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© 2016. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2016) 219, 445-456 doi:10.1242/jeb.121871

RESEARCH ARTICLE Feast or flee: bioelectrical regulation of feeding and predator evasion behaviors in the planktonic Favella sp. (Spirotrichia) Michael L. Echevarria1,*, Gordon V. Wolfe2 and Alison R. Taylor1

ABSTRACT preferred prey efficiently and reject toxic and unpalatable prey, Alveolate ( and ) grazers are integral components while avoiding predators (Taniguchi and Takeda, 1988; Buskey and of the marine and must therefore be able to sense a range of Stoecker, 1989; Broglio et al., 2001). To effectively capture prey mechanical and chemical signals produced by prey and predators, and avoid , alveolate grazers must be able to sense a range integrating them via signal transduction mechanisms to respond with of hydrodynamic, mechanical, chemical, and electrostatic signals effective prey capture and predator evasion behaviors. However, the produced by prey and predators, and integrate these signals via sensory biology of alveolate grazers is poorly understood. Using novel signal transduction mechanisms to respond with effective prey techniques that combine electrophysiological measurements and capture or predator evasion behaviors. Predators and prey of high-speed videomicroscopy, we investigated the sensory biology of alveolate grazers can generate similar mechanical stimuli so the Favella sp., a model alveolate grazer, in the context of its trophic sensory mechanisms of alveolate grazers must also be sophisticated ecology. Favella sp. produced frequent rhythmic depolarizations enough to discern between the two. Despite their importance, the (∼500 ms long) that caused backward swimming and are responsible sensory mechanisms of alveolate grazers are poorly understood. for endogenous swimming patterns relevant to foraging. Contact of The marine Favella sp. (Fig. 1A) is a cosmopolitan member both prey cells and non-prey polystyrene microspheres at the cilia of the plankton community that has well-described trophic behaviors produced immediate mechanostimulated depolarizations (∼500 ms (Stoecker and Sanders, 1985; Buskey and Stoecker, 1988, 1989; long) that caused backward swimming, and likely underlie aggregative Taniguchi and Takeda, 1988; Stoecker et al., 1995, 2013; Strom swimming patterns of Favella sp. in response to patches of prey. et al., 2007) and has morphological and behavioral characteristics Contact of particles at the peristomal cavity that were not suitable for that are representative of marine planktonic ciliates; they are therefore ingestion resulted in depolarizations after a lag of ∼600 ms, allowing an excellent model for studying the sensory mechanisms underlying time for particles to be processed before rejection. Ingestion the trophic ecology of marine alveolate grazers (Montagnes, 2013; of preferred prey particles was accompanied by transient Echevarria et al., 2014). [Note: the Favella has been recently hyperpolarizations (∼1 s) that likely regulate this step of the feeding redescribed and some former Favella spp. have been placed in process. Predation attempts by the copepod Acartia tonsa elicited fast the new genus Schmidingeralla by Agatha and Strüder-Kypke (∼20 ms) animal-like action potentials accompanied by rapid (2012). For consistency with the ecological literature, and because of contraction of the to avoid predation. We have shown that the uncertainties in -level identification in previous studies, we sensory mechanisms of Favella sp. are finely tuned to the type, retain the name Favella throughout this paper.] Favella sp. exhibit location, and intensity of stimuli from prey and predators. aggregative swimming patterns in the presence of chemical and mechanical stimuli from preferred prey that are believed to allow KEY WORDS: Favella, Schmidingerella arcuata, Action potential, them to increase residence time in prey patches that typically occur in Ciliate, Electrophysiology, Signal transduction the environment (Buskey and Stoecker, 1989). Increased frequency of ciliary reversals that cause transient backward swimming and INTRODUCTION turning events in response to mechanical stimuli appear to underlie Alveolate grazers (ciliates and dinoflagellates) are key members these aggregative swimming patterns (Buskey and Stoecker, 1988; of marine plankton communities, and as members of the Stoecker et al., 1995). However, the sensory mechanisms that microzooplankton consume ∼60% of global marine primary underlie these ciliary reversals are unknown. Once food particles production in the world’s oceans (Schmoker et al., 2013). They have been successfully contacted, they are processed at the therefore mediate regeneration of nutrients in the surface oceans peristomal cavity where they are either rejected or consumed (Dolan, 1997; L’Helguen et al., 2005) and act as a trophic link depending on the characteristics of the prey surface (Tanguchi and between and larger metazoan consumers in marine Takeda, 1988; Stoecker et al., 1995; Montagnes et al., 2008). Favella food webs. The ecological success of alveolate grazers is sp. are also capable of performing predator avoidance behaviors. In underpinned by complex behaviors they have evolved to capture response to predation attempts by a predatory , Favella azorca contract into the lorica, although dinoflagellates are still able to extract them (Uchida et al., 1997). The lorica may be effective as 1Department of Biology and Marine Biology, University of North Carolina, Wilmington, 601 South College Road, Wilmington, NC 28403, USA. 2Department protection from copepods; in feeding experiments with a copepod, of Biological Sciences, California State University, 1205 W. 7th Street, Chico, crushed loricas containing intact cells were observed at the end of the CA 95929-0515, USA. experiment (Stoecker and Sanders, 1985). *Author for correspondence ([email protected]) Although Favella sp. exhibit complex behavioral responses to predators and prey, the cellular mechanisms that underlie these

Received 18 June 2015; Accepted 20 October 2015 responses are unknown. In contrast to marine ciliates, the sensory Journal of Experimental Biology

