Effect of 16S rRNA Gene Hypervariable Region on the microbiome taxonomic profile and diversity in the endangered fish Totoaba macdonaldi

Understanding the intricate dynamics of fish microbiota through 16S rRNA amplicon sequencing is pivotal for ecological insights and effective disease management. However, this approach faces challenges including the co-amplification of host mitochondrial sequences and the variability in bacterial composition influenced by the selected 16S rRNA gene regions. To overcome these limi-tations, we conducted a comprehensive investigation to identify the most suitable 16S rRNA region for bacterial microbial analysis in endangered fish Totoaba macdonaldi, an endemic species of significant ecological and economic importance in Mexico. Targeting four distinct regions (V1-V2, V2-V3, V3-V4, and V5-V7) of the 16S rRNA gene, we determined the microbial composition within the distal intestine. A total of 40 microbiomes were sequenced. Our findings underscore the critical impact of region selection on the accuracy of microbiota analysis, with the V3-V4 region demon-strating the highest taxonomic certainty. Furthermore, our results unveiled statistically significant differences in alpha and beta diversity metrics among the selected regions. Notably, the V3-V4 re-gion exhibited the highest bacterial diversity, followed by V1-V2. Moreover, disparities in bacterial phyla classification were observed, exemplified by Firmicutes predominance in the V3-V4 region contrasting with Protobacteria abundance in V1-V2. This study emphasizes the necessity of metic-ulous 16S rRNA region selection for fish microbiota analysis, particularly in native species of eco-logical and economic significance such as the endangered T. macdonaldi, where information is lim-ited. Such optimization enhances the reliability and applicability of microbiota studies in fisheries management and conservation efforts.