© 2021. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2021) 224, jeb239855. doi:10.1242/jeb.239855

RESEARCH ARTICLE Efficient high-voltage protection in the electric Georg Welzel* and Stefan Schuster*

ABSTRACT against predators (Bauer, 1968; Belbenoit et al., 1979; Rankin and For thousands of years, starting with detailed accounts from ancient Moller, 1992) or to stir up prey fish (Bauer, 1968; Belbenoit et al., Egypt, the African electric catfish (Malapteruridae) has been renowned 1979). Such defensive discharges can be elicited by mechanically for its ability to hunt and to defend itself with powerful electric shocks. stimulating the skin of electric catfish (Ballowitz, 1899; Bauer, Surprisingly, the degree to which electric catfish are protected against 1968; Bennett, 1971). In addition, electric organ discharges may their own or external electric shocks, how specific any protection would also play a role in social interactions between conspecifics (Rankin be to the -specific waveform and whether a discharging catfish and Moller, 1986) or in intraspecific communication (Kastoun, has to actively prepare for the onset of its high-voltage discharges has 1971; Rankin and Moller, 1992). never been analysed. Here, we used digital high-speed video to record High-voltage electric shocks have massive effects on the nervous catfish during their own discharges or as they were exposed to external system (Beaumont, 2016; Nordgreen et al., 2008; Robb and Roth, discharges, employing goldfish to carefully calibrate the efficiency of all 2003; Sharber and Sharber Black, 1999) as well as the skeletal discharges. Electric catfish show a remarkable degree of protection muscles of fish (Beaumont, 2016; Catania, 2014, 2015). However, up against high voltages: both self-produced and external electric shocks to now it has only been speculated whether electric catfish are self- that heavily affected control goldfish failed to evoke involuntary muscle protected against their own high-voltage electric shocks (Babucchin, contraction or to affect sensorimotor processing. Even a commercial 1877; Bilharz, 1857; Du Bois-Reymond, 1887; Norris, 2002; Rankin electrofishing device, set to efficiently immobilise and narcotise fish, and Moller, 1986). Despite centuries of interest, the extent to which failed to have any effect on the electric catfish. Our findings rule out skeletal muscles or other electrically vulnerable tissues of electric several protective mechanisms and demonstrate a highly efficient and catfish are affected by their own high-voltage discharges has not been versatile shielding whose nature is presently unclear. analysed. Moreover, it is unknown whether a discharging catfish prepares for the high voltages similar to the way in which KEY WORDS: Electronarcosis, Strongly , Electric shock, echolocating bats avoid deafening by their intense calls (Suga and Predator, Novelty response Jen, 1975). In this study, we exposed electric catfish to different types of high-voltage electric shocks and used digital high-speed video INTRODUCTION recordings to examine to what extent these remarkable fish are Starting with the remarkably detailed accounts of the ancient protected against their own and external electric shocks. Egyptians (Gamer-Wallert, 1970; Westendorf, 1999), the African electric catfish (Malapteruridae) has been renowned for its ability to MATERIALS AND METHODS generate powerful electric shocks. These are emitted by a specialised electric organ which is composed of a thin layer of We used two electric catfish ( beniensis Murray 1855, electrocytes located directly below the skin that covers nearly the Malapteruidae) and five goldfish [Carassius auratus (Linnaeus whole body (Fig. 1A,B) (Johnels, 1956). Each electric organ 1758), Cypriniformes]. All fish were obtained commercially from discharge produces a strong monophasic pulse with a duration of an authorised specialist retailer (Aquarium Glaser GmbH, Rodgau, 2–3 ms and an amplitude of up to 300 V (Bauer, 1968; Remmler, Germany). The electric catfish (6 and 16 cm) were kept individually 1929). Similar to other strongly electric fish, such as the electric eel, in 120 l glass aquaria and the goldfish (5–15 cm) were kept in a electric catfish use their high-voltage pulses as a defensive and 240 l glass aquarium under a 12 h:12 h light:dark cycle (lights on at offensive weapon (Bennett, 1971). During prey capture, electric 07:00 h and off at 19:00 h). The size range of electric catfish and catfish emit volleys of electric organ discharges (Fig. 1C) with an goldfish was chosen so that we could have control goldfish that initial high-frequency phase (up to 500 Hz) and up to 500 pulses matched the experimental catfish in size. Temperature (22–24°C), (Bauer, 1968; Belbenoit et al., 1979) that paralyse the muscles of pH (6.5–7.0) and conductivity (250–300 µS cm−1) of the water prey fish. These volleys can last for several seconds and stop when [based on 50% tap water and 50% demineralised water, enriched the immobilised prey is snapped up and swallowed (Bauer, 1968). with 7% (w/v) NaHCO3, 4% (w/v) CaSO4 and 1% (w/v) marine Electric catfish are also able to produce brief discharges composed salt] were kept constant during the experimental period. care of several high-voltage pulses (Fig. 1D–F) (Ballowitz, 1899; Bauer, and all experimental procedures were conducted in accordance with 1968) that are thought to be emitted during defensive behaviour the German Animal Welfare Act (Tierschutzgesetz) as well as the German Regulation for the protection of animals used for experimental purposes or other scientific purposes (Tierschutz- Department of Animal Physiology, University of Bayreuth, Universitätsstrasse 30, Versuchstierordnung) and were approved by the government of 95447 Bayreuth, Germany. Lower Franconia (Regierung Unterfranken, Würzburg, Germany). *Authors for correspondence ([email protected]; georg.welzel@ uni-bayreuth.de) Natural and artificial defensive discharges G.W., 0000-0002-7017-604X; S.S., 0000-0002-0873-8996 Defensive high-voltage discharges can be elicited by touching the electric catfish as described previously (Bauer, 1968; Bennett,