445 RESEARCH ARTICLE Journal of Experimental Biology (2016) 219, 445-456 doi:10.1242/jeb.121871

known that ionotropic mechanisms – those governed by voltage-, List of symbols and abbreviations chemo-, or mechano-sensitive ion channels – are important in AP action potential regulating the behaviors of ciliates (Preston and Van Houten, 1987; AP-RD action potential–rhythmic depolarization Shiono and Naitoh, 1997; Grønlien et al., 2011). For example, ASW artificial seawater ionotropic mechanisms are known to be important in sensory CBF ciliary beat frequency processes that relate to prey capture in ciliates. Contact of the GPCR G-protein coupled receptor freshwater ciliate vorax with prey cells elicits ciliary Im membrane current MSD mechanostimulated depolarization reversal and backward swimming, which are regulated by transient RD rhythmic depolarization depolarizations (Grønlien et al., 2011). Ionotropic mechanisms may ROI region of interest also be important in regulating predator evasion behaviors. Vm membrane potential Contractile behavior of the freshwater ciliate sp. upon mechanostimulation is regulated by rapid all-or-nothing action potentials (Shiono and Naitoh, 1997). Many ciliates contain mechanisms of model freshwater ciliates such as and extrusomes, membrane-bound organelles that can be ejected from Tetrahymena have been well studied. Although Favella sp. may be the cell to deter predators and immobilize prey, whose release is expected to utilize similar conserved sensory mechanisms, it is likely coordinated by the bioelectrical activity of the cell (Iwadate et al., that they implement them in different ways due to their different 1997; Harumoto et al., 1998; Rosati and Modeo, 2003). Similar morphological and ecological characteristics. Nevertheless, it is ionotropic mechanisms may function in regulating predator evasion and prey capture behaviors in Favella sp. To investigate the sensory capabilities of Favella sp., A Stalk Lorica Oral membranelles electrophysiological measurements and high-speed video microscopy were performed in the presence of artificial and live prey, and predatory copepodsto investigate the bioelectrical regulation of prey capture and predator evasion behaviors. Using this powerful combination of techniques, we investigated signal transduction from environmental stimuli to behavioral response in an ecologically Cell relevant context, providing a deeper understanding of the sensory Electrode mechanisms possessed by marine alveolate grazers.

Peristomal cavity MATERIALS AND METHODS Maintenance of Favella sp., Acartia tonsa, and prey cultures B Favella sp. and the phytoplankton cultures Heterocapsa triquetra, Mantoniella squamata,andIsochrysis galbana were obtained from 0 mV the Shannon Point Marine Center, Anacortes, WA. The phytoplankton Tetraselmis sp. was obtained from Michael Finiguerra at the University of Connecticut, Groton, CT. Favella sp. cultures were maintained in 200 ml batches at 14–16°C in filtered –70 mV seawater (30 ppt) supplemented with a dilute trace metal solution (Strom et al., 2007). Favella sp. cultures were fed with prey and sub- 600 ms cultured every 3–4 days. Phytoplankton were maintained in 40– C 1000 ml batches in filtered 36 ppt seawater supplemented with f/2 0 mV nutrients and Guillard’s vitamins (Guillard, 1975) at 14–16°C and 50– 80 μmol photons m−2 s−1 on a 12 h:12 h light:dark cycle. Phytoplankton were sub-cultured every 1–2 weeks. The copepod predator of Favella sp., Acartia tonsa, was obtained from the –65 mV aquaculture supplier AlgaGen (Vero Beach, FL, USA). Acartia tonsa were maintained in 2 liter batches at 14–16°C in filtered 33 ppt 300 ms 30 ms − − D seawater at 14–16°C and 50–80 μmol photons m 2 s 1 ona12h:12h 0 nA light:dark cycle. They were fed Tetraselmis sp. and I. galbana every 3–4 days. For physiological experiments examining interactions of 4 nA Favella sp. and H. triquetra, the latter were prepared immediately prior 1 s to use by gentle filtration through 3 μm pore size Whatman polycarbonate filters (General Electric, Pittsburgh, PA, USA) −1 Fig. 1. Spontaneous electrical activity of Favella sp. (A) Brightfield followed by two rinses in artificial seawater (ASW; 450 mmol l −1 −1 −1 micrograph showing Favella sp. cell tethered by the lorica with a glass suction NaCl, 30 mmol l MgCl2,16mmoll MgSO4,8mmoll KCl, μ −1 −1 −1 micropipette for electrophysiological recording (scale bar=50 m). (B) Typical 10 mmol l CaCl2, 2 mmol l NaHCO3,10mmoll HEPES, pH current clamp trace of free-running membrane potential (Vm) showing two main adjusted to 8.0 with NaOH) to remove any dissolved chemical cues types of spontaneous electrical activity, rhythmic depolarizations (RDs) and present in the supernatant. all-or-nothing action potentials (APs) (indicated by red boxes). (C) Detailed example of an RD and an AP from the V trace in B. (D) Voltage clamp m Electrophysiology recording showing free-running membrane current (Im) of a cell voltage clamped at −62 mV. Spontaneous inward currents of up to 10 nA and similar in Electrophysiological measurements of the bioelectrical activity of duration to RDs are observed. Favella sp. were obtained using sharp electrode current clamp and Journal of Experimental Biology

