Electric organ discharge frequency jamming during social interactions in brown ghost knifefish, Apteronotus leptorhynchus

ANIMAL BEHAVIOUR, 2005, 70, 1355–1365 doi:10.1016/j.anbehav.2005.03.020

Electric organ discharge frequency jamming during social interactions in brown ghost knifefish, Apteronotus leptorhynchus SARA K. TALLAR OVIC * & H AROLD H . Z AKON †

*Department of Biology, University of the Incarnate Word ySection of Neurobiology, University of Texas at Austin (Received 21 May 2003; initial acceptance 21 August 2003; final acceptance 16 March 2005; published online 8 November 2005; MS. number: A9617)

In most communication systems, animals avoid jamming when similar signals from two individuals might overlap and cause interference. Most species of wave-type electric fish avoid jamming by shifting their electric organ discharge (EOD) frequencies away from those of conspecifics or away from mimics of sine wave EOD frequencies presented in playback experiments. Both male and female brown ghosts, however, appear to intentionally jam rivals during competitive interactions if the rival has a higher EOD frequency. We studied this behaviour in competitive interactions between two fish in a neutral arena, and in playback studies with single fish in resident–intruder and neutral arena models. During competitive interactions, females jammed the rival’s EOD signal only when the rival’s EOD frequency was within w30 Hz of their own EOD frequency, whereas males tried to jam EOD signals of rivals that were 70 Hz higher than their own EOD frequency. In the resident–intruder model experiment, fish tended to raise their EOD frequencies to within potential jamming range in response to a simulated intruder with a higher EOD frequency than their own. In a neutral arena model, fish directed the most aggressive attacks in response to simulated rivals with lower EOD frequencies, yet were less likely to attack the playback device if it played a frequency elevation that mimicked a fish with a lower EOD frequency attempting to jam them. This is the first evidence that electric fish may actively use jamming as an aggressive behaviour. Ó 2005 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

In animal communication systems, interference between signals from two different senders may occur, causing a phenomenon known as jamming (Heiligenberg 1986). Such interference is generally considered to be detrimental to both senders, because information reaching the receiver is ambiguous, and thus neither individual is communicating effectively. In most cases, senders will modify their communication behaviour in some way to avoid this interference. Several notable exceptions to this are Smilisca treefrogs (Ryan 1986) and Magicada cicadas (Cooley & Marshall 2001). In both of these cases, calling males alter their courtship calls to overlap those of rival males. Active navigation systems present yet another consideration in terms of jamming. When an animal such as an electric fish uses a signal for both electrolocation and communication, jamming can occur (Heiligenberg 1973). Wave-type electric fish generate a quasisinusoidal electric organ discharge (EOD) from a specialized organ in the

Correspondence: S. K. Tallarovic, University of the Incarnate Word, Department of Biology, 4301 Broadway, San Antonio, TX 78209, U.S.A. (email: [email protected]). 0003–3472/05/$30.00/0

tail. The resulting electric field around the body is used for electrolocation via electroreceptors that are distributed over the entire body surface, but more densely clustered around the head. During electrolocation, electric fish perceive nearby objects by the minute perturbations (microvolt changes in EOD amplitude, microsecond changes in EOD timing) that objects make in the electric field. The EOD frequencies of wave-type electric fish remain steady over long periods (Bullock 1970; Moortgat et al. 1998), with each fish maintaining its own unique frequency. However, individuals can rapidly modulate EOD frequency for communication purposes (see below). When the EOD frequencies of two fish are within about 2–10 Hz of each other, their EODs interact, causing large modulations in each fish’s EOD frequency (Heiligenberg 1973). These modulations mask small perturbations caused by nearby objects and have a detrimental effect on the fish’s electrolocation ability (Heiligenberg 1973; Bastian 1987a, b). To circumvent this jamming, most species of wave-type electric fish studied display a behaviour known as the jamming avoidance response (JAR) in which one or both individuals shift their EOD frequencies away from that of the other fish (review in Heiligenberg 1986). In the