Received 23 October 2020; Accepted 24 December 2020 1971). In this study, all defensive discharges of the electric catfish Journal of Experimental Biology

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speed digital video camera (HotShot 2300cc, NAC Image A B Skin Technology; 20 mm f/1.8 EX DG lens, Sigma). To optimise the Spinal cord contrast, the tank was illuminated from below with two LED Muscles floodlights (IP FL-50 COB 6400K, Eurolite). Four control goldfish – Electric organ (5 10 cm) and two electric catfish (16 cm) were monitored while exposed to either defensive discharges generated by the electric C Prey capture Cross-section catfish or artificially generated discharges delivered by a pair of stimulation electrodes (silver wire) that were positioned 60 cm apart and perpendicular to the longitudinal axis of the Plexiglas channel. 20 V As the exact field strength and spatial properties of the defensive Electric organ discharges discharges within our setup were unknown, special care was taken to 50 ms ensure that the positions of the test fish were similar in each experiment. More precisely, the discharging electric catfish was always positioned behind (at 5–10 cm distance) the receiving goldfish or catfish, respectively. Additionally, only experiments in D Defence which the discharging catfish and the receiving fish swam in line 10 V Threat (with a tolerated maximal deviation of about 5 deg) were included in 10 ms the analysis. All electrical discharges were recorded using two carbon electrodes in the water placed at opposite ends of the test E F aquaria. Each electrode was first fed into a voltage divider to reduce the output voltage to a quarter of the input voltage and connected to 20 300 a differential amplifier (EXT 02F-2, npi electronic). Its output was 200 used to trigger the high-speed video system so that it recorded the 10

Total no. Total 100 catfish 100 ms before and for another 100 ms after onset of its

