Axenic and Gnotobiotic Aquatic Models: Methods, Husbandry, and Applications

Abstract

Axenic and gnotobiotic aquatic animals provide direct experimental access to the causal roles of microorganisms in host physiology, nutrition, immunity and disease. Fish are particularly suitable vertebrate models because fertilization and embryogenesis commonly occur externally, allowing embryos to be disinfected before hatching and maintained under microbiologically controlled conditions. Current approaches combine embryo or egg surface disinfection, sterile water and culture systems, sterilized formulated feeds or axenic live feeds, as well as controlled colonization with defined microorganisms. These systems enable direct testing of microbial effects on immunity, metabolism, pathogen resistance, and nutritional responses. However, their interpretation is constrained by contamination risk, species- and developmental-specific husbandry requirements, changes in feed and water chemistry caused by sterilization, low-biomass limitations in sterility testing, and potential confounding effects of prolonged microbial absence. Zebrafish (Danio rerio) is currently the most studied and well-established axenic or gnotobiotic fish model, but methods have also been established for medaka (Oryzias latipes) and aquaculture species such as Rainbow trout (Oncorhynchus mykiss), European sea bass (Dicentrarchus labrax) and Atlantic cod (Gadus morhua). This narrative review evaluates the practical and conceptual basis of axenic aquatic animal research, with emphasis on fish production, rearing and handling. It compares representative derivation procedures, sterile culture formats, feeding strategies, water-quality constraints and sterility assays, and it examines how these methodological choices affect biological interpretation. A central conclusion is that “axenic” is an operational microbiological state rather than an absolute biological property, as confidence intervals depend on sampling design and orthogonal detection. The review therefore proposes minimum reporting and quality-control practices, including vessel-level replication, longitudinal sterility testing, feed validation, contamination rules and appropriate recolonization controls. These standards are essential if axenic or gnotobiotic aquatic models are to produce reproducible mechanistic results. Future progress will depend on standardized validation and reporting criteria, improved sterile nutrition, water management and longer-term husbandry systems.