446 RESEARCH ARTICLE Journal of Experimental Biology (2016) 219, 445-456 doi:10.1242/jeb.121871

voltage clamp recordings of whole-cell membrane potential (Vm) triggered with a transistor–transistor logic pulse from the and currents, respectively (Fig. 1A). Favella sp. were prepared for electrophysiology software for simultaneous electrophysiology electrophysiology by rinsing cultures three times in ASW by reverse and behavioral recordings. To quantify spontaneous and evoked filtration through a 40 μm nylon mesh cell strainer (Corning Life behavioral characteristics of Favella sp., videos were analyzed using Sciences, ME, USA) before being placed in a recording chamber MiDAS Player (Ver. 2.2.1.1., Xcitex, Cambridge, MA, USA) and equipped with gravity-fed perfusion that was secured to the stage of Metamorph Basic (Ver. 7.7.4.0., Molecular Devices) software to an Olympus IX 71 microscope. A Peltier cooled stage (fabricated define regions of interest (ROIs). ROIs were drawn around areas in-house) maintained the recording chamber at 14–16°C for occupied by structures of interest, e.g. , cell body, peristomal experiments where behaviors and electrical activity of Favella sp. cavity, or stalk. Threshold analysis tools in Clampfit 11.0 were recorded simultaneously. All other experiments were (Molecular Devices) were used to measure changes in average performed at room temperature (∼20°C). Cells were tethered by pixel intensity value as the cell structure moved into and out of the the base of the lorica (Fig. 1A) using a micro suction pipette ROI. Pixel intensity values were normalized to a scale of 100 and fabricated from non-filamented borosilicate glass capillaries inverted for the purposes of data presentation. (1.5 mm outer diameter×0.86 mm inner diameter, Sutter Instruments, Novato, CA, USA) using a microelectrode puller Responses to natural and artificial prey (Narishge, Tokyo, Japan) and bent into a position that allowed To determine how the bioelectrical activity of Favella sp. coordinates optimal imaging and access to cells with sharp electrodes. Sharp feeding behaviors, electrophysiological and high-speed video recording electrodes (10–20 MΩ) were fabricated from filamented recordings were obtained in the presence of natural and artificial borosilicate glass capillaries (1.5 mm outer diameter×0.86 mm prey (polystyrene microspheres). Favella sp. were exposed to either inner diameter, Sutter Instruments), coated in beeswax to minimize the preferred prey dinoflagellate H. triquetra or 15 μm polystyrene − stray capacitance, filled with 1 mol l 1 KCl and inserted into an microspheres (Polysciences, Warrington, PA, USA) at concentrations electrode holder (Harvard Apparatus, Holliston, MA, USA) that was of 15,000–30,000 ml−1 while membrane potential and behavior were mounted onto the headstage of an Axon 900 A voltage clamp recorded simultaneously. Microspheres were coated with amplifier (Molecular Devices, Sunnyvale, CA, USA). The electrode methylcellulose to neutralize surface charge (Stoecker et al., 1995) was positioned using a Sutter MP-285 motorized micromanipulator and were the same equivalent spherical diameter as H. triquetra,and (Sutter Instruments) until contact was made with the lateral surface therefore simulated the mechanical stimulus from prey while of the cell, just posterior to the ring of membranelles. Electrical controlling for chemical prey cues. Interactions of Favella sp. with access to the cell was accomplished using a brief (<30 ms) microspheres and H. triquetra were manually scored via frame-by- capacitance overcompensation. frame observations to determine the sequence of events from time and Single electrode current and voltage clamp experiments were location of particle contact to bioelectrical response and behavioral recorded using Clampex software (Molecular Devices). Single event. electrode current clamp switching frequency was generally between 5 and 15 kHz and voltage clamp gain was generally between 0.3 and Interactions of Favella sp. with copepod predators 3nAmV−1. To determine the voltage response of Favella sp. to Behavioral interactions of free-swimming Favella sp. with the negative current injection and the passive membrane properties of copepod predator A. tonsa were investigated using high-speed Favella sp., hyperpolarizing step protocols were used that injected (125 frames s−1) video microscopy. Prior to experiments, A. tonsa 1 nA pulses of negative current for 120 ms. The passive membrane cultures were rinsed twice with ASW by reverse filtration through properties of Favella sp. were calculated by calculating resistance and 40 μm pore size nylon cell strainers and starved for 15–20 h. the time constant (τ) from the voltage response to injection of negative Immediately prior to experiments, ciliates and copepods were current. Action potentials (APs) and rhythmic depolarizations (RDs) rinsed twice with ASW by reverse filtration through 40 μm pore (two stereotypical patterns of bioelectrical activity in Favella sp.) size nylon cell strainers (Corning Life Sciences). Thirty individuals were elicited by injecting 5–10 nA current for 10–20 ms. Cells were of Favella sp. and six late-stage copepodite to adult female A. tonsa − perfused with 0.8, 4, 8, and 40 mmol l 1 KCl ASW while measuring were combined in a 3.5 ml spectrophotometry cuvette and the + free-running Vm to determine the role of K in regulating resting volume was adjusted to 1 ml with ASW. Cuvettes were imaged potential and on spontaneous electrical activity. Experiments that through the side using an Olympus SZX12 dissecting microscope at examined the relationship between electrical activity and behaviors 12.5× magnification. High-speed (125 frames s−1) video recordings were performed without perfusion to avoid mechanostimulation by were accomplished using a Fastec Inline 250 frames s−1 camera the flow of media around the cell. (Fastec Imaging) with 640×478 pixel resolution. Cuvettes were Data were analyzed off-line in Clampfit 11.0 (Molecular observed for 0.5 h and all events where ciliates were captured by Devices). The characteristics of spontaneous electrical activity copepods were recorded. Behavioral interactions between copepods and electrical activity in the presence of prey were determined using and ciliates were manually scored as ‘captures and ingests Favella the threshold analysis tools of the software. The characteristics of sp.’ or ‘captures and releases Favella sp.’. The amount of time it APs evoked by positive current injection were measured using took Favella sp. to emerge from the lorica post-release was also manual cursors. measured.

Behavioral observations and analysis Statistical tests Simultaneous electrophysiological and video acquisition allowed us All differences in means were tested using a one-way ANOVA to determine how the bioelectrical phenomena described above followed by multiple comparisons with Tukey’s HSD test. To coordinate behavior in Favella sp. High-speed (up to determine the relationship between the length of rhythmic 250 frames s−1) video recordings were accomplished using a depolarizations and behavioral events, linear regressions were Fastec Inline 250 frames s−1 camera (Fastec Imaging, San Diego, performed. All statistical analyses were performed with Sigmaplot

CA, USA) with 320×228 pixel resolution. Camera acquisition was Software version 11 (Systat Software). All quantitative measures Journal of Experimental Biology

447 RESEARCH ARTICLE Journal of Experimental Biology (2016) 219, 445-456 doi:10.1242/jeb.121871 are given as means±s.d. (n=number of cells, events analyzed = produced spontaneous RDs (n=34; Fig. 1B,C), and occasionally total number of events analyzed for all cells) unless noted rapid ‘all-or-nothing’ APs were observed (n=4, events analyzed=4; otherwise. Fig. 1B,C). RDs had a frequency of 0.153± 0.1 Hz, a peak amplitude of 27.4±2.2 mV and an average length of 618± 183 ms (n=5, events analyzed=58). When cells were voltage RESULTS − Bioelectrical characteristics of Favella sp. clamped at resting Vm ( 62 mV), spontaneous inward currents The passive membrane properties of Favella sp. were similar to were recorded that underlie the RDs observed in current clamp (Fig. 1D). previously investigated ciliates (Table S1). All Favella sp. cells + A strong dependence of Vm upon external [K ] was observed, with a Nernstian depolarization (50 mV change in Vm per decadal + A change in external [K ]) of Vm, over the concentration range 10 mV 40 mmol l–1 KCl −1 between 4 and 40 mmol l KCl ASW (Fig. 2A,B). Vm deviated 20 s from the Nernstian relationship in <4 mmol l−1 KCl ASW, with cells exhibiting only a slight hyperpolarization when perfused with 0.8 mmol l−1 KCl ASW (Fig. 2A,B). Interestingly, changing 28 mV

–61 mV 3 mV A –10 mV 0.8 mmol l–1 KCl

B –20 n=4 –30 –60 mV 10.7 nA –40 n=28 –50 m n=5 1 s

V n –60 =3 B 40 mV –70

–80 0.1 1 10 100 [K+] (mmol l–1)

