DOI: 10.5061/dryad.v15dv427m
title: Alternative proteins remodel the microbiome and support growth and development in zebrafish

description: Protein intake is indispensable to growing animals. As populations and
 resource demands expand, animal protein-based diets cannot be sustained.
 In order to validate a move completely away from these diets, we fed a
 model vertebrate alternative proteins during development. Diets were based
 on pea, milk and whey, as well as fishmeal (control). All diets supported
 growth to some degree, with the exception of those high in milk and whey
 proteins. The most promising diets were associated with the upregulation
 of genes associated with insulin sensitivity and fat storage. The
 microbiome reflected the dietary changes, with a shift occurring from
 Fusobacteriota to Proteobacteria. Correlations were also noted with
 health, as Cetobacterium positively affected development, while Aeromonas
 did the opposite. Our findings may help direct the selection of specific
 animal protein-free diets that can be introduced early into development to
 maximize vertebrate health.
description: # Alternative proteins remodel the microbiome and support growth and
 development in zebrafish Dataset DOI:
 [10.5061/dryad.v15dv427m](10.5061/dryad.v15dv427m) ## Description of the
 data and file structure This file set contains the data required to
 replicate our analyses in "alternative proteins support somatic and
 muscular development while remodeling the microbiome in zebrafish".
 This dataset contains survivorship, growth measurements, gene expression
 values, and gut microbiota data from zebrafish fed various alternative
 protein diets including pea, milk, and wheat-based formulations over a ~6
 month period. ### Files and variables #### File: Amino_Acid_Data.csv
 **Description:** Amino acid profiles of the dietary treatments compared to
 recommended amounts. ##### Variables * Diet: Diet treatment group (FM,
 Fishmeal; P1, Pea-1; P2, Pea-2; M, Milk; M/P2, Milk/Pea-2; W/P2,
 Wheat/pea-2). * Percent: Proportion of the specific amino acid within the
 total protein content of the diet (expressed as %). * AminoAcid: Standard
 abbreviation for the amino acid. #### File: Biological_Replicate_Data.csv
 **Description:** qPCR gene expression results from biological replicates
 of zebrafish fed alternative protein diets. Each row represents the mean
 expression data for a target gene in a specific diet group (biological
 group), based on pooled technical replicates. Expression levels were
 normalized to the endogenous control gene E1a (eef1a1l1). Note that ΔΔCt =
 0 and RQ = 1 for the FM (reference) group by definition, and missing RQ
 values indicate control genes or normalization baselines. Note that this
 file includes intentional empty cells created by the software. #####
 Variables * BiogroupName: Name of the dietary treatment group (FM,
 Fishmeal; P1, Pea-1; P2, Pea-2; M, Milk; M/P2, Milk/Pea-2; W/P2,
 Wheat/pea-2). * Target: Name of the gene being measured. * Omitted:
 Indicates whether any technical replicates were omitted due to QC issues
 (True/False/mixed). * TechReplicates: Number of technical replicates used
 in the calculation. * RQ: Relative Quantification (fold change compared to
 FM control). * RQMin/RQMax: Lower and upper confidence bounds for RQ. *
 CTMean: Mean cycle threshold (Ct) value across technical replicates. *
 ΔCTMean: Difference in Ct between the target gene and endogenous control.
 * ΔCTSE: Standard error of the ΔCt. * ΔΔCT: Normalized expression
 difference compared to FM control. #### File: Microbiome_Data.zip
 **Description:** This archive contains 16S rRNA sequencing data from the
 gut microbiome of zebrafish fed alternative protein diets. The data
 include alpha and beta diversity metrics, principal coordinate analyses
 (PCoA), and taxonomic summaries across multiple levels. The study compares
 microbiome outcomes across six diet treatment groups: (FM, Fishmeal; P1,
 Pea-1; P2, Pea-2; M, Milk; M/P2, Milk/Pea-2; W/P2, Wheat/pea-2). **File
 Contents:** **1)** alpha_shannon.csv Description: Shannon diversity index
 (alpha diversity) per sample. It summarizes microbial richness and
 evenness for each sample across groups. Structure:  Row: individual
 zebrafish sample Columns: * Group: Diet treatment group. * Samples: Unique
 sample ID (e.g., S00OL_XXXX). * Variable: Always "Shannon"
 (alpha diversity metric used). * Value: Calculated Shannon diversity index
 for that sample. * se: Standard error (not available in this dataset).
