Omics and imaging combinatorial approach reveals butyrate-induced inflammatory effects in the zebrafish gut

Adrià López Nadal; Jos Boekhorst; Carolien Lute; Frank van den Berg; Michelle A. Schorn; Tommy Bergen Eriksen; David Peggs; Charles McGurk; Detmer Sipkema; Michiel Kleerebezem; Geert F. Wiegertjes; Sylvia Brugman

doi:10.1186/s42523-023-00230-2

Omics and imaging combinatorial approach reveals butyrate-induced inflammatory effects in the zebrafish gut

López Nadal, Adrià; Boekhorst, Jos; Lute, Carolien; van den Berg, Frank; Schorn, Michelle A.; Bergen Eriksen, Tommy; Peggs, David; McGurk, Charles; Sipkema, Detmer; Kleerebezem, Michiel; Wiegertjes, Geert F.; Brugman, Sylvia 2023-03-03 00:00:00 Background Prebiotic feed additives aim to improve gut health by influencing the microbiota and the gut barrier. Most studies on feed additives concentrate on one or two (monodisciplinary) outcome parameters, such as immunity, growth, microbiota or intestinal architecture. A combinatorial and comprehensive approach to disclose the complex and multifaceted effects of feed additives is needed to understand their underlying mechanisms before making health benefit claims. Here, we used juvenile zebrafish as a model species to study effects of feed additives by inte - grating gut microbiota composition data and host gut transcriptomics with high-throughput quantitative histological analysis. Zebrafish received either control, sodium butyrate or saponin-supplemented feed. Butyrate-derived compo - nents such as butyric acid or sodium butyrate have been widely used in animal feeds due to their immunostimulant properties, thereby supporting intestinal health. Soy saponin is an antinutritional factor from soybean meal that promotes inflammation due to its amphipathic nature. Results We observed distinct microbial profiles associated with each diet, discovering that butyrate (and saponin to a lesser extent) affected gut microbial composition by reducing the degree of community-structure (co-occurrence network analysis) compared to controls. Analogously, butyrate and saponin supplementation impacted the tran- scription of numerous canonical pathways compared to control-fed fish. For example, both butyrate and saponin increased the expression of genes associated with immune response and inflammatory response, as well as oxidore - ductase activity, compared to controls. Furthermore, butyrate decreased the expression of genes associated with histone modification, mitotic processes and G-coupled receptor activity. High-throughput quantitative histological analysis depicted an increase of eosinophils and rodlet cells in the gut tissue of fish receiving butyrate after one week of feeding and a depletion of mucus-producing cells after 3 weeks of feeding this diet. Combination of all datasets indicated that in juvenile zebrafish, butyrate supplementation increases the immune and the inflammatory response to a greater extent than the established inflammation-inducing anti-nutritional factor saponin. Such comprehen- sive analysis was supplemented by in vivo imaging of neutrophil and macrophage transgenic reporter zebrafish (mpeg1:mCherry/mpx:eGFPi ) larvae. Upon exposure to butyrate and saponin, these larvae displayed a dose- dependent increase of neutrophils and macrophages in the gut area. *Correspondence: Sylvia Brugman Sylvia.brugman@wur.nl Full list of author information is available at the end of the article © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. López Nadal et al. Animal Microbiome (2023) 5:15 Page 2 of 21 Conclusion The omics and imaging combinatorial approach provided an integrated evaluation of the effect of butyrate on fish gut health and unraveled inflammatory-like features not previously reported that question the usage of butyrate supplementation to enhance fish gut health under basal conditions. The zebrafish model, due to its unique advantages, provides researchers with an invaluable tool to investigate effects of feed components on fish gut health throughout life. Keywords Microbiome, Transcriptome, Omics, Imaging, Zebrafish, Butyrate, Soy saponin, Gut, Inflammation Background inflammation to decipher the underlying diet-microbe- In the last decades, the implications of the microbiome host interactions in the zebrafish gut and to assess feed in human and animal health have gained interest among compounds that can potentially protect the gut from and beyond the scientific community. As a consequence, becoming inflamed [77]. Experimental designs of fish food ingredients able to modulate the microbiome, such feed studies are often based on specific outcome param - as prebiotics, became increasingly popular and accepted eters, including fish growth, expression of a limited set of among the general public and have been utilized in genes, plasma levels of antioxidants, semi-quantitative human dietary supplements as well as in animal feed [17, scoring of histological parameters, profiling of the gut 39]. A prebiotic is a substrate that is selectively utilized bacteria composition or pathogenic challenges. Habitu- by host microorganisms and thereby proposed to confer ally, only end-point analysis have been performed, ignor- a health benefit on the host (reviewed in [26]. ing the kinetics of the responses. Most of these studies Butyrate is a short-chain fatty acid (SCFA) derived base eventual gut health claims of dietary treatments on from fiber fermentation by the gut bacteria that exhibits one or two of these parameters. For instance, the anti- some prebiotic properties, playing a role in the interac- inflammatory properties of supplemented microalgae tion between bacterial population dynamics and host were assessed by only quantifying the neutrophils in the gut homeostasis [44]. Butyrate has a direct impact on zebrafish gut larvae [10]. The expression of a small set the immune system via signaling G-protein coupled of genes were proposed to support immune-boosting, receptors (GPCR) on epithelial and immune cells and anti-inflammatory and antioxidative stress properties of also induces epigenetic changes via regulation of histone phytates after soybean-meal feeding in zebrafish larvae acetylase and histone deacetylase enzymes (reviewed in [74]. Although quantitative assessment of health associ- [31]. In the last years, butyrate has been extensively used ated phenotypes is critical to support health claims, bas- in animal feed, including its supplementation to several ing these on the determination of a single or only few fish diets in the form of butyric acid or sodium butyrate parameters may lead to overstated conclusions. There - due to its growth-promoting, immuno-stimulating and fore, to appropriately assess and understand the complex antioxidative properties (reviewed in [1] and to mitigate and multifaceted effects of feed supplements on fish gut detrimental effects of sub-optimal plant-containing diets health more integrated and holistic approaches are war- [23, 48, 70, 83]. ranted (as reviewed in [50]). In order to achieve this, we Plant-based protein ingredients have been replac- propose that a combination of high-throughput readouts ing fish meal in feed due to their more favorable price may be employed to supply unbiased datasets that could and availability. However, several anti-nutritional com- lead to a detailed description of the host gut health status ponents derived from plant-based protein sources are upon feed interventions. reported detrimental for fish health (reviewed in [73]. For Many gut functions and immune genes are conserved instance, soy saponin is an anti-nutritional component between different species. Moreover, zebrafish larvae are of soybean meal that interacts with cell membranes and optically transparent and together with the development promotes pore formation, vesiculation and membrane of several transgenic fish lines that express fluorescent domain disruption [4]. Various studies linked the pres- proteins in specific cell-lineages facilitates in vivo track - ence of soy saponin to inflammatory responses in the ing of certain immune cells, which empowered the use of intestinal mucosa, enteritis as well as microbiota modula- the zebrafish model to examine intestinal inflammation tion in several fish species [13, 16, 40], including zebrafish (reviewed in [12] as well as a model organism to evaluate [49]. In zebrafish larvae, the number of neutrophils novel feeds for farmed fish [79]. However, the zebrafish increased in the gut after soybean meal feeding [30] or gastrointestinal tract presents several particularities. For exposure to soy saponin in solution [49]. After assessing example, zebrafish lack a stomach and instead employ the the inflammatory effect of soy saponin, soy-containing anterior gut segment, named intestinal bulb, as a reser- diets have been employed as a model for feed-induced voir for feed. Although this intestinal bulb lacks gastric L ópez Nadal et al. Animal Microbiome (2023) 5:15 Page 3 of 21 glands, it produces digestive enzymes and mimics what tank after 3 weeks of the feeding experiment per each may occur in the stomach [22, 57]. The zebrafish gut epi - diet. The feeding experiment was performed blind and thelial layer also lacks intestinal crypts that are typically fish were fed until slightly before satiation twice a day. found in other fish species or in mammals and rather Each tank received one of the following: a control diet, forms protrusions called folds that decrease in size from a saponin-supplemented diet or a butyrate-supplemented anterior to posterior gut segments [85]. Nevertheless, the diet. Full diet composition is listed in Table 1. canonical intestinal epithelial cells (IECs) such as entero- cytes, mucin-producing goblet cells and enteroendocrine Experimental design cells are present in the zebrafish gut. Moreover, zebrafish Water quality was set to standard values by replacing half gut segments presented analogous expression to their of the water in the zebrafish system before the start of mammalian counterparts [87] and when transcriptom- the experiment and monitored twice a week during the ics where performed on IECs from zebrafish, stickle - whole experiment (Additional file 1: Fig. S1). A pH meter back, mouse and human a highly conserved expression (Hanna Instruments, Nieuwegein, The Netherlands) was was found between zebrafish and mammals [46]. Like in used to measure the pH and the water conductivity. Kits many animal models for inflammation, mucus-producing to measure ammonium, nitrite and nitrate (Merck KGaA, cells (Goblet cells) decrease and granulocytes (mainly Darmstadt, Germany) were used according to manufac- neutrophils and eosinophils), macrophages and lympho- turer’s instructions. Additionally, nitrite, nitrate, general cytes increase upon inflammation in the zebrafish gut hardness, carbonate hardness, pH and chlorine were (re) [11, 49]. Due to its shared expression and functionality measured by using Tetra Test 6in1 (Tetra, Melle, Ger- the zebrafish is an excellent model to understand host- many) according to manufacturer’s instructions. Fish microbe-immune interactions.Research on feed supple- survival and standard length -from the tip of the head ments and their effect on gut health are usually based on until the bifurcation of the caudal fin- were assessed by few readouts parameters which may not reflect the com - using a digital caliper (Sylvac, Yverdon, Switzerland) dur- plexity of fish gut health. The main goal of this present ing the experiment (Additional file 2: Fig. S2). The dietary study is to provide an comprehensive investigation based intervention consisted of three diets identical in com- on the integration of several high-throughput readouts position except the supplementation with 1 g/kg feed of of the gut mucosa to depict a more holistic view of the sodium butyrate in the butyrate diet and 3.3 g/kg of 95% effects of feed supplements on zebrafish gut health. Material and methods Table 1 Formulation of experimental diets analysed once the feed intervention was performed to check which compositions Ethics statement corresponded to the blinded diets The present study was approved by the Dutch Commit - tee on Animal Welfare (2017.W-0034) and the Animal A: Control B: Butyrate C: Saponin diet (%) diet (%) diet (%) Welfare Body (IvD) of the Wageningen University (The Netherlands). Furthermore, we adhered to standard bios- Wheat 7.00 6.99 6.67 ecurity and institutional safety procedures at Wagenin- Wheat gluten 16.00 16.00 16.00 gen University and Research. Sunflower meal 1.68 1.68 1.68 Soy protein concentrate 15.16 15.16 15.16 Zebrafish and diets Fish meal 52.00 52.00 52.00 Adult double transgenic (mpeg1:mCherry/mpx:eGFPi ) Fish oil 4.40 4.40 4.40 expressing mCherry under the macrophage-specific Rapeseed oil 2.00 2.00 2.00 mpeg1 promotor and GFP under the neutrophil-specific Vitamin mix 0.35 0.35 0.35 mpx promotor fish were housed and fed as previously Mineral mix 1.92 1.92 1.92 described [49]. Embryos were obtained by natural spawn- Butyrate 0.00 0.01 0.00 ing. Fish were fed as follows: weeks 1 and 2 with rotifers Saponin 0.00 0.00 0.33 (× 4/day from 5 days post fertilization -dpf-), week 3 with [ VOLUME] 100.0 100.0 100.0 rotifers and Artemia Nauplii 230.000 npg (Nauplii per Dry matter 92.2 92.0 92.0 gram) (Ocean Nutrition Europe, Essen, Belgium) (× 2/ Crude protein 56.0 57.4 57.4 day), week 4 with Artemia (× 2/day) and until 40 dpf Crude fat 13.5 13.8 13.8 Artemia and Tetramin Flakes (Tetra, Melle, Germany) Ash 8.9 8.9 8.9 (× 2/day). When fish reached the juvenile stage, at 40 dpf The three diets are similar in composition (dry-matter, protein, fat and ash). 95% [76], fish were randomly distributed into 6 tanks, 2 per ultrapure soy saponin was kindly provided by Trond Kortner NMBU Oslo Norway, each diet: one was sampled after 1 week and the other origin: Organic Technologies, Coshocton, OH, [40] López Nadal et al. Animal Microbiome (2023) 5:15 Page 4 of 21 ultrapure soy saponin in the saponin diet (Table 1). We using the Qsep100 Bio-Fragment Analyzer (Bioptic inc., st ™ sampled fish guts after 1 week (54 dpf, 1 timepoint) and New Taipei City, Taiwan) and the Qubit RNA BR Assay after 3 weeks (68dpf, 2nd timepoint) after the start of the Kit (ThermoFisher Scientific, MA, USA). A schematic dietary intervention. Fish were fed twice daily until satia- pipeline of the whole process from sample collection to tion and the amount of feed provided was quantified with results analyses is depicted in Fig. 2. a micro-spoon, feeding 15.1 mg of feed per tank per day (averaging to 0.46 mg of feed per day per fish). A sum - Microbiome: 16S rRNA profiling and sequencing data mary of the experiment design is depicted in Fig. 1. analysis Samples from juvenile zebrafish fed the three differ - Single gut and water samples RNA extraction ent diets were sampled from individual separate tanks. Guts were extracted from juvenile zebrafish, rinsed in Amplicon libraries of the V4 region of the 16S rRNA sterile PBS, snap frozen in liquid nitrogen and preserved were generated from the cDNA synthetized from sin- at − 80 °C for total gut RNA extraction. RNA extrac- gle gut and water samples, using barcoded and modified tion was performed from single intestines as previously F515-806R primers [86]. The PCRs were performed in described [35]. Water samples were obtained by filter - triplicate, purified, and quantified as previously described ing 2L of water from each fish tank using Nalgene [29]. Purified PCR amplicons were pooled in an equimo - Rapid-Flow Sterile Disposable Bottle Top Filters with lar mix and sent for library preparation and sequencing PES Membrane 0.45 µm (ThermoFisher Scientific, MA, using the Illumina NovaSeq 6000 S2 PE150 XP technol- USA). Aliquots of total RNA were used for cDNA syn- ogy at Eurofins Genomics Germany GmbH (Eurofins thesis with the Maxima H minus First Strand cDNA Syn- Genomic, Ebersberg, Germany). Raw paired-end reads thesis Kit (ThermoFisher Scientific, MA, USA), following were analyzed using the standard parameters of NG-Tax the standard protocol using random hexamer primers to 2.0 [66], with the exception of using 100 bp as the for- create cDNA. This cDNA was used for 16S rRNA gene ward and reverse read length, as implemented in Gal- profiling of the bacterial communities and the extracted axy [2], to obtain Amplicon Sequence Variants (ASVs). RNA was used for metatranscriptomic analysis. The Taxonomy was assigned to ASVs using the Silva_132 quantity, quality and purity of total RNA was determined database [67]. Two synthetic “mock communities” with Fig. 1 Experimental design. Fish were bred and raised as described in the section ‘Zebrafish and diets’. At 40 dpf ( juvenile stage) they were fed diet A for 1 week for acclimatisation to dry feed pellets. At 47 dpf, fish were randomly distributed into tanks and fed one of the diets (A, B or C) for 3 weeks. Survival and