C 10 mV D 10 mV 0.5 s –85 mV 15 ms 10 nA –61 mV 0.8 mmol l–1 KCl 10 ms

–61 mV C –1 nA 4 mmol l–1 KCl

–40 mV –61 mV 40 mmol l–1 KCl –60 mV 8 mmol l–1 KCl

+ Fig. 2. Effect of [K ]onVm and bioelectrical activity of Favella sp. (A) Effect + −1 20 mV of [K ]ext on Vm of Favella sp. Perfusion with 0.8 mmol l KCl artificial seawater −1 (ASW) causes a slight hyperpolarization in Vm, from 61 mV in 8 mmol l KCL − 20 ms ASW (dashed line), whereas perfusion with 40 mmol l 1 KCl ASW causes a ∼ + dramatic 30 mV depolarization in Vm. (B) Relationship between [K ]ext and −1 +−1 Vm. A roughly linear and Nernstian slope (of 1.2 mV mmol l K ) is observed −1 + for concentrations above 4 mmol l K while Vm is relatively insensitive to − concentration changes below 4 mmol l 1. (C) Examples of spontaneous bioelectrical activity in Favella sp. under various K+ treatments. From top to − bottom, traces show examples of RDs in 0.8, 4, and 8 mmol l 1 KCl ASW, respectively. Their shape is similar in all solutions, but after-hyperpolarization + – (arrows) amplitudes increase with decreasing [K ]ext. (D) An action potential rhythmic depolarization (AP-RD) associated with the rising phase of the RDs −1 + that frequently occur in 40 mmol l KCl ASW. Unlike in lower [K ]ext ASW, AP- Fig. 3. Evoked bioelectrical properties of Favella sp. (A) RD (top) elicited by − RDs in 40 mmol l 1 KCl ASW do not exhibit hyperpolarizations. The change in positive current injection (bottom). (B) AP (top) elicited by positive current + spontaneous electrical activity from RDs in low [K ]ext to AP-RDs in higher injection (bottom). (C) Voltage response (bottom) to injection of negative + [K ]ext is likely due to Vm depolarization closer to the AP threshold. current during hyperpolarizing step protocol (top). Journal of Experimental Biology

448 RESEARCH ARTICLE Journal of Experimental Biology (2016) 219, 445-456 doi:10.1242/jeb.121871 external [K+] changed the characteristics of RDs. The duration and Under normal ionic conditions, positive current injection pulses + peak depolarization of RDs in all [K ]ext concentrations was similar. elicited depolarization and either RDs or APs (Fig. 3A,B) that were However, decreased external [K+] led to decreased amplitude of the identical to spontaneous RDs and APs. Over the range of positive small hyperpolarizations that occurred at the end of RDs (P<0.05, current injection protocols tested there did not appear to be a ANOVA, Tukey’s HSD; Fig. 2C, Table S1). In contrast, high correlation between length (10–20 ms) or amplitude (5–10 nA) of external [K+] (40 mmol l−1 KCl ASW) caused APs to be generated injection and type of response elicited (AP or RD). In contrast, at the rising phase of the RDs (Fig. 2D). These AP-RDs were much negative current injection elicited a typical RC voltage response shorter than RDs and did not exhibit hyperpolarizations following (Fig. 3C). APs were elicited in seven cells where 5–10 nA current the depolarized phase of the event (n=4, events analyzed=51; was injected for 10–20 ms (Fig. 3B). APs did not occur until Vm Fig. 2D, Table S1). They almost always preceded bursts of APs reached an average depolarization threshold of −3.7±17.10 mV induced by the depolarized state (up to 3 Hz; Fig. S1) that were (n=3, events analyzed=3) and Vm at peak after-hyperpolarization accompanied by whole-cell contraction into the lorica (not shown). was −76.50±6.75 mV (n=3, events analyzed=3). The time to peak depolarization from start of the evoked AP was very rapid at 6.92± 2.40 ms (n=7, events analyzed=7). A n=4 Ciliary reversal 1600 y=–58.03+1.12x r 2=0.89 P<0.001 1200

800 B 2 1 3 –35 mV 400

0 n=4 –70 mV y=0.61x+323.82 Peristomal contraction r 2 C 100 1600 =0.23 P<0.001

1200

0 800 D 100

400 Duration of behavior (ms)

0 0 n E =4 Stalk bend 100 y x 2000 =0.61 +214.15 r 2=0.17 P=0.007 1600

0 1 s 1200

Fig. 4. Spontaneous bioelectrical activity and associated synchronous 800 behaviors of Favella sp. (A) Micrographs of Favella sp. cell immediately prior to a RD depolarization (left) and during a depolarization (right; scale bar=35 μm). Regions of interest (ROIs) are drawn around areas covered by an 400 area passed through by a single cilium during normal forward beating (red), peristomal cavity (blue), and a portion of the stalk (green) at resting Vm. – 0 (B) Free-running Vm of cell showing RDs. (C E) Normalized average gray level 300 600 900 1200 pixel intensity values that are color-coded to correspond with ROIs in A. Pixel RD time length (ms) intensity values from each grayscale image were inverted and normalized such that values of 100 represent maximal coverage of ROI by cell feature, and 0 Fig. 5. Correlations between length of RDs and length of associated represents minimal coverage of ROI by cell feature. (C) Normalized pixel behaviors of Favella sp. A total of 58 RD events from four cells were intensity values derived from ROI passed through by a single cilium during analyzed, with each color representing values derived from a single cell. Linear normal forward swimming. Troughs in graph represent periods of ciliary regressions were performed to determine the degree of correlation between reversals. (D) Pixel intensity values derived from the ROI over the RD length and length of behaviors, and were performed on event lengths for all peristomal cavity of the cell. Troughs in graph represent periods where cells pooled together. There was a strong 1:1 correlation between length of the contractions of the peristomal cavity occur. (E) Pixel intensity values derived RD and the length of time spent in ciliary reversal (top). Correlations between from an ROI over a portion of the stalk. Troughs in graph represent periods of length of RD and length of peristomal contraction (middle) and stalk bending stalk bending. (bottom) events were weaker. Journal of Experimental Biology

449 RESEARCH ARTICLE Journal of Experimental Biology (2016) 219, 445-456 doi:10.1242/jeb.121871

A Rhythmic depolarizations n=4 Events analyzed=58 Rhythmic depolarization Ciliary reversal Peristomal cavity contraction Stalk bend

B H. triquetra contact with cilia n=4 Events analyzed=17 Time to depolarization start Rhythmic depolarization Ciliary reversal Peristomal cavity contraction Stalk bend No data C Microsphere contact with cilia n=5 Time to depolarization start Events analyzed=56 Rhythmic depolarization Ciliary reversal Peristomal cavity contraction Stalk bend

