Butyrates

SCFAs including acetate, propionate, butyrate, isobutyrate, valerate, and isovalerate were analysed as described previously67 (link). To ensure the homogenicity of the intestine content sample, the freeze-dried samples were prepared using a Vacuum freeze-dryer (Hrist ALPHA 2-4/LSC, Germany) at −80 °C. Briefly, freeze-dried samples (0.5–0.6 g) were weighed into 10 ml centrifuge tubes and mixed with 8 ml ddH₂O, homogenised, and centrifuged in sealed tube at 7,000 g and 4 °C for 10 min. A mixture of the supernatant fluid and 25% metaphosphoric acid solution (0.9 and 0.1 ml, respectively) was centrifuged at 20,000 g and 4 °C for 10 min after standing in a 2 ml sealed tube at 4 °C for over 2 h. The supernatant portion was then filtered through a 0.45-μm polysulfone filter and analysed using Agilent 6890 gas chromatography (Agilent Technologies, Inc, Palo Alto, CA, USA) with a flame ionisation detector and a 1.82 m × 0.2 mm I.D. glass column that was packed with 10% SP-1200/1% H₃PO₄ on the 80/100 Chromosorb W AW (HP, Inc., Boise, ID, USA). The concentration of NH₃-N in the supernatant fluid was measured at 550 nm using a UV-2450 spectrophotometer (Shimadzu, Kyoto, Japan)68 . The bioamines including 1,7-heptyl diamine, cadaverine, phenylethylamine, putrescine, trytamine, tyramine, spermidine, and spermine, as well as the indoles and skatoles, were analysed as described previously69 .

Fecal SCFAs were measured by gas chromatography as previously described (Clarke et al., 2011 (link)). Thawed fecal material was spiked with three times the volume of internal standard (1.68 mM heptanoic acid), homogenized and centrifuged (2000 g, 10 min, 4°C). After centrifugation, 300 μl of supernatant was added to a 0.2 μm filter vial containing 10 μl of 1 M phosphoric acid. The vials were then analysed for SCFA content via gas chromatography. Samples were analysed using an Agilent GC6890 gas chromatography coupled to a flame-ionisation detector (FID), with helium used as the carrier gas. An Agilent free fatty acid phase (FFAP) column (30 m × 0.53 mM (internal diameter) x 1.00 μM (film thickness) was installed for analysis. A splitless injection technique was used, with 0.2 μl of sample injected. A constant flow rate of 4.0 ml/min was used on the column. Upon injection, the oven was initially held at 90°C for 1 min, then raised to 190°C at 20°C/min and held for 3 min. Samples were run in triplicate to ensure accurate and replicable data were obtained. A CV <10% within triplicate samples was used as a quality control measure.
Plasma samples (heparin) were analysed in duplicate for SCFA content using gas-chromatography, as previously described (Gill et al., 2020 (link)). Briefly, 300 µl of plasma was spiked with 50 µl of 200 µM heptanoic acid and acidified with the addition of 50 µl of 10% sulfosalicylic acid before the addition of 3 ml diethyl ether solvent. The mixture was vortexed and centrifuged so that the organic layer could be clarified and transferred into 50 µl 0.2 M NaOH. The alkaline solution containing SCFA was concentrated by evaporation using nitrogen, dissolved in 30 µl 1 M phosphoric acid and transferred into a cold GC glass vial for analysis using an Agilent GC6890 coupled to FID. Concentrations for acetate, propionate and butyrate were determined by the average of the triplicate results, where the CV was <20%. Total SCFA was calculated by the sum of the individual SCFA. Results were expressed as µmol/L.

Adult double transgenic (mpeg1:mCherry/mpx:eGFPi¹¹⁴) expressing mCherry under the macrophage-specific mpeg1 promotor and GFP under the neutrophil-specific mpx promotor fish were housed and fed as previously described [49 (link)]. Embryos were obtained by natural spawning. Fish were fed as follows: weeks 1 and 2 with rotifers (× 4/day from 5 days post fertilization -dpf-), week 3 with rotifers and Artemia Nauplii 230.000 npg (Nauplii per gram) (Ocean Nutrition Europe, Essen, Belgium) (× 2/day), week 4 with Artemia (× 2/day) and until 40 dpf Artemia and Tetramin Flakes (Tetra, Melle, Germany) (× 2/day). When fish reached the juvenile stage, at 40 dpf [76 (link)], fish were randomly distributed into 6 tanks, 2 per each diet: one was sampled after 1 week and the other tank after 3 weeks of the feeding experiment per each diet. The feeding experiment was performed blind and fish were fed until slightly before satiation twice a day. Each tank received one of the following: a control diet, a saponin-supplemented diet or a butyrate-supplemented diet. Full diet composition is listed in Table 1.Table 1