growth were measured before and during the whole experiment. After 1 week (54 dpf ) and after 3 weeks (68 dpf ) of feeding the fish. Gut samples were collected for histological, metatranscriptomic and microbiome analyses L ópez Nadal et al. Animal Microbiome (2023) 5:15 Page 5 of 21 Fig. 2 Combinatorial approach employed: total RNA was extracted from single zebrafish gut fed on different diets for both timepoints. Aliquots of total RNA were used for cDNA. For the 16S rRNA gene profiling, amplicon libraries of the V4 region of the 16S RNA gene were generated from the cDNA synthetized. NG-Tax 2.0 Galaxy was sued to obtain the ASVs. Several packages of R v4.1.2., Canoco v5.15 and Cytoscape v3.9.1 were used for results visualization. For transcriptomics, the cDNA libraries were sent to NovaSeq 6000 PE150 for sequencing. MetaPhlAn 3.0 (Beghini et al. 2021) and KneadData were used to trim the overrepresented sequences. Nf-core/rnaseq Nextflow pipeline was used for processing of the reads with the GRCz11 genome assembly. The results were visualized by R v.4.1.2, Canoco v5.15 and ErmineJ was used for the GO Enrichment analysis. The histological samples were extracted and embedded in paraffin and sectioned using a microtome. AB-PAS and HIC stains were automated. Samples were digitally scanned and an automated quantification of the histological parameters was performed using VIS v.2019.07 and Canoco v5.15 and GraphPad Prism v9.0.0 to visualize the results. The data integration was performed using heatmaps of normalized relevant parameters from all datasets, both timepoints and all diets known compositions were amplified and sequenced with Canoco v5.15 [9] using analysis type “constrained” as positive controls and a no-template control was also or “unconstrained”, respectively. Response variables were included as a negative control [68]. The distribution of log-transformed with the formula log(10,000*relative_ reads per sample and the variance in ASVs were assessed abundance + 1). RDA p-values were determined through and Alpha- and Beta-diversity measurements were per- permutation testing (500 permutations). Boxplots were formed using R v4.1.2 and RStudio [43], using packages generated using Prism v.9.0.0 (GraphPad Software, San ggplot2, [89], ape, [61], plyr, [93], vegan, [59], RColor- Diego, California USA). Cytoscape v3.9.1 [75] was used Brewer, [58], reshape2, [90], scales [91], data.table, [19], to visualize the diet-specific co-occurrence of ASVs based microbiome, [42], dplyr, [92], phyloseq, [55], ggdendro, on their relative abundances. Additional data handling [84] and DT [95]. The analysis yielded 17,203,234 high- and format conversions were done in Python (https:// quality reads. We excluded one sample (54 dpf butyrate www. python. org/). diet) because it had 2 reads only and we kept all the other samples (> 30.000 reads). Rarefaction curves for Zebrafish gut transcriptome analyses all samples reached a plateau, indicating that sufficient Total RNA (n = 5 diet/timepoint) was sent to Novogene sequencing depths was achieved (data not shown). For (Cambridge, UK), where quality control was done, rRNA the calculation of alpha-diversity indices, data was rare- was depleted and the metatranscriptome libraries were fied against the sample containing the lowest number of prepared. Paired-end reads were generated by NovaSeq reads (31,814 reads). Redundancy analysis (RDA) and 6000 PE150. For the host reads we used nf-core/rnaseq principal component analysis (PCA) were performed Nextflow pipeline [21] and the zebrafish (Danio rerio) López Nadal et al. Animal Microbiome (2023) 5:15 Page 6 of 21 genome assembly GRCz11 (NCBI) and according to the USA, diluted 1:300) antibodies to study proliferating cells MultiQC reports generated, the quality check param- (epithelial renewal) as well as NK-like cells and T cells. eters were satisfactory for all samples. “Salmon” was Samples were scanned at 20× magnification using Pan - used to quantify the expression of the transcripts [62] noramic SCAN II (3DHISTECH, Budapest, Hungary) to and DEseq2 [52], ggplot2, [89], scales [91], viridis [24] in produce digital whole slide images and analyzed using RStudio to investigate the differentially expressed genes Visiopharm v. 2019.07 image analysis software (Visiop- (DEG) in our diet treatments and timepoints. PCA analy- harm, Hoersholm, Denmark). Specialized automated ses were performed in the Canoco v5.15 software suite image analysis protocols were developed for each stain- (v5.02, [9]. Gene Score Resampling (GSR) analyses was ing type. Before employing the quantitative histology, tis- performed ErmineJ (v3.1.2) [27] with the annotation file sue regions were manually defined on the images to select of zebrafish (Danio rerio; genome assembly GRCz11) representative tissue (avoiding artefacts). The automated generated by Gemma [97]. GSR used DEG scores from analysis was then preformed only within those regions. DEseq2 from all genes in the dataset and calculated a Making an automated protocol involves selecting pre- p-value for each Gene Ontology (GO) term. The fold- processing steps, such as median filters to reduce noise change of each GO term across dietary interventions was and enhance structures, training the Bayesian classifier calculated by collapsing individual transcripts per million algorithm by annotating examples of the image back- (tpm) of each gene to the belonging GO term(s). Differ - ground, tissue, and target cells, utilizing post-processing entially expressed GO terms were visualized as a network steps based on shape, size and pixel colour to enhance by using Cytoscape v3.9.1 [75]: the nodes contained the the final image segmentation, and define calculations to fill depicting the log2 fold change (FC) of the control vs give the output data (area, counts, and perimeters). This butyrate at T2 and the border depicting the log2 FC of method for image analysis allowed us to perform quan- the control vs saponin at T2. The nodes with an abso - titative histology which differs from the commonly used lute FC ≥ 0.5 and p value ≤ 0.1 between dietary interven- semi-quantitative scoring. The latter involves a patholo - tions were taken into account. The edges connected the gist ascribing a subjective scoring with ordinal data, relevant nodes if the GO term contains at least 10 genes which is strongly operator-biased and time consuming. and shared at least half of them with the connecting GO Quantitative histology is automated, more detailed, thor- term(s) with FC ≥ 0.2 and p value ≤ 0.05 between dietary ough, and consistent, producing numerical data that can interventions. All data and files used to generate these detect subtle differences between states. The cell types visualisations can be found in Additional file 9. imaged were mucus (goblet) cells, PAS + cells (granulo- cytes), rodlet cells; PCNA + cells (proliferative cells) and High‑throughput quantitative histology Zap-70 cells (NK-like and T lymphocytes) [56]. The histo - At 54 dpf and 68 dpf zebrafish were euthanized in buff - logical parameters quantified were as follows. Absorptive ered MS222 overdose [88] 250 mg/L Tricaine (Sigma- capacity (AC): which was formulated as (interface length Aldrich, DL, United States). Intestines were removed, between mucosa and lumen / interface length between rinsed in PBS, placed in 4% paraformaldehyde over- serosa and exterior of the gut). Cell area fraction (%) (tis- night and transferred to 70% ethanol on the next day. sue area made up of cells) formula: area cell type A/total After subsequent dehydration steps, total intestines tissue area *100. Cell density: cell number/total tissue were embedded in paraffin blocks. Five-micrometer sec - area. Cell size: area of cells/cell number. Cell distance: the tions were stained with one of the following: hematoxy- distance of the cells from the outer serosal layer, where a lin and eosin (H&E) or Alcian blue periodic acid-Schiff higher distance would indicate a cell migrating towards (ABPAS) as previously described in [11] or by immuno- the mucosal fold (villus) end towards the lumen. The histochemistry (IHC). For the latter, antigen retrieval was imaging and the histological quantification were per - performed using the PT Link automatic antigen retrieval formed at the facilities of Skretting Aquaculture Innova- machine (Dako Agilent, CA, USA): samples were placed tion (Skretting, Stavanger, Norway). Further downstream into citrate buffer pH 6.1 (Dako Agilent, CA, USA) at processing of the multivariate analysis was performed by 60 °C, heated to 97 °C in 20 min, kept at 97 °C for 20 min, using Canoco v.5.12 (v5.02 [9], using principal coordinate and cooled down to 60 in 20 min. Samples were stained (PCoA) and Redundancy (RDA) analyses, performing using an automated staining machine (Autostainer Link analysis type “unconstrained” and “constrained”, respec- 48, Dako Agilent, CA, USA) with anti-proliferating cell tively. Response variables (histological parameters) were nuclear antigen (PCNA mouse mAb Clone PC10, M0879, scaled (0–1) and biplots were generated. RDA p-values Dako A/S, Denmark, diluted 1:10.000) or with anti-Zeta were determined through permutation testing (500 per- chain of T cell receptor associated protein kinase 70 mutations). Boxplots were generated using Prism v.9.0.0 (ZAP70 Rabbit mAb 99F2, Cell Signaling Technology (GraphPad Software, San Diego, California USA). L ópez Nadal et al. Animal Microbiome (2023) 5:15 Page 7 of 21 Complete pipeline of the combinatorial approach: omics the Phylogenetic Diversity slightly increased for butyrate and quantitative histology fed fish overtime (Fig. 3). Fluorescent in vivo imaging experiment A principal component analysis (PCA) analysis shows Adult Tg (mpeg1:mCherry/mpx:eGFPi ) were housed that time (from 54 to 68 dpf ) explains ~ 17% of the vari- and fed as previously described [49]and embryos ation observed in the microbial communities (x-axis obtained by natural spawning and raised with E3 water Fig. 4A). To analyze the effect of diet on the microbial (0.10 mM NaCl in demineralized water, pH 7.6) in petri communities, we performed redundancy analysis (RDA) dishes at 28 °C (12/12-h light/dark cycle) [88]. Larvae separately for each timepoint. After one week on the dif- were randomly distributed in 6 well plates (n = 20 fish/ ferent diets (54 dpf), the gut microbiota composition well) and exposed to different concentrations [0.005, was not significantly different between the different diet 0.01 mg/ml] of butyrate and [0.5, 0.7 mg/ml] saponin groups (p = 0.24) (Additional file 3: Fig. S3), whereas, dissolved in E3 water (10 ml solution/well) from 3 to 6 after prolongation of the diet intervention (3 weeks, 68 dpf. Larvae were anaesthetized and in vivo imaged as dpf ) a significant association between the diet and the previously described in [49]. Pictures were analyzed with gut microbiota was detected (p = 0.018). The top-15 most ImageJ software (United States National Institutes of discriminant genera associated with the diet induced Health, Bethesda, United States): the intestinal area was microbiota difference were further investigated (Fig. 4B), selected manually for each fish from the bright field and revealing that these genera were absent in all fish after copied to the other channels and fluorescent cells quan - one week on the distinctive diets (Additional file 4: Fig. tified and boxplots were generated using Prism v.9.0.0 S4 and Additional file 5: Fig. S4 and Additional file 5: Fig. (GraphPad Prism Software, San Diego, California, USA). S4_2). This finding implies that short term diet exposure (1 week) is insufficient to elicit the diet induced micro - Results biota changes. The relative abundances of the most Butyrate and saponin diets did not affect survival nor fish discriminating genera of the gut samples were consist- growth ently zero or extreme low except for Rhodobacter and All of the fish survived the dietary intervention and fish Pseudomonas (Additional file 4: Fig. S4), indicating that growth was comparable regardless of the diet provided microbiota fluctuations in the zebrafish gut were not (Additional file 1: Fig. S1). The water quality indicators influenced to a larger extend by the surrounding water measured: water pH, water conductivity (µS/m), nitrite microbiota composition. RDA of the genera composi- − + − (NO in mM), ammonia (NH in mM), nitrate (NO tion at 68 dpf associated ZOR006 and unclassified Des - 2 4 3 − 2+ in mM), chlorine (Cl in mM), general hardness (Ca ulfovibrionaceae with fish fed a control diet, whereas 2+ and Mg per volume of water) and carbonate hardness associated Mycobacterium, Vibrio, Aeromonas and Meth- (CaCO and MgCO per volume of water) were consist- ylobacterium with fish fed a saponin-supplemented diet 3 3 ently within the recommended range (Additional file 2: and associated Flavobacterium, unclassified Sutterel - Fig. S2). Moreover, water quality indicators remained laceae, Bacteroides, Pandoraea, Rhodobacter, unclassified constant during the whole experiment, indicating that Barnesiellaceae and Plesiomonas with fish fed butyrate- the diet-related changes described below result from supplemented diet. The relative abundances of the most the dietary intervention and not from differences in fish discriminative genera detected by the RDA (Fig. 4B, sub- growth rates or fluctuations in water quality. set of boxplots around the RDA) together with the heat- map of the relative abundances of most important taxa Butyrate‑ and saponin‑supplemented diets altered gut (Additional file 6: Fig. S5) demonstrated distinct micro- microbiota composition over time bial profiles associated with butyrate and saponin-sup - Ten gut samples per diet per timepoint were used to plemented diets. determine prokaryotic community composition based on amplicon sequencing of 16S rRNA. The samples yielded Butyrate reduced taxa connectivity in the zebrafish gut 17,203,234 high-quality reads, with an average of 286,720 After assessing distinct microbiota composition due to reads per sample, ranging from 31,814 to 577,719. The diets after 3 weeks of feeding (68 dpf ), taxa connectivity reads resulted in 579 amplicon sequence variants (ASVs) was analyzed by network analyses of co- and anti-occur- which were reduced to 204 ASVs after filtering out the rence of each pair of taxa at 68 dpf, based on the relative ones occurring in ≤ 2 counts. Alpha-diversity indexes for abundances (Fig. 5). The gut microbiota in fish fed the richness (observed ASVs and Chao1) and diversity (Shan- control diet presented a higher degree of taxa connec- non, Inverse Simpson, Fisher and Phylogenetic Diversity) tivity when compared to the gut microbiota of fish that within the samples did not reveal any significant differ - were fed either the butyrate- or saponin-supplemented ences between diets and timepoints for all samples. Only diet. Quantification of pairs of taxa with significant López Nadal et al. Animal Microbiome (2023) 5:15 Page 8 of 21 Fig. 3 Alpha-diversity indexes for richness (observed ASVs and Chao1) and for diversity (Shannon, Inverse Simpson, Fisher and Phylogenetic Diversity). *p ≤ 0.05, Ordinary one-way ANOVA after confirming normally distributed data by Shapiro–Wilk test. Whiskers: min. to max. shall all points with median connectivity were compared using the cumulative fre- taxa connectivity from 54 to 68 dpf and not saponin and quency histogram (p < 0.05; p.log < 1.30, Additional butyrate fed fish (Additional file 7 : Fig. S6B). file 7: Fig. S6A), showing an increase of connecting pairs of taxa in the control fed fish compared to sapo - Gut transcriptome analysis reveals unique and shared nin and butyrate fed fish at 68 dpf. These differences in effects of butyrate and saponin taxa connectivity were not present after 1 week of feed- After observing substantial dietary induced differences ing (54 dpf ) and occurred exclusively after 3 weeks of in bacterial composition and taxa connectivity, transcrip- feeding (68 dpf ) where only control fed fish increased tome profiles of the same zebrafish gut samples were ana - lyzed. This analysis (pipeline described in Fig. 2) resulted (See figure on next page.) Fig. 4 A Principal component analysis exploring the interaction of diet and time. The x axis separates the samples after 1 week feeding (54 dpf ) from samples after 3 weeks feeding (68 dpf ) and explains 16.87% of the variation observed. B Redundancy analysis of samples after 3 weeks of feeding (68 dpf ), the x axis separates saponin from butyrate fed fish and explains 7.94% of the microbial differences observed and the y axis separates the control from the saponin fed fish and explains 3.45% of the microbial differences observed. The microbial communities changed significantly due to diets (p = 0.018). The relative abundance of the most discriminative genera are depicted with boxplots around the RDA. In