D H. triquetra contact with peristomal cavity n=4 Events analyzed=8 Time to depolarization start Rhythmic depolarization Ciliary reversal Peristomal cavity contraction Stalk bend No data n E Microsphere contact with peristomal cavity =5 Events analyzed=6 Time to depolarization start Rhythmic depolarization Ciliary reversal Peristomal cavity contraction Stalk bend

0 200 400 600 800 1000 1200 1400 1600 Time (ms) F Ingestion of H. triquetra n=3, events analyzed=3 Time to hyperpolarization start n=7, events analyzed=7 Hyperpolarization n=7, events analyzed=7 Ingestion

0 12345

G Contraction n=7, events analyzed=7 Time to peak depolarization n=5, events analyzed=5 Cilia stop beating n=5, events analyzed=5 Contraction

02468101214 16 Time (s)

Fig. 6. Temporal relationships between bioelectrical events and behaviors they regulate in Favella sp. (A–G) Gray lines represent start and end time of respective events. Error bars at start and end times represent s.d. of start times of events relative to 0 s, and s.d. of length of events, respectively. In D and E, asterisks indicate that there was high variability in the lag between Heterocapsa triquetra (s.d.=±0.552 s) and 15 μm polystyrene microsphere (s.d.=±0.777) contact with the peristomal cavity and time to the start of depolarization; therefore, error bars are omitted for clarity of presentation. (G) Red diamond represents average time of 95% contraction into the lorica. Journal of Experimental Biology

450 RESEARCH ARTICLE Journal of Experimental Biology (2016) 219, 445-456 doi:10.1242/jeb.121871

A Regulation of swimming behavior by rhythmic depolarizations Spontaneous RDs always resulted in ciliary reversals, peristomal cavity contractions, and stalk bending events (Figs 4, 5, 6A), – –30 mV which all began within 70 110 ms of the start of RDs (Fig. 6A). RDs lasted approximately the same length of time as the events they regulated (between 600 and 750 ms, n=4, events analyzed=58; Fig. 6A): there was a strong 1:1 correlation 2 –70 mV (r =0.89, P<0.001) between the length of RDs and the period B 50 ms of time cilia were in reverse beating. There were weaker correlations between length of RDs and duration of peristomal cavity contractions (r2=0.23, P<0.001) and stalk bending (r2=0.17, P=0.007; Fig. 5). During forward beating, the instantaneous ciliary beat frequency (CBF) was 27.7±1.5 Hz –30 mV (n=5, events analyzed >10,000) and during reversed beating it was slightly higher at 33.3±7.5 Hz (n=5, events analyzed >500; Fig. S2). Forward beating transitioned to reverse beating very rapidly (within approximately 45 ms; n=5, events analyzed=48). –70 mV 50 ms Favella C Behavioral interactions of sp. with prey are differently regulated by contact with cilia and the peristome Contact of H. triquetra with the cilia of Favella sp. invariably resulted in depolarizations and behaviors (ciliary reversals, peristomal cavity contractions, and stalk bending; n=4, events –30 mV analyzed=13) that were very similar to RDs (Figs 6B, 7A, Movie S1). Identical responses were observed when H. triquetra-

–70 mV A 0 s B 0.064 s D 50 ms

–30 mV

–70 mV 50 ms E

C 0.432 s D 13.872 s

–55 mV

–63 mV 200 ms

Fig. 7. Bioelectrical regulation of prey processing and ingestion in Favella sp. Top panels. (A–E) Micrographs showing interactions between Favella sp. μ and particles (scale bars=50 m). Bottom panels. Vm traces showing bioelectrical activity that regulates the behavioral interactions shown in top panels. Red arrows on Vm traces indicate times at which the corresponding images were taken. In A–D, blue arrows indicate the direction in which the particle is moving. Contact of H. triquetra (A) and 15 μm polystyrene Fig. 8. Favella sp. responses to predation attempt by Acartia tonsa. microspheres (B) with cilia results in immediate mechanostimulated (A) Immediately prior to attack jump by copepod. (B) Immediately after attack depolarizations (MSDs) and ciliary reversals. (C,D) Contact of H. triquetra jump. Ciliate has been captured and is being processed. (C) Immediately after (C) and 15 μm polystyrene microspheres (D) with the peristomal cavity results release of ciliate by copepod. Ciliate is contracted into lorica. (D) Immediately in MSDs and ciliary reversals after a lag. (E) Ingestion of H. triquetra results in after ciliate has reemerged from lorica and has started swimming again. Red hyperpolarization. Red circle indicates position of H. triquetra cell prior to (left arrows show location of ciliate. Time values indicate time elapsed relative to A micrograph) and after (right micrograph) ingestion. (0 s). Scale bars=500 μm. Journal of Experimental Biology