1355 Ó 2005 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

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best-studied species, Eigenmannia virescens, the higher-frequency fish raises its EOD frequency while the lower-frequency fish shifts downward. In species that are unable to lower the EOD frequency, such as the brown ghost, Apteronotus leptorhynchus, only the higher-frequency individual raises its EOD frequency. The JAR can be elicited from an artificially generated sine wave presented through playback electrodes (Bullock et al. 1972). Most studies of the JAR are performed while the fish is restrained in a tube or nylon mesh and presented with a sine wave perpendicular to its body; often the playback signal is frequency-clamped to the fish’s EOD frequency (as the fish changes its EOD frequency, the playback signal follows it, such that the fish is continuously jammed) (Bullock et al. 1972; Bastian et al. 2001). Brown ghosts readily produce a JAR in these circumstances, but their behaviour under more natural circumstances (free-swimming and/or interacting with another live fish) is not as well understood. Brown ghost knifefish are sexually dimorphic in both body form and EOD frequency (Hagedorn & Heiligenberg 1985). Males are larger, with a longer, broader snout, and typically have higher EOD frequencies than females (800– 1100 Hz versus 600–800 Hz at 26–28
C). Males are typically territorial and have a linear dominance hierarchy in which the largest males also have the highest EOD frequency (Hagedorn & Heiligenberg 1985; Dunlap 2002; K. D. Dunlap & H. H. Zakon, unpublished data). Female social behaviour is not well understood, and there is not as clear a relation between EOD frequency and size or dominance. In spite of the extreme regularity of the EOD frequency in Apteronotus, these fish are able to modulate the signal for communicative purposes. Their communication signals generally fall into two classes: chirps and rises. Chirps are rapid, transient frequency excursions (Larimer & McDonald 1968; Hopkins 1974; Zupanc & Maler 1994) that can be categorized into as many as four types ranging from tens of hertz to several hundred hertz, and lasting from tens of milliseconds to about 200 ms (Engler et al. 2000; Engler & Zupanc 2001; Triefenbach & Zakon 2003). Rises show less modulation in frequency than chirps (typically under 30 Hz), last from several hundred milliseconds to many seconds, and can be categorized into three types (Tallarovic & Zakon 2002). Both sexes produce chirps and rises, but males mainly produce chirps (Engler et al. 2000) and females mainly produce rises. Female chirps, when they do occur, are also produced at much lower magnitude than those of males (Hagedorn & Heiligenberg 1985; Dulka & Maler 1994; Triefenbach & Zakon 2003). In a pilot study of social behaviour and dominance in female brown ghosts, we noted that pairs of fish competing over a shelter often overlapped in EOD frequency while fighting, even when their baseline EOD frequencies were within 20–30 Hz (Tallarovic et al. 2002). Surprisingly, and against most predictions of the JAR literature, the fish with the lower baseline EOD frequency often raised its EOD frequency, apparently into the jamming range of the other fish. This unusual result led us to ask in this study whether this behaviour is observed in both sexes, and whether fish elevate their EOD frequency to the