both analyses, the top 15 most distinctive genera are represented with black arrows. The direction of the arrows correlated with the dietary treatments and the timepoints and their length correlate with the strength of the correlation. **p ≤ 0.01, *** p ≤ 0.005 one-way ANOVA test or Kruskal–Wallis test after testing for normality on data distribution by Shapiro–Wilk test. No false discovery rate performed. Whiskers: min. to max. shall all points with median L ópez Nadal et al. Animal Microbiome (2023) 5:15 Page 9 of 21 Fig. 4 (See legend on previous page.) in a total of 47,046 genes expressed in transcripts per 3 weeks of feeding (68 dpf ) and not after 1 week of feed- million (tpm). Within-group transcriptome differences ing (54 dpf ). At 68 dpf fish fed the butyrate diet presented across diets and timepoints revealed a significant differ - more significantly homogeneous gut transcriptomic ence of dissimilarity of the transcriptomic samples after López Nadal et al. Animal Microbiome (2023) 5:15 Page 10 of 21 Fig. 5 Taxa connectivity: taxa included when prevalence is ≥ in 3/10 samples, abundance is ≥ 10 counts in 1 M and significance ≤ 0.1. The lines inform about the nature of the taxa interaction: the thickness of the lines represents the strength of the correlation (r-score value) and the shape of the lines represents the direction of the correlation, straight lines mean positive correlation (co-occurrence) whereas dashed lines mean negative correlation of the pairs of taxa (anti-occurrence). A Pairs of taxa co- and anti-occurring for all the diets: in black the interactions occurring in all three diets whereas in grey the interactions not occurring in all diets. Node size corresponds to average abundance of taxa for all diets at 68 dpf. B In red the interactions occurring in the control fed fish and not in the other two diets. Node size corresponds to average abundance of taxa for control diet at 68 dpf. C In green the interactions occurring in the butyrate fed fish and not in the other two diets. Node size corresponds to average abundance of taxa for butyrate diet at 68 dpf. D In yellow the interactions occurring in the saponin fed fish and not in the other two diets. Node size corresponds to average abundance of taxa for saponin diet at 68 dpf profile than fish fed the control and the saponin diets 893 GO terms while significantly up-regulated 40 GO (Fig. 6A). terms out of a total of 6111 GO terms (Additional file 9). Since the transcriptomic profiles were most dissimilar The transcriptomic network depicts a shared down-reg - after 3 weeks of feeding the unique and shared effects of ulation of the transcription and mitotic processes as well butyrate and saponin on the host gut were examined by as histone acetylation and histone methylation, that is creating a transcriptome network analysis (Fig. 6B, all most prominently observed in butyrate fed fish (Fig. 6B). raw data in Additional file 9). Compared to control fed Compared to control fed fish, both butyrate and sapo - fish, butyrate and saponin significantly down-regulated nin up-regulated the carboxylic catabolic processes, the L ópez Nadal et al. Animal Microbiome (2023) 5:15 Page 11 of 21 Fig. 6 Eec ff ts of butyrate and saponin on the host gut transcriptome. A Bray–Curtis distances to examine the dissimilarity of the host transcriptome across diets and timepoints. ** p ≤ 0.01, Kruskal–Wallis test after testing for non-normally distributed data by Shapiro–Wilk test. Whiskers: min. to max. shall all points with median. B Network depicting transcriptomic regulation of butyrate and saponin supplemented diets vs control diet at 68 dpf. Each node is a GO term and the node border represent the log2 fold-change of the control diet vs the saponin supplemented diet and the node fill represent the log2 fold-change of the control diet vs the butyrate supplemented diet. The edges connect nodes containing at least 10 genes and sharing 50% of the contained genes. Related GO terms are encircled encompassing canonical pathways. Shared effects on the gut transcriptome can be observed when edge and fill of a node have the same color in the network: up-regulation -in red- and down-regulation -in blue- compared to the control feed. C Immune response-associated GO terms and particularly inflammatory response analysed in fish fed a control, butyrate and saponin diet. Genes are expressed in tpm and scaled colored per individual gene value. The heatmap contained genes color-scaled per individual gene that reflect the individual within group fish-to-fish variation López Nadal et al. Animal Microbiome (2023) 5:15 Page 12 of 21 Fig. 6 continued oxidoreductase activity, response to estradiol and the activity which were down-regulated in butyrate fed fish immune response although some specific GO terms (Fig. 6B). G-protein receptor activity is the GO term within these processes present differential modulation category that shows the strongest opposite regulation (Fig. 6B). between saponin (up-regulated) and butyrate (down-reg- Compared to control fed fish, saponin up-regulated ulated) and encompassed GO terms associated to pho- 37 GO terms that were down-regulated in butyrate fed toreceptor activity, serotonin receptors activity as well fish while butyrate up-regulated 79 GO terms that were as synaptic signaling (Fig. 6B). To explore the immune- down-regulated in saponin fed fish (Additional file 9). related effects of butyrate and saponin, the immune Saponin up-regulated genes associated to GTPase activ- response of the transcriptome network was zoomed in ity, potassium channel activity and G-protein receptor on (Fig. 6C). In particular, the GO term “inflammatory L ópez Nadal et al. Animal Microbiome (2023) 5:15 Page 13 of 21 response” was examined for butyrate and saponin fed week of exposure) of the fish fed the butyrate diet by fish. Compared to controls, saponin and butyrate up- increased eosinophils, rodlet cells and a decrease of the regulated genes involved in the “inflammatory response” AC. The inflammatory condition remain unresolved after associated to chemokine activity as well as leukocyte and 3 weeks of feeding (68 dpf ) as the fish fed the butyrate innate cell recruitment (ccl19b, ccl25b, csfr1ra, cxcl18b. diet still presented increased eosinophils and rodlet cells cxcl19, cxcl8a, cxcl8b.1, cxcl8b.3, fpr1, mpx, mst1 and tlr- and a depletion of mucus cells compared to saponin and family) (Fig. 6C). control fed fish (black arrows and boxplots Fig. 7C, rep- resentative pictures Fig. 7A). Gut quantitative histological analysis depicted distinct gut architectural profiles for butyrate and saponin Combinatorial approach reveals distinct profiles The zebrafish gut samples collected were analyzed using for saponin and butyrate fed fish high-throughput quantitative histological analysis. While In order to define robust and multi-parameter supported microbiota and transcriptomic data showed differential effects of butyrate and saponin supplementation, the key as well as similar effects of the butyrate and the saponin findings of the different datasets were integrated in a supplementation, the tissue make-up and topography heatmap (Fig. 8). Control fed fish did not present extreme provided further insight on whether changes in gene microbiota fluctuations over time. Butyrate fed fish pre - expression and microbiota also coincide with morpho- sented the most divergent microbiota composition (with logical indications of disturbed intestinal host gut health. increased relative abundance of Bacteroides, Rhodobac- Whole images were obtained from scanned slides and ter, Pandoraea and Flavobacterium) and the lowest taxa quantification was automated for several parameters: the connectivity compared to the other diets, which might AC and the cell area fraction, cell density, cell size, and the be indicative of disturbed ecosystem stability. Saponin distance of each individual cell to the outer serosal layer fed fish presented an increased number of Vibrio con - for cell lineages of particular interest, including mucus trasting with butyrate fed fish. Compared to control fish, cells (goblet cells), eosinophils (PAS + granulocytes), rod- butyrate and saponin shared an increased expression of let cells (PAS +), proliferative cells (PCNA + cells) and T genes associated to immune responses, inflammatory and NK-like cells (Zap70 + cells). Representative pictures responses and oxidoreductase activity. Besides, butyrate of all cell-types and time-points are shown in Fig. 7A. fed fish presented down-regulated genes in GO terms The effect of time did not correlate to any of the his - associated with histone acetylation, histone methyla- tological parameters analyzed (black arrows in the RDA tion, mitotic processes and G-protein coupled receptor graph, Fig. 7B, p value = 0.066) except for the increase activity. These differential gene expressions patterns were of the PCNA area over time, indicative that the relative stronger after 3 weeks of feeding (68 dpf ) compared to number of proliferative cells increased during fish devel - 1 week of feeding (54 dpf ). In terms of histology, after opment. Significant differences on the histological gut 1 week of feeding butyrate, fish already showed increased parameters were found due to the dietary interventions area of eosinophils and rodlet cells compared to sapo- (p = 0.036) (Fig. 7C). The absorptive capacity of the fish nin or control fed fish, which is consistent after 3 weeks gut was decreased for the butyrate fed fish compared of feeding. Butyrate fed fish at 68 dpf showed decreased to saponin and control fed fish at 54 dpf, although dis - area of mucus cells compared to control fed fish. The played similar values at 68 dpf (boxplots around Fig. 7C). histological parameters for saponin fed fish appeared to The area of the eosinophils and rodlet cells increased be less pronounced than those of butyrate fed fish. Col - in butyrate fed fish compared to saponin and control lectively, these observations, showed fish fed a butyrate fed fish after 1 week of feeding (54 dpf ), suggesting an supplemented diet elicited a stronger response in terms inflammatory condition which was partly alleviated but of changes in the microbial composition, expression of not fully resolved at 68 dpf. In addition, compared to genes associated to immune activation processes as well controls, fish fed the butyrate diet showed a clear mucus- as the presence of (pro)inflammatory-like cells such as producing cell depletion after 3 weeks of feeding (68 dpf ). eosinophils and rodlet cells and depletion of mucus cells. Saponin fed fish presented a reduced proliferative cell area compared to butyrate and control fed fish. To illus - Butyrate and saponin increased neutrophil trate these differences in histological parameters, accept - and macrophage recruitment in the gut of zebrafish larvae ing the biological fish to fish variation within each group, Since the data clearly indicated an unexpected induction a heatmap of each individual fish and all the histological of immunity related functions upon butyrate addition to parameters per each RDA axis was generated (Additional the feed (Fig. 6B), the advantages of the zebrafish model file 8: Fig. S7). The combination of these observations system were used to validate the results by in vivo imag- suggested an acute inflammatory response (after one ing of fluorescently labeled neutrophils and macrophages López Nadal et al. Animal Microbiome (2023) 5:15 Page 14 of 21 Fig. 7 High-throughput quantitative histological analysis. A Representative pictures of all cell-types analyzed for all the diets and timepoints with cells of interest in dashed black lines per each group. B Redundancy analysis to examine the effect of time on the histological parameters analyzed. The x axis separated the samples by timepoints and explained 5.76% of the variation observed. The link of time and variation of the histological parameters was not significant (p = 0.066) C Redundancy analysis to examine the effect of diet on the histological parameters analyzed. The x axis separated the samples of butyrate fed fish from saponin and control fed fish and explained 5.86% of the variation explained. The y axis separated saponin fed fish from control fed fish and explained 5.07% of the variation observed. The link of time and variation of the histological parameters was significant (p = 0.036). The top 10 most distinctive histological parameters are depicted in black arrows. The direction of the arrows correlate with the dietary intervention and the length of the arrows represents the strength of the correlation. The boxplots around the RDA depicted the absorptive capacity and the percentage area of cells of interest compared to the total gut area per diet and timepoint. *p ≤ 0.05, one-way ANOVA test or Kruskal–Wallis test after testing for normality on data distribution by Shapiro–Wilk test. No false discovery rate performed. Whiskers: min. to max. shall all points with median L ópez Nadal et al. Animal Microbiome (2023) 5:15 Page 15 of 21 Fig. 7 continued upon butyrate and saponin exposure in zebrafish larvae. of genes associated with immune response together Double Tg(mpeg1:mCherry/mpx:eGFPi ) zebrafish with an increased eosinophil and rodlet cell presence larvae were exposed to butyrate and saponin in differ - and mucus-producing cell depletion in the gut tissue. ent doses for 3 days (3-6dpf ) and were (in vivo) imaged Moreover, butyrate increased the neutrophil and mac- at 6dpf. Fish treated with butyrate as well as saponin rophage in vivo recruitment to the gut area in transgenic presented a dose-dependent increase of neutrophils and zebrafish larvae. Zebrafish fed a saponin-supplemented macrophages in the intestinal area (Fig. 9A). The quan - diet showed differentially modulated microbial composi - tification of the cells present in the gut area showed that tion from butyrate and low taxa connectivity as well as butyrate as well as saponin significantly increased neu - increased expression of immune response while the his- trophils and macrophages in the gut of zebrafish larvae tological parameters comparable to control fed fish. The (Fig. 9B). combinatorial approach of bacterial microbiome profil - ing, host gut transcriptomics, automated high-through- Discussion put quantitative histology (novel in zebrafish research) In the present study the effects of butyrate and saponin- together with in vivo innate cell recruitment in the gut supplemented feed in the zebrafish gut were assessed area in zebrafish larvae revealed evidence of the pro- following a combinatorial approach that integrates sev- inflammatory effects exerted by butyrate supplementa - eral datasets and validating the results by in vivo imag- tion which were partly shared with the well-establish ing. Juvenile zebrafish fed a butyrate-supplemented feed pro-inflammatory saponin supplementation, indicating for 3 weeks presented a modulated microbial compo- detrimental effects of butyrate in the zebrafish intestinal sition and low taxa connectivity, increased expression milieu. López Nadal et al. Animal Microbiome (2023) 5:15 Page 16 of 21 Fig. 8 The heatmap brings together the main observations of each analysis and compare them per diet and timepoint. The more representative genera are illustrated with the average relative abundance per timepoint and diet. The taxa connectivity contained the amount of pairs of taxa that correlate to each other in a significant fashion ( p ≤ 0.05). The GO terms contain the transcripts per million (tpm) of all genes expressed in the dataset that collapsed under that GO term. All histological parameters are normalized and scaled (from 0 to 1). Each individual feature within the heatmap is normalized and colored from red (more present) to white (absent) While saponin and soybean meal have been consist- the host during a bacterial challenge [65]. No baseline ently associated with gut inflammation in several fish studies of butyrate-supplemented feed are reported in species, including carp [80, 81, 96], salmon [5, 32, 37, 40, the (zebra)fish literature to our knowledge. In the present 80, 81] and zebrafish [30, 49], butyrate has been reported study, we used for the first time a butyrate-supplemented to convey beneficial effects when supplemented to fish diet for zebrafish and our combinatorial approach feed such as intestinal growth enhancement [71] and showed that 0.01% inclusion of butyrate induced a immunostimulant and antioxidant properties [20, 47], pleiotropic damaging response comparable to the well- (reviewed in [1]. However, previously published stud- establish pro-inflammatory anti-nutritional factor soy ies with histological and gene expression redouts com- saponin. In mammals, colonocytes located along the gut pared butyrate supplementation to other challenges such crypts take up the butyrate produced by the microbiota, as high