451 RESEARCH ARTICLE Journal of Experimental Biology (2016) 219, 445-456 doi:10.1242/jeb.121871

A Fig. 9. Evoked APs cause prolonged whole-cell contraction and cessation of ciliary beating. (A) Micrographs of Favella sp. cell immediately prior to an AP (left), after an AP contraction (middle) and after recovery from an AP-mediated contraction (right). ROIs are drawn around the area covered by the uncontracted cell body (red), a fully contracted cell body (blue), and an area passed μ through by a single cilium (green; scale bar=50 m). (B) Vm 12 3 before (1), immediately after (2) and at recovery (3) from B * AP-mediated contraction. Asterisks indicate injection of 0 mV 10 nA current for 20 ms to elicit AP. (C) Expanded section – of Vm trace from between cursors 1 and 2. (D F) Pixel –80 mV intensity values from each ROI in the grayscale image were C inverted and normalized such that values of 100 represent maximal coverage of ROI by cell feature, and 0 represent 0 mV minimal coverage of ROI by cell feature. Normalized –80 mV average gray level pixel intensity values are color-coded to 10 ms correspond with ROIs in D. (D) ROI for uncontracted cell. D 100 The beginning of contraction is measured as the point at which threshold values decrease sharply to minimal values from the maximal normalized value of 100. Recovery from 0 contraction is measured as the point at which threshold values return to maximal normalized values. (E) ROI for E 100 contracted cell. The time of full contraction is measured as the point at which these values reach their maximum amplitude. (F) ROI for cilium. Time that cilia cease beating 0 is measured as the time at which high-frequency F 100 oscillations in threshold intensity due to ciliary beating cease. Time that cilia resume beating is measured as the time at which these oscillations resume. 1 s 0 sized polystyrene microspheres (15 μm) that were used as AP-regulated contraction serves as predator evasion artificial prey to control for the effect of chemical cues present behavior in Favella sp. on the surface of natural prey particles (Figs 6C, 7B), and these Capture of Favella sp. by A. tonsa resulted in contraction of Favella events are thus termed mechanostimulated depolarizations sp. into the lorica (Fig. 8). Favella sp. were released 73±25% of the (MSDs). There was a very short lag of 29.8±17.9 ms (n=4, time and ingested the rest of the time (six experiments, n=16 events analyzed=13) between contact of H. triquetra with cilia events). Released ciliates were invariably contracted into the lorica, and MSDs, with a nearly identical lag observed with beads but re-emerged and resumed swimming after 10.6±3.8 s (six (Figs 6B–C, 7A–B, Movie S1). experiments, n=5 events; Fig. 8). Polystyrene microspheres were never observed to be ingested on The bioelectrical basis of this predator evasion behavior was contact with the peristomal cavity (n=5, events analyzed=6) investigated using sharp electrode current clamp recordings and (although one cell contained a microsphere that was ingested simultaneous high-speed video microscopy. Spontaneous and before video recording started; Fig. 6B,D). Instead, a significant lag evoked APs caused cessation of cilia beating and rapid occurred before a depolarization was elicited that resulted in ciliary contraction into the lorica (Figs 6F, 9, Movie S3). The coupling reversal and ejection of microspheres from the peristomal cavity between electrical event and behavioral response was extremely (Figs 6E, 7D). In some cases when H. triquetra contacted the rapid, with contraction starting 12.2±5.9 ms after the initiation of peristomal cavity, they also elicited this response (Fig. 6D, Fig. 7C). AP (n=7, events analyzed=7), with cells reaching 95% of their full Although the lag between particle contact at the peristomal cavity contraction at 121±77 ms (n=5, events analyzed=5). Cells gradually and subsequent MSD was longer for H. triquetra (750±552 ms) recovered to their pre-AP state after 8.8±2.0 s (n=5, events than for microspheres (596±777 ms), lag times were highly variable analyzed=5; Figs 6F, 9, Movie S3). and therefore not statistically significant. However, in other instances, contact of H. triquetra with the peristomal cavity DISCUSSION resulted in ingestion after a processing period of 1.01±0.84 s Passive membrane properties and resting membrane (n=7, events analyzed=7) that invariably resulted in potential of Favella sp. hyperpolarization that began 212.1±384.7 ms (n=3, events Favella sp. exhibited a resting membrane potential that was more analyzed=3) after ingestions started, lasted 1700±800 ms (n=8, negative than previously studied marine and freshwater ciliates events analyzed=8), and had peak amplitudes of 3.5±0.8 mV (n=8, reported in the literature (Table S2). This indicates that their resting events analyzed=8) (Figs 6F, 7E, Movie S2). No change in CBF was permeability and the electromotive force across the plasma observed during hyperpolarizations associated with prey ingestion membrane are determined by somewhat different conductances. (n=4, data not shown), suggesting that swimming was unaffected. The passive membrane properties of Favella sp. show a high input The striking discrimination against ingesting microspheres suggests capacitance in comparison with other ciliates (Table S2) that likely that ingestion and associated hyperpolarization are mediated reflects their large cell size and large membrane surface area. by chemical cues present on the cell surface, but not on the Additionally, their input resistance was rather low compared with microspheres. that of other ciliates (Table S2), suggesting a high resting ionic Journal of Experimental Biology

452 RESEARCH ARTICLE Journal of Experimental Biology (2016) 219, 445-456 doi:10.1242/jeb.121871 permeability. Similar to other , resting permeability to K+ A Behaviors and ecological Bioelectrical activity was important in determining resting Vm in Favella sp. The near- + processes Nernstian relationship of Vm and [K ]ext at concentrations above −1 8 mmol l KCl indicated that Vm was primarily determined by permeability to K+ ions. However, below 8 mmol l−1 KCl the non-linear curve suggests that permeability to ions other than K+ contributes to resting Vm.

Bioelectrical regulation of swimming behavior The bioelectrical activity of Favella sp. is crucial in regulating endogenous swimming patterns (Fig. 10). Favella sp. exhibited Backward swimming regulates Rhythmic frequent spontaneous RDs that regulated ciliary reversals, stalk endogenous swimming patterns depolarization bending, and peristomal cavity contractions. Ciliary reversals result B in periods of backward swimming in free-swimming cells, and stalk bending changes the orientation of the cell and would therefore affect swimming angle. Peristomal cavity contractions may function in clearing the surface of the peristomal cavity of particles during ciliary reversals. The strong correlation between length of RDs and length of ciliary reversals indicates that Favella sp. can modify the length of RDs and ciliary reversals based on environmental and Backward swimming regulates endogenous signals. aggregative swimming patterns Mechanostimulated RDs have been observed in several ciliates (Machemer, 1970; and rejection of non-preferred prey depolarization Lueken et al., 1996), including the marine benthic C vannus, in which RDs mediate alternating periods of slow and fast walking and backward walking behavior (Machemer, 1970; Lueken et al., 1996). Increases in inward conductance of Ca2+ and decreases in outward conductance of K+ appear to be important in mediating RDs in E. vannus (Lueken et al., 1996). The specific ionic conductances responsible for RDs in Favella sp. are unknown, but the importance of voltage-gated channels in mediating RDs in Ingestion of prey Hyperpolarization Favella sp. was suggested by the fact that transient depolarizations D and ciliary reversals were elicited by the injection of positive current (Fig. 3A). We observed increased after-hyperpolarization amplitude + + with decreased [K ]ext, indicating that K channels were important + in returning the cell to resting Vm following RDs (decreased [K ]ext resulted in an increased electromotive force of K+ from Favella sp.). The cellular mechanisms that regulate the rhythmicity of RDs in Favella sp. and other ciliates have not been identified. The high frequency of RDs and backward swimming events in Favella sp. and presence of these mechanisms in multiple taxa indicates that they most likely play an important role in structuring ciliate search Contractile defense behavior in patterns for food particles and in determining encounter rates with response to capture by predators Action potential prey and predators. Fig. 10. Relationships between behaviors and ecological processes (left The rate of ciliary beating in Favella sp. was also regulated by column) and the bioelectrical events that regulate them (right column). bioelectrical activity of the cell. The increased reverse CBF of (A) Backward swimming events during endogenous swimming (left) are Favella sp. during RDs will correspond to increases in swimming regulated by RDs (right). (B) Backward swimming occurs upon contact of prey speed in free-swimming cells. Intracellular Ca2+ dynamics are particles with cilia and non-preferred prey particles (red particle) with the most likely involved in regulating ciliary beating in Favella sp. In peristomal cavity (left). These behaviors may regulate aggregative swimming behaviors in patches of preferred prey and rejection of non-preferred prey. Paramecium sp., Ca2+ dynamics determine the frequency and 2+ MSDs (right) regulate these backward swimming behaviors. (C) Ingestion direction of ciliary beating, with Ca concentrations greater than occurs upon contact of preferred prey particles at the peristomal cavity (left). −1 1 μmol l causing a reversal of ciliary beating in Paramecium sp. Hyperpolarizations (right) underlie ingestion events. (D) Contraction into the (Nakaoka et al., 1984; Plattner et al., 2006). Powerful Ca2+ lorica occurs upon capture of Favella sp. by predators (left). Contractile buffering systems must also be present in the cilia that return cilia behavior is mediated by APs (right). to resting Ca2+ levels following depolarization-induced ciliary reversals (Husser et al., 2004). The cyclic nucleotides cAMP and cGMP are also important in regulating CBF and direction, and Bioelectrical activity regulates behavioral responses of have interactive effects with Ca2+ (Bonini and Nelson, 1988; Favella sp. to prey cues Noguchi et al., 2004). Similar mechanisms may regulate ciliary Although earlier studies linked RDs and ciliary movements, a novel beating in Favella sp. Higher CBF will lead to increased observation in the present study is the coordination of RDs with swimming velocities and increased encounter rates with prey, other behaviors such as peristomal contractions and stalk bending but will also increase the susceptibility of Favella sp. to detection (Fig. 4). Moreover, the differences in bioelectrical signaling at the by predators. cilia and peristome during prey handling indicate that Favella sp. are Journal of Experimental Biology