same frequency as a rival, presumably to jam it, or raise their EOD frequency higher than a rival’s, possibly to appear more dominant. We also wished to know whether frequency elevations are associated with offensive or defensive aggression, or both. The experiments reported here include (1) behavioural observations of live fish competing in a neutral arena for a resource (shelter tube); (2) a modified resident–intruder study using playbacks to fish in home tanks; and (3) a neutral arena study using playbacks to observe aggressive behaviour. GENERAL METHODS All fish used in these experiments were obtained from a commercial dealer. Females were 13.3–18.5 cm long, weighed 6.64–13.7 g, and had EOD frequencies between 670 and 799 Hz at 26
C. Males were 13.8–21.3 cm long, weighed 6.0–16.8 g, and had EOD frequencies between 810 and 948 Hz at 26
C. All fish were sexually mature adults and were sexed by EOD frequency, body shape, gravidity and the presence or absence of a white spot on the cloaca (Hagedorn & Heiligenberg 1985; Bastian et al. 2001). Fish were housed individually in 28-litre tanks (23 ! 50 ! 24 cm high) in two 660-litre recirculating freshwater systems. We monitored water quality weekly and maintained conductivity at approximately 150 mS/ cm during all experiments. Water temperature for all experiments was between 26 and 28
C. Daylength in the laboratory was maintained on a 12:12 h light:dark cycle. All experiments occurred during the dark cycle, and were conducted under red light (to which fish had been acclimatized) or infrared illumination. Fish were fed frozen bloodworms (Chironomus sp. larvae) every other day. Experiments performed in a tank other than the fish’s home tank used water from the recirculating systems, thus maintaining similar water quality to that of the home tank. Paired silver wire electrodes were used for all recordings and playbacks during the experiments. EOD frequency recordings (8-kHz sampling rate and 16-bit resolution, gain C18 dB) were made using a Terratec EWS88MT A/D converter and CoolEdit Pro 1.2 software (Syntrillium Software Corp., Phoenix, Arizona, U.S.A.). Each recording was then viewed as a spectrogram in CoolEdit Pro to visually assess frequency modulations. Communication signals were visualized on a spectrogram in CoolEdit Pro using a fast Fourier transformation size of 4096. Frequency modulations of only a few hertz were clearly visible in the spectrogram view, and only nonchirp modulations were counted (rises, as defined in Tallarovic & Zakon 2002, were lumped together). We determined the EOD frequency of the subject by plotting a line at the midpoint of the EOD frequency trace on the spectrogram, and aligning it with the frequency axis. Video recordings were made using Sony digital video cameras with Nightshot IR illumination.

Statistical Analysis All fish in this study produced a variety of communication signals including chirps and EOD rises (we did not

TALLAROVIC & ZAKON: EOD JAMMING IN ELECTRIC FISH

classify rise or chirp types in this study). Sometimes these were superimposed on a gradual elevation in frequency. Since EOD rises and chirps can also be produced without this gradual increase in frequency, we treat them independently. Analyses were performed using Statview 5.0 software (SAS Institute Inc., Cary, North Carolina, U.S.A.), and all tests are two tailed.

Live Interactions between Females (Neutral Arena) Methods To examine whether frequency elevations in EOD are associated with offensive and/or defensive aggression in females, we observed paired female brown ghosts in a neutral arena where neither fish had a territorial advantage. Fish (N Z 16) were paired such that no pair was closer in EOD frequency than 15 Hz at the beginning of the experiment. Eight pairs were observed. We placed fish on either side of a mesh barrier in a 76-litre aquarium and allowed them to acclimate for 5 min. After the acclimation period, we monitored the tanks for 10 min, then removed the barrier and placed a clear acrylic shelter tube in the centre of the tank (to provide a limited resource for which the fish could compete; Dunlap & Oliveri 2002). Fish were allowed to interact for 10 min, during which time they were videotaped. After 10 min, a light was switched on, which generally caused the fish to stop interacting, or greatly decreased the amount of interaction (brown ghosts are nocturnal and become relatively inactive under light). After 10 min the light was again switched off and the recording continued for an additional 10 min. We named these four 10-min observation periods (1) Separated, (2) Light Off 1st, (3) Light On and (4) Light Off 2nd, respectively. We examined spectrograms of each paired interaction to compare the EOD frequencies of each fish during each time period. For each 10-min period, we determined the EOD frequency of each fish at five time points, 1 min apart, and calculated a mean EOD frequency for each fish. We then compared the difference between the mean EOD frequencies of the two fish during each time period in the trial, as well as how large a frequency elevation each fish made in relation to its baseline EOD at the start of the trial. Comparisons were made using ANOVA and Fisher’s protected least significant difference (PLSD).

Results During staged interactions, both females made EOD rises, as expected from a previous study of aggressive interactions in this species (Tallarovic & Zakon 2002), but the fish with the lower baseline EOD frequency often made a slower frequency elevation. EOD elevations occurred during periods where the fish were physically interacting in the dark (Fig. 1a). The fish with the higher EOD frequency sometimes responded with small elevations of EOD frequency, probably JARs, as the EOD frequency of the lower-frequency fish approached to within a few hertz of its own. Often the EOD frequencies of the two fish fluctuated in synchrony as the lower-frequency fish’s EOD

