Optimization of bacterial colonization in the gut of axenic and conventional zebrafish larvae using live food
Abstract
The zebrafish increasingly serves as a model for studying the influence of the gut microbiota on the physiology of the host. In an experimental setting, specific bacteria can be used to colonize the gut of zebrafish at an early stage (larvae), affecting their development, physiology, behavior, and overall health. However, the efficiency of gut microbial colonization protocols in zebrafish larvae depends on several factors, such as bacterial strains, zebrafish lines, pre-colonization status, timing of colonization, the type of food vector, and assay methods. In this study, we tested how different parameters influence bacterial colonization in the gut larvae from various genetic lines, both axenic and conventional. To demonstrate gut colonization in transparent larvae up to 9 days post-fertilization (dpf), we used an Escherichia coli strain expressing fluorescent proteins and fluorescence microscopy. Additionally, we utilized plate counts to quantify the extent of bacterial colonization. We found that introducing live food ( Tetrahymena thermophila ) loaded with bacterial cells strongly facilitated colonization, increasing the initial bacterial load by one log. We also examined the impact of larval density and timing of exposure to bacteria, which together enabled successful bacterial colonization of the zebrafish larvae’s gut with increasing bacterial cell counts (from 10 4 to 10 6 ) over four days. We also successfully colonized the zebrafish larvae with strains of Pseudomonas aeruginosa , Aeromonas veronii , and Vibrio cholerae . Implementing standardized and optimized bacterial colonization protocols in zebrafish larvae should facilitate the use of this model to study the interactions between the gut microbiota and host physiology. IMPORTANCE Zebrafish larvae hold potential as a model for studying gut microbiota. However, standardized protocols for controlling bacterial colonization of the larval zebrafish gut are required. This article presents the analysis of various parameters that led to an effective method for colonizing zebrafish larvae with various bacteria. The proposed detailed protocol enables the qualitative, quantitative, and reproducible observation of bacterial colonization in the zebrafish larvae’s gut.
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BibTeX
@article{byatt2026optimization,
title = {{Optimization of bacterial colonization in the gut of axenic and conventional zebrafish larvae using live food}},
author = {Gabriel Byatt and Cédric Goffard and Jeffrey K. Cornuault and Mado Lemieux and Paul de Koninck and Marie-Eve Paquet and Sylvain Moineau},
journal = {Microbiology Spectrum},
year = {2026},
month = jun,
doi = {10.1128/spectrum.02853-25},
url = {https://hal.science/hal-05782915v1},
note = {CC BY},
}
RIS
TY - JOUR
TI - Optimization of bacterial colonization in the gut of axenic and conventional zebrafish larvae using live food
AU - Gabriel Byatt
AU - Cédric Goffard
AU - Jeffrey K. Cornuault
AU - Mado Lemieux
AU - Paul de Koninck
AU - Marie-Eve Paquet
AU - Sylvain Moineau
PY - 2026
DA - 2026/06/02
JO - Microbiology Spectrum
DO - 10.1128/spectrum.02853-25
UR - https://hal.science/hal-05782915v1
AB - The zebrafish increasingly serves as a model for studying the influence of the gut microbiota on the physiology of the host. In an experimental setting, specific bacteria can be used to colonize the gut of zebrafish at an early stage (larvae), affecting their development, physiology, behavior, and overall health. However, the efficiency of gut microbial colonization protocols in zebrafish larvae depends on several factors, such as bacterial strains, zebrafish lines, pre-colonization status, timing of colonization, the type of food vector, and assay methods. In this study, we tested how different parameters influence bacterial colonization in the gut larvae from various genetic lines, both axenic and conventional. To demonstrate gut colonization in transparent larvae up to 9 days post-fertilization (dpf), we used an Escherichia coli strain expressing fluorescent proteins and fluorescence microscopy. Additionally, we utilized plate counts to quantify the extent of bacterial colonization. We found that introducing live food ( Tetrahymena thermophila ) loaded with bacterial cells strongly facilitated colonization, increasing the initial bacterial load by one log. We also examined the impact of larval density and timing of exposure to bacteria, which together enabled successful bacterial colonization of the zebrafish larvae’s gut with increasing bacterial cell counts (from 10 4 to 10 6 ) over four days. We also successfully colonized the zebrafish larvae with strains of Pseudomonas aeruginosa , Aeromonas veronii , and Vibrio cholerae . Implementing standardized and optimized bacterial colonization protocols in zebrafish larvae should facilitate the use of this model to study the interactions between the gut microbiota and host physiology. IMPORTANCE Zebrafish larvae hold potential as a model for studying gut microbiota. However, standardized protocols for controlling bacterial colonization of the larval zebrafish gut are required. This article presents the analysis of various parameters that led to an effective method for colonizing zebrafish larvae with various bacteria. The proposed detailed protocol enables the qualitative, quantitative, and reproducible observation of bacterial colonization in the zebrafish larvae’s gut.
SN - 2165-0497
ER -