453 RESEARCH ARTICLE Journal of Experimental Biology (2016) 219, 445-456 doi:10.1242/jeb.121871 able to discriminate the type and location of stimuli from prey. the cilia and peristomal cavity; for example, Favella sp. may have Contact of H. triquetra and 15 μm microspheres at the cilia elicited more sensitive or higher densities of mechanosensors located on almost instantaneous MSD-mediated ciliary reversals, whereas cilia than the peristomal cavity. The spatial distribution of ion contact of H. triquetra with the peristomal cavity elicited variable channels is known to be important in regulating complex behaviors responses, sometimes resulting in hyperpolarization-mediated in Euplotes sp. and Paramecium (Machemer and Ogura, 1979; ingestions and sometimes resulting in MSD-mediated ciliary Krüppel et al., 1993). Particle processing at the peristome most reversals preceded by a lag period. In contrast, contact of 15 μm likely includes crosstalk between mechanosensory and microspheres at the peristomal cavity invariably elicited ciliary chemosensory signaling pathways. The lag between particle reversals preceded by a lag period (Fig. 10B,C). We hypothesize contact and depolarization-mediated ciliary reversals may give that spatially distinct classes of ion channels and receptors, allowing ciliates sufficient time to assess particles with chemosensors on the for sophisticated prey-handling behaviors, regulate interactions of peristomal cavity and determine whether they are suitable for planktonic , such as Favella sp., with prey. ingestion. Further work is now required to understand the spatial MSD-mediated ciliary reversals upon particle contact at the cilia distribution of sensory mechanisms and how this allows alveolate may allow the cell to briefly re-orientate to a particle on the grazers to perceive mechanical and chemical prey cues during periphery of the cell that could not otherwise be directly ingested. processing and ingestion. MSD-mediated reversals could also result in increased encounter Unlike cilia, detection of particles at the peristomal cavity likely rates of Favella sp. with prey because of the patchy small-scale involves chemical information, as 15 μm microspheres were distributional patterns of phytoplankton in nature (Mitchell et al., rejected, unlike similarly sized H. triquetra (Fig. 10B). Chemical 2008). We propose that the ciliary reversals produced by MSDs recognition of prey particles is known to be important in allowing increase residence time of Favella sp. within food patches by Favella sp. to identify and ingest favorable prey. For example, decreasing their diffusivity, and thus may contribute to Favella sp. are able to recognize and reject toxic Heterosigma mechanostimulus-induced aggregative swimming behavior akashiwo upon contact, indicating that unfavorable surface previously reported for Favella sp. (Buskey and Stoecker, 1988, compounds may be present on H. akashiwo (Taniguchi and 1989) (Fig. 10B). Therefore, this is the first demonstration of the Takeda, 1988), while ingesting similarly sized H. triquetra. The cellular basis of population-level responses in Favella sp. mechanism by which chemical recognition occurs in Favella sp. is Mechanostimulated aggregative swimming behaviors may be an unknown, but is likely similar to those known for other ciliates. important planktonic alveolate foraging strategy. Sugar–lectin interactions at the interface of prey and predator cell The production of MSDs is presumably due to the activation surfaces seem to be a common and important method of prey of mechanosensitive ion channels (Fig. 10B). In E. vannus, particle identification by ciliates and other planktonic alveolates the bioelectrical regulation of depolarizations and backward (Scott and Hufnagel, 1983; Esteve, 1984; Casci and Hufnagel, movement events induced by mechanical stimulus has been 1988; Sakaguchi et al., 2001; Wilks and Sleigh, 2004; Roberts et al., well described (albeit in a non-ecological context). In these cells, 2006; Wootton et al., 2007). Surface proteins that are attached to the mechanical stimulus at the anterior results in an influx of Ca2+ extracellular surface by glycosylphosphatidylinositol are also and Mg2+ through stretch-activated ion channels and Na+ through important in predator–prey recognition processes in freshwater Ca2+- dependent Na+ channels, resulting in a reversal of the cirri ciliates (Simon and Kusch, 2013). (compound cilia responsible movement) beating direction that Recognition of favorable prey particles most likely involves results in backward movement (Krüppel and Lueken, 1990; cross-talk between metabotropic (enzyme-linked or second Krüppel et al., 1995). The Vm of E. vannus is returned to resting messenger-linked) signaling and ionotropic signaling pathways. level by the efflux of K+ from slower voltage-gated K+ channels G-protein coupled receptor (GPCR) pathways may be important in (Krüppel et al., 1995). As with RDs, the involvement of voltage- linking these signal transduction mechanisms (Echevarria et al., gated channels in mediating MSDs in Favella sp. is suggested by 2014). GPCR signaling pathways are crucial for chemosensation of the fact that transient depolarizations and reversals may be prey in ciliates, as demonstrated in experiments with the marine elicited by the injection of positive current (Echevarria et al., ciliate Uronema sp., where chemosensory responses to prey were 2014). decreased by application of pharmacological compounds that Ciliary reversals are rarely elicited upon contact of Favella sp. inhibited the GPCR signal transduction pathway (Hartz et al., with smaller non-preferred prey; therefore, smaller particles 2008). These GPCR signaling pathways provide a feedback link to (<4 µm) do not elicit aggregative changes in swimming in modulate activity of ion channels through the activity of cyclic swimming behavior (Buskey and Stoecker, 1988; Stoecker et al., AMP, and thus the behaviors described above that these channels 1995). This suggests that smaller particles do not produce enough regulate. For example, genes for adenylyl cyclase were cloned from force on contact with membranelles to activate mechanosensitive P. tetraurelia and found to localize to cilia, where they potentially ion channels. The mechanosensitivity of Favella sp. and other regulate K+ channels that regulate ciliary movements (Weber et al., alveolates may therefore be tuned to sense optimally sized prey 2004). One potential consequence of such a pathway may be the items, supporting the role of MSD-mediated ciliary reversals in ingestion-mediated hyperpolarization observed in Favella sp., capturing and encountering prey. which to our knowledge has not been described in the literature. In contrast with ciliary contact by particles, direct peristomal Hyperpolarization may make the cell less excitable, decreasing the contact resulted in a lag period before initiation of MSD (Fig. 10B). likelihood that either RDs or MSDs will be activated that would This may allow particles in the peristomal cavity to undergo a cause particle ejection from the peristomal cavity during ingestion, processing period during which they are sampled for their surface as observed by Taniguchi and Takeda (1988), where toxic properties and are subsequently ingested, or rejected via ciliary H. akashiwo cells were rejected by Favella sp. (Fig. 10C). Thus, reversal and backward swimming. The longer response time to membrane hyperpolarization during ingestion may be a general particles that contact the peristomal cavity indicates that Favella sp. mechanism for allowing planktonic alveolates to process food possess different mechanisms that regulate mechanosensitivity at particles. Journal of Experimental Biology