Frequency (Hz) (90) (89) (83) (73) (49) (36) discharges. 0 24681012 152 3 4 6 We analysed the pectoral fin angle relative to the body, body No. of pulses Inter-pulse interval bending and apparent body length using ImageJ software. Body bending was defined as the angle between the tip of the fish’s snout, Fig. 1. The high-voltage electric organ discharges of electric catfish. the ventral midline between the pectoral fins and the caudal peduncle. (A) Schematic side view and (B) cross-section of an electric catfish to illustrate ’ the location of the electric organ (yellow). The dashed line in A indicates the Body length was defined as the distance between the tip of the fish s position of the cross-section shown in B. The electric organ covers nearly the snout and the caudal peduncle (Fig. 2A) and was determined by using whole body and is located directly below the skin. (C) High-frequency volley of the line selection tool in the ImageJ toolbar. The pectoral fin angle and electric organ discharges typically emitted to immobilise and catch prey fish. body bending were determined by using the angle tool in the ImageJ (D) Representative defensive discharge of an electric catfish elicited by toolbar. It was sufficient to determine all parameters every 5 ms in a mechanical stimulation. Each discharge consists of several monophasic period from 50 ms before (control interval) to 50 ms after the pulses. Note that the measured voltages depend on the distance and the orientation of the recording electrodes. The scale bars only indicate the order of beginning of a discharge. Body length was normalised to the mean magnitude of the discharges. (E) Frequency distribution of the number of value before the discharge. Movie 1 illustrates the effect of high- pulses in n=90 defensive discharges emitted by an electric catfish (N=1). voltage defensive discharges on an electric catfish and a goldfish. (F) Reciprocal of the inter-pulse interval (frequency; 272±20 Hz) between individual pulses within the defensive discharges (numbers analysed as Effect of high voltages on acoustically elicited electric indicated). startle responses The novelty response in electric catfish was discovered and analysed were elicited by slight mechanical stimulation of the tail with a paint in a test tank (60×30×25 cm) filled with 15 l of water. To produce an brush as described by Bauer (1968). Additionally, the effect of acoustically elicited electric startle response, we used an intense artificial high-voltage defensive discharges was tested. The artificial auditory stimulus that consisted of 300 Hz sine waves of 16.66 ms discharges consisted of monopolar square wave pulses (99 V) duration. The stimulus was generated using a function generator delivered at frequencies of 200 Hz (4 pulses of 3 ms duration each), (DS345, Stanford Research Systems) and delivered by a large 300 Hz (5 pulses of 1.5 ms duration) or 600 Hz (10 pulses of 1 ms loudspeaker (112 MA, Thomann) placed next to the tank. The duration) by an isolated stimulator (DS2A, Digitimer) driven by a maximum sound pressure level was 190 dB re. 1 µPa, as measured pulse generator (TGP-110, TTi). with a hydrophone at the position of the fish (hydrophone: Bruel and Kjaer, type 8106; amplifier: Bruel and Kjaer, type 2610). High-speed video analysis of the effect of defensive Latency of the defensive discharges generated by the fish in discharges response to the acoustic stimulus was defined as the time between To analyse the impact of natural and artificial high-voltage onset of the acoustic stimulus and onset of the discharge. To avoid defensive discharges, two fish (either 2 electric catfish or 1 habituation, acoustic stimuli were delivered at intervals of at least electric catfish and 1 goldfish) swam in an oval custom-made 5 min. In the tests, artificial defensive discharges (5 pulses, 1.5 ms, Plexiglas channel. The channel was separated into two halves by 99 V, 300 Hz) were delivered simultaneously to the acoustic plastic mesh. In the centre of one half, a paddle wheel powered by an stimulus and the effect of pairing on latency of the acoustically electric motor created a water current against which the two fish induced startle response was examined. The artificial discharges swam in the other half of the channel (12 cm diameter and 90 cm were delivered by a pair of stimulation electrodes (silver wire, length long). A porous filter pad in the channel separated the two fish. The 30 cm) that were positioned 60 cm apart and perpendicular to the −1 fish were monitored from below at ≥1000 frames s using a high- longitudinal axis of the aquarium and the test fish. Efficiency of the Journal of Experimental Biology

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Fig. 2. High-speed video showing the absence of any muscle twitches in discharging electric catfish. (A) Experimental approach with fish swimming A Electric organ stationary against a water current to closely examine involuntary muscle Water discharge contractions that might accompany the firing of discharges in the electric current +− recording catfish. The electric catfish was always positioned behind the goldfish at a defined distance. Discharges were elicited by touch and their detrimental effect Tactile was calibrated using similar-sized goldfish. Pale red lines schematically stimulus indicate the electric dipole field produced by the discharging electric catfish. (B) Time course of the pectoral fin angle velocity and (C) the body length of an electric catfish (n=10 trials, N=1 fish) compared with goldfish (n=18 trials, N=3 fish). Body length was normalised to the mean value of the control interval Goldfish Electric catfish (50 ms interval before the discharge). The defensive discharges started at time zero. In every trial, they induced involuntary muscle contractions in goldfish Fin spreading within the first 20 ms (red areas). Pectoral fin angle and body length were determined every 5 ms. Each line represents the mean±s.e.m. (D) Effect of the discharges on maximal pectoral fin angle velocity and (E) minimal body length before (control interval) and during electric shocks (yellow warning signs) in Body shortening High-speed video analysis catfish (n=10 trials) and goldfish (n=18 trials). The decrease in goldfish body Involuntary No involuntary length was the result of whole-body muscle contractions but not a change in muscle contractions muscle contractions body bending (Fig. S1). Red points represent the mean. ***P<0.001; ns, not significant; paired two-tailed t-test. B 2 Electric catfish

) Goldfish positioned 70 cm apart and perpendicular to the longitudinal –1 axis at the ends of the tank covering the whole surface at each end. One goldfish (15 cm in length) and one electric catfish 1 (16 cm in length) were exposed to a homogeneous electric field Fin spreading (100 Hz pulsed direct current, peak voltage gradients of −1

