Blastocystis load mediates the gut microbiome associations with within-host diversity of Blastocystis in non-human primates.

Blastocystis is a prevalent gut eukaryote intricately associated with the gut microbiota. This genetically diverse protozoan exhibits significant intra-host subtype heterogeneity, yet the implications of this diversity for the host gut microbiome remain poorly understood. Here, we investigated the interactions between Blastocystis and gut microbiota in non-human primates at the level of subtypes, using a comprehensive investigation of gut microbiota for Blastocystis carriers of captive Macaca fascicularis (discovery cohort, n = 100) and Macaca mulatta (validation cohort, n = 26). We identified highly prevalent intra-host co-occurrence patterns of Blastocystis SSU rRNA-based subtypes, primarily dominated by Subtype 1 (ST1) or ST3. These patterns were associated with compositional and structural variations in the gut microbiome but were not significantly influenced by host covariates such as sex, age, or BMI. Specifically, Ruminococcaceae-enterotype was enriched in the patterns dominated by ST1, whereas Limosilactobacillus-enterotype was predominantly identified in the patterns dominated by ST3. Variance partitioning and mediation analyses revealed that the absolute abundance of Blastocystis was a critical determinant in elucidating this microbiota association across subtype concurrent patterns. In vivo experiments in a new cohort (n = 11) demonstrated that lactic acid bacteria, enriched in the Limosilactobacillus-enterotype, were sufficient to reduce Blastocystis load. We validated the strong association between gut microbiome composition and Blastocystis load in M. mulatta, confirming that specific microbial features could quantitatively predict Blastocystis status in both species. These findings highlight the close links of the gut microbiome with within-host subtype diversity patterns and absolute abundance of Blastocystis.