with the respective yeast at 4 dpf and challenged with V. anguillarum at 5 dpf. Data represent three independent experiments.

Bacterial diversity analyses of Tg(Bacmpx:GFP)ⁱ¹¹⁴ larvae inoculated with yeasts and/or V. anguillarum. (A) Alpha diversity indexes Chao1, Shannon, and Simpson. (B) Principal coordinate analysis (PCoA) based on weighted Unifrac distance matrix plot. Analysis of the bacterial microbiota was performed in Tg(Bacmpx:GFP)ⁱ¹¹⁴ zebrafish larvae at 8 days post fertilization (dpf), corresponding to 3 days post V. anguillarum infection. Control group: non-inoculated conventionally raised larvae (CONV-R). Dh97 and Yl242 groups: CONV-R larvae inoculated by immersion with 5 × 10⁶ CFU/mL of D. hansenii 97 or Y. lipolytica 242, respectively, at 4 dpf. Va group: CONV-R larvae challenged at 5 dpf by immersion with 10⁷ CFU/mL of V. anguillarum. Dh97 + Va and Yl242 + Va groups: CONV-R larvae inoculated by immersion with both yeasts at 4 dpf and challenged with V. anguillarum at 5 dpf. ^∗p < 0.05.

Bacterial composition of Tg(Bacmpx:GFP)ⁱ¹¹⁴ larvae inoculated with yeasts and/or Vibrio anguillarum. (A) Phylum level, (B) genus level, and (C) venn diagram showing the number of shared and unique genera among groups. Analysis of the bacterial microbiota was performed in Tg(Bacmpx:GFP)ⁱ¹¹⁴ zebrafish larvae at 8 days post fertilization (dpf), corresponding to 3 days post V. anguillarum infection. Control group: non-inoculated conventionally raised larvae (CONV-R). Dh97 and Yl242 groups: CONV-R larvae inoculated by immersion with 5 × 10⁶ CFU/mL of D. hansenii 97 or Y. lipolytica 242, respectively, at 4 dpf. Va group: CONV-R larvae challenged at 5 dpf by immersion with 10⁷ CFU/mL of V. anguillarum. Dh97 + Va and Yl242 + Va groups: CONV-R larvae inoculated by immersion with both yeasts at 4 dpf and challenged with V. anguillarum at 5 dpf.

Relative abundance of bacterial genera affected by infection with V. anguillarum. Analysis of the bacterial microbiota was performed in Tg(Bacmpx:GFP)ⁱ¹¹⁴ zebrafish larvae at 8 days post fertilization (dpf), corresponding to 3 days post V. anguillarum infection. Control group: non-inoculated conventionally raised larvae (CONV-R). Dh97 and Yl242 groups: CONV-R larvae inoculated by immersion with 5 × 10⁶ CFU/mL of D. hansenii 97 or Y. lipolytica 242, respectively, at 4 dpf. Va group: CONV-R larvae challenged at 5 dpf by immersion with 10⁷ CFU/mL of V. anguillarum. Dh97 + Va and Yl242 + Va groups: CONV-R larvae inoculated by immersion with both yeasts at 4 dpf and challenged with V. anguillarum at 5 dpf. Significant differences between groups and the control group are marked with black asterisks. Green asterisks indicated significant differences between Dh97 + Va or Yl242 + Va with Va group (p < 0.05, Kruskal–Wallis followed by Dunn’s test).