Pectoral fin angle 3Vcm ) generated by an electrofishing device (ELT61NGI, velocity (deg ms Hans Grassl GmbH). In all experiments shown in this study, the 0 C electric catfish and the goldfish swam in parallel to the electric field lines (tolerated maximal deviation ∼5 deg) at the onset of the electric shock. 1.0 First, the fish were exposed to electric shocks for about 1 s to identify any involuntary muscle contractions in the electric catfish. The responses of the fish were recorded from below at Body shortening 1000 frames s−1 using a high-speed digital video camera 0.9 Normalised body length –40 –20 02040 (HotShot 2300cc, NAC Image Technology; 20 mm f/1.8 EX DG Time (ms) lens, Sigma). To illustrate the normal swimming of the electric catfish during the electric shocks, we analysed the pectoral fin angle relative to the body every 10 ms in a period from 1 s before (control D Electric catfish E Goldfish interval) to 2 s after the beginning of an electric shock by using the 3 ns17 ns angle tool of the ImageJ software. ) *** *** – –1 7 Additionally, the responses of the fish to longer (3 4 s) electric shocks that usually narcotise fish were recorded from the side at 2 16 30 frames s−1 using a video camera (Blackmagic Cinema Camera 6 MFT, Blackmagic Design; 16 mm f/2.2 lens, Walimex Pro). The swimming speed of the fish was measured every 200 ms in a period 15 1 from 30 s before to 100 s after the electric shock by determining

Maximal pectoral fin 5 their position every 200 ms using ImageJ software. The timing of angle velocity (deg ms Minimal body length (cm) 0 14 mouth or opercula opening was defined as the timing of respiration. Control Control Control Control The recovery time of the narcotised goldfish was defined as the period from the end of the electric shock until respiration started again. The effect of the narcotising electric shocks is shown in artificial defensive discharge was confirmed in controls run on Movie 2. goldfish: these demonstrated that the high-voltage pulses were equally efficient in goldfish as the natural defensive discharges of Data analysis and statistics the electric catfish. Statistical analyses were carried out using Graph Pad Prism version 5.01 (Graph Pad Software) and Excel (Microsoft). Normal Electrofishing distribution of data was tested using the D’Agostino–Pearson To analyse the effect of stronger electric shocks that are typically normality test. Pairwise comparisons were performed using the used in electrofishing operations, the fish were placed in a test paired two-tailed t-test. The effect of high voltages on startle aquarium (75×10×40 cm) filled with water to a height of 30 cm. response latency and startle response probability was tested by using Two electrofishing electrodes (each a braided copper strip the Mann–Whitney U-test and two-tailed Fisher’s exact test, connected to plate aluminium electrodes; 10×30 cm) were respectively. Pearson’s correlation was used to test the Journal of Experimental Biology

3 RESEARCH ARTICLE Journal of Experimental Biology (2021) 224, jeb239855. doi:10.1242/jeb.239855 relationship between body length and body bending. Values of A Electric organ P<0.05 were considered statistically significant. All data are discharge N n Water +− recording presented as means±s.e.m. and denote the number of animals current and trials, respectively. Movies were edited using Adobe Premiere TactileT i Pro 2.0 (Adobe Systems Incorporated). ststimulusmull

RESULTS Electric catfish do not twitch in response to their own defensive discharges Receiver Sender We first explored whether high-voltage pulses fired by an electric catfish might cause any involuntary contraction of its own skeletal High-speed No involuntary muscles. We used digital high-speed video to closely monitor an video analysis electric catfish that swam steadily in a countercurrent arrangement muscle contractions and that could reliably be induced to fire defensive discharges (4–7 high-voltage pulses of about 250 Hz; Fig. 1E,F) by mechanical stimulation. These defensive discharges had massive effects on goldfish that swam in the same countercurrent setup. We identified B 2 the two most reliable effects of the defensive discharges on the Receiver )

control goldfish (Fig. 2; Movie 1). First, the defensive discharges –1 caused the fast spreading of the pectoral fins (increase in pectoral fin angle velocity: paired t-test: P<0.001; Fig. 2B,D). Second, whole- body muscle contractions (see Fig. S1) caused a typical and 1 significant body shortening (paired t-test: P<0.001; Fig. 2C,E). Normal swimming Pectoral fin angle

However, neither of these effects could ever be detected in the velocity (deg ms electric catfish that fired the discharges (Movie 1). A close analysis based on digital high-speed videos failed to show any significant 0 changes in pectoral fin angle velocity (paired t-test: P=0.413; C Fig. 2B,D) or in body length (paired t-test: P=0.500; Fig. 2C,E). 1.0 Insensitivity to electric shocks does not require any preparedness The electric catfish that fired a defensive discharge in the preceding Receiver experiment controlled the timing of its discharges and the number of 0.9 Normalised body length pulses and could thus potentially have prepared in some way for the –40 –20 02040 expected effects of the high-voltage discharge, much as a bat must Time (ms) prepare for its intense echolocation calls (Suga and Jen, 1975) or an DEReceiver elephantnose fish must prepare the processing of its exquisitely 3 ) sensitive ampullary electroreceptors for its own discharges (Bell, –1 7 1981). Perhaps similar mechanisms are essential for the apparent insensitivity of a discharging electric catfish. To test this, we simply 2 confronted two electric catfish using the same experimental 6 ns approach as described in Fig. 2. By touching one electric catfish of the pair, it could be induced to fire its defensive discharges with 1 ns

arbitrary timing. Hence, the non-discharging test fish, whose Maximal pectoral fin 5 angle velocity (deg ms response we then evaluated, had no control over the timing and Minimal body length (cm) number of pulses that it received (Fig. 3A). However, again we did 0 not observe any twitches of the skeletal muscles or any other Control Control reactions caused by the defensive discharges emitted by the Fig. 3. Electric catfish are insensitive to electric shocks of conspecifics. conspecific (Fig. 3B–D). Neither pectoral fin angle velocity (A) Experimental approach with catfish swimming stationary against a water (paired t-test: P=0.559) nor body length (paired t-test: P=0.401) current to detect involuntary muscle contractions in an electric catfish was affected by the discharges that all strongly affected the control (receiver) after touch-elicited defensive discharges of a conspecific (sender). goldfish (see Fig. 2). The discharging electric catfish was always positioned behind the receiver at the same distance as used in the goldfish experiments. Pale red lines We next replaced the discharges of a conspecific catfish with schematically indicate the electric dipole field produced by the discharging artificial discharges from a generator (Fig. 4A). These discharges electric catfish. (B) Time course of pectoral fin angle velocity and (C) body differed in slope, duration of the individual pulse and rate from those length of the non-discharging electric catfish (n=5 trials). Body length was of the electric catfish (see Fig. 1) and all had demonstrable effects on normalised to the mean value of the control interval (50 ms interval before the goldfish: all artificial discharge mimics induced whole-body muscle discharge). The defensive discharge started at time zero. The red areas contractions in the goldfish (Fig. 4). Again, in each experiment, the indicate the time interval in which the involuntary muscle contractions in goldfish were induced. Pectoral fin angle and body length were determined artificial discharges induced a significant increase in pectoral fin t P every 5 ms. Each line represents the mean±s.e.m. (D) Effect of the electric angle velocity (paired -test: <0.001), resulting in a fast spreading shocks on the maximal pectoral fin angle velocity and (E) the minimal body of the pectoral fins (Fig. 4B,F–H) and caused a typical and length before (control interval) and during the electric shocks (n=5 trials). The significant body shortening (paired t-test: P<0.001; Fig. 4D,I–K). red points represent the mean. ns, not significant; paired two-tailed t-test. Journal of Experimental Biology

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A Isolated Artificial B C 4 n n n 4 n n n voltage stimulator defensive discharge ) =5 =8 =5 =5 =10 =6 –1

AMPLITUDE DURATION *** 3 *** 3 Water + − current + − 2 *** 2 + − + − + − ns + − 1 1 ns ns + − Maximal pectoral fin + − + − angle velocity (deg ms + − 0 0 + Goldfish Electric catfish − + − D E n=6 n=6 n=5 n=5 n=10 n=6 High-speed 16 ns video analysis ns *** ns Fin spreading 9 *** ***

15 Body shortening 8 Minimal body length (cm) Involuntary No involuntary muscle contractions muscle contractions Control 200 Control 300 Control 600 Control 200 Control 300 Control 600 Hz Hz Hz Hz Hz Hz F I Electric catfish (n=5) 200 Hz 200 Hz 2 Goldfish (n=5) 1.0

1 Body shortening Fin spreading 0.9 Electric catfish (n=5) Goldfish (n=6) 0 G J Electric catfish (n=10) 300 Hz 300 Hz ) Goldfish (n=8)

–1 2 1.0

1 length 0.9 Pectoral fin angle Electric catfish (n=10) velocity (deg ms Normalised body Goldfish (n=6) 0 HK Electric catfish (n=6) 600 Hz 600 Hz 2 Goldfish (n=5) 1.0

1 0.9 Electric catfish (n=6) Goldfish (n=5) 0 –40 –20 02040 –40 –20 02040 Time (ms) Time (ms)

Fig. 4. See next page for legend.

Nevertheless, the electric catfish skeletal muscles were insensitive Sensorimotor processing in electric catfish remains to the tested artificial discharge mimics (Fig. 4B–K). These findings unchanged during exposure to high voltages suggest that electric catfish not only need not prepare for high- The above experiments only monitored the effect of high-voltage voltage discharges but that they also tolerate at least some deviation discharges on muscle contractions. However, the discovery of a from their species-specific discharges. robust novelty response allowed us to also test whether the Journal of Experimental Biology

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Fig. 4. Electric catfish are also insensitive to calibrated artificial electric A Sensory input Integration Motor output shocks. (A) Experimental approach with fish swimming stationary against a water current to closely monitor involuntary muscle contractions in an electric catfish (N=1) and a control goldfish (N=1). Artificial discharges consisted of several monopolar square wave pulses (99 V) at different frequencies (200, 300 and 600 Hz; duration of individual pulse: 1, 1.5, 3 ms, respectively) generated by an isolated voltage stimulator. Pale red lines schematically Mechanoreceptor Brain Electric organ indicate the electric field produced by the artificial discharges. (B,C) Effect of the artificial discharges on the maximal pectoral fin angle velocity in (B) B Electric organ C goldfish and (C) electric catfish. (D,E) Effect of the artificial discharges on the discharge recording +− Electric minimal body length in (D) goldfish and (E) electric catfish. The 50 ms control startle interval is indicated. The red points represent the mean. ***P<0.001; ns, not t response significant; paired two-tailed t-test. Note the strong effect of the discharges on the control goldfish but the absence of effects in the electric catfish. (F–H) Time course of the pectoral fin angle velocity before, during and after the fish were exposed to monopolar square wave pulses at frequencies of (F) 200 Hz, Control (G) 300 Hz and (H) 600 Hz. (I–K) Time course of the body length before, during Acoustic and after the fish were exposed to monopolar square wave pulses at stimulus 190 dB frequencies of (I) 200 Hz, (J) 300 Hz and (K) 600 Hz. Body length was 10 ms normalised to the mean value of the control interval (50 ms interval before the discharge). The artificial discharges started at time zero and their strength D Electric organ E was selected to induce involuntary muscle contractions in every trial in the discharge recording +− Electric control goldfish (red areas). Pectoral fin angle and the body length were t startle determined every 5 ms. Each line represents the mean±s.e.m. response transduction of sensory signals, the subsequent integration and processing of sensory information in the brain as well as the production of an appropriate motor output were equally immune to high-voltage electric shocks (Fig. 5A). The electric startle response Acoustic stimulus 190 dB could be reliably elicited by intense acoustic stimuli to which the fish 10 ms respond with a defensive discharge (Fig. 5B,C), fired at a low latency F G of 12.40±2.78 ms (mean±s.d., n=200 trials; Fig. 5F). Producing this 25 ns 100 ns response requires the functioning of mechanosensory cells of the 80 inner ear and the lateral line system (Higgs and Radford, 2013), and 20 circuits that can elicit the response, perhaps similar to the well-known 60 escape response of fish (Mirjany et al., 2011; Zottoli, 1977). 15 If high voltages interfered with any component of the process, one 40

would predict a decrease in startle response probability or an increase 10 Probability (%) in latency. To test these predictions, we presented the acoustic stimuli 20 while the electric catfish were simultaneously exposed to high-

Startle response latency (ms) 5 voltage discharges. These experiments could be run by using the 0 artificial discharges introduced in Fig. 4 that all had strong effects on Control Control similar-sized control goldfish (Fig. 4) and that could easily be paired Fig. 5. No effect of high-voltage discharges on a sensorimotor novelty with the acoustic stimuli (Fig. 5D,E). Our results clearly show that the circuit of the electric catfish. (A) Schematic representation of the basic electric shocks neither changed response latency (12.46±2.90 ms; pathway of the electric startle response we discovered in the electric catfish. P=0.979, Mann–Whitney U-test; Fig. 5F) nor affected the capacity of High-voltage discharges might impact each stage of the circuit from sensory the acoustic stimulus to cause the usual behavioural response transduction in mechanosensory cells of the inner ear and the lateral line, to P ’ integration of the sensory information in the brain as well as the reflexive ( =0.888, two-tailed Fisher s exact test; Fig. 5G). discharge of the electric organ. (B) The electric startle response in an electric catfish (N=1) was elicited by an intense acoustic stimulus (190 dB re. 1 µPa, Electric catfish are neither immobilised nor narcotised 300 Hz sine wave) and consists (C) of a defensive discharge being fired after a by electrofishing brief latency (Δt; defined as the time between stimulus onset and the peak of The above findings demonstrate that electric catfish are insensitive the first reflexive discharge). (D) To determine the impact of high-voltage to short high-voltage discharges. But electric catfish additionally discharges on the circuits that underlie the startle response, an artificial defensive discharge mimic consisting of five monopolar square wave pulses emit high-voltage volleys that can last for seconds (Fig. 1B) (Bauer, (1.5 ms, 99 V, 300 Hz; see Fig. 4 for calibration with goldfish) was presented 1979). Therefore, we next examined whether electric catfish can simultaneously with the acoustic stimulus and (E) the startle response was also resist electric shocks with an increased duration and intensity recorded. (F) Effect of the artificial high-voltage discharges on startle response by challenging the fish with a commercial electric fishing device latency (n=200 trials; horizontal lines indicate the mean±s.e.m.) and (G) startle (Fig. 6A). This was set as in electrofishing operations (100 Hz response probability [n=153 and 151 trials before (control) and during pulsed direct current, peak voltage gradient of 3 V cm−1) to cause an stimulation, respectively]. ns, not significant. immediate immobilisation following whole-body muscle contractions, loss of consciousness (Beaumont, 2016; Robb and or during an electric shock of 1 s duration delivered by the Roth, 2003; Schwartz and Herricks, 2004) and cardiac arrest in fish electrofishing device. However, instead of being immobilised (Schreer et al., 2004) (Fig. 6A). As in our previous experiments instantaneously as a result of tetanic muscle contractions, the (Figs 2–4), we first used high-speed videos to analyse whether we electric catfish showed undisturbed swimming behaviour and could identify any involuntary muscle contractions at the beginning normal movements of their fins (Fig. 6B,C; Figs S2 and S3). Journal of Experimental Biology

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A 100 Hz Electrofishing D Before After F 400 device 1 s

Before + − Video 200 After 5 m s–1

+ − no. Total Swimming analysis + − speed + − 10 s + − Swimming + Electronarcosis − 0 + − + − 0 01020 + − + − EG Before After 400 High-speed video analysis Before 200 After Electric catfish

200 speed B Goldfish no. Total Swimming 100 0 0 0 10 20 Pectoral fin angle (deg) 0 Swimming speed (m s–1) ) 2 C –1 H Before After

Goldfish 1 (deg ms Electric catfish Respiration

Pectoral fin angle 0 velocity −10 1 −300 30 Time (s) Time (s)

Fig. 6. A commercial electrofishing device fails to narcotise electric catfish. (A) Experimental approach to analyse the response of an electric catfish (N=1) and a goldfish (N=1) to stronger and longer lasting (1–4 s) electric shocks (100 Hz pulsed direct current, peak voltage gradient of 3 V cm−1) that are typically used in electrofishing operations. Pale red lines schematically indicate the homogeneous electric field produced by the electrofishing device. (B) Representative examples of pectoral fin movement in goldfish and electric catfish before and during an electric shock. The duration of the electric shock (1 s) is indicated by the red area. For additional examples, see Fig. S2. (C) The electric shocks had no effect on the electric catfish but immediately caused spreading and subsequent immobilisation of the fins of the goldfish. Solid lines represent the mean±s.e.m. (n=4 trials). (D) Swimming speed of a goldfish and (E) an electric catfish before, during (red area) and after a narcotising electric shock (3–4 s). Swimming speed was determined every 200 ms. (F,G) Distribution of swimming speed for (F) goldfish and (G) electric catfish before and after the narcotising electric shock (n=6 trials). (H) Timing of respiration of goldfish (n=6 trials) and electric catfish (n=4 trials) before and after the electric shocks. Each vertical line indicates the timing of respiration events. The red areas indicate the duration of the electric shocks.

Impressively, there was also no electronarcosis even after longer catfish are protected against their high-voltage discharges. They also electric shocks (3–4 s) that always instantaneously narcotised the exclude several mechanisms that have been proposed for this and control goldfish (Fig. 6E,G; Fig. S3 and Movie 2). Goldfish show that the protection is far more powerful and extends even to immediately stopped moving (Fig. 6D,F; Fig. S3 and Movie 2) and external high voltages. breathing (Fig. 6H), and resumed breathing only 24.0±3.2 s (n=6) Although we simply used the novelty response as a tool to assess after the discharges had ended. In summary, these results the effects of external discharges on sensorimotor processing, we demonstrate that the electric catfish is immune to high-voltage would like to briefly comment on the conspicuously short latency of electric shocks from a commercial device that reliably immobilises only 12.40±2.78 ms. This latency is in the range of latencies known and narcotises other fish. for mechanically induced Mauthner neuron-driven escape C-starts of teleost fish (e.g. Korn and Faber, 2005). In the electromotor DISCUSSION system of the catfish, two large electromotorneurons command the Our results demonstrate for the first time that electric catfish are high-voltage discharges (Bennett et al., 1967; Schikorski et al., completely immune not only to their own high-voltage electrical 1992) and so these neurons might either directly receive at least shocks but also to external shocks delivered by a commercial mechanosensory information or, more plausibly, be directly driven electrofishing device. We show that the catfish’s skeletal muscles by the Mauthner neurons. A connection between the Mauthner are not activated by discharges that induced massive muscle neuron and an electromotor pathway has been suggested in the contractions in goldfish. In none of our experiments did we see any South American weakly electric fish Gymnotus carapo (Falconi involuntary muscle twitch in electric catfish, either in response to et al., 1995). their own discharges, generated by their own electric organ or that of In fish, electric shocks of increasing intensity elicit the startle another electric catfish, or after strong artificial discharges from an response, complete muscle tetanus and finally electronarcosis electrofishing device. Moreover, the findings obtained in our (Beaumont, 2016). High-intensity shocks are used in experiments using the novelty response suggest that the nervous electrofishing operations to immobilise and catch fish. Depending system and sensorimotor processing within the brain were also on fish size, species, water conductivity or duration of the electric unaffected by the high-voltage electric shocks used in this study. shock, field strengths of 0.5–1.0 V cm−1 (pulsed direct current, as

These findings confirm the centuries-long speculation that electric used in this study) are sufficient to immediately cause whole-body Journal of Experimental Biology

7 RESEARCH ARTICLE Journal of Experimental Biology (2021) 224, jeb239855. doi:10.1242/jeb.239855 muscle tetanus (Beaumont, 2016). To affect the nervous system of Funding fish (Beaumont, 2016; Nordgreen et al., 2008; Robb and Roth, 2003; The project was funded by overheads from the Deutsche Forschungsgemeinschaft Sharber and Sharber Black, 1999) and subsequently induce (DFG grant Schu1470/8). – −1 electronarcosis, field strengths of 0.5 2.0 V cm are necessary Supplementary information (Beaumont, 2016; Gaikowski et al., 2001; Taylor et al., 1957; Walker Supplementary information available online at et al., 1994). It is therefore remarkable that even stronger electric https://jeb.biologists.org/lookup/doi/10.1242/jeb.239855.supplemental shocks (3 V cm−1) caused neither involuntary muscle contractions nor any changes in the processing within the central nervous system References Babucchin, A. (1877). Beobachtungen und Versuche am Zitterwelse und of the electric catfish. 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Biol. 216, 1484-1490. doi:10.1242/jeb.078816 and arrhythmia are the most serious and most common injuries Johnels, A. G. (1956). On the origin of the electric organ in Malapterus electricus. Q. J. Microsc. Sci. 97, 455-464. following electrical shock in humans (Waldmann et al., 2017), the Kastoun, E. (1971). Elektrische Felder als Kommunikationsmittel beim Zitterwels. present findings make the powerful high-voltage protection of the Naturwissenschaften 58, 459. doi:10.1007/BF00624628 electric catfish a promising subject to explore further. Korn, H. and Faber, D. S. (2005). The Mauthner cell half a century later: a neurobiological model for decision-making? Neuron 47, 13-28. doi:10.1016/j. Acknowledgements neuron.2005.05.019 We thank Laura Angerer, Laura Löhner, Sebastian Steibl and Antje Halwas for their Mirjany, M., Preuss, T. and Faber, D. S. (2011). Role of the lateral line help, and our reviewers for many stimulating ideas, including a scheme of how active mechanosensory system in directionality of goldfish auditory evoked escape compensation might be useful and emphasising the short latency of the electrical response. J. Exp. Biol. 214, 3358-3367. doi:10.1242/jeb.052894 novelty response. Nelson, M. E. (2011). Electric fish. Curr. Biol. 21, R528-R529. doi:10.1016/j.cub. 2011.03.045 Competing interests Nordgreen, A. H., Slinde, E., Møller, D. and Roth, B. (2008). Effect of various The authors declare no competing or financial interests. electric field strengths and current durations on stunning and spinal injuries of Atlantic herring. J. Aquat. Anim. Health 20, 110-115. doi:10.1577/H07-010.1 Author contributions Norris, S. M. (2002). A revision of the African Electric , Family Conceptualization: G.W., S.S.; Methodology: G.W.; Validation: G.W., S.S.; Formal Malapteruridae (Teleostei, Siluriformes), with Erection of a New and analysis: G.W., S.S.; Investigation: G.W.; Resources: S.S.; Writing - original draft: Descriptions of Fourteen New Species, and an Annotated Bibliography. Tervuren:

G.W., S.S.; Visualization: G.W. Koninklijk Museum voor Midden-Afrika. Journal of Experimental Biology

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