bioRxiv2023
Marine medaka (Oryzias melastigma) animal models play critical roles in environmental and human health by facilitating evaluation of pollutant toxicity and building of disease models. The fish gut microbiota contributes to host health and physiological metabolism, especially special bacterial strains and their functions in marine organisms. However, the distribution of the gut microbiota during medaka growth and development is still unclear, and successful generation of a germ-free (GF) marine medaka model has not been reported to date. In this study, we investigated the microbial composition with the major phyla and genera of marine fish at different life stages, as well as the isolated culturable intestinal bacteria, and then identified them by sequencing of the16S rRNA V3-V4 region. Importantly, the early stage model (larvae) of GF marine medaka without feeding and long-term (from juvenile to early adult stages) GF fish fed GF brine shrimp (Artemia sp.) were first generated. Moreover, the basic indexes and behavioral ability of GF fish showed weaker and delayed developmental changes compared to conventionally raised (CR) marine medaka at the same life stages. Notably, the significant differences in the histopathological characteristics of immune organs, intestinal tissues and the reproductive system were observed between GF and CR early-adult and adult fish. Furthermore, the transcriptomic profiles of the screened critical genes in signaling pathways in GF and CR marine medaka were also explored to illustrate the developmental impacts of the absence of the intestinal microbiota during the host growth. Comprehensively, our study provided novel insights into the intestinal microbiota distribution of CR fish during growth, and GF marine medaka from the larval to adult stages via GF fish food preparation. The histopathological and transcriptomic differences indicated the potential microbial regulation on growth, and application prospects of GF medaka fish models to clarify the relationships of intestinal bacterial functions to host health in the future. SignificanceThe generation and application of germ-free (GF) fish models are mostly limited to the early life stages with innate immunity and without feeding. Marine medaka (Oryzias melastigma) is a critical animal for evaluating environmental toxicity and human disease models. The gut microbiota contributes to host growth and development, but GF model of this organism has not been successfully generated. In this study, we revealed for the first time the distribution of the gut microbiota in marine medaka during growth and generated GF fish from the larval to adult stages with GF Artemia provided daily as food. According to the basic indexes, weaker behavioral ability, smaller immune organs, reproductive system, intestinal tissues, and transcriptome, the delayed development and differences indicated the negative influences of the absence of the microbiota in GF medaka, compared to conventionally raised (CR) fish at the same life stages. All these results provided novel insights into the application of GF medaka models to define intestinal bacterial functions in the host. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC="FIGDIR/small/536225v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@15625b5org.highwire.dtl.DTLVardef@c5cf62org.highwire.dtl.DTLVardef@b264c5org.highwire.dtl.DTLVardef@1f5e7d3_HPS_FORMAT_FIGEXP M_FIG C_FIG This work revealed the distribution of the gut microbiota in marine medaka during growth, and successfully generated GF marine medaka models from larvae to adults with GF Artemia as food, which indicated the delayed development in the absence of the microbiota in GF fish. Moreover, the histopathological analysis presented further evidence of developmental differences in immune organs, intestinal villi, goblet cells, gonad tissues and cell maturation between GF and CR fish at various life stages. Finally, the transcriptomic profile showed the significantly differentially regulated genes, which combined with the major bacteria can be potential "biomarkers" to explore the inner mechanisms or signaling pathways of GF fish models for studying host development and health.