Abstract:The intestinal microbiota plays a crucial role in the nutritional absorption, immune regulation, and overall health of aquatic animals. In recent years, increasing attention has been focus on the composition and developmental dynamics of gut microbial communities in fish, particularly during early life stages. However, limited information exists on the gut microbiota of economically important marine fish such as the greenfin horse-faced filefish (Thamnaconus septentrionalis), especially across different developmental timepoints. Understanding the microbiota succession in this species is crucial for improving larval rearing success and optimizing feed strategies. This study systematically investigated the temporal changes and community structure of intestinal microbiota in T. septentrionalis larvae and juveniles at eight representative developmental stages (G0 to G50), from fertilized eggs to advanced juvenile stages. The primary objective was to explore the dynamic succession of gut microbiota and to evaluate its relationship with host age, physiological state, and feed transitions (rotifer, Artemia nauplii, and frozen Artemia). The study also aimed to identify dominant and stage-specific bacterial taxa associated with different gut ecological environments during development. High-throughput 16S rRNA gene sequencing was performed on 24 gut samples across the eight timepoints. Alpha diversity was assessed using Chao1 and Shannon indices to quantify richness and diversity. Beta diversity analysis, including NMDS (Non-Metric Multidimensional Scaling) and Unweighted UniFrac distances, was used to evaluate community dissimilarities across stages. LEfSe (Linear Discriminant Analysis Effect Size) analysis was employed to identify significantly enriched bacterial taxa at different stages. In addition, heatmaps and cluster analyses were used to visualize taxonomic shifts and abundance gradients of key genera. The results revealed that the gut microbial community of T. septentrionalis underwent distinct compositional shifts during development, exhibiting a phased and directed successional pattern. Alpha diversity indices showed that microbial richness was lowest in the G0 stage (fertilized eggs), characterized by sparse colonization mostly originating from the egg surface and surrounding water. Following the initiation of exogenous feeding in G1 (rotifer ingestion), both Chao1 and Shannon indices increased sharply, peaking at G3. This increase corresponded with microbial input from live feeds. Diversity began to fluctuate during the G9–G30 stages and gradually stabilized by G40–G50 (frozen Artemia stage), suggesting the establishment of a selective and stable gut microenvironment. Beta diversity analyses further supported these findings. Samples from G0 were clearly separated from other stages, reflecting a unique early-stage microbial signature. A transitional shift was observed from G3 to G30, and samples from G40–G50 clustered more tightly, indicating microbiota convergence and ecological stabilization in the later stages. These trends align with previous findings in other marine fish species and emphasize the interaction between gut microbiota and host development. At the genus level, the dominant bacterial taxa changed substantially throughout development. In G0, Vibrio and Tenacibaculum were dominant, likely originating from environmental or egg-associated communities. In G1–G9, genera such as Acinetobacter, Cupriavidus, and Rubritalea increased in relative abundance, indicating microbial adaptation to rotifer ingestion and gut environmental changes. In G30, Cupriavidus, Pseudobacteriovorax, and Legionella were significantly enriched, suggesting strong selection pressure from Artemia nauplii and associated metabolites. In G50, Photobacterium became the predominant genus (up to 63.8% relative abundance), likely due to the prolonged feeding of frozen Artemia and altered gut nutrient availability. The enrichment of species such as P. aphoticum and P. damselae may also indicate potential health risks related to pathogenicity, as reported in other marine fish. LEfSe analysis confirmed the presence of stage-specific characteristic genera. Tenacibaculum was a biomarker for G0, Epibacterium and Xanthomarina for G3, Lactobacillus and Streptococcus for G9, while Massilia and Pseudobacteriovorax were indicators of G20–G30. These findings suggest that different feeding regimes and developmental stages exert selective pressures shaping unique microbial niches within the gut. The dynamic pattern of appearance and disappearance of key taxa implies an adaptive microbial response to host nutritional status and gut maturation. This study provides the first comprehensive profile of the intestinal microbial succession in T. septentrionalis during early ontogeny. The results highlight the essential role of diet and age in shaping gut microbial ecology. Maintaining microbial diversity, particularly during critical feed transitions, may enhance host resistance and survival. Moreover, insights into dominant and beneficial taxa lay the groundwork for future development of probiotic or microbiome-targeted functional feeds. These findings contribute valuable knowledge to larval rearing practices and microbiota management in marine aquaculture.