concentration of plant-based meal or a patho- preventing high concentrations of this SCFA to reach gen challenge. As a matter of fact, butyrate-supplemen- the proliferating stem cells at the bottom of the crypts. tation effects depended on co-treatment(s) employed In fact, high concentrations (1.5–2 mM) of butyrate and duration of the feeding intervention. For instance, were shown to be toxic to mouse pluripotent stem cells 0.8% inclusion of sodium butyrate in a low percentage in vitro [45]. This is especially relevant in cryptless organ - plant-containing diet in gilthead sea bream for 10 weeks isms such as fish [3, 41, 82], where higher concentrations resulted in a mild inflammatory reaction whereas in the of butyrate can reach the stem cells localized between same study, 0.4% inclusion of sodium butyrate in high the intestinal folds (villi). Mechanistic studies in mouse percentage plant-based diet for a longer period protected and zebrafish larvae suggested that butyrate at high L ópez Nadal et al. Animal Microbiome (2023) 5:15 Page 17 of 21 Fig. 9 A Representative pictures of the fluorescent in vivo imaging of the gut area of Tg(mpeg1:mCherry / mpx:eGFPi ) larvae exposed to control media, 0.005 mg/ml and 0.01 mg/ml butyrate and 0.5 mg/ml and 0.7 mg/ml saponin. B Quantification of neutrophils and macrophages in the gut area of the zebrafish larvae (n = 10 in all groups except 0.7 mg/ml saponin where n = 3). *p ≤ 0.5, **** p ≤ 0.0001 Kruskal–Wallis test after testing for non-normally distributed data by Shapiro–Wilk test. Whiskers: min. to max. shall all points with median concentrations inhibits stem cell proliferation via FoxO3 composition, more information might be extracted in cryptless organisms such as zebrafish [25]. Taking from the analysis of microbial networks. For example, these observations together, the butyrate-supplemented in inflammatory bowel disease patients, topological diet showed a compromised intestinal epithelial barrier properties of the co-occurring bacterial networks iden- function, coinciding with a disrupted microbiota com- tified anti- and pro-inflammatory key organisms that position with decreased taxa connectivity, and increased defined the degree of structure of the ecosystem [6]. expression levels of genes associated with inflammatory In fish, recent studies validated the usage of co-occur - and immune responses. The inflammatory response due rence and anti-occurrence taxa networks to identify the to butyrate was confirmed by enhanced innate immune core gut European seabass microbiota [38] as well as to cell recruitment in vivo to the zebrafish gut. Our data reveal microbial interactions due to prebiotics and pro- warrants that further research should investigate the long biotics [53]. In the present study, zebrafish fed 3 weeks term effects of butyrate-supplemented feed and suscep - a butyrate-supplanted feed presented altered the micro- tibility towards infectious or inflammatory challenges biota composition as well as reduced taxa connectivity which were not investigated here. Potentially, butyrate- (co- and anti-occurrence) compared to control (and to associated immuno-stimulation early in life, could boost a lesser extent to saponin)-fed fish (Figs. 4, 5 and Addi- immunity and strengthen disease resistance in later life tional file 7: Fig. S6). We had 6 tanks, 2 per each diet stages (trained immunity) [64]. and 1 tank per diet was sampled after 1 week of feeding Disruption of the gut microbiota homeostasis, often intervention and the other after 3 week of feeding inter- caused by an imbalance in the microbial community (or vention. This could have an effect on the sample clus - dysbiosis), is commonly associated with inflammatory tering of the fish microbiota. However, the water from conditions in the zebrafish gut [8, 11] also (reviewed in the recirculating system was the same for all tanks and [12, 50]). However, whether these disruptions of micro- its quality remained comparable in all tanks across the bial community cause gut inflammation in a direct man - study (Additional file 2: Fig. S2). Furthermore microbiota ner or via disruptions in the microbiota is a matter of composition of the water samples of the fish tanks were discussion and current research in the fish immunity comparable among themselves and very different from and nutrition field. Next to investigating the community the gut samples (Additional file 4: Fig. S4 and Additional López Nadal et al. Animal Microbiome (2023) 5:15 Page 18 of 21 file 5: Fig. S4_2) suggesting that the differences in micro - IL8 did not affect human eosinophils in vitro [63], eosino - bial communities found across dietary treatments did phils are able (via granule proteins) to stimulate neutrophils not originate due to the separate environments per treat- that produce IL8 and superoxide contributing to gastroin- ment. Butyrate increased the relative abundance of the testinal pathologies [72]. However, eosinophil research in genera Rhodobacter, Flavobacterium and Bacteroides that the context of gastrointestinal health is limited in humans were previously associated with gut inflammation in fish and mice [34] as well as (zebra)fish [7 ]. Butyrate increased [51, 78, 94] whereas saponin increased the relative abun- eosinophil and rodlet cell area even after 1 week of feeding, dance of the Vibrio genus, which contains several patho- while reduced the presence of mucus cells overtime (Fig. 7), biont species which might become pathogenic upon features associated with (chemically-induced) intesti- challenge of the gut barrier integrity (reviewed in [15]. nal inflammation in zebrafish [11]. Rodlet cells were first In mammals, butyrate is produced by fermenting bac- reported to act against fish parasites and later studies dis - teria in the intestinal tract and until now scientists were closed their granulocyte nature and include them as part of not able to measure any naturally occurring concentra- the innate fish immune system, increasing in number when tions of butyrate in the zebrafish gut [14]. Since it is not exogeneous stressors were present [18, 33, 54, 69]. More certain whether fish gut may produce butyrate, exogene - research into this well-known but often forgotten cell type ous butyrate supplementation may disrupt the growth of may elucidate its role in (zebra)fish mucosal immunology. bacteria since they may not be used to metabolize such To reinforce the observation that saponin and butyrate substrate. In butyrate-fed fish increased abundance of recruited immune cells to the gut we used transgenic Bacteroides correlated with lower abundance of Vibrio. zebrafish larvae to in vivo visualize neutrophil and Interestingly, in vitro studies have revealed that butyrate macrophage presence in the gut. The fact that saponin exposure can negatively impact the colonization of spe- induced a stronger cell recruitment than butyrate could cific Vibrio campbellii PUGSK8 by its effect on biofilm be explained by the fact that lower concentrations of formation capacity in these bacteria [36]. Taken together, butyrate were used (mimicking the ones employed in the these findings warrant further studies to understand diets) (Fig. 9). Other studies showed decreased neutrophil the mechanisms by which butyrate influences microbial recruitment after tail wounding when zebrafish larvae ecosystems. were immersed to butyrate [14]. However, such studies Inflammatory-associated taxa in butyrate-fed fish briefly immersed zebrafish larvae to extremely high con - matched with an increased expression of genes belonging centrations of sodium butyrate (30 mM = 3303 mg/ml) to inflammatory and immune responses (Fig. 8). While and such study design may greatly differ from the natu - targeted gene expression is commonly used in (fish) nutri - rally occurring physiological situation in the zebrafish tion studies, this approach is often hypothesis-driven and gut. We hypothesize that the increased chemokine the discovery risk of novel premises is relatively low com- expression in butyrate fed fish might be the driving force pared to more comprehensive transcriptome analyses. In for the increased leucocyte recruitment in the gut and the present study, butyrate down-regulated genes associ- further research may disclose specific butyrate modes of ated with mitotic and transcription processes which is in action in the (zebra)fish gut. line with the inhibition of stem cell proliferation previously In the present study butyrate-supplemented feed reported [25], although proliferative cells (PCNA +) were appeared to modulate the microbial composition as not decreased in butyrate-fed fish as shown by the histo - indicated by low taxa connectivity, increased expression logical dataset. Butyrate down-regulated genes associated of gene associated to inflammatory processes as well as with histone modifications (acetylation and methylation) in increased presence of rodlet cells, and eosinophils while line with previously described epigenetic effects of butyrate decreasing Goblet cells. Moreover, we supplemented this in mammals (reviewed in [31]. Further research may elu- data with in vivo observations of the increased recruit- cidate whether there is an effect of butyrate supplemented ment of the neutrophil and macrophage population in feed on epigenetic markers and in the affirmative case the gut upon butyrate and saponin exposure. The com - whether such epigenetic modifications can be passed on bination of these datasets indicate that butyrate has fish- the fish offspring. A clear subset of chemokines within the specific effects on the gut homeostasis that differ from inflammatory response appeared to be up-regulated after the mammalian counterparts [28]. The particular fish gut butyrate-supplemented feeding (Fig. 6C) among which structure, lacking intestinal crypts could play an impor- cxcl8a, cxcl8b.1 and cxcl8b.3. Cxcl8 (or il8) is known as one tant role on the absorption and the effect of the butyrate of the most potent chemoattractant molecules for recruit- on the epithelial lining where chemokines might orches- ing neutrophils (expressing CXCR1/2 receptors for Cxcl8) trate the inflammatory-like response. However, in the and other leukocytes upon inflammation [60]. Although present study butyrate absorption by the enterocytes in L ópez Nadal et al. Animal Microbiome (2023) 5:15 Page 19 of 21 the zebrafish gut has not been quantified and should be Additional file 4: Fig. S4_1. Relative abundances of the top 15 most addressed in future research. While more mechanistic distinctive genera for all diets at both timepoints, including water samples from all fish tanks at both timepoints. studies are needed to shed light on the specific modes Additional file 5: Fig. S4_2. Figure S4_1 continued. of action of butyrate on the fish gut health, the present combined study (omics, histology and imaging) provides Additional file 6: Fig. S5. Heatmap of the relative abundance (relative to 1) of the most distinctive and important taxa for all diets at the 2nd evidence to support non-beneficial effect of butyrate- timepoint. Importance was calculated as (sqrt( CorS1^2+CorS2^2)), i.e., supplemented feed on growing juvenile zebrafish. the length of the arrows in Figure 4B. In conclusion, combining several high throughput Additional file 7: Fig. S6. Normalized cumulative frequency histogram approaches we provide a more comprehensive and gran- depicting the amount of significant pairs of taxa correlations per each diet A) at 68 dpf, B) at 54 dpf; (dotted line represents logarithmic p value =1.30 ular view of the effects of dietary interventions on fish and p =0.05). gut health. Translation to aquaculture species is possible Additional file 8: Fig. S7. Heatmaps of each individual gut fish sample since our redouts do not depend on any species-specific (n=5 diet / timepoint) for both axis of the redundancy analysis. Despite antibodies. However, integration of multi-layered high- of the fish to fish variation present dietary effects are visible for both timepoints. Values are normalized and scaled from 0-1. throughput studies remain a challenge in fish because of various reasons. On the one hand, there are difficulties to Additional file 9: Gene expression of transcripts from zebrafish gut fed either a control, butyrate- or saponin-supplemented diets at 54 and 69 fully comprehend the connections between the complex dpf. layers of data deriving from high-throughput methods and the most relevant outcomes (fish health biomark - Acknowledgements ers). On the other hand, scientist may not have yet the The authors would like to thank Steven Aalvink and Ineke Heikamp-de Jong technology to adequately obtain multi-omics data with for extracting RNA and the PCRs for the molecular analyses and Trond Kortner to supply to us the 95% ultra-pure saponin. sufficient resolution (lack of noise) and reproducibility that facilitates omics datasets combination. In the pre- Author contributions sent study, the detrimental effects of butyrate towards ALN performed research, analyzed and integrated all data, drafted manuscript and figures. JB analyzed and integrated all data, drafted manuscript and the zebrafish gut were congruent throughout all the data - figures. CL and FvdB and TBE collected and analyzed histological data. MAS sets in our combinatorial approach strengthening the analyzed 16S and transcriptomics data and drafted manuscript. DP and CMcG biologically relevant observation that butyrate appears drafted and edited manuscript and developed and provided feeds. DS MK drafted and edited manuscript. GW and SB drafted and edited manuscript and detrimental to the zebrafish gut. Steps towards observa - provided the funding. All authors review the manuscript. All authors read and tional scientific studies with an integrative view, combin - approved the final manuscript. ing high-throughput datasets with imaging techniques to Funding understand complex multifactorial biological processes This study was funded by NWO-TTW Applied and Engineering Sciences such as fish gut health may help researchers to evaluate (Project Number 15566). novel diets for healthier fish generations. Availability of data and materials Raw data of the transcriptomic analyses can be found in Additional file 9. All Supplementary Information raw data will be uploaded to an open access repository. The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s42523- 023- 00230-2. Declarations Additional file 1: Fig. S1. Standard length (mm) was measured at 40, 54 Ethics approval and consent to participate and 68 dpf for the 3 diets by using a digital calliper. The present study was approved by the Dutch Committee on Animal Welfare Additional file 2: Fig. S2. Water quality values just before and during the (2017.W-0034) and the Animal Welfare Body (IvD) of the Wageningen Univer- experiment at 38, 45, 50, 56, 62 and 65 dpf. In green the range of prefer- sity ( The Netherlands). Furthermore, we adhered to standard biosecurity and able values for the measurements and in red the values above which institutional safety procedures at Wageningen University and Research. the water quality is considered to be detrimental for the fish according to manufacturer’s instructions: A) pH (accepted range 6.6-8.4), B) Water Consent for publication conductivity (accepted range 300-1500 µS/m), C) Nitrite (accepted range All authors revised and agreed on the submitted version of this manuscript. 0-7 mM), D) Ammonium (only 0 mM accepted), E) Nitrate (accepted range 0-70 mM), F) Chlorine (only 0 mM accepted), G) General hard- Competing interests ness (accepted range 2-16) and H) Carbonate hardness (accepted range The authors declare to have no competing interests. 1.5-10). Author details Additional file 3: Fig. S3. Redundancy Analysis (RDA) at the 1st timepoint Cell Biology and Immunology Group, Wageningen University and Research, to examine the effect of the diets on the gut microbiota. The x axis sepa- Wageningen, The Netherlands. Aquaculture and Fisheries Group, Wagenin- rates saponin form control fed fish and explains 5.82% of the microbial dif- gen University and Research, Wageningen, The Netherlands. Host-M icrobe ferences observed and the y axis separates the butyrate form the saponin Interactomics, Wageningen University and Research, De Elst 1, 6708 WD Wage- fed fish and explains 2.28% of the microbial differences observed. The top ningen, The Netherlands. Laboratory of Microbiology, Wageningen University 15 most distinctive genera are depicted as supplementary variables in and Research, Wageningen, The Netherlands. Skretting Aquaculture Innova- black arrows, p=0.24. tion, Stavanger, Norway. López Nadal et al. 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Publisher: Springer Journals
Copyright: Copyright © The Author(s) 2023
eISSN: 2524-4671
DOI: 10.1186/s42523-023-00230-2
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Abstract