frequency moved into jamming range (2–10 Hz) of the EOD of the higher-frequency fish (Fig. 1b). When all trials (N Z 8) were analysed together, no consistent pattern of change in baseline EOD frequency was apparent. However, it was evident that when baseline EOD frequencies of females were less than 30 Hz apart, the female with the lower baseline EOD frequency usually elevated her EOD frequency to within the jamming range of the other fish. The mean difference in frequency of these interacting females was significantly less than that during the Light Off 1st and Light Off 2nd phases than during the Separated and Light On phases (Fig. 2a). For female pairs whose baseline EOD frequencies differed by more than 30 Hz, there was no significant difference in mean EOD frequency for any phase of the experiment (Fig. 2b), and these females did not appear to produce the large elevations in EOD frequency seen in the other trials.

Live Interactions between Males (Neutral Arena) Methods To examine whether frequency elevations in EOD are associated with offensive and/or defensive aggression in males, we observed paired male brown ghosts in a neutral arena where neither fish had a territorial advantage. Trials (N Z 8) involving males (N Z 16) were similar in design to the female trials, with the following exception: because males continued to fight under visible light, it was necessary to replace the barrier to interrupt the fighting. Therefore, the time periods used were (1) Separated 1st; (2) Together; and (3) Separated 2nd.

Results During the Light Off phase, males produced many chirps as expected. Because the male with the lower EOD frequency elevated it during these encounters, males also showed less difference in EOD frequency during physical interactions, and the average EOD frequency difference was significantly smaller during the Together phase than during the Separated 1st and Separated 2nd phases of the experiment (Fig. 3). Unlike the females, there was no behavioural difference in pairs whose EOD frequencies were closer (N Z 4) or further apart (N Z 4) in frequency at the start of the trial. All pairs of males behaved similarly to the female pairs whose baseline frequencies differed by less than 30 Hz. In addition to the frequency elevation, males also produced EOD rises and chirps while interacting.

Modified Resident–Intruder Model Methods One disadvantage of staged aggressive encounters with two live animals is that it is difficult to differentiate between offensive and defensive aggressive behaviours, because each animal is responding to the other’s behaviour. To circumvent this problem we simulated an intruder

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Time elapsed (mm:ss) Figure 1. Spectrograms of two female brown ghosts interacting during a 40-min trial in a neutral arena. (a) During the first 10 min, fish were separated by a mesh barrier. At 10 min, the barrier was removed and fish were allowed to compete over a shelter, during which time their EOD frequencies overlapped as a result of a frequency elevation by the lower-frequency fish. At 20 min, a light was switched on to interrupt fighting, and the females’ EOD frequencies drifted apart. At 30 min, the light was turned off again and the females’ EOD frequencies overlapped again as they resumed fighting. (b) Close-up showing each fish’s EOD frequency while fighting.

fish by playback of a sine wave from inside a plastic shelter that was placed in a fish’s tank. By testing a fish in its home tank rather than a neutral arena, conditions were further optimized for a fish to make offensive aggressive signals. Stimuli from 2 to 10 Hz are effective for jamming (Heiligenberg 1973; Bastian 1987a), with 4 Hz being optimal in this species (Dye 1987). Therefore, playback signals of 15 Hz above or below the resident fish’s own EOD frequency were used to minimize jamming. We also chose the G15 Hz stimuli as signals to examine whether fish would match or surpass the stimulus frequency. Both males and females can make JARs and EOD rises of at least 30 Hz (Engler et al. 2000; Bastian et al. 2001; Tallarovic & Zakon 2002). Thus, if a fish were attempting to jam a rival, it would raise its EOD frequency to within a few hertz of the intruder’s EOD frequency. If, on the other hand, it were attempting to appear more dominant (EOD frequency correlates with size and dominance in male brown ghosts, Dunlap 2002), it ought to attempt to raise its EOD frequency past that of the intruder. Based on the results of our earlier experiment, we expected males and females to behave similarly to the stimuli, and therefore analysed them as a single data set. However, we also present statistical analyses for each sex separately.

