Integrated Multi-Omics Analysis Provides No Evidence for Pathogenicity of Balantidium Ctenopharyngodoni in Grass Carp Hindgut

Balantidium ctenopharyngodoni Chen, 1955 is the only ciliate species known to inhabit the digestive tract of grass carp, where it predominantly resides in the spaces between mucosal folds and within the intestinal contents of the hindgut with a high prevalence. Despite its high prevalence, the pathogenicity remains controversial. The intestinal bacterial microbiota interacts closely with enteric protozoa, and such interactions may alter host–parasite dynamics, with consequences for host susceptibility and overall health. In this study, we conducted a feeding experiment with three different dietary regimes to explore the effects of B. ctenopharyngodoni colonization on grass carp, using an integrative approach that combined microbial amplicon sequencing, metabolomics, and intestinal transcriptomics. Our results showed that balantidia colonization was associated with slightly lower body length and body weight, although these differences were not statistically significant. Balantidia colonization was also related to altered microbiota composition and diversity: the relative abundance of Bacteroidetes was more abundant in the balantidia-positive fish with a plant-based diet, while alphaproteobacteria and betaproteobacteria were enriched in the balantidia-positive fish with commercial feed. Although B. ctenopharyngodoni moderately perturbed the microbial profiles and metabolic processes of the grass carp hindgut, intestinal transcriptomic analysis did not detect significant transcriptional changes in immune-related genes between balantidia-positive and balantidia-negative groups. Collectively, under the tested dietary and physiological conditions, our findings provide no evidence of overt pathogenicity, further supporting the view that B. ctenopharyngodoni acts as a commensal ciliate in the grass carp hindgut.