Background Prebiotic feed additives aim to improve gut health by influencing the microbiota and the gut barrier. Most studies on feed additives concentrate on one or two (monodisciplinary) outcome parameters, such as immunity, growth, microbiota or intestinal architecture. A combinatorial and comprehensive approach to disclose the complex and multifaceted effects of feed additives is needed to understand their underlying mechanisms before making health benefit claims. Here, we used juvenile zebrafish as a model species to study effects of feed additives by inte - grating gut microbiota composition data and host gut transcriptomics with high-throughput quantitative histological analysis. Zebrafish received either control, sodium butyrate or saponin-supplemented feed. Butyrate-derived compo - nents such as butyric acid or sodium butyrate have been widely used in animal feeds due to their immunostimulant properties, thereby supporting intestinal health. Soy saponin is an antinutritional factor from soybean meal that promotes inflammation due to its amphipathic nature. Results We observed distinct microbial profiles associated with each diet, discovering that butyrate (and saponin to a lesser extent) affected gut microbial composition by reducing the degree of community-structure (co-occurrence network analysis) compared to controls. Analogously, butyrate and saponin supplementation impacted the tran- scription of numerous canonical pathways compared to control-fed fish. For example, both butyrate and saponin increased the expression of genes associated with immune response and inflammatory response, as well as oxidore - ductase activity, compared to controls. Furthermore, butyrate decreased the expression of genes associated with histone modification, mitotic processes and G-coupled receptor activity. High-throughput quantitative histological analysis depicted an increase of eosinophils and rodlet cells in the gut tissue of fish receiving butyrate after one week of feeding and a depletion of mucus-producing cells after 3 weeks of feeding this diet. Combination of all datasets indicated that in juvenile zebrafish, butyrate supplementation increases the immune and the inflammatory response to a greater extent than the established inflammation-inducing anti-nutritional factor saponin. Such comprehen- sive analysis was supplemented by in vivo imaging of neutrophil and macrophage transgenic reporter zebrafish (mpeg1:mCherry/mpx:eGFPi ) larvae. Upon exposure to butyrate and saponin, these larvae displayed a dose- dependent increase of neutrophils and macrophages in the gut area. *Correspondence: Sylvia Brugman Sylvia.brugman@wur.nl Full list of author information is available at the end of the article © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. López Nadal et al. Animal Microbiome (2023) 5:15 Page 2 of 21 Conclusion The omics and imaging combinatorial approach provided an integrated evaluation of the effect of butyrate on fish gut health and unraveled inflammatory-like features not previously reported that question the usage of butyrate supplementation to enhance fish gut health under basal conditions. The zebrafish model, due to its unique advantages, provides researchers with an invaluable tool to investigate effects of feed components on fish gut health throughout life. Keywords Microbiome, Transcriptome, Omics, Imaging, Zebrafish, Butyrate, Soy saponin, Gut, Inflammation Background inflammation to decipher the underlying diet-microbe- In the last decades, the implications of the microbiome host interactions in the zebrafish gut and to assess feed in human and animal health have gained interest among compounds that can potentially protect the gut from and beyond the scientific community. As a consequence, becoming inflamed [77]. Experimental designs of fish food ingredients able to modulate the microbiome, such feed studies are often based on specific outcome param - as prebiotics, became increasingly popular and accepted eters, including fish growth, expression of a limited set of among the general public and have been utilized in genes, plasma levels of antioxidants, semi-quantitative human dietary supplements as well as in animal feed [17, scoring of histological parameters, profiling of the gut 39]. A prebiotic is a substrate that is selectively utilized bacteria composition or pathogenic challenges. Habitu- by host microorganisms and thereby proposed to confer ally, only end-point analysis have been performed, ignor- a health benefit on the host (reviewed in [26]. ing the kinetics of the responses. Most of these studies Butyrate is a short-chain fatty acid (SCFA) derived base eventual gut health claims of dietary treatments on from fiber fermentation by the gut bacteria that exhibits one or two of these parameters. For instance, the anti- some prebiotic properties, playing a role in the interac- inflammatory properties of supplemented microalgae tion between bacterial population dynamics and host were assessed by only quantifying the neutrophils in the gut homeostasis [44]. Butyrate has a direct impact on zebrafish gut larvae [10]. The expression of a small set the immune system via signaling G-protein coupled of genes were proposed to support immune-boosting, receptors (GPCR) on epithelial and immune cells and anti-inflammatory and antioxidative stress properties of also induces epigenetic changes via regulation of histone phytates after soybean-meal feeding in zebrafish larvae acetylase and histone deacetylase enzymes (reviewed in [74]. Although quantitative assessment of health associ- [31]. In the last years, butyrate has been extensively used ated phenotypes is critical to support health claims, bas- in animal feed, including its supplementation to several ing these on the determination of a single or only few fish diets in the form of butyric acid or sodium butyrate parameters may lead to overstated conclusions. There - due to its growth-promoting, immuno-stimulating and fore, to appropriately assess and understand the complex antioxidative properties (reviewed in [1] and to mitigate and multifaceted effects of feed supplements on fish gut detrimental effects of sub-optimal plant-containing diets health more integrated and holistic approaches are war- [23, 48, 70, 83]. ranted (as reviewed in [50]). In order to achieve this, we Plant-based protein ingredients have been replac- propose that a combination of high-throughput readouts ing fish meal in feed due to their more favorable price may be employed to supply unbiased datasets that could and availability. However, several anti-nutritional com- lead to a detailed description of the host gut health status ponents derived from plant-based protein sources are upon feed interventions. reported detrimental for fish health (reviewed in [73]. For Many gut functions and immune genes are conserved instance, soy saponin is an anti-nutritional component between different species. Moreover, zebrafish larvae are of soybean meal that interacts with cell membranes and optically transparent and together with the development promotes pore formation, vesiculation and membrane of several transgenic fish lines that express fluorescent domain disruption [4]. Various studies linked the pres- proteins in specific cell-lineages facilitates in vivo track - ence of soy saponin to inflammatory responses in the ing of certain immune cells, which empowered the use of intestinal mucosa, enteritis as well as microbiota modula- the zebrafish model to examine intestinal inflammation tion in several fish species [13, 16, 40], including zebrafish (reviewed in [12] as well as a model organism to evaluate [49]. In zebrafish larvae, the number of neutrophils novel feeds for farmed fish [79]. However, the zebrafish increased in the gut after soybean meal feeding [30] or gastrointestinal tract presents several particularities. For exposure to soy saponin in solution [49]. After assessing example, zebrafish lack a stomach and instead employ the the inflammatory effect of soy saponin, soy-containing anterior gut segment, named intestinal bulb, as a reser- diets have been employed as a model for feed-induced voir for feed. Although this intestinal bulb lacks gastric L ópez Nadal et al. Animal Microbiome (2023) 5:15 Page 3 of 21 glands, it produces digestive enzymes and mimics what tank after 3 weeks of the feeding experiment per each may occur in the stomach [22, 57]. The zebrafish gut epi - diet. The feeding experiment was performed blind and thelial layer also lacks intestinal crypts that are typically fish were fed until slightly before satiation twice a day. found in other fish species or in mammals and rather Each tank received one of the following: a control diet, forms protrusions called folds that decrease in size from a saponin-supplemented diet or a butyrate-supplemented anterior to posterior gut segments [85]. Nevertheless, the diet. Full diet composition is listed in Table 1. canonical intestinal epithelial cells (IECs) such as entero- cytes, mucin-producing goblet cells and enteroendocrine Experimental design cells are present in the zebrafish gut. Moreover, zebrafish Water quality was set to standard values by replacing half gut segments presented analogous expression to their of the water in the zebrafish system before the start of mammalian counterparts [87] and when transcriptom- the experiment and monitored twice a week during the ics where performed on IECs from zebrafish, stickle - whole experiment (Additional file 1: Fig. S1). A pH meter back, mouse and human a highly conserved expression (Hanna Instruments, Nieuwegein, The Netherlands) was was found between zebrafish and mammals [46]. Like in used to measure the pH and the water conductivity. Kits many animal models for inflammation, mucus-producing to measure ammonium, nitrite and nitrate (Merck KGaA, cells (Goblet cells) decrease and granulocytes (mainly Darmstadt, Germany) were used according to manufac- neutrophils and eosinophils), macrophages and lympho- turer’s instructions. Additionally, nitrite, nitrate, general cytes increase upon inflammation in the zebrafish gut hardness, carbonate hardness, pH and chlorine were (re) [11, 49]. Due to its shared expression and functionality measured by using Tetra Test 6in1 (Tetra, Melle, Ger- the zebrafish is an excellent model to understand host- many) according to manufacturer’s instructions. Fish microbe-immune interactions.Research on feed supple- survival and standard length -from the tip of the head ments and their effect on gut health are usually based on until the bifurcation of the caudal fin- were assessed by few readouts parameters which may not reflect the com - using a digital caliper (Sylvac, Yverdon, Switzerland) dur- plexity of fish gut health. The main goal of this present ing the experiment (Additional file 2: Fig. S2). The dietary study is to provide an comprehensive investigation based intervention consisted of three diets identical in com- on the integration of several high-throughput readouts position except the supplementation with 1 g/kg feed of of the gut mucosa to depict a more holistic view of the sodium butyrate in the butyrate diet and 3.3 g/kg of 95% effects of feed supplements on zebrafish gut health. Material and methods Table 1 Formulation of experimental diets analysed once the feed intervention was performed to check which compositions Ethics statement corresponded to the blinded diets The present study was approved by the Dutch Commit - tee on Animal Welfare (2017.W-0034) and the Animal A: Control B: Butyrate C: Saponin diet (%) diet (%) diet (%) Welfare Body (IvD) of the Wageningen University (The Netherlands). Furthermore, we adhered to standard bios- Wheat 7.00 6.99 6.67 ecurity and institutional safety procedures at Wagenin- Wheat gluten 16.00 16.00 16.00 gen University and Research. Sunflower meal 1.68 1.68 1.68 Soy protein concentrate 15.16 15.16 15.16 Zebrafish and diets Fish meal 52.00 52.00 52.00 Adult double transgenic (mpeg1:mCherry/mpx:eGFPi ) Fish oil 4.40 4.40 4.40 expressing mCherry under the macrophage-specific Rapeseed oil 2.00 2.00 2.00 mpeg1 promotor and GFP under the neutrophil-specific Vitamin mix 0.35 0.35 0.35 mpx promotor fish were housed and fed as previously Mineral mix 1.92 1.92 1.92 described [49]. Embryos were obtained by natural spawn- Butyrate 0.00 0.01 0.00 ing. Fish were fed as follows: weeks 1 and 2 with rotifers Saponin 0.00 0.00 0.33 (× 4/day from 5 days post fertilization -dpf-), week 3 with [ VOLUME] 100.0 100.0 100.0 rotifers and Artemia Nauplii 230.000 npg (Nauplii per Dry matter 92.2 92.0 92.0 gram) (Ocean Nutrition Europe, Essen, Belgium) (× 2/ Crude protein 56.0 57.4 57.4 day), week 4 with Artemia (× 2/day) and until 40 dpf Crude fat 13.5 13.8 13.8 Artemia and Tetramin Flakes (Tetra, Melle, Germany) Ash 8.9 8.9 8.9 (× 2/day). When fish reached the juvenile stage, at 40 dpf The three diets are similar in composition (dry-matter, protein, fat and ash). 95% [76], fish were randomly distributed into 6 tanks, 2 per ultrapure soy saponin was kindly provided by Trond Kortner NMBU Oslo Norway, each diet: one was sampled after 1 week and the other origin: Organic Technologies, Coshocton, OH, [40] López Nadal et al. Animal Microbiome (2023) 5:15 Page 4 of 21 ultrapure soy saponin in the saponin diet (Table 1). We using the Qsep100 Bio-Fragment Analyzer (Bioptic inc., st ™ sampled fish guts after 1 week (54 dpf, 1 timepoint) and New Taipei City, Taiwan) and the Qubit RNA BR Assay after 3 weeks (68dpf, 2nd timepoint) after the start of the Kit (ThermoFisher Scientific, MA, USA). A schematic dietary intervention. Fish were fed twice daily until satia- pipeline of the whole process from sample collection to tion and the amount of feed provided was quantified with results analyses is depicted in Fig. 2. a micro-spoon, feeding 15.1 mg of feed per tank per day (averaging to 0.46 mg of feed per day per fish). A sum - Microbiome: 16S rRNA profiling and sequencing data mary of the experiment design is depicted in Fig. 1. analysis Samples from juvenile zebrafish fed the three differ - Single gut and water samples RNA extraction ent diets were sampled from individual separate tanks. Guts were extracted from juvenile zebrafish, rinsed in Amplicon libraries of the V4 region of the 16S rRNA sterile PBS, snap frozen in liquid nitrogen and preserved were generated from the cDNA synthetized from sin- at − 80 °C for total gut RNA extraction. RNA extrac- gle gut and water samples, using barcoded and modified tion was performed from single intestines as previously F515-806R primers [86]. The PCRs were performed in described [35]. Water samples were obtained by filter - triplicate, purified, and quantified as previously described ing 2L of water from each fish tank using Nalgene [29]. Purified PCR amplicons were pooled in an equimo - Rapid-Flow Sterile Disposable Bottle Top Filters with lar mix and sent for library preparation and sequencing PES Membrane 0.45 µm (ThermoFisher Scientific, MA, using the Illumina NovaSeq 6000 S2 PE150 XP technol- USA). Aliquots of total RNA were used for cDNA syn- ogy at Eurofins Genomics Germany GmbH (Eurofins thesis with the Maxima H minus First Strand cDNA Syn- Genomic, Ebersberg, Germany). Raw paired-end reads thesis Kit (ThermoFisher Scientific, MA, USA), following were analyzed using the standard parameters of NG-Tax the standard protocol using random hexamer primers to 2.0 [66], with the exception of using 100 bp as the for- create cDNA. This cDNA was used for 16S rRNA gene ward and reverse read length, as implemented in Gal- profiling of the bacterial communities and the extracted axy [2], to obtain Amplicon Sequence Variants (ASVs). RNA was used for metatranscriptomic analysis. The Taxonomy was assigned to ASVs using the Silva_132 quantity, quality and purity of total RNA was determined database [67]. Two synthetic “mock communities” with Fig. 1 Experimental design. Fish were bred and raised as described in the section ‘Zebrafish and diets’. At 40 dpf ( juvenile stage) they were fed diet A for 1 week for acclimatisation to dry feed pellets. At 47 dpf, fish were randomly distributed into tanks and fed one of the diets (A, B or C) for 3 weeks. Survival and growth were measured before and during the whole experiment. After 1 week (54 dpf ) and after 3 weeks (68 dpf ) of feeding the fish. Gut samples were collected for histological, metatranscriptomic and microbiome analyses L ópez Nadal et al. Animal Microbiome (2023) 5:15 Page 5 of 21 Fig. 2 Combinatorial approach employed: total RNA was extracted from single zebrafish gut fed on different diets for both timepoints. Aliquots of total RNA were used for cDNA. For the 16S rRNA gene profiling, amplicon libraries of the V4 region of the 16S RNA gene were generated from the cDNA synthetized. NG-Tax 2.0 Galaxy was sued to obtain the ASVs. Several packages of R v4.1.2., Canoco v5.15 and Cytoscape v3.9.1 were used for results visualization. For transcriptomics, the cDNA libraries were sent to NovaSeq 6000 PE150 for sequencing. MetaPhlAn 3.0 (Beghini et al. 2021) and KneadData were used to trim the overrepresented sequences. Nf-core/rnaseq Nextflow pipeline was used for processing of the reads with the GRCz11 genome assembly. The results were visualized by R v.4.1.2, Canoco v5.15 and ErmineJ was used for the GO Enrichment analysis. The histological samples were extracted and embedded in paraffin and sectioned using a microtome. AB-PAS and HIC stains were automated. Samples were digitally scanned and an automated quantification of the histological parameters was performed using VIS v.2019.07 and Canoco v5.15 and GraphPad Prism v9.0.0 to visualize the results. The data integration was performed using heatmaps of normalized relevant parameters from all datasets, both timepoints and all diets known compositions were amplified and sequenced with Canoco v5.15 [9] using analysis type “constrained” as positive controls and a no-template control was also or “unconstrained”, respectively. Response variables were included as a negative control [68]. The distribution of log-transformed with the formula log(10,000*relative_ reads per sample and the variance in ASVs were assessed abundance + 1). RDA p-values were determined through and Alpha- and Beta-diversity measurements were per- permutation testing (500 permutations). Boxplots were formed using R v4.1.2 and RStudio [43], using packages generated using Prism v.9.0.0 (GraphPad Software, San ggplot2, [89], ape, [61], plyr, [93], vegan, [59], RColor- Diego, California USA). Cytoscape v3.9.1 [75] was used Brewer, [58], reshape2, [90], scales [91], data.table, [19], to visualize the diet-specific co-occurrence of ASVs based microbiome, [42], dplyr, [92], phyloseq, [55], ggdendro, on their relative abundances. Additional data handling [84] and DT [95]. The analysis yielded 17,203,234 high- and format conversions were done in Python (https:// quality reads. We excluded one sample (54 dpf butyrate www. python. org/). diet) because it had 2 reads only and we kept all the other samples (> 30.000 reads). Rarefaction curves for Zebrafish gut transcriptome analyses all samples reached a plateau, indicating that sufficient Total RNA (n = 5 diet/timepoint) was sent to Novogene sequencing depths was achieved (data not shown). For (Cambridge, UK), where quality control was done, rRNA the calculation of alpha-diversity indices, data was rare- was depleted and the metatranscriptome libraries were fied against the sample containing the lowest number of prepared. Paired-end reads were generated by NovaSeq reads (31,814 reads). Redundancy analysis (RDA) and 6000 PE150. For the host reads we used nf-core/rnaseq principal component analysis (PCA) were performed Nextflow pipeline [21] and the zebrafish (Danio rerio) López Nadal et al. Animal Microbiome (2023) 5:15 Page 6 of 21 genome assembly GRCz11 (NCBI) and according to the USA, diluted 1:300) antibodies to study proliferating cells MultiQC reports generated, the quality check param- (epithelial renewal) as well as NK-like cells and T cells. eters were satisfactory for all samples. “Salmon” was Samples were scanned at 20× magnification using Pan - used to quantify the expression of the transcripts [62] noramic SCAN II (3DHISTECH, Budapest, Hungary) to and DEseq2 [52], ggplot2, [89], scales [91], viridis [24] in produce digital whole slide images and analyzed using RStudio to investigate the differentially expressed genes Visiopharm v. 2019.07 image analysis software (Visiop- (DEG) in our diet treatments and timepoints. PCA analy- harm, Hoersholm, Denmark). Specialized automated ses were performed in the Canoco v5.15 software suite image analysis protocols were developed for each stain- (v5.02, [9]. Gene Score Resampling (GSR) analyses was ing type. Before employing the quantitative histology, tis- performed ErmineJ (v3.1.2) [27] with the annotation file sue regions were manually defined on the images to select of zebrafish (Danio rerio; genome assembly GRCz11) representative tissue (avoiding artefacts). The automated generated by Gemma [97]. GSR used DEG scores from analysis was then preformed only within those regions. DEseq2 from all genes in the dataset and calculated a Making an automated protocol involves selecting pre- p-value for each Gene Ontology (GO) term. The fold- processing steps, such as median filters to reduce noise change of each GO term across dietary interventions was and enhance structures, training the Bayesian classifier calculated by collapsing individual transcripts per million algorithm by annotating examples of the image back- (tpm) of each gene to the belonging GO term(s). Differ - ground, tissue, and target cells, utilizing post-processing entially expressed GO terms were visualized as a network steps based on shape, size and pixel colour to enhance by using Cytoscape v3.9.1 [75]: the nodes contained the the final image segmentation, and define calculations to fill depicting the log2 fold change (FC) of the control vs give the output data (area, counts, and perimeters). This butyrate at T2 and the border depicting the log2 FC of method for image analysis allowed us to perform quan- the control vs saponin at T2. The nodes with an abso - titative histology which differs from the commonly