Fish in their home tanks (N Z 19; 10 females, 9 males) were presented with a simulated intruder: an acrylic shelter tube inside which a pair of carbon playback electrodes was embedded. The ends of the tube were covered with mesh to prevent the resident fish from entering the tube, but the resident fish was able to swim freely around and investigate the playback device. The tube was placed inside the subject’s tank approximately 5 min before the playback began. Fish were either presented with a computergenerated sine wave 15 Hz higher than the subject fish’s starting EOD frequency, called the C15-Hz stimulus, or a stimulus frequency 15 Hz below the subject fish’s starting EOD frequency, called the 15-Hz stimulus. Stimulus order was randomly determined, and the following week, fish received the other treatment. The sine wave was presented in five continuous 1-min loops with a 2-s fade in/ out amplitude envelope for each 1-min loop to avoid noise artefacts (clicks or pops) when the signal began or ended. We used paired t tests to compare the maximum frequency elevation reached by each fish (in relation to its own baseline) during each presentation, as well as the number of frequency elevations made (excluding chirps), and the smallest frequency difference reached between the playback signal and the fish’s EOD frequency.

TALLAROVIC & ZAKON: EOD JAMMING IN ELECTRIC FISH

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Results In the combined data set, both sexes made EOD rises and elevated their EOD frequencies in response to G15Hz stimuli, however, the overall magnitude of the elevations in hertz was significantly larger in response to the C15-Hz stimulus than in response to the 15-Hz stimulus (paired t test: t18 Z 2.59, P ! 0.05; Fig. 4a). This did not appear to be an attempt at a JAR, because EOD frequencies of fish were significantly closer in frequency to the stimulus signal during the C15-Hz stimulus presentation than

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Figure 2. Mean G SE difference in EOD frequencies of female brown ghost knifefish during staged encounters in a neutral arena when the baseline EOD frequencies of females differed by (a) less than 30 Hz and (b) more than 35 Hz. *P ! 0.05.

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Figure 4. Mean G SE (a) maximum EOD frequency elevation and (b) difference in baseline EOD frequency of brown ghost knifefish presented with playbacks in their home tanks of computer-generated sine waves either 15 Hz above or below their own EOD frequency. *P ! 0.05; **P ! 0.01.

during the 15-Hz stimulus presentation (t18 Z 4.16, P ! 0.01; Fig. 4b). Although fish made the largest frequency elevations in response to the C15-Hz stimulus, they did not make significantly more EOD rises towards it (t18 Z 1.64, P Z 0.118). When females and males were analysed separately, females made significantly larger EOD frequency elevations in response to the C15-Hz stimulus than in response to the 15-Hz stimulus, whereas males’ responses to the two stimuli did not differ significantly (paired t test: females: t9 Z 2.346, P ! 0.05; males: t8 Z 1.270, P Z 0.239). Although the EOD frequencies of both sexes were closer to C15-Hz stimulus than they were to the 15-Hz stimulus, only females’ EOD frequencies were significantly closer to C15-Hz stimulus during presentation of the signal (females: t9 Z 4.386, P ! 0.01; males: t8 Z 2.033, P Z 0.076).

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Figure 3. Mean G SE difference in EOD frequencies of male brown ghost knifefish during staged encounters in a neutral arena. *P ! 0.05.

Playbacks in Neutral Arena Methods Our observations of interactions between live fish in a neutral arena and of residents’ responses to computersimulated intruders suggested that elevation of baseline