Formulation of experimental diets analysed once the feed intervention was performed to check which compositions corresponded to the blinded diets

	A: Control diet (%)	B: Butyrate diet (%)	C: Saponin diet (%)
Wheat	7.00	6.99	6.67
Wheat gluten	16.00	16.00	16.00
Sunflower meal	1.68	1.68	1.68
Soy protein concentrate	15.16	15.16	15.16
Fish meal	52.00	52.00	52.00
Fish oil	4.40	4.40	4.40
Rapeseed oil	2.00	2.00	2.00
Vitamin mix	0.35	0.35	0.35
Mineral mix	1.92	1.92	1.92
Butyrate	0.00	0.01	0.00
Saponin	0.00	0.00	0.33
[VOLUME]	100.0	100.0	100.0
Dry matter	92.2	92.0	92.0
Crude protein	56.0	57.4	57.4
Crude fat	13.5	13.8	13.8
Ash	8.9	8.9	8.9

The three diets are similar in composition (dry-matter, protein, fat and ash). 95% ultrapure soy saponin was kindly provided by Trond Kortner NMBU Oslo Norway, origin: Organic Technologies, Coshocton, OH, [40 (link)]

Samples from juvenile zebrafish fed the three different diets were sampled from individual separate tanks. Amplicon libraries of the V4 region of the 16S rRNA were generated from the cDNA synthetized from single gut and water samples, using barcoded and modified F515-806R primers [86 (link)]. The PCRs were performed in triplicate, purified, and quantified as previously described [29 (link)]. Purified PCR amplicons were pooled in an equimolar mix and sent for library preparation and sequencing using the Illumina NovaSeq 6000 S2 PE150 XP technology at Eurofins Genomics Germany GmbH (Eurofins Genomic, Ebersberg, Germany). Raw paired-end reads were analyzed using the standard parameters of NG-Tax 2.0 [66 (link)], with the exception of using 100 bp as the forward and reverse read length, as implemented in Galaxy [2 (link)], to obtain Amplicon Sequence Variants (ASVs). Taxonomy was assigned to ASVs using the Silva_132 database [67 (link)]. Two synthetic “mock communities” with known compositions were amplified and sequenced as positive controls and a no-template control was also included as a negative control [68 (link)]. The distribution of reads per sample and the variance in ASVs were assessed and Alpha- and Beta-diversity measurements were performed using R v4.1.2 and RStudio [43 ], using packages ggplot2, [89 ], ape, [61 (link)], plyr, [93 ], vegan, [59 ], RColorBrewer, [58 ], reshape2, [90 ], scales [91 ], data.table, [19 ], microbiome, [42 ], dplyr, [92 ], phyloseq, [55 (link)], ggdendro, [84 ] and DT [95 ]. The analysis yielded 17,203,234 high-quality reads. We excluded one sample (54 dpf butyrate diet) because it had 2 reads only and we kept all the other samples (> 30.000 reads). Rarefaction curves for all samples reached a plateau, indicating that sufficient sequencing depths was achieved (data not shown). For the calculation of alpha-diversity indices, data was rarefied against the sample containing the lowest number of reads (31,814 reads). Redundancy analysis (RDA) and principal component analysis (PCA) were performed with Canoco v5.15 [9 ] using analysis type “constrained” or “unconstrained”, respectively. Response variables were log-transformed with the formula log(10,000*relative_abundance + 1). RDA p-values were determined through permutation testing (500 permutations). Boxplots were generated using Prism v.9.0.0 (GraphPad Software, San Diego, California USA). Cytoscape v3.9.1 [75 (link)] was used to visualize the diet-specific co-occurrence of ASVs based on their relative abundances. Additional data handling and format conversions were done in Python (https://www.python.org/).