Effects of Inducible Nitric Oxide Synthase (iNOS) Gene Knockout on the Diversity, Composition, and Function of Gut Microbiota in Adult Zebrafish

Simple Summary The gut microbiome, a complex microbial community, is intricately linked to the host's genetic background. The regulatory role of host genes on the microbiome has garnered considerable attention due to its impact not only on physiological and immune responses but also on the composition and functionality of intestinal microbes and the overall immune system. Nitric oxide synthase (NOS), through the production of nitric oxide (NO), participates in host defense, immune regulation, inflammatory responses, and autoimmune diseases. However, the effects of NOS on the composition and function of the zebrafish gut microbiota and the regulation of its homeostasis remain unclear. Genomic editing technologies have enabled the creation of zebrafish with specific genetic defects, making them a recognized model for studying host-microbiome-immune interactions. In this study, we characterized the impact of inducible nitric oxide synthase (iNOS) deficiency on the gut microbiota of zebrafish through 16S rRNA amplicon sequencing. Results revealed significant alterations in the microbial diversity and abundance in iNOS-deficient zebrafish, notably a reduction in Vibrio and an increase in Aeromonas. Transcriptomic sequencing of the gut confirmed functional changes, showing significant alterations in pathways related to the complement and coagulation cascades, PPAR signaling, cell adhesion molecules, Staphylococcus aureus infection, steroid synthesis, and bile acid synthesis. These pathways are crucial in pathogen clearance, inflammatory responses, and immune regulation, highlighting the significant role of the iNOS gene not only in microbial composition but also in gut immune and metabolic functions.Abstract The gut microbiota constitutes a complex ecosystem that has an important impact on host health. In this study, genetically engineered zebrafish with inducible nitric oxide synthase (iNOS or NOS2) knockout were used as a model to investigate the effects of nos2a/nos2b gene single knockout and nos2 gene double knockout on intestinal microbiome composition and function. Extensive 16S rRNA sequencing revealed substantial changes in microbial diversity and specific taxonomic abundances, yet it did not affect the functional structure of the intestinal tissues. Notably, iNOS-deficient zebrafish demonstrated a decrease in Vibrio species and an increase in Aeromonas species, with more pronounced effects observed in double knockouts. Further transcriptomic analysis of the gut in double iNOS knockout zebrafish indicated significant alterations in immune-related and metabolic pathways, including the complement and PPAR signaling pathways. These findings underscore the crucial interplay between host genetics and gut microbiota, indicating that iNOS plays a key role in modulating the gut microbial ecology, host immune system, and metabolic responses.