used lute FC ≥ 0.5 and p value ≤ 0.1 between dietary interven- semi-quantitative scoring. The latter involves a patholo - tions were taken into account. The edges connected the gist ascribing a subjective scoring with ordinal data, relevant nodes if the GO term contains at least 10 genes which is strongly operator-biased and time consuming. and shared at least half of them with the connecting GO Quantitative histology is automated, more detailed, thor- term(s) with FC ≥ 0.2 and p value ≤ 0.05 between dietary ough, and consistent, producing numerical data that can interventions. All data and files used to generate these detect subtle differences between states. The cell types visualisations can be found in Additional file 9. imaged were mucus (goblet) cells, PAS + cells (granulo- cytes), rodlet cells; PCNA + cells (proliferative cells) and High‑throughput quantitative histology Zap-70 cells (NK-like and T lymphocytes) [56]. The histo - At 54 dpf and 68 dpf zebrafish were euthanized in buff - logical parameters quantified were as follows. Absorptive ered MS222 overdose [88] 250 mg/L Tricaine (Sigma- capacity (AC): which was formulated as (interface length Aldrich, DL, United States). Intestines were removed, between mucosa and lumen / interface length between rinsed in PBS, placed in 4% paraformaldehyde over- serosa and exterior of the gut). Cell area fraction (%) (tis- night and transferred to 70% ethanol on the next day. sue area made up of cells) formula: area cell type A/total After subsequent dehydration steps, total intestines tissue area *100. Cell density: cell number/total tissue were embedded in paraffin blocks. Five-micrometer sec - area. Cell size: area of cells/cell number. Cell distance: the tions were stained with one of the following: hematoxy- distance of the cells from the outer serosal layer, where a lin and eosin (H&E) or Alcian blue periodic acid-Schiff higher distance would indicate a cell migrating towards (ABPAS) as previously described in [11] or by immuno- the mucosal fold (villus) end towards the lumen. The histochemistry (IHC). For the latter, antigen retrieval was imaging and the histological quantification were per - performed using the PT Link automatic antigen retrieval formed at the facilities of Skretting Aquaculture Innova- machine (Dako Agilent, CA, USA): samples were placed tion (Skretting, Stavanger, Norway). Further downstream into citrate buffer pH 6.1 (Dako Agilent, CA, USA) at processing of the multivariate analysis was performed by 60 °C, heated to 97 °C in 20 min, kept at 97 °C for 20 min, using Canoco v.5.12 (v5.02 [9], using principal coordinate and cooled down to 60 in 20 min. Samples were stained (PCoA) and Redundancy (RDA) analyses, performing using an automated staining machine (Autostainer Link analysis type “unconstrained” and “constrained”, respec- 48, Dako Agilent, CA, USA) with anti-proliferating cell tively. Response variables (histological parameters) were nuclear antigen (PCNA mouse mAb Clone PC10, M0879, scaled (0–1) and biplots were generated. RDA p-values Dako A/S, Denmark, diluted 1:10.000) or with anti-Zeta were determined through permutation testing (500 per- chain of T cell receptor associated protein kinase 70 mutations). Boxplots were generated using Prism v.9.0.0 (ZAP70 Rabbit mAb 99F2, Cell Signaling Technology (GraphPad Software, San Diego, California USA). L ópez Nadal et al. Animal Microbiome (2023) 5:15 Page 7 of 21 Complete pipeline of the combinatorial approach: omics the Phylogenetic Diversity slightly increased for butyrate and quantitative histology fed fish overtime (Fig. 3). Fluorescent in vivo imaging experiment A principal component analysis (PCA) analysis shows Adult Tg (mpeg1:mCherry/mpx:eGFPi ) were housed that time (from 54 to 68 dpf ) explains ~ 17% of the vari- and fed as previously described [49]and embryos ation observed in the microbial communities (x-axis obtained by natural spawning and raised with E3 water Fig. 4A). To analyze the effect of diet on the microbial (0.10 mM NaCl in demineralized water, pH 7.6) in petri communities, we performed redundancy analysis (RDA) dishes at 28 °C (12/12-h light/dark cycle) [88]. Larvae separately for each timepoint. After one week on the dif- were randomly distributed in 6 well plates (n = 20 fish/ ferent diets (54 dpf), the gut microbiota composition well) and exposed to different concentrations [0.005, was not significantly different between the different diet 0.01 mg/ml] of butyrate and [0.5, 0.7 mg/ml] saponin groups (p = 0.24) (Additional file 3: Fig. S3), whereas, dissolved in E3 water (10 ml solution/well) from 3 to 6 after prolongation of the diet intervention (3 weeks, 68 dpf. Larvae were anaesthetized and in vivo imaged as dpf ) a significant association between the diet and the previously described in [49]. Pictures were analyzed with gut microbiota was detected (p = 0.018). The top-15 most ImageJ software (United States National Institutes of discriminant genera associated with the diet induced Health, Bethesda, United States): the intestinal area was microbiota difference were further investigated (Fig. 4B), selected manually for each fish from the bright field and revealing that these genera were absent in all fish after copied to the other channels and fluorescent cells quan - one week on the distinctive diets (Additional file 4: Fig. tified and boxplots were generated using Prism v.9.0.0 S4 and Additional file 5: Fig. S4 and Additional file 5: Fig. (GraphPad Prism Software, San Diego, California, USA). S4_2). This finding implies that short term diet exposure (1 week) is insufficient to elicit the diet induced micro - Results biota changes. The relative abundances of the most Butyrate and saponin diets did not affect survival nor fish discriminating genera of the gut samples were consist- growth ently zero or extreme low except for Rhodobacter and All of the fish survived the dietary intervention and fish Pseudomonas (Additional file 4: Fig. S4), indicating that growth was comparable regardless of the diet provided microbiota fluctuations in the zebrafish gut were not (Additional file 1: Fig. S1). The water quality indicators influenced to a larger extend by the surrounding water measured: water pH, water conductivity (µS/m), nitrite microbiota composition. RDA of the genera composi- − + − (NO in mM), ammonia (NH in mM), nitrate (NO tion at 68 dpf associated ZOR006 and unclassified Des - 2 4 3 − 2+ in mM), chlorine (Cl in mM), general hardness (Ca ulfovibrionaceae with fish fed a control diet, whereas 2+ and Mg per volume of water) and carbonate hardness associated Mycobacterium, Vibrio, Aeromonas and Meth- (CaCO and MgCO per volume of water) were consist- ylobacterium with fish fed a saponin-supplemented diet 3 3 ently within the recommended range (Additional file 2: and associated Flavobacterium, unclassified Sutterel - Fig. S2). Moreover, water quality indicators remained laceae, Bacteroides, Pandoraea, Rhodobacter, unclassified constant during the whole experiment, indicating that Barnesiellaceae and Plesiomonas with fish fed butyrate- the diet-related changes described below result from supplemented diet. The relative abundances of the most the dietary intervention and not from differences in fish discriminative genera detected by the RDA (Fig. 4B, sub- growth rates or fluctuations in water quality. set of boxplots around the RDA) together with the heat- map of the relative abundances of most important taxa Butyrate‑ and saponin‑supplemented diets altered gut (Additional file 6: Fig. S5) demonstrated distinct micro- microbiota composition over time bial profiles associated with butyrate and saponin-sup - Ten gut samples per diet per timepoint were used to plemented diets. determine prokaryotic community composition based on amplicon sequencing of 16S rRNA. The samples yielded Butyrate reduced taxa connectivity in the zebrafish gut 17,203,234 high-quality reads, with an average of 286,720 After assessing distinct microbiota composition due to reads per sample, ranging from 31,814 to 577,719. The diets after 3 weeks of feeding (68 dpf ), taxa connectivity reads resulted in 579 amplicon sequence variants (ASVs) was analyzed by network analyses of co- and anti-occur- which were reduced to 204 ASVs after filtering out the rence of each pair of taxa at 68 dpf, based on the relative ones occurring in ≤ 2 counts. Alpha-diversity indexes for abundances (Fig. 5). The gut microbiota in fish fed the richness (observed ASVs and Chao1) and diversity (Shan- control diet presented a higher degree of taxa connec- non, Inverse Simpson, Fisher and Phylogenetic Diversity) tivity when compared to the gut microbiota of fish that within the samples did not reveal any significant differ - were fed either the butyrate- or saponin-supplemented ences between diets and timepoints for all samples. Only diet. Quantification of pairs of taxa with significant López Nadal et al. Animal Microbiome (2023) 5:15 Page 8 of 21 Fig. 3 Alpha-diversity indexes for richness (observed ASVs and Chao1) and for diversity (Shannon, Inverse Simpson, Fisher and Phylogenetic Diversity). *p ≤ 0.05, Ordinary one-way ANOVA after confirming normally distributed data by Shapiro–Wilk test. Whiskers: min. to max. shall all points with median connectivity were compared using the cumulative fre- taxa connectivity from 54 to 68 dpf and not saponin and quency histogram (p < 0.05; p.log < 1.30, Additional butyrate fed fish (Additional file 7 : Fig. S6B). file 7: Fig. S6A), showing an increase of connecting pairs of taxa in the control fed fish compared to sapo - Gut transcriptome analysis reveals unique and shared nin and butyrate fed fish at 68 dpf. These differences in effects of butyrate and saponin taxa connectivity were not present after 1 week of feed- After observing substantial dietary induced differences ing (54 dpf ) and occurred exclusively after 3 weeks of in bacterial composition and taxa connectivity, transcrip- feeding (68 dpf ) where only control fed fish increased tome profiles of the same zebrafish gut samples were ana - lyzed. This analysis (pipeline described in Fig. 2) resulted (See figure on next page.) Fig. 4 A Principal component analysis exploring the interaction of diet and time. The x axis separates the samples after 1 week feeding (54 dpf ) from samples after 3 weeks feeding (68 dpf ) and explains 16.87% of the variation observed. B Redundancy analysis of samples after 3 weeks of feeding (68 dpf ), the x axis separates saponin from butyrate fed fish and explains 7.94% of the microbial differences observed and the y axis separates the control from the saponin fed fish and explains 3.45% of the microbial differences observed. The microbial communities changed significantly due to diets (p = 0.018). The relative abundance of the most discriminative genera are depicted with boxplots around the RDA. In both analyses, the top 15 most distinctive genera are represented with black arrows. The direction of the arrows correlated with the dietary treatments and the timepoints and their length correlate with the strength of the correlation. **p ≤ 0.01, *** p ≤ 0.005 one-way ANOVA test or Kruskal–Wallis test after testing for normality on data distribution by Shapiro–Wilk test. No false discovery rate performed. Whiskers: min. to max. shall all points with median L ópez Nadal et al. Animal Microbiome (2023) 5:15 Page 9 of 21 Fig. 4 (See legend on previous page.) in a total of 47,046 genes expressed in transcripts per 3 weeks of feeding (68 dpf ) and not after 1 week of feed- million (tpm). Within-group transcriptome differences ing (54 dpf ). At 68 dpf fish fed the butyrate diet presented across diets and timepoints revealed a significant differ - more significantly homogeneous gut transcriptomic ence of dissimilarity of the transcriptomic samples after López Nadal et al. Animal Microbiome (2023) 5:15 Page 10 of 21 Fig. 5 Taxa connectivity: taxa included when prevalence is ≥ in 3/10 samples, abundance is ≥ 10 counts in 1 M and significance ≤ 0.1. The lines inform about the nature of the taxa interaction: the thickness of the lines represents the strength of the correlation (r-score value) and the shape of the lines represents the direction of the correlation, straight lines mean positive correlation (co-occurrence) whereas dashed lines mean negative correlation of the pairs of taxa (anti-occurrence). A Pairs of taxa co- and anti-occurring for all the diets: in black the interactions occurring in all three diets whereas in grey the interactions not occurring in all diets. Node size corresponds to average abundance of taxa for all diets at 68 dpf. B In red the interactions occurring in the control fed fish and not in the other two diets. Node size corresponds to average abundance of taxa for control diet at 68 dpf. C In green the interactions occurring in the butyrate fed fish and not in the other two diets. Node size corresponds to average abundance of taxa for butyrate diet at 68 dpf. D In yellow the interactions occurring in the saponin fed fish and not in the other two diets. Node size corresponds to average abundance of taxa for saponin diet at 68 dpf profile than fish fed the control and the saponin diets 893 GO terms while significantly up-regulated 40 GO (Fig. 6A). terms out of a total of 6111 GO terms (Additional file 9). Since the transcriptomic profiles were most dissimilar The transcriptomic network depicts a shared down-reg - after 3 weeks of feeding the unique and shared effects of ulation of the transcription and mitotic processes as well butyrate and saponin on the host gut were examined by as histone acetylation and histone methylation, that is creating a transcriptome network analysis (Fig. 6B, all most prominently observed in butyrate fed fish (Fig. 6B). raw data in Additional file 9). Compared to control fed Compared to control fed fish, both butyrate and sapo - fish, butyrate and saponin significantly down-regulated nin up-regulated the carboxylic catabolic processes, the L ópez Nadal et al. Animal Microbiome (2023) 5:15 Page 11 of 21 Fig. 6 Eec ff ts of butyrate and saponin on the host gut transcriptome. A Bray–Curtis distances to examine the dissimilarity of the host transcriptome across diets and timepoints. ** p ≤ 0.01, Kruskal–Wallis test after testing for non-normally distributed data by Shapiro–Wilk test. Whiskers: min. to max. shall all points with median. B Network depicting transcriptomic regulation of butyrate and saponin supplemented diets vs control diet at 68 dpf. Each node is a GO term and the node border represent the log2 fold-change of the control diet vs the saponin supplemented diet and the node fill represent the log2 fold-change of the control diet vs the butyrate supplemented diet. The edges connect nodes containing at least 10 genes and sharing 50% of the contained genes. Related GO terms are encircled encompassing canonical pathways. Shared effects on the gut transcriptome can be observed when edge and fill of a node have the same color in the network: up-regulation -in red- and down-regulation -in blue- compared to the control feed. C Immune response-associated GO terms and particularly inflammatory response analysed in fish fed a control, butyrate and saponin diet. Genes are expressed in tpm and scaled colored per individual gene value. The heatmap contained genes color-scaled per individual gene that reflect the individual within group fish-to-fish variation López Nadal et al. Animal Microbiome (2023) 5:15 Page 12 of 21 Fig. 6 continued oxidoreductase activity, response to estradiol and the activity which were down-regulated in butyrate fed fish immune response although some specific GO terms (Fig. 6B). G-protein receptor activity is the GO term within these processes present differential modulation category that shows the strongest opposite regulation (Fig. 6B). between saponin (up-regulated) and butyrate (down-reg- Compared to control fed fish, saponin up-regulated ulated) and encompassed GO terms associated to pho- 37 GO terms that were down-regulated in butyrate fed toreceptor activity, serotonin receptors activity as well fish while butyrate up-regulated 79 GO terms that were as synaptic signaling (Fig. 6B). To explore the immune- down-regulated in saponin fed fish (Additional file 9). related effects of butyrate and saponin, the immune Saponin up-regulated genes associated to GTPase activ- response of the transcriptome network was zoomed in ity, potassium channel activity and G-protein receptor on (Fig. 6C). In particular, the GO term “inflammatory L ópez Nadal et al. Animal Microbiome (2023) 5:15 Page 13 of 21 response” was examined for butyrate and saponin fed week of exposure) of the fish fed the butyrate diet by fish. Compared to controls, saponin and butyrate up- increased eosinophils, rodlet cells and a decrease of the regulated genes involved in the “inflammatory response” AC. The inflammatory condition remain unresolved after associated to chemokine activity as well as leukocyte and 3 weeks of feeding (68 dpf ) as the fish fed the butyrate innate cell recruitment (ccl19b, ccl25b, csfr1ra, cxcl18b. diet still presented increased eosinophils and rodlet cells cxcl19, cxcl8a, cxcl8b.1, cxcl8b.3, fpr1, mpx, mst1 and tlr- and a depletion of mucus cells compared to saponin and family) (Fig. 6C). control fed fish (black arrows and boxplots Fig. 7C, rep- resentative pictures Fig. 7A). Gut quantitative histological analysis depicted distinct gut architectural profiles for butyrate and saponin Combinatorial approach reveals distinct profiles The zebrafish gut samples collected were analyzed using for saponin and butyrate fed fish high-throughput quantitative histological analysis. While In order to define robust and multi-parameter supported microbiota and transcriptomic data showed differential effects of butyrate and saponin supplementation, the key as well as similar effects of the butyrate and the saponin findings of the different datasets were integrated in a supplementation, the tissue make-up and topography heatmap (Fig. 8). Control fed fish did not present extreme provided further insight on whether changes in gene microbiota fluctuations over time. Butyrate fed fish pre - expression and microbiota also coincide with morpho- sented the most divergent microbiota composition (with logical indications of disturbed intestinal host gut health. increased relative abundance of Bacteroides, Rhodobac- Whole images were obtained from scanned slides and ter, Pandoraea and Flavobacterium) and the lowest taxa quantification was automated for several parameters: the connectivity compared to the other diets, which might AC and the cell area fraction, cell density, cell size, and the be indicative of disturbed ecosystem stability. Saponin distance of each individual cell to the outer serosal layer fed fish presented an increased number of Vibrio con - for cell lineages of particular interest, including mucus trasting with butyrate fed fish. Compared to control fish, cells (goblet cells), eosinophils (PAS + granulocytes), rod- butyrate and saponin shared an increased expression of let cells (PAS +), proliferative cells (PCNA + cells) and T genes associated to immune responses, inflammatory and NK-like cells (Zap70 + cells). Representative pictures responses and oxidoreductase activity. Besides, butyrate of all cell-types and time-points are shown in Fig. 7A. fed fish presented down-regulated genes in GO terms The effect of time did not correlate to any of the his - associated with histone acetylation, histone methyla- tological parameters analyzed (black arrows in the RDA tion, mitotic processes and G-protein coupled receptor graph, Fig. 7B, p value = 0.066) except for the increase activity. These differential gene expressions patterns were of the PCNA area over time, indicative that the relative stronger after 3 weeks of feeding (68 dpf ) compared to number of proliferative cells increased during fish devel - 1 week of feeding (54 dpf ). In terms of histology, after opment. Significant differences on the histological gut 1 week of feeding butyrate, fish already showed increased parameters were found due to the dietary interventions area of eosinophils and rodlet cells compared to sapo- (p = 0.036) (Fig. 7C). The absorptive capacity of the fish nin or control fed fish, which is consistent after 3 weeks gut was decreased for the butyrate fed fish compared of feeding. Butyrate fed fish at 68 dpf showed decreased to saponin and control fed fish at 54 dpf, although dis - area of mucus cells compared to control fed fish. The played similar values at 68 dpf (boxplots around Fig. 7C). histological parameters for saponin fed fish appeared to The area of the eosinophils and rodlet cells increased be less pronounced than those of butyrate fed fish. Col - in butyrate fed fish compared to saponin and control lectively, these observations, showed fish fed a butyrate fed fish after 1 week of feeding (54 dpf ), suggesting an supplemented diet elicited a stronger response in terms inflammatory condition which was partly alleviated but of changes in the microbial composition, expression of not fully resolved at 68 dpf. In addition, compared to genes associated to immune activation processes as well controls, fish fed the butyrate diet showed a clear mucus- as the presence of (pro)inflammatory-like cells such as producing cell depletion after 3 weeks of feeding (68 dpf ). eosinophils and rodlet cells and depletion of mucus cells. Saponin fed fish presented a reduced proliferative cell area compared to butyrate and control fed fish. To illus - Butyrate and saponin increased neutrophil trate these differences in histological parameters, accept - and macrophage recruitment in the gut of zebrafish larvae ing the biological fish to fish variation within each group, Since the data clearly indicated an unexpected induction a heatmap of each individual fish and all the histological of immunity related functions upon butyrate addition to parameters per each RDA axis was generated (Additional the feed (Fig. 6B), the advantages of the zebrafish model file 8: Fig. S7). The combination of these observations system were used to validate the results by in vivo imag- suggested an acute inflammatory response (after one ing of fluorescently