Abstract
Babesiosis is an emerging and re-emerging zoonosis that is prevalent worldwide, caused by over 100 Babesia species. These intracellular parasites efficiently invade host red bloods cells, replicate rapidly, and exit the infected cells to cause clinical symptoms. However, the underlying mechanisms of regulating this asexual stage is largely unknown. Here, we generated a chromosome-level reference assembly of a novel Babesia species, Babesia xinjiangensis. Using single-copy orthologous genes, we confirmed its phylogenetic relationships with other apicomplexan parasites and estimated its speciation time. We identified species-specific gene families and core gene families that could be responsible for species speciation and immune evasion. Furthermore, we also used a single-cell RNA-sequencing (scRNA-seq) protocol to uncover hidden transcriptional variations in the asexual stages of this unicellular Babesia parasite and its cell-to-cell heterogeneity. We inferred the replication cycle and performed a pseudotime analysis to speculate the gene expression profiles. Although the peak expression times of most epigenetic markers and transcription factors were confined to specific phases, BxAP2-M2 (GWHPERCV002055) is constantly expressed during asexual development progression. Genetic analyses revealed that BxAP2-M2 directly or indirectly regulates the expression of rhoptry proteins and membrane proteins, which may play critical roles in the parasite's invasion of red blood cells and the merozoite morphology. Our findings provide valuable markers of asexual replication, including some that are specific to Babesia gametocytes, and regulators specific to distinct cell-cycle phases.