Intestinal inflammation induced by soybean meal alters intestinal physiology and is independent of the presence of microbiota. (A,B) Lateral view of the mid-intestine of 9 dpf larvae showing the diffusion of dextran in control (A) and inflamed (B) larvae. Scale bar, 200 um. (C) Normalized dextran fluorescence quantification in the trunk of control and inflamed larvae. (D) Relative mRNA expression of several tight junction proteins. All data was normalized against rpl13a and compared to the control condition (dotted line). (E–L) Transversal cryosection of the midintestine of 9 dpf control and inflamed larvae. (E–J) Immunofluorescence labeling the brush border (E,F), mucus (G,H), and Claudin 15 (I,J); nuclei were stained with DAPI (blue). (K,L) Merge of the four channels in control and inflamed larvae. Scale bar, 5 um. (M,N) Quantification of brush border interruptions (white arrowheads in E,F) and goblet cells (white asterisks in G and H). (O,P) Representative images of the area of the midintestine showing HRP absorption in control and inflamed larvae. Scale bar, 100 um. (Q) Quantification of the area covered by HRP in the midintestine of control and inflamed larvae. (R,S) Representative images showing the number of cell that proliferate during 16 h in control and inflamed larvae. Scale bar, 100 um. (T) Quantification of EdU⁺ cells in the midintestine per larva. (U–X) Lateral view of the midintestine showing immunohistochemistry against the neutrophil marker Mpx on those conventionally raised. The area quantified is delimited by the red dotted rectangle (U,V) and germ-free larvae (W,X). Scale bar, 100 um. (Y) Quantification of the amount of neutrophils present in the midintestine in conventionally raised and germ-free larvae. Permeability, immunofluorescence, protein absorption, proliferation assay, and immunohistochemistry were performed at least in three biological replicates with 15 larvae of 9 dpf per condition. Statistical analysis was performed using the Mann–Whitney U-test, ***p < 0.001, ****p > 0.0001. RT-qPCR was performed at least in three biological replicates with 100 intestines from 9 dpf larvae per condition. Statistical analysis was analyzed using one-way ANOVA and Tukey multiple comparison test. ns, non-significant, *p < 0.05, **p < 0.01, and ****p < 0.0001.

Intestinal inflammation induced by soybean meal increases neutrophil turnover. (A) Experimental strategy used in (B,C,H–P). Larvae at 5 dpf were fed with control or inflammatory diet for 6 days. Then, 1st and 2nd photoconversion of neutrophils present in the intestine was performed at 8 and 9 days post-fertilization (dpf), respectively. Quantification of red- and green-neutrophils present at midintestine (purple dotted line) at 0, 24, and 48 h post-photoconversion (hpc) was performed. (B) Quantification of the total amount of neutrophils present in the intestine at 0, 24, and 48 h post-photoconversion (hpc) in control and inflamed larvae. (C) Quantification of the number of neutrophils replaced at 24 and 48 hpc in control and inflamed larvae. (D) Experimental strategy used in (E–G). The head (circle) or caudal hematopoietic tissue (CHT, rectangle) region was photoconverted at 8 dpf larvae. Later, at 24 hpc red-head-neutrophils and red-CHT-neutrophils present in the intestine were quantified. (E,F) Representative images showing red-head-neutrophil or red-CHT-neutrophils infiltrated in the intestine at 24 hpc in inflamed larvae. (G) Quantification of head and CHT photoconverted neutrophils in the intestine after 24 hpc. (H–O) Representative images showing the region of the body (head, caudal fin, anterior intestine, trunk) where intestine-derived neutrophils were found. (P) Quantification of the number of neutrophils that left the intestine at 24 h. Statistical analysis was performed with the Mann–Whitney U test. ns., non-significant, *p < 0.05, **p < 0.01, and ****p < 0.001. Scale bar, 200 um.

Gut microbiota changes induced by soybean meal diet. (A) Cladogram showing significant changes in genus relative abundance in 9 dpf zebrafish larvae labeled [phylum, (genera)] unless otherwise noted. Circle color indicates change in abundance: yellow, no change; red, elevated in fishmeal (FM); and green, elevated in soybean meal (SBM). Linear discriminant analysis (LDA) effect size analysis (LEfSe) score >3 was considered significant. QIIME data analysis was used. (B–E) The four most significant taxa. The horizontal solid straight line in each panel indicates the group means, and the dotted line indicates the group medians. (F–H) Analysis of cultivable microbiota, relative abundance of phyla. (I–K) Venn diagrams showing exclusive or shared phyla, genera and species respectively. Tables S2–S8 are detailed in Supplementary Material.