labeled neutrophils and macrophages López Nadal et al. Animal Microbiome (2023) 5:15 Page 14 of 21 Fig. 7 High-throughput quantitative histological analysis. A Representative pictures of all cell-types analyzed for all the diets and timepoints with cells of interest in dashed black lines per each group. B Redundancy analysis to examine the effect of time on the histological parameters analyzed. The x axis separated the samples by timepoints and explained 5.76% of the variation observed. The link of time and variation of the histological parameters was not significant (p = 0.066) C Redundancy analysis to examine the effect of diet on the histological parameters analyzed. The x axis separated the samples of butyrate fed fish from saponin and control fed fish and explained 5.86% of the variation explained. The y axis separated saponin fed fish from control fed fish and explained 5.07% of the variation observed. The link of time and variation of the histological parameters was significant (p = 0.036). The top 10 most distinctive histological parameters are depicted in black arrows. The direction of the arrows correlate with the dietary intervention and the length of the arrows represents the strength of the correlation. The boxplots around the RDA depicted the absorptive capacity and the percentage area of cells of interest compared to the total gut area per diet and timepoint. *p ≤ 0.05, one-way ANOVA test or Kruskal–Wallis test after testing for normality on data distribution by Shapiro–Wilk test. No false discovery rate performed. Whiskers: min. to max. shall all points with median L ópez Nadal et al. Animal Microbiome (2023) 5:15 Page 15 of 21 Fig. 7 continued upon butyrate and saponin exposure in zebrafish larvae. of genes associated with immune response together Double Tg(mpeg1:mCherry/mpx:eGFPi ) zebrafish with an increased eosinophil and rodlet cell presence larvae were exposed to butyrate and saponin in differ - and mucus-producing cell depletion in the gut tissue. ent doses for 3 days (3-6dpf ) and were (in vivo) imaged Moreover, butyrate increased the neutrophil and mac- at 6dpf. Fish treated with butyrate as well as saponin rophage in vivo recruitment to the gut area in transgenic presented a dose-dependent increase of neutrophils and zebrafish larvae. Zebrafish fed a saponin-supplemented macrophages in the intestinal area (Fig. 9A). The quan - diet showed differentially modulated microbial composi - tification of the cells present in the gut area showed that tion from butyrate and low taxa connectivity as well as butyrate as well as saponin significantly increased neu - increased expression of immune response while the his- trophils and macrophages in the gut of zebrafish larvae tological parameters comparable to control fed fish. The (Fig. 9B). combinatorial approach of bacterial microbiome profil - ing, host gut transcriptomics, automated high-through- Discussion put quantitative histology (novel in zebrafish research) In the present study the effects of butyrate and saponin- together with in vivo innate cell recruitment in the gut supplemented feed in the zebrafish gut were assessed area in zebrafish larvae revealed evidence of the pro- following a combinatorial approach that integrates sev- inflammatory effects exerted by butyrate supplementa - eral datasets and validating the results by in vivo imag- tion which were partly shared with the well-establish ing. Juvenile zebrafish fed a butyrate-supplemented feed pro-inflammatory saponin supplementation, indicating for 3 weeks presented a modulated microbial compo- detrimental effects of butyrate in the zebrafish intestinal sition and low taxa connectivity, increased expression milieu. López Nadal et al. Animal Microbiome (2023) 5:15 Page 16 of 21 Fig. 8 The heatmap brings together the main observations of each analysis and compare them per diet and timepoint. The more representative genera are illustrated with the average relative abundance per timepoint and diet. The taxa connectivity contained the amount of pairs of taxa that correlate to each other in a significant fashion ( p ≤ 0.05). The GO terms contain the transcripts per million (tpm) of all genes expressed in the dataset that collapsed under that GO term. All histological parameters are normalized and scaled (from 0 to 1). Each individual feature within the heatmap is normalized and colored from red (more present) to white (absent) While saponin and soybean meal have been consist- the host during a bacterial challenge [65]. No baseline ently associated with gut inflammation in several fish studies of butyrate-supplemented feed are reported in species, including carp [80, 81, 96], salmon [5, 32, 37, 40, the (zebra)fish literature to our knowledge. In the present 80, 81] and zebrafish [30, 49], butyrate has been reported study, we used for the first time a butyrate-supplemented to convey beneficial effects when supplemented to fish diet for zebrafish and our combinatorial approach feed such as intestinal growth enhancement [71] and showed that 0.01% inclusion of butyrate induced a immunostimulant and antioxidant properties [20, 47], pleiotropic damaging response comparable to the well- (reviewed in [1]. However, previously published stud- establish pro-inflammatory anti-nutritional factor soy ies with histological and gene expression redouts com- saponin. In mammals, colonocytes located along the gut pared butyrate supplementation to other challenges such crypts take up the butyrate produced by the microbiota, as high concentration of plant-based meal or a patho- preventing high concentrations of this SCFA to reach gen challenge. As a matter of fact, butyrate-supplemen- the proliferating stem cells at the bottom of the crypts. tation effects depended on co-treatment(s) employed In fact, high concentrations (1.5–2 mM) of butyrate and duration of the feeding intervention. For instance, were shown to be toxic to mouse pluripotent stem cells 0.8% inclusion of sodium butyrate in a low percentage in vitro [45]. This is especially relevant in cryptless organ - plant-containing diet in gilthead sea bream for 10 weeks isms such as fish [3, 41, 82], where higher concentrations resulted in a mild inflammatory reaction whereas in the of butyrate can reach the stem cells localized between same study, 0.4% inclusion of sodium butyrate in high the intestinal folds (villi). Mechanistic studies in mouse percentage plant-based diet for a longer period protected and zebrafish larvae suggested that butyrate at high L ópez Nadal et al. Animal Microbiome (2023) 5:15 Page 17 of 21 Fig. 9 A Representative pictures of the fluorescent in vivo imaging of the gut area of Tg(mpeg1:mCherry / mpx:eGFPi ) larvae exposed to control media, 0.005 mg/ml and 0.01 mg/ml butyrate and 0.5 mg/ml and 0.7 mg/ml saponin. B Quantification of neutrophils and macrophages in the gut area of the zebrafish larvae (n = 10 in all groups except 0.7 mg/ml saponin where n = 3). *p ≤ 0.5, **** p ≤ 0.0001 Kruskal–Wallis test after testing for non-normally distributed data by Shapiro–Wilk test. Whiskers: min. to max. shall all points with median concentrations inhibits stem cell proliferation via FoxO3 composition, more information might be extracted in cryptless organisms such as zebrafish [25]. Taking from the analysis of microbial networks. For example, these observations together, the butyrate-supplemented in inflammatory bowel disease patients, topological diet showed a compromised intestinal epithelial barrier properties of the co-occurring bacterial networks iden- function, coinciding with a disrupted microbiota com- tified anti- and pro-inflammatory key organisms that position with decreased taxa connectivity, and increased defined the degree of structure of the ecosystem [6]. expression levels of genes associated with inflammatory In fish, recent studies validated the usage of co-occur - and immune responses. The inflammatory response due rence and anti-occurrence taxa networks to identify the to butyrate was confirmed by enhanced innate immune core gut European seabass microbiota [38] as well as to cell recruitment in vivo to the zebrafish gut. Our data reveal microbial interactions due to prebiotics and pro- warrants that further research should investigate the long biotics [53]. In the present study, zebrafish fed 3 weeks term effects of butyrate-supplemented feed and suscep - a butyrate-supplanted feed presented altered the micro- tibility towards infectious or inflammatory challenges biota composition as well as reduced taxa connectivity which were not investigated here. Potentially, butyrate- (co- and anti-occurrence) compared to control (and to associated immuno-stimulation early in life, could boost a lesser extent to saponin)-fed fish (Figs. 4, 5 and Addi- immunity and strengthen disease resistance in later life tional file 7: Fig. S6). We had 6 tanks, 2 per each diet stages (trained immunity) [64]. and 1 tank per diet was sampled after 1 week of feeding Disruption of the gut microbiota homeostasis, often intervention and the other after 3 week of feeding inter- caused by an imbalance in the microbial community (or vention. This could have an effect on the sample clus - dysbiosis), is commonly associated with inflammatory tering of the fish microbiota. However, the water from conditions in the zebrafish gut [8, 11] also (reviewed in the recirculating system was the same for all tanks and [12, 50]). However, whether these disruptions of micro- its quality remained comparable in all tanks across the bial community cause gut inflammation in a direct man - study (Additional file 2: Fig. S2). Furthermore microbiota ner or via disruptions in the microbiota is a matter of composition of the water samples of the fish tanks were discussion and current research in the fish immunity comparable among themselves and very different from and nutrition field. Next to investigating the community the gut samples (Additional file 4: Fig. S4 and Additional López Nadal et al. Animal Microbiome (2023) 5:15 Page 18 of 21 file 5: Fig. S4_2) suggesting that the differences in micro - IL8 did not affect human eosinophils in vitro [63], eosino - bial communities found across dietary treatments did phils are able (via granule proteins) to stimulate neutrophils not originate due to the separate environments per treat- that produce IL8 and superoxide contributing to gastroin- ment. Butyrate increased the relative abundance of the testinal pathologies [72]. However, eosinophil research in genera Rhodobacter, Flavobacterium and Bacteroides that the context of gastrointestinal health is limited in humans were previously associated with gut inflammation in fish and mice [34] as well as (zebra)fish [7 ]. Butyrate increased [51, 78, 94] whereas saponin increased the relative abun- eosinophil and rodlet cell area even after 1 week of feeding, dance of the Vibrio genus, which contains several patho- while reduced the presence of mucus cells overtime (Fig. 7), biont species which might become pathogenic upon features associated with (chemically-induced) intesti- challenge of the gut barrier integrity (reviewed in [15]. nal inflammation in zebrafish [11]. Rodlet cells were first In mammals, butyrate is produced by fermenting bac- reported to act against fish parasites and later studies dis - teria in the intestinal tract and until now scientists were closed their granulocyte nature and include them as part of not able to measure any naturally occurring concentra- the innate fish immune system, increasing in number when tions of butyrate in the zebrafish gut [14]. Since it is not exogeneous stressors were present [18, 33, 54, 69]. More certain whether fish gut may produce butyrate, exogene - research into this well-known but often forgotten cell type ous butyrate supplementation may disrupt the growth of may elucidate its role in (zebra)fish mucosal immunology. bacteria since they may not be used to metabolize such To reinforce the observation that saponin and butyrate substrate. In butyrate-fed fish increased abundance of recruited immune cells to the gut we used transgenic Bacteroides correlated with lower abundance of Vibrio. zebrafish larvae to in vivo visualize neutrophil and Interestingly, in vitro studies have revealed that butyrate macrophage presence in the gut. The fact that saponin exposure can negatively impact the colonization of spe- induced a stronger cell recruitment than butyrate could cific Vibrio campbellii PUGSK8 by its effect on biofilm be explained by the fact that lower concentrations of formation capacity in these bacteria [36]. Taken together, butyrate were used (mimicking the ones employed in the these findings warrant further studies to understand diets) (Fig. 9). Other studies showed decreased neutrophil the mechanisms by which butyrate influences microbial recruitment after tail wounding when zebrafish larvae ecosystems. were immersed to butyrate [14]. However, such studies Inflammatory-associated taxa in butyrate-fed fish briefly immersed zebrafish larvae to extremely high con - matched with an increased expression of genes belonging centrations of sodium butyrate (30 mM = 3303 mg/ml) to inflammatory and immune responses (Fig. 8). While and such study design may greatly differ from the natu - targeted gene expression is commonly used in (fish) nutri - rally occurring physiological situation in the zebrafish tion studies, this approach is often hypothesis-driven and gut. We hypothesize that the increased chemokine the discovery risk of novel premises is relatively low com- expression in butyrate fed fish might be the driving force pared to more comprehensive transcriptome analyses. In for the increased leucocyte recruitment in the gut and the present study, butyrate down-regulated genes associ- further research may disclose specific butyrate modes of ated with mitotic and transcription processes which is in action in the (zebra)fish gut. line with the inhibition of stem cell proliferation previously In the present study butyrate-supplemented feed reported [25], although proliferative cells (PCNA +) were appeared to modulate the microbial composition as not decreased in butyrate-fed fish as shown by the histo - indicated by low taxa connectivity, increased expression logical dataset. Butyrate down-regulated genes associated of gene associated to inflammatory processes as well as with histone modifications (acetylation and methylation) in increased presence of rodlet cells, and eosinophils while line with previously described epigenetic effects of butyrate decreasing Goblet cells. Moreover, we supplemented this in mammals (reviewed in [31]. Further research may elu- data with in vivo observations of the increased recruit- cidate whether there is an effect of butyrate supplemented ment of the neutrophil and macrophage population in feed on epigenetic markers and in the affirmative case the gut upon butyrate and saponin exposure. The com - whether such epigenetic modifications can be passed on bination of these datasets indicate that butyrate has fish- the fish offspring. A clear subset of chemokines within the specific effects on the gut homeostasis that differ from inflammatory response appeared to be up-regulated after the mammalian counterparts [28]. The particular fish gut butyrate-supplemented feeding (Fig. 6C) among which structure, lacking intestinal crypts could play an impor- cxcl8a, cxcl8b.1 and cxcl8b.3. Cxcl8 (or il8) is known as one tant role on the absorption and the effect of the butyrate of the most potent chemoattractant molecules for recruit- on the epithelial lining where chemokines might orches- ing neutrophils (expressing CXCR1/2 receptors for Cxcl8) trate the inflammatory-like response. However, in the and other leukocytes upon inflammation [60]. Although present study butyrate absorption by the enterocytes in L ópez Nadal et al. Animal Microbiome (2023) 5:15 Page 19 of 21 the zebrafish gut has not been quantified and should be Additional file 4: Fig. S4_1. Relative abundances of the top 15 most addressed in future research. While more mechanistic distinctive genera for all diets at both timepoints, including water samples from all fish tanks at both timepoints. studies are needed to shed light on the specific modes Additional file 5: Fig. S4_2. Figure S4_1 continued. of action of butyrate on the fish gut health, the present combined study (omics, histology and imaging) provides Additional file 6: Fig. S5. Heatmap of the relative abundance (relative to 1) of the most distinctive and important taxa for all diets at the 2nd evidence to support non-beneficial effect of butyrate- timepoint. Importance was calculated as (sqrt( CorS1^2+CorS2^2)), i.e., supplemented feed on growing juvenile zebrafish. the length of the arrows in Figure 4B. In conclusion, combining several high throughput Additional file 7: Fig. S6. Normalized cumulative frequency histogram approaches we provide a more comprehensive and gran- depicting the amount of significant pairs of taxa correlations per each diet A) at 68 dpf, B) at 54 dpf; (dotted line represents logarithmic p value =1.30 ular view of the effects of dietary interventions on fish and p =0.05). gut health. Translation to aquaculture species is possible Additional file 8: Fig. S7. Heatmaps of each individual gut fish sample since our redouts do not depend on any species-specific (n=5 diet / timepoint) for both axis of the redundancy analysis. Despite antibodies. However, integration of multi-layered high- of the fish to fish variation present dietary effects are visible for both timepoints. Values are normalized and scaled from 0-1. throughput studies remain a challenge in fish because of various reasons. On the one hand, there are difficulties to Additional file 9: Gene expression of transcripts from zebrafish gut fed either a control, butyrate- or saponin-supplemented diets at 54 and 69 fully comprehend the connections between the complex dpf. layers of data deriving from high-throughput methods and the most relevant outcomes (fish health biomark - Acknowledgements ers). On the other hand, scientist may not have yet the The authors would like to thank Steven Aalvink and Ineke Heikamp-de Jong technology to adequately obtain multi-omics data with for extracting RNA and the PCRs for the molecular analyses and Trond Kortner to supply to us the 95% ultra-pure saponin. sufficient resolution (lack of noise) and reproducibility that facilitates omics datasets combination. In the pre- Author contributions sent study, the detrimental effects of butyrate towards ALN performed research, analyzed and integrated all data, drafted manuscript and figures. JB analyzed and integrated all data, drafted manuscript and the zebrafish gut were congruent throughout all the data - figures. CL and FvdB and TBE collected and analyzed histological data. MAS sets in our combinatorial approach strengthening the analyzed 16S and transcriptomics data and drafted manuscript. DP and CMcG biologically relevant observation that butyrate appears drafted and edited manuscript and developed and provided feeds. DS MK drafted and edited manuscript. GW and SB drafted and edited manuscript and detrimental to the zebrafish gut. Steps towards observa - provided the funding. All authors review the manuscript. All authors read and tional scientific studies with an integrative view, combin - approved the final manuscript. ing high-throughput datasets with imaging techniques to Funding understand complex multifactorial biological processes This study was funded by NWO-TTW Applied and Engineering Sciences such as fish gut health may help researchers to evaluate (Project Number 15566). novel diets for healthier fish generations. Availability of data and materials Raw data of the transcriptomic analyses can be found in Additional file 9. All Supplementary Information raw data will be uploaded to an open access repository. The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s42523- 023- 00230-2. Declarations Additional file 1: Fig. S1. Standard length (mm) was measured at 40, 54 Ethics approval and consent to participate and 68 dpf for the 3 diets by using a digital calliper. The present study was approved by the Dutch Committee on Animal Welfare Additional file 2: Fig. S2. Water quality values just before and during the (2017.W-0034) and the Animal Welfare Body (IvD) of the Wageningen Univer- experiment at 38, 45, 50, 56, 62 and 65 dpf. In green the range of prefer- sity ( The Netherlands). Furthermore, we adhered to standard biosecurity and able values for the measurements and in red the values above which institutional safety procedures at Wageningen University and Research. the water quality is considered to be detrimental for the fish according to manufacturer’s instructions: A) pH (accepted range 6.6-8.4), B) Water Consent for publication conductivity (accepted range 300-1500 µS/m), C) Nitrite (accepted range All authors revised and agreed on the submitted version of this manuscript. 0-7 mM), D) Ammonium (only 0 mM accepted), E) Nitrate (accepted range 0-70 mM), F) Chlorine (only 0 mM accepted), G) General hard- Competing interests ness (accepted range 2-16) and H) Carbonate hardness (accepted range The authors declare to have no competing interests. 1.5-10). Author details Additional file 3: Fig. S3. Redundancy Analysis (RDA) at the 1st timepoint Cell Biology and Immunology Group, Wageningen University and Research, to examine the effect of the diets on the gut microbiota. The x axis sepa- Wageningen, The Netherlands. Aquaculture and Fisheries Group, Wagenin- rates saponin form control fed fish and explains 5.82% of the microbial dif- gen University and Research, Wageningen, The Netherlands. Host-M icrobe ferences observed and the y axis separates the butyrate form the saponin Interactomics, Wageningen University and Research, De Elst 1, 6708 WD Wage- fed fish and explains 2.28% of the microbial differences observed. The top ningen, The Netherlands. Laboratory of Microbiology, Wageningen University 15 most distinctive genera are depicted as supplementary variables in and Research, Wageningen, The Netherlands. Skretting Aquaculture Innova- black arrows, p=0.24. tion, Stavanger, Norway. López Nadal et al. 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Journal

