Aneuploidy is considered a widespread and potentially ancestral characteristic of trypanosomatids, the exception being the stably diploid Trypanosoma brucei. Although aneuploidy and polyploidy in T. brucei appear to be rare, chromosomic trisomies and polyploid hybrids have been reported. However, what aspect of T. brucei genome duplication largely limits its genome to a diploid state is unclear. 

Here we describe a stable, fully tetraploid, procyclic T. brucei cell line, recovered during unrelated genetic manipulation. Morphologically, the tetraploid cells are marginally larger than the parental diploid cells and have an ~1.4x increase in nuclear area. Their nucleus to kinetoplast ratio, DNA replication, and growth in culture are not substantially affected, suggesting the cell cycle is unperturbed. Flow cytometry indicates that the cell line is fully tetraploid, showing double the DNA content of its parent at days 8 and 57 of continuous growth in culture. Whole genome sequencing is consistent with whole genome tetraploidy, showing uniform coverage across all chromosome cores and subtelomeres, with the only evidence of aneuploidy seen being in a transcriptionally silent bloodstream VSG expression site. Single nucleotide polymorphism (SNP) analysis revealed no shift in allele frequency, and therefore no evidence of genetic drift between the 4 copies of the genome, suggesting that these cells arose from a recent whole genome duplication event. Remarkably, however, transcriptome analysis revealed virtually no differentially expressed genes between the tetraploid and parental diploid cells, suggesting the existence of a robust process to maintain transcription homeostasis after an increase in genome copy number.