454 RESEARCH ARTICLE Journal of Experimental Biology (2016) 219, 445-456 doi:10.1242/jeb.121871

Bioelectrical activity regulates behavioral responses to Conclusions predator cues Favella sp. exhibited complex and diverse bioelectrical and Our research represents the first demonstration of behavioral behavioral properties, with responses to stimuli dependent on interactions of Favella sp. with copepod predators. Previous their type and location. Additionally, bioelectrical responses and research suggests that copepods may extract Favella sp. from the resulting behaviors were intensity-dependent: from single-particle- lorica during predation (Stoecker and Sanders, 1985), indicating triggered MSD and ciliary reversal, to high-frequency APs that that the lorica is an awkward size or shape for ingestion or is non- resulted in extended contraction into the lorica. This may allow fine- nutritive (the lorica is highly refractory in nature and does not tuned behavioral responses based on the strength of stimuli. Overall, degrade in the presence of strong acids and bases; Agatha et al., these findings show that Favella sp. have sophisticated cellular 2013). We showed that upon capture, Favella sp. contracted into the mechanisms that enable them to perceive predators and prey, lorica and were often subsequently released. Therefore, contraction discriminate between them and respond with appropriate behaviors into the lorica may prevent copepods from detecting the nutritive (Echevarria et al., 2014). cell inside and impede extraction. Upon release by copepods, With the techniques developed in this study, it is now possible to Favella sp. remained contracted for several seconds, not beating examine the behavioral and bioelectrical responses of Favella sp. to a their cilia, instead sinking through the water column. This might range of chemical, mechanical (contact) and hydromechanical (non- decrease their hydrodynamic signature and decrease their encounter contact) cues from both predators and prey, and ultimately the ionic rate with predators that still occupy the area. These results suggest mechanisms and downstream signaling processes such as GPCR that mechanostimulated contraction into the lorica is a predator signal transduction pathways and calcium-induced calcium release evasion behavior that could be common in the plankton given the mechanisms that link bioelectrical responses to behavior. These prevalence of loricated organisms. mechanisms underpin the integration of multiple environmental cues to Contraction into the lorica was directly regulated by APs maximize fitness in these single-celled organisms that are crucial (Fig. 10D). Evoked AP-mediated contractions in tethered cells and consumers of phytoplankton and play important roles in the marine contractions caused by predation attempts in free-swimming microbial food web. cells were approximately the same length of time, indicating that similar molecular mechanisms mediate both processes. The Acknowledgements ionic mechanisms underlying changes in V during AP production We thank Suzanne Strom for helpful discussion and for Favella sp. strains. We thank m Chris Finelli for loan of the Fastec camera for high-speed video microscopy and are unknown, although voltage-activated channels are most Michael Finiguerra for providing Tetraselmis sp. cultures. likely involved because current injection resulted in rapid (ms) excitation–contraction coupling. In addition, spontaneous Competing interests + The authors declare no competing or financial interests. depolarizations in low [K ]ext changed into AP-RDs in higher + [K ]ext, a change that is likely due to a shift in Vm sufficient to reach the threshold for voltage-gated cation channels responsible for APs. Author contributions M.L.E. participated in development, design and execution of experiments, data How predation attempts trigger the escape response is unclear, analysis, interpretation of results, and manuscript preparation. G.V.W. participated in although similar classes of mechanosensitive ion channels that are initial experimental design and in writing the manuscript. A.R.T. participated in involved in production of MSDs during interactions with prey are development and design of experiments, supervision of data collection and analysis, most likely activated on contact of Favella sp. with predators. interpretation of results and writing the manuscript. However, stronger mechanostimulus by predators may activate more Funding ion channels, or a different class of mechanoreceptors, resulting in a This work was supported by National Science Foundation [IOS 0949744 to A.R.T.] greater depolarization that reaches the threshold necessary for all- and National Science Foundation Doctoral Dissertation Award [IOS 1407059 to or-nothing APs (Fig. 10D). Such threshold-dependent sensory M.L.E. and A.R.T.]. mechanisms likely underpin distinct prey capture and predator evasion behaviors exhibited by alveolate grazers. Supplementary information Although there is a clear relationship between fast APs and Supplementary information available online at http://jeb.biologists.org/lookup/suppl/doi:10.1242/jeb.121871/-/DC1 whole-cell contractions in Favella sp., the mechanism is yet to be determined. 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