Biologia 66/4: 648—653, 2011 Section Zoology DOI: 10.2478/s11756-011-0061-y The changes in the predatory behavior of the microturbellarian Stenostomum sphagnetorum on two species of toxin-secreting ciliates of the genus Spirostomum Federico Buonanno Department of Educational Sciences and Training, University of Macerata, P.le L. Bertelli 1, 62100 Macerata, Italy; e-mail: federico.buonanno@unimc.it Abstract: It is known that the microturbellarian Stenostomum sphagnetorum, a common ciliate predator, is very sensitive against different types of toxins produced by other ciliates for chemical defense, and consequently, it is not able to capture and ingests these ciliates. In particular, when the predator tries to attack one of these toxin-secreting ciliates, it is forced to regurgitate the captured prey in response to the toxin discharged from the ciliates. In this study it is shown that after repeated attacks by S. sphagnetorum against two species of toxic ciliates (Spirostomum ambiguum and Spirostomum teres), the predator acquires a behavior of prey selection that leads to the exclusion of these ciliates among the possible prey and to the distinction between edible and inedible (toxic) ciliates. This learned behavior, which is maintained for days, is lost only after the asexual reproduction of the microturbellarian. In addition, S. sphagnetorum learns to recognize and avoid specimens of S. ambiguum and S. teres artificially deprived of their toxins, strongly suggesting that the toxins are not exclusively associated in the prey recognition of the microturbellarian. Key words: Microturbellaria; ciliates; predatory behaviour Introduction Recent studies have shown that the freshwater micro- turbellarian Stenostomum sphagnetorum Luther, 1960, which is a common predator of a large number of pro- tists, in particular species living as bottom dwellers in lakes and ponds, can interact with different cili- ate species both as predator and as prey (Buonanno 2005, 2009). Among these kinds of interactions some ciliates have evolved different defensive strategies to avoid the predation by microturbellarians. An example is given by the inducible defenses where ciliates in the genus Euplotes may show phenotypic and behavioral changes induced by the release of an active kairomone from the predator (Kuhlmann & Heckmann 1985, 1994; Kush 1993; Hammil et al. 2009). Instead other cili- ates are able to release some secondary metabolites (toxins) from peculiar organelles called “extrusomes” to ward off potential predators including microturbel- larians (Harumoto et al. 1998; Buonanno 2005; Buo- nanno et al. 2005). Extrusomes are membrane-bound organelles that ciliates evolved in association with their cortex, and have the unique capacity to be discharged in response to a variety of environmental stimuli (Haus- mann 1978). Recently, it was shown that S. sphagneto- rum is “immune” to several extrusomal toxins such as stentorin from Stentor coeruleus Ehrenberg, 1830 (Buo- nanno 2005), climacostol from Climacostomum virens Ehrenberg, 1833 (Buonanno 2005; Buonanno et al. 2008; Pucciarelli et al. 2008; Buonanno & Ortenzi 2010) or blepharismin from Blepharisma japonicum Suzuki, 1954 (unpublished data) and, consequently, the preda- tor is able to capture and ingest these ciliates. On the contrary, S. sphagnetorum is very sensitive to lox- odesin(s) from Loxodes striatus Ehrenberg, 1830 (Buo- nanno 2005; Buonanno et al. 2005) or spirostomin from Spirostomum teres Clapar` ede et Lachmann, 1858 (Buo- nanno 2005). In particular when the microturbellarian tries to attack a specimen of L. striatus or S. teres, it is forced to regurgitate the captured prey due to the toxin discharge from the ciliates temporarily trapped into the predator’s pharynx (Buonanno 2005). In gen- eral, a successful attack of S. sphagnetorum against a toxin-secreting ciliate seems to be related to the sen- sitivity of the predator for different extrusomal toxins. However, with regard to this last type of interaction, it was recently observed that S. sphagnetorum, after few attempts, stops the attacks against toxic ciliates ignoring the subsequent encounters. On the basis of these preliminary observations, the present study exam- ined the possible involvement of learning in predatory behavior of S. sphagnetorum on two species of toxin- secreting ciliates, S. teres and Spirostomum ambiguum Ehrenberg, 1835. Material and methods Organisms and cultures The catenulid microturbellarian Stenostomum sphagneto- rum (approx. 1400 × 110 μm) was kindly provided by Dr. c 2011 Institute of Zoology, Slovak Academy of Sciences