Abstract
The progressive impact of Enterocytozoon hepatopenaei (EHP) infection on gut microbial function in Litopenaeus vannamei remains poorly understood beyond static comparisons between healthy and infected individuals. To close this knowledge gap, metagenomic sequencing was used to characterize the gut microbiomes of normal, long, medium, and short-sized adult shrimp categorized by increasing severity of infection. EHP infection suppressed digestive activity while inducing immune responses compared with healthy shrimp. Increasing infection severity was associated with a gradual decline in gut α-diversity and an expansion of potential pathogens and virulence factors (VFs). In addition, dysbiosis in gut microbiota composition and function, as well as reduced network stability among differential species, intensified with infection severity. Accordingly, we identified 24 EHP-discriminatory species that contributed an overall 83.3% accuracy in diagnosing infection severity without false negatives. Functional pathway analysis revealed significant suppression of metabolic, degradative, and biosynthetic processes in EHP-infected shrimp compared with healthy controls. Among them, map00630 glyoxylate and dicarboxylate metabolism and map00280 valine, leucine and isoleucine degradation were consistently depleted in infected individuals, thereby impairing their digestive function and anti-inflammatory responses. Additionally, EHP infection diversified VFs directly affecting shrimp gut microbiome. These findings support a conceptual model linking EHP pathogenesis to the parasite-gut microbiome-shrimp physiology axis.