Effect of intestinal microbiota on adaptation to overcrowding stress in grouper (Epinephelus fuscoguttatus♀×E. lanceolatus♂)

Density is an important aquaculture parameter. When the pearl gentian grouper (Epinephelus fuscoguttatus♀ × E. lanceolatus♂) is farmed intensively, it could lead to a degradation in genetic resources and an increase in disease outbreaks. The composition of the intestinal microbiota plays a key role in creating a specific intestinal microecosystem, which is essential for the survival, growth, and immune response of the host under environmental stress like overcrowding. This study utilized 16S rRNA sequencing and metabolomics analysis techniques to investigate the differences in intestinal microbial community stability of grouper under different stocking time and density pressure conditions. The research results showed that compared to the low-density group, the high-density group of groupers experienced an increase in mortality rate and feed coefficient in the early stages of culture, while the weight gain rate decreased. Differential analysis of intestinal microbial communities revealed significant differences in the gut microbiota of grouper between different density groups after 10 days of culture, but no significant differences were observed after 20 days of culture. At the same time, intestinal histopathology showed that the high-density group of groupers exhibited a reduction in intestinal villi length and thickness of the intestinal wall after 10 days of culture. However, the intergroup differences had reduced after 20 days of culture. Furthermore, high density cultivation upregulated the expression of inflammatory factors like IL-1β, TNF-α, IL-8, and IL-6 in the intestinal tract of groupers after 10 days of culture. However, after 20 days of culture, the expression levels of intestinal inflammatory factors in both the high-density and low-density groups of groupers were significantly reduced, and the differences between the intergroup diminished. Through correlation analysis of differential metabolites and species in the intestine, multiple metabolites significantly upregulated and associated with the upregulation of the Staphylococcus genus were identified in the intestinal tract of groupers after 20 days of high-density cultivation. The selected four associated metabolites (including creatine, fosinopril, 4-aminobutyric acid, and guanidinopropanoic acid) were validated to significantly reduce the expression of cellular inflammatory factors using the self-established grouper head kidney (HK) cell line. In conclusion, density pressure in the early culture period could affect the stability of the intestinal microbial environment of grouper. As aquaculture time increases, the intestinal microbial community of grouper drives the body's anti-inflammatory response and enhanced its adaptation to density pressure by regulating own structure and secretion of metabolites.