Meiosis is regulated by phase-specific genes to orchestrate nuclear and cytoskeletal dynamics essential for sexual reproduction. The ciliate Paramecium tetraurelia exhibits nuclear dimorphism, harboring two germline micronuclei (MICs) and one somatic macronucleus (MAC) within a single cell during vegetative growth. During sexual reproduction, the MICs undergo meiosis and the MAC deforms and fragments, providing a unique model to study the regulation of diverse nuclear events. Transcription factor DP (TFDP), which heterodimerizes with E2Fs, can bind to specific DNA motifs in promoters of cell cycle-regulated genes to activate or repress their expression. Here, we identified 16 TFDP homologs in P. tetraurelia, representing an exceptional gene family expansion accompanied by functional domain diversification. The functions of Tfdp1a and Tfdp1b, which are specifically expressed during sexual reproduction and localize in the old and new MACs, were further investigated. Their depletion resulted in meiotic arrest at metaphase in the MIC, failure of old MAC fragmentation, and abortive cytokinesis. Transcriptomic analysis revealed that TFDP1A/1B knockdown primarily causes gene downregulation, with > 60% of downregulated genes being specifically highly expressed during sexual reproduction. Functional annotation and enrichment analyses demonstrated significant downregulation of proteins involved in meiosis, DNA replication, and DNA repair. Critically, multiple downregulated meiotic regulators are essential for proper homologous chromosome segregation and sister chromatid separation, providing a mechanistic basis for the observed MIC meiotic arrest. This study uncovers Tfdp1a/1b as essential regulators of the distinctive meiotic process in P. tetraurelia, providing crucial insights into nuclear dynamics and the regulation of sexual reproduction in binucleate systems.

Scuticociliatia is one of the most species-rich subclasses in the phylum Ciliophora. The evolutionary relationships among Scuticociliatia groups have long been very unclear due to the homogeneity of morphology and insufficiency of molecular data. With morphological and multi-gene-based molecular data presented here, the evolutionary phylogeny of several Scuticociliatia taxa that were hitherto especially poorly defined is analyzed and discussed. The results indicate: (1) all scuticociliates cluster into two well supported and one poorly supported group, representing three order-level taxa; (2) with the support of both morphological and molecular data, a new family Homalogastridae fam. nov. is proposed in the order Philasterida; (3) Parauronema is formally transferred to Uronematidae and Potomacus is treated as incertae sedis in the order Philasterida, therefore Parauronematidae is proposed to be a junior synonym of Uronematidae; (4) the genus Madsenia and the species Parauronema longum and Pseudocyclidium longum are treated as incertae sedis, while the genus Protophyra should be maintained in the family Ancistridae. In addition, the putative secondary structure of internal transcribed spacer 2 (ITS2) of representative taxa from the three orders of Scuticociliatia are analyzed, and consensus structures and nucleotide composition in each order are exhibited.