Effects of Different Preservation Methods on the Structure and Diversity of Intestinal Microbiota of Marine Fishes

The fish intestine is a complex ecosystem where microbial communities are dynamic and influenced by various factors. Preservation conditions during field collection can introduce biases affecting the microbiota amplified during sequencing. Therefore, establishing effective, standardized methods for sampling fish intestinal microbiota is crucial. This study used hybrid groupers (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂) to examine the effects of six preservation methods: dry ice (1 day), dry ice (1 day) followed by - 80 °C storage (5 days), liquid nitrogen (1 day), liquid nitrogen (1 day) with subsequent - 80 °C storage (5 days), refrigeration at 4 °C (3 days), and freezing at - 20 °C (3 days), with fresh samples as controls. High-throughput 16S rRNA sequencing assessed microbial diversity, community structure, dominant species, and OTU abundance across treatments. Results indicated that dry ice and liquid nitrogen methods, especially with - 80 °C storage, had minimal impact on microbial diversity and structure. Compared to other preservation methods, refrigeration at 4 °C and freezing at - 20 °C may result in suboptimal reproducibility and altered community structure, particularly affecting rare microbial taxa. This study underscores the need for standardized preservation techniques to ensure accurate fish intestinal microbiota analysis and provides a foundation for future research.