Toxoplasma gondii, a distinctive intracellular parasite, is capable of infecting almost all warm-blooded animals. Its merozoite stage, restricted to epithelial cells of the intestinal of final hosts (felines), is categorized into five distinct types——types A through E, but the kinetic development processes through A to E and the variation in gene expression levels among these five types have not been extensively explored.

To elucidate the transcriptional patterns of single individuals of the five types，we harvested merozoites 35 hours and 7 days post tissue cyst infection and conducted single-cell RNA sequencing. Through this process, we were able to effectively get 6,762 parasites and categorize them into 7 clusters after clustering analysis. Cluster 4, consisting of 467 parasites, was identified as tachyzoites due to their specific expression of Sag1. Cluster 0, with 2,635 parasites, exhibited specific expression of both Sag2A and LDH2 (but not Gra11b), such being defined as tachyzoite-bradyzoite intermediates. Other five clusters all expressed a merozoite marker gene, GRA80, with Cluster 1, 2, 3, and 6 also expressing Gra11b. Additionally, Cluster 1 highly expressed SRS22E (1,184 parasites), Cluster 2 expressed AT1 but not TGME49_287040 (1,055 parasites), Cluster 3, expressed TGME49_231880 without LDH2 (993 parasites), Cluster 5, specifically expressed SRS22b (330 parasites), and Cluster 6, specifically expressed TGME49_278410 (124 parasites).

To correlate our transcriptional findings with Types A to E, we constructed a reporter strain employing Eno2, Bag1, and SRS22b as promoters, which resulted in Eno2-induced green fluorescence in the nucleus, LDH2-induced red fluorescence in the cytoplasm, and SRS22b-induced green fluorescence on the parasite membrane or cytoplasm. Intestinal smears from cats 7 days post infection of this strain showed that Eno2 was present in B through E, with Type D also expressing SRS22b.

Furthermore, one reporter strain with promoters for Sag2A, LDH2, and Gra81, and another with promoters for Sag1, Gra11b, and Gra80 are also constructed to pinpoint distinct biomarkers corresponding to Type A through E. Ongoing single-cell RNA sequencing investigations will be conducted to investigate earlier stages of presexual development, aiming to provide valuable insights into merozoite marker typing and the presexual evolution of T. gondii within feline intestines.