Comparative Genomic and Transcriptomic Profiling Revealed the Molecular Basis of Starch Promoting the Growth and Proliferation of Balantioides coli

**Background:** Balantioides coli is a zoonotic ciliated parasite that colonizes the cecum and colon of humans and pigs, causing diarrhea—particularly in weaned piglets transitioning from sow milk to starch-containing pellet feed. Previous work showed that starch is essential for B. coli survival in culture, but the molecular mechanisms were unknown. This study aimed to elucidate how starch promotes B. coli growth and proliferation at the genomic and transcriptomic levels.

**Methods:** B. coli strain P011 was isolated from a diarrheic 43-day-old weaned piglet in Henan Province, China. Trophozoites were cultured in modified DMEM medium at 28°C. Using mouth pipette single-cell isolation under a stereomicroscope, 300 trophozoites were placed in medium with 5 mg/mL starch (T group) and 300 in medium without starch (N group). For transcriptomic analysis, 10 cells were collected at peak growth (IS subgroup), at 72 h post-starch addition (TS subgroup), and at 72 h from the N group (NS subgroup). cDNA libraries were constructed using the SMART-seq2 single-cell RNA-seq method, and 150 bp paired-end sequencing was performed on an Illumina HiSeq 4000. Clean reads were aligned to the B. coli genome (GWHBOZN00000000). DEGs were identified using edgeR (fold change ≥ 2, FDR < 0.05). GO and KEGG enrichment analyses were performed. Eight DEGs were validated by RT-qPCR. Comparative genomic analysis was conducted against eight other ciliate species using Orthofinder v2.4 and café v4.2 to identify specific and expanded gene families.

**Key Results:** A total of 22.27 Gb of clean data were generated. Clean read percentages were 92.63% (IS), 93.61% (TS), and 87.32% (NS). Q20 ranged from 89.04–93.69%, Q30 > 86.70%, and GC content averaged 30%. Mapping rates were 68.2–76.1%. In the TS vs. NS comparison, 5508 genes were upregulated and 2231 downregulated. In the NS vs. IS set, most of the 8876 DEGs were downregulated. In the TS vs. IS set, 6493 DEGs were identified. Hierarchical clustering identified 3690 genes in cluster 4 with highest expression in TS, enriched for calcium ion binding, cell cycle regulation, and phosphorylation. KEGG analysis showed upregulation of starch/sucrose metabolism genes (AMY, log2FC=3.58, p=1.49×10⁻⁹; MA, log2FC=2.32, p=2.68×10⁻²; malQ, log2FC=8.10, p=1.30×10⁻⁹) and glycolysis genes (PYG, log2FC=4.89, p=3.68×10⁻³⁹; GCK, log2FC=5.25, p=1.10×10⁻¹⁸; GPI, log2FC=3.59, p=1.27×10⁻⁷). Cell cycle genes were upregulated (Cdc25, log2FC=3.19, p=2.01×10⁻³; Cyr1, log2FC=7.29, p=4.54×10⁻⁶; CDK1, log2FC=8.13, p=7.43×10⁻¹⁰; CDK2, log2FC=6.58, p=6.67×10⁻⁴; CDK4, log2FC=11.44, p=1.27×10⁻³; CCNA, log2FC=7.74, p=7.04×10⁻⁸; CCNE, log2FC=2.40, p=4.23×10⁻⁶; AKT, log2FC=1.56, p=1.32×10⁻²; PDK1, log2FC=2.04, p=1.91×10⁻³; SGK1, log2FC=3.27, p=2.40×10⁻⁶). Autophagy-related genes were downregulated (AMPK, log2FC=−3.25, p=1.11×10⁻¹²; ATG1, log2FC=−4.59, p=3.38×10⁻²⁸; PI3K, log2FC=−3.31, p=4.44×10⁻³). RT-qPCR validation showed high correlation with RNA-seq data (R²=0.86). Comparative genomics identified 1436 specific gene families (4461 genes) and 36 expanded gene families (510 genes) in B. coli. Expanded families were enriched for endocytosis, phagocytic vesicles, glucose transport, carbohydrate utilization, and the cAMP signaling pathway. Specific families were enriched for endocytosis, ubiquitin-mediated proteolysis, MAPK signaling, and transmembrane transporter activities. In culture, the T group reached peak concentration at 96 h, while the N group showed declining numbers with trophozoites disappearing by 96 h. At 72 h, the T group had significantly more trophozoites than the N group (p < 0.0001; average 10,400 cells/mL vs. 30 cells/mL).

**Clinical Implications:** This study provides the first single-cell transcriptomic analysis of B. coli and reveals the molecular basis for starch-dependent growth. Starch is ingested via endocytosis, hydrolyzed to glucose by amylases, and metabolized through glycolysis. The glycolytic intermediate fructose-1,6-bisphosphate activates the cAMP/PKA signaling pathway via Cdc25 and Cyr1, promoting cell cycle progression through upregulation of Cyclins and CDKs. Concurrently, the PI3K/AKT/mTOR pathway suppresses autophagy via downregulation of AMPK and ATG1. These mechanisms explain why weaned piglets—whose diet shifts abruptly to starch-rich pellet feed—are particularly susceptible to B. coli proliferation and balantidial diarrhea. The findings suggest that adjusting starch levels in feed could serve as a control strategy for balantiosis in young animals. The authors note limitations including the small sample size and the need for further multi-omics studies and gene knock-out experiments to confirm key gene functions.