Pun, Ka IengKa IengPun2025-11-202025-11-202025-08https://dspace.usj.edu.mo/handle/123456789/6662The intestinal microbiota plays a key role in the physiological functions of fish. It not only participates in digestion and absorption, but also regulates immune response, disease resistance, nervous system development, behavioural performance, and may even affect social interactions and reproductive behaviour. However, research on the reverse direction of communication, i.e. how the social environment affects the gut microbiota, remains relatively scarce. This study uses the Siamese fighting fish (Betta splendens), known for its highly aggressive behaviour, as a model to explore the effects of different sampling methods on intestinal microbial diversity, and further examines the association between social environment, genotype, gut microbiota and social behaviour. The study first compared the effects of traditional intestinal sampling with a non-lethal oral swabbing method to collect the gut microbiota. DNA was extracted from intestinal samples or oral mouth swabs, and the 16S rRNA V3–V4 segment sequenced for microbiome analysis. Alpha diversity analysis revealed no significant differences in species diversity or richness between the two sampling methods. However, beta diversity metrics that incorporate phylogenetic relationships demonstrated significant differences, with the oral swab method capturing greater diversity in microbial composition. Taken together, these findings support the oral swab method as a feasible and ethically advantageous alternative, especially for longitudinal studies that require repeated sampling. After validation of the oral swab method, the work explored the effects of the social environment and genotype on gut microbiota composition. For this, males of B. splendens from two different strains, wildtype and a fighter strain selected for high aggression, were exposed for a period of 21 days to three different social contexts: i) mating; ii) aggression; iii) no social stimuli. The results showed that genotype played a significant role in shaping the gut microbiota, with fighter and wildtype fish having different gut bacterial profiles, as measured both by alpha and beta diversity indexes. There were no within-strain differences in alpha diversity, showing that average gut bacterial diversity within individuals was not affected by treatment. Contrarily, beta diversity index revealed an impact of treatment, with fish exposed to the aggression context, but not to the mating context, differing in bacterial community composition as compared with controls. This study not only validates the non-lethal oral swab method as a reliable and ethical tool for gut microbiota research in fish but also highlights the significant influence of the social environment on gut microbial composition. The findings suggest that genotype plays a key role in shaping gut microbiota profiles, with distinct bacterial communities identified in the wildtype and fighter strains of B. splendens. Notably, exposure to aggression significantly altered bacterial composition, underscoring the importance of considering social interactions when studying microbiota dynamics. Future research could focus on identifying specific microbial groups and investigating how they contribute to behavioural regulation in fishengut microbiotaSiamese fighting fishswab collection methodnon- invasive methodaggressiontext::thesis::master thesis