Animal Microbiome – Springer Journals

Published: Mar 3, 2023

Keywords: Microbiome; Transcriptome; Omics; Imaging; Zebrafish; Butyrate; Soy saponin; Gut; Inflammation

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Lickwar, CR
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Liu, Y
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López Nadal, A
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López Nadal, A
Characterization of isolates of Flavobacterium psychrophilum associated with coldwater disease or rainbow trout fry syndrome I: phenotypic and genomic studies

Lorenzen, E; Dalsgaard, I; Bernardet, J-F
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Love, MI; Huber, W; Anders, S
Exogenous enzymes and probiotics alter digestion kinetics, volatile fatty acid content and microbial interactions in the gut of Nile tilapia

Maas, RM
Rodlet cells in teleosts: a new insight into their nature and functions

Manera, M; Dezfuli, BS
phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data

McMurdie, PJ; Holmes, S
T Cell Immune Deficiency in zap70 Mutant Zebrafish

Moore, JC
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Cxcl8 (IL-8) mediates neutrophil recruitment and behavior in the zebrafish inflammatory response

Oliveira, S
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Salmon provides fast and bias-aware quantification of transcript expression

Patro, R
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Petering, H
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Petit, J; Wiegertjes, GF
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Poncheewin, W
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Reite, OB; Evensen, Ø
Butyrate and taurine exert a mitigating effect on the inflamed distal intestine of European sea bass fed with a high percentage of soybean meal

Rimoldi, S
Effect of partially protected butyrate used as feed additive on growth and intestinal metabolism in sea bream (Sparus aurata)

Robles, R
Eosinophils: changing perspectives in health and disease

Rosenberg, HF; Dyer, KD; Foster, PS
The effect of fish meal replacement by soyabean products on fish growth: a meta-analysis

Sales, J
Probiotic expressing heterologous phytase improves the immune system and attenuates inflammatory response in zebrafish fed with a diet rich in soybean meal

Santos, KO
Cytoscape: a software environment for integrated models of biomolecular interaction networks

Shannon, P
Growth and maturation in the zebrafish, danio Rerio : a staging tool for teaching and research

Singleman, C; Holtzman, NG
Intestinal inflammation induced by soybean meal ingestion increases intestinal permeability and neutrophil turnover independently of microbiota in zebrafish

Solis, CJ
Altered gut microbiota associated with intestinal disease in grass carp (Ctenopharyngodon idellus)

Tran, NT
Zebrafish as animal model for aquaculture nutrition research

Ulloa, PE; Medrano, JF; Feijo, CG
Soybean meal-induced enteritis in Atlantic salmon (Salmo salar L.) at different temperatures

Urán, PA
Soybean meal induces intestinal inflammation in common carp (Cyprinus carpio L.)

Urán, PA
The 3d pattern of the rainbow trout (Oncorhynchus mykiss) enterocytes and intestinal stem cells

Verdile, N
Effects of butyrate glycerides supplementation in high soybean meal diet on growth performance, intestinal morphology and antioxidative status of juvenile black sea bream, Acanthopagrus schlegelii

Volatiana, JA
Unique and conserved aspects of gut development in zebrafish

Wallace, KN; Pack, M
Improved bacterial 16S rRNA Gene (V4 and V4–5) and fungal internal transcribed spacer marker gene primers for microbial community surveys

Walters, W
Morphological and molecular evidence for functional organization along the rostrocaudal axis of the adult zebrafish intestine

Wang, Z
Effects of short term lead exposure on gut microbiota and hepatic metabolism in adult zebrafish

Xia, J
Effect of replacing soybean meal with cottonseed meal on growth, hematology, antioxidant enzymes activity and expression for juvenile grass carp, Ctenopharyngodon idellus

Zheng, Q
‘Gemma: a resource for the reuse, sharing and meta-analysis of expression profiling data

Zoubarev, A

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Omics and imaging combinatorial approach reveals butyrate-induced inflammatory effects in the zebrafish gut

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Omics and imaging combinatorial approach reveals butyrate-induced inflammatory effects in the zebrafish gut

Omics and imaging combinatorial approach reveals butyrate-induced inflammatory effects in the zebrafish gut

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