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EOD frequencies and production of EOD rises both occur during aggressive encounters. To confirm this, we videotaped fish and recorded their EODs during aggressive encounters in which the fish were able to attack the playback electrodes. The neutral arena was used since it was not possible to videotape fish in their home tanks. We presented sine wave playback stimuli with no frequency elevations, and sine wave playback stimuli with simulated frequency elevations to examine whether these frequency elevations influenced a fish’s behaviour. Fish (N Z 14; 7 males, 7 females) were presented with a playback stimulus in a neutral arena. The simulated intruder playback device described above was used for presentation of the stimulus signals, but the mesh was removed so that the fish could enter the tube and bite the electrodes, providing a measure of aggression (Dunlap & Larkins-Ford 2003). The playback arena (100 ! 55 cm, water depth of 10 cm) contained a 100-W thermostat heater attached to one side of the tank that kept the water temperature between 26 and 27
C. Because the EOD frequency is sensitive to temperature, we placed a plastic mesh barrier that spanned the width of the arena at each end of the tank to create two compartments that contained air-driven, flow-through filters to circulate the water and maintain an even temperature gradient. The centre of the arena contained a mesh basket that could be lifted out of the water to release a fish into the rest of the arena. The playback device was placed at one end of the arena. To facilitate scoring the locations of fish, we visually divided the tank into three zones by placing black masking tape on the outside bottom of the tank to delineate (1) a near zone immediately surrounding the playback device (25 cm), (2) a neutral zone in the centre of the tank (50 cm), and (3) a far zone along the opposite end of the tank from the playback device (25 cm). A camera was positioned over the arena and digital video images were recorded at a rate of two frames/s. We placed a fish in the centre of the mesh basket and allowed it to acclimate for 5 min. After the acclimation period, a stimulus signal was presented once while the fish was still confined in the basket. Immediately after the first presentation, we lifted the basket and released the fish into the arena. The stimulus was then repeated 10 s after the first presentation. We noted the fish’s location in the tank during the stimulus and recorded the time that the fish spent in each zone. We positioned a second camera over the arena and counted the number of bites at the electrodes from the videotapes. We used four 60-s playback stimuli that differed in frequency from that of the subject’s baseline EOD: (1) a C15-Hz stimulus (see above); (2) a 15-Hz stimulus; (3) a C15-Hz stimulus with a 14-Hz elevation (i.e. elevation of 29 Hz above the test fish’s own EOD frequency): a C15Hz C elevation stimulus; and (4) a 15-Hz with a 14-Hz elevation (i.e. 1 Hz below the test fish’s own EOD frequency): 15-Hz C elevation stimulus. The stimuli containing the elevations were presented at G15 Hz for 5 s, with a 5-s transition time as it ramped up to C29 Hz, remained at C29 Hz for 40 s, ramped back down to G15 Hz for 5 s and then remained at G15 Hz for 5 s.

Each stimulus signal was generated for each fish using a Matlab program written especially for generation of artificial electric fish signals by JeeHyun Kim, University of Texas, Department of Electrical and Computer Engineering. Fish received randomly assigned treatments the first day of the experiments, and then were assigned the next three treatments such that no two fish were given the same presentation order of the four signals. Each fish was used in only one trial per day. Data collected included the time spent in each zone of the playback arena, how many times the fish entered the playback tube to investigate the electrodes, how many times the fish bit the electrodes, how many EOD rises the fish made (we did not count chirps), and the magnitude (in Hz) of the largest rise in EOD frequency. Because fish often made EOD rises in addition to a slower frequency elevation, we used the fish’s baseline EOD immediately before the stimulus presentation as the baseline when measuring the maximum rise in EOD frequency. We normalized the number of EOD rises and bites made by each fish (percentage of its total response), because some fish responded aggressively to the stimuli (readily approached all stimulus signals), whereas others showed only weak responses to all stimuli. We analysed the data using a MANCOVA to compare the response variables for each treatment stimulus, and included each fish’s baseline EOD frequency, body length and weight as independent covariables. We made two additional close-up digital video recordings of fish interacting with the playback electrodes during the presentation of a 15-Hz signal. These recordings contained an additional track of EOD data synchronized to the digital video to determine whether the fish was making a frequency elevation during a biting attack at the electrodes.

Results The locations of fish in the playback arena did not differ during presentation of any of the stimulus signals (i.e. fish did not affiliate with or avoid any signal more than any other). However, fish produced more EOD rises in response to both the 15-Hz signals, with and without the additional elevation (Fig. 5a). Fish entered the playback tube significantly more often (Fig. 5b) and made significantly more bites at the electrodes (Fig. 5c) only in response to the 15-Hz signal without the elevation. We found a positive correlation between the number of EOD rises made by fish and the number of biting attacks on the electrodes (Z test: correlation coefficient Z 0.88, Z Z 10.374, N Z 60, P ! 0.01) for all stimuli combined (Fig. 6). Video analysis of the two recordings with synchronized soundtrack and digital video revealed that fish elevated EOD frequency while biting. Although fish also elevated EOD frequency without attacking, we did not observe a fish biting when it was not also elevating its EOD frequency.

