Citation: Buonanno, F.; Trenti, F.; Achille, G.; Vallesi, A.; Guella, G.; Ortenzi, C. Chemical Defence by Sterols in the Freshwater Ciliate Stentor polymorphus. Biology 2022, 11, 1749. https://doi.org/10.3390 /biology11121749 Academic Editors: Fengli Zhang and Michel Denis Received: 7 November 2022 Accepted: 29 November 2022 Published: 30 November 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). biology Article Chemical Defence by Sterols in the Freshwater Ciliate Stentor polymorphus Federico Buonanno 1, * ,† , Francesco Trenti 2, * ,† , Gabriele Achille 1 , Adriana Vallesi 3 , Graziano Guella 2 and Claudio Ortenzi 1 1 Laboratory of Protistology and Biology Education, Department of Education, Cultural Heritage, Tourism (ECHT), University of Macerata, 62100 Macerata, Italy 2 Bioorganic Chemistry Laboratory, Department of Physics, University of Trento, 38050 Trento, Italy 3 Laboratory of Eukaryotic Microbiology and Animal Biology, School of Biosciences and Veterinary Medicine, University of Camerino, 62032 Camerino, Italy * Correspondence: federico.buonanno@unimc.it (F.B.); f.trenti@unitn.it (F.T.) † These authors contributed equally to this work. Simple Summary: Predator-prey interactions are widely observed in nature and involve not only animals but also unicellular organisms. In this context, the basic techniques for offence or defence adopted by predators and prey can be mediated by mechanical/morphological and/or chemical strategies. Among unicellular organisms, ciliated protozoa represent a paradigmatic model for the investigation of both mechanisms. Therefore, in this study, we choose to investigate the defensive functions adopted by the ciliate Stentor polymorphus against predators that share the same aquatic ecosystem. On the basis of the experimental observations, we found that the defensive strategy evolved by this ciliate is essentially chemical and mediated by a mix of sterols. The defensive function of these molecules appears as a novelty, at least among the ciliated protozoa. Overall, our study represents an advance in the knowledge of the ethology and ecology of ciliates, with particular regard to the natural compounds that this group of organisms has selected in the last 1.8 billion years. Abstract: Heterotrich ciliates typically retain toxic substances in specialized ejectable organelles, called extrusomes, which are used in predator-prey interactions. In this study, we analysed the chem- ical defence strategy of the freshwater heterotrich ciliate Stentor polymorphus against the predatory ciliate Coleps hirtus, and the microturbellarian flatworm Stenostomum sphagnetorum. The results showed that S. polymorphus is able to defend itself against these two predators by deploying a mix of bioactive sterols contained in its extrusomes. Sterols were isolated in vivo and characterized by liquid chromatography-mass spectrometry (LC-MS), and nuclear magnetic resonance (NMR), as ergosterol, 7-dehydroporiferasterol, and their two peroxidized analogues. The assessment of the toxicity of ergosterol and ergosterol peroxide against various organisms, indicated that these sterols are essential for the effectiveness of the chemical defence in S. polymorphus. Keywords: Stentor; predator-prey interactions; sterols; ergosterol; ergosterol peroxide 1. Introduction Stentor polymorphus is a large trumpet-shaped contractile (stretched individuals grow up to 2 mm long) colourless freshwater ciliate, belonging to the order Heterotrichida. Commonly present in ponds, wells, and lakes, this ciliate is also capable of attaching itself to aquatic objects by means of a specialized posterior holdfast organelle secreting a sticky substance [1]. As in the case of other heterotrich ciliates, the cortex of S. polymorphus is characterized by the presence of membrane-bound, colourless cortical granules (a kind of extrusomes), with a diameter of ~0.5–1 μm[1]. As is common in heterotrichs, extrusomes discharge Biology 2022, 11, 1749. https://doi.org/10.3390/biology11121749 https://www.mdpi.com/journal/biology