PHYLOGENETIC RELATIONSHIP AMONG POLYMORPHIC OLIGOHYMENOPHOREAN CILIATES, WITH GENE EXPRESSION IN LIFE-HISTORY STAGES OF MIAMIENSIS AVIDUS (CILIOPHORA, OLIGOHYMENOPHOREA)
Gebler, Glenn Frederick
Small, Eugene B.
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The Class Oligohymenophorea is a monophyletic group possessing polymorphic taxa. Thus far, relationships within subclasses of oligohymenophorean ciliates and between polymorphic taxa within families are not well resolved. Here, nuclear small subunit rRNA (SSU rRNA) gene sequences from 63 representative taxa, including several polymorphic species, were used to construct phylogenies and test monophyly of the subclass Scuticociliatia and of the polymorphic taxa within the Oligohymenophorea. In addition, suppression subtraction hybridization (SSH) was used to test the hypothesis that genes are differentially expressed during microstome-to-macrostome and tomite-to-microstome transformation in the polymorphic scuticociliate Miamiensis avidus. Phylogenetic analyses confirmed monophyly of the subclasses Peritrichia and Hymenostomatia. The monophyletic scuticociliates encompassed most, but not all, taxa included in this study. The conditional acceptance of the hypothesis supporting monophyly of the Scuticociliatia was due to the ambiguous placement of three taxa, the apostome Anoplophrya marylandensis, the scuticociliate Dexitrichides pangi, and the peniculine Urocentrum turbo. The polymorphic trait most likely arose on at least four, and perhaps on as many as six, separate occasions within the oligohymenophorean ciliates. Several genes previously implicated in morphogenetic processes in eukaryotes were upregulated during microstome-to-macrostome transformation in M. avidus. Those genes were, elongation factor-1 alpha (Ef-1α), Constans, Constans-like TOC1 (CCT) transcription factor, a disulfide isomerase, heat shock protein 70, step II splicing factor (Slu7), U1 zinc finger protein, and WD40-16 repeat protein. A similar analysis for M. avidus undergoing tomite-to-microstome transformation identified genes previously linked to transformation processes in other protists: two cysteine protease genes lacking formal description (papain-family and XCP1 cysteine protease), two described cysteine protease genes, cathepsin B and cathepsin L, and one cysteine protease inhibitor (cystatin-1) gene. The roles of candidate genes for regulation of M. avidus life-history stages (Ef-1α for microstome-to-macrostome transformation; cathepsin B and cathepsin L for tomite-to-microstome transformation) were examined using pharmacological inhibition experiments. Drug treatments significantly reduced transformation of M. avidus microstomes into macrostomes within 6 h and prevented tomite-to-microstome transformation after 2.5 h. Results indicated that genes specifically linked to oral transformation in M. avidus are differentially expressed during microstome-macrostome and tomite-microstome transformation. Thus, this study used molecular techniques to understand the evolutionary history and development of polymorphism within the Oligohymenophorean ciliates.