 **2)** beta_pcoa_score.csv Description: Principal coordinate analysis
 (PCoA) scores from beta diversity analysis. Each row corresponds to one
 sample. Structure: Row: individual zebrafish sample. Columns: * Axis.1:
 First principal coordinate (PCoA axis 1). * Axis.2: Second principal
 coordinate (PCoA axis 2). * MBI.Sample.ID: Internal microbiome ID used in
 sequencing analysis. * Your.Sample.ID: Experimental sample ID. *
 Sampling.Date: Date when sample was collected. * Sample.Type: Tissue type
 (viscera). * Group: Diet treatment group. * Reads: Total sequencing read
 count. * Shannon: Shannon index value (alpha diversity). **3)**
 CLR_input.data.csv Description: Centered log-ratio (CLR) transformed OTU
 abundance data used for multivariate analyses. Structure: * Row:
 individual zebrafish sample - First column A (unnamed): Sample IDs (e.g.,
 S00OL_XXXX). - Columns B onwards: CLR transformed abundances of individual
 OTUs (e.g., Otu0002, Otu0003,...). Each column represents one OTU
 feature.  - Values: Log-ratio transformed abundance values for each OTU,
 standardized across all samples. **4)** GroupM_OTU_raw.result.csv, 
 GroupM_P2_OTU_raw.result.csv, 
 GroupP1_raw.result.csv, GroupP2_OTU_raw.result.csv, GroupW_P2_OTU_raw.result.csv. Description: Differential abundance results for individual OTUs in the diet groups, generated from statistical analysis using DESeq2 comparing OTU-level microbiome profiles across treatment groups. Structure: Row: one OTU (e.g., Otu0001, Otu0002, etc.), with statistical test results evaluating whether that OTU is significantly differentially abundant in the given diet group (GroupX) relative to others. Columns:  * GroupX.baseMean: Mean normalized abundance of the OTU across all samples. * GroupX.log2FoldChange: Log2 fold change in abundance between GroupX and reference group. * GroupX.lfcSE: Standard error of the log2 fold change. * GroupX.stat: Test statistic. * GroupX.pvalue: Raw p-value for the test. * GroupX.padj: Adjusted p-value. * GroupX.reject: Significance flag (TRUE is significant at FDR threshold). * GroupX.df: Degrees of freedom used. **5)** proportions_Class.csv, proportions_Family.csv, proportions_Genus.csv, proportions_Order.csv, proportions_Phylum.csv Description: These files contain the taxonomic composition of the zebrafish gut microbiome at the Phylum, Class, Order, Family, and Genus levels, expressed as proportional abundances. Structure: Rows: Individual zebrafish gut samples Columns: Bacterial taxonomy. Cell values: proportion of reads assigned to each bacteria classification in eachsample (range 0 to 1). **6)** relative_abundance_OTUs_input.data.csv Description: This file contains the relative abundance data of OTUs for each zebrafish gut sample. The abundance values are normalized to proportions (range from 0 to 1), reflecting the fraction of total reads assigned to each OTU per sample.  Structure: Rows: individual zebrafish samples. Columns: OTUs. Cell values: Proportional abundance of each OTU in sample. #### File: Muscle_Cell_Area_Data.csv **Description:** This dataset contains individual skeletal muscle fiber cross-sectional area measurements from adult zebrafish fed various protein diets for 145 days. ##### Variables * Tank: Numeric identifier for each experimental tank. * Diet: Diet treatment group (FM, Fishmeal; P1, Pea-1; P2, Pea-2; M, Milk; M/P2, Milk/Pea-2; W/P2, Wheat/pea-2). * Fish: Identifier for each individual fish sampled. * Sex: Biological sex of each fish (F= female, M=male). * Cell #: Index number for each measured muscle fiber from a muscle section. * Area: Cross-sectional area of the muscle cells (µm²). #### File: Nuclei_Data.csv **Description:** Nuclei per muscle section in adult zebrafish fed alternative proteins for 145 days. ##### Variables * FM: Fishmeal * P1: Pea-1 * P2: Pea-2 * M: Milk * M/P2: Milk/Pea-2 * W/P2: Wheat/pea-2 #### File: Standard_Length_Data.csv **Description:** Standard body length (mm) of zebrafish following 145 days of dietary treatment on alternative protein diets. ##### Variables * Tank: Numeric identifier for each experimental tank. * Diet: Diet treatment group (FM, Fishmeal; P1, Pea-1; P2, Pea-2; M, Milk; M/P2, Milk/Pea-2; W/P2, Wheat/pea-2). * Length: Mean standard body length of fish in each tank (mm). #### File: Survivorship_Data.csv **Description:** Survivorship was recorded at multiple timepoints to evaluate how different dietary treatments influence mortality across development. ##### Variables * Days: Number of days since the start of dietary treatment (day 0 marks the beginning of alternative diet feeding). * Treatment: Diet treatment group (FM, Fishmeal; P1, Pea-1; P2, Pea-2; M, Milk; M/P2, Milk/Pea-2; W/P2, Wheat/pea-2). * Survive: Percent survival of fish in each treatment group at the indicated time point. #### File: Total_Length_Data.csv **Description:** Total body length (mm) of zebrafish fed alternative protein diets. ##### Variables * Tank: Numeric identifier for each experimental tank. * Diet: Diet treatment group (FM, Fishmeal; P1, Pea-1; P2, Pea-2; M, Milk; M/P2, Milk/Pea-2; W/P2, Wheat/pea-2). * Day: Number of days on the diet at the time of measurement (alternative diet feeding began at 30 dpf). * Length: Mean total body length of fish in each tank (mm). #### File: Technical_Replicate_Data.csv **Description:** qPCR gene expression results from zebrafish fed alternative protein diets. Data were generated using SYBR green chemistry. Target genes were normalized against the endogenous control gene E1a (eef1a1l1). Reference group is FM (fishmeal diet). Each row represents a technical replicate for a specific gene in a fish from a given diet group. Data are grouped by sample name and target gene, and include relative quantification (RQ) values, as well as error and Ct metrics. Note that each sample is derived from one individual fish, and that this file includes intentional empty cells created by the software. ##### Variables * Sample Name: Diet identifier for each biological replicate (FM, Fishmeal; P1, Pea-1; P2, Pea-2; M, Milk; M/P2, Milk/Pea-2; W/P2, Wheat/pea-2). * Target Name: Gene measured by qPCR. * Omitted: Indicates whether the replicate was omitted (true/false). * RQ: Relative quantification value (fold change vs reference group FM). * RQ Min/RQ Max: Confidence interval bounds for RQ. * Ct Mean: Average cycle threshold (Ct) value from technical replicates. * ΔCt Mean: Difference between Ct of target and endogenous control. * ΔCt SE: Standard error of ΔCt. * ΔΔCt: Normalized expression difference between treatment and reference group. ## Code/software Microsoft Excel or other spreadsheet software Raw microbiome data (.fastq) can be analyzed using QIIME2, DADA2, or other microbiome analysis pipelines.