<i>Blastocystis</i> load mediates the gut microbiome associations with within-host diversity of <i>Blastocystis</i> in non-human primates

<i>Blastocystis</i> 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 <i>Blastocystis</i> and gut microbiota in non-human primates at the level of subtypes, using a comprehensive investigation of gut microbiota for <i>Blastocystis</i> carriers of captive <i>Macaca fascicularis</i> (discovery cohort, n = 100) and <i>Macaca mulatta</i> (validation cohort, n = 26). We identified highly prevalent intra-host co-occurrence patterns of <i>Blastocystis SSU</i> 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, <i>Ruminococcaceae</i>-enterotype was enriched in the patterns dominated by ST1, whereas <i>Limosilactobacillus</i>-enterotype was predominantly identified in the patterns dominated by ST3. Variance partitioning and mediation analyses revealed that the absolute abundance of <i>Blastocystis</i> was a critical determinant in elucidating this microbiota association across subtype concurrent patterns. <i>In vivo</i> experiments in a new cohort (n = 11) demonstrated that lactic acid bacteria, enriched in the <i>Limosilactobacillus</i>-enterotype, were sufficient to reduce <i>Blastocystis</i> load. We validated the strong association between gut microbiome composition and <i>Blastocystis</i> load in <i>M. mulatta</i>, confirming that specific microbial features could quantitatively predict <i>Blastocystis</i> status in both species. These findings highlight the close links of the gut microbiome with within-host subtype diversity patterns and absolute abundance of <i>Blastocystis</i>.