DISCUSSION The results of these experiments indicate that brown ghosts often elevate their EOD frequencies during social interactions within jamming range of another fish, rather

TALLAROVIC & ZAKON: EOD JAMMING IN ELECTRIC FISH

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Treatment Figure 5. Mean G SE number of (a) EOD rises, (b) entries into the playback tube and (c) bites directed at the playback electrodes in response to each of four computer-generated playback stimuli presented to individual brown ghost knifefish in a neutral arena. Number of entries into the playback tube and number of bites were normalized as the percentage of each fish’s total response. yP ! 0.056; *P ! 0.05; **P ! 0.01.

than performing a JAR. These frequency elevations occur during aggressive encounters and almost certainly play a role in communicating intent or threat.

Frequency Elevations and Aggression Brown ghost electric fish raised their EOD frequencies towards the EOD frequency of a higher-, and less often a lower-, frequency fish during agonistic encounters. In

addition, brown ghosts elevated their EOD frequencies to an EOD mimic sine wave stimulus in playback experiments in their home tank or a neutral arena. When they had access to the playback electrodes, they bit the electrodes, and these bite attacks were preceded by and were concurrent with EOD frequency elevations. Furthermore, when we added a synthetic EOD frequency elevation to a stimulus signal that otherwise provokes aggressive attacks (15-Hz), fish were less likely to attack it, suggesting that perceiving this signal may inhibit aggression. These observations suggest that EOD frequency elevations are aggressive signals. Males made frequency elevations to EOD frequencies up to 42 Hz higher than their own, whereas females only made elevations to EOD frequencies within 15 Hz of their own. This is consistent with observations of greater aggressive behaviour in males of this species. Both sexes acted most aggressively in terms of biting attacks towards the 15-Hz playback signal in the neutral arena study. Although they produced EOD rises at both the 15-Hz stimulus and the 15-Hz C elevation stimulus, they mainly attacked the 15-Hz and not the 15Hz C elevation stimulus. Two explanations for this difference seem equally likely. The frequency elevation itself could have been interpreted by the fish as threatening, causing them to be more cautious or less likely to attack. Alternatively, because the stimulus paradigm created a jamming situation, a fish may have chosen not to attack if its electrolocation abilities were compromised. In either case, the frequency elevation added to the 15-Hz signal seemed to reduce biting behaviour but not aggressive communication. An additional consideration is whether the fish is equally jammed by C15-Hz and 15-Hz signals. In theory, both signals should jam the fish equally by creating the same interference frequency. In a related species, the black ghost, A. albifrons, EOD frequency elevations are primarily made by the submissive member of a pair following establishment of dominance (Serrano-Ferna´ndez 2003). We do not believe that frequency elevations in brown ghosts indicate submissiveness,

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because brown ghosts in this study elevated EOD frequency (1) before and during bite attacks, the most aggressive behaviour made by electric fish, (2) in response to playback electrodes that were harmless to them, (3) in response to other males, which are more aggressive than females and (4) in response to a larger range of EOD frequencies than has been reported for black ghosts. Rather, we postulate that the behavioural significance of this display differs between brown and black ghosts. Black ghost males have lower EOD frequencies than females (Dunlap et al. 1998), the pattern observed in all the other wellstudied wave-type gymnotiforms except for brown ghosts (Zakon & Smith 2002). It is possible that as the reversed sexual dimorphism in EOD frequency evolved in brown ghosts, so did a reversal in the function of the frequency elevation.

What is the Function of Frequency Elevations? We consider three mutually exclusive hypotheses to explain why brown ghosts make frequency elevations during agonistic encounters: electrical puffing up, phase coupling and jamming. EOD frequency is strongly correlated with size, aggressiveness (as measured by the number of chirps produced in aggressive encounters) and blood androgen level in male brown ghosts (Dunlap 2002; Dunlap & Oliveri 2002). The relationship between EOD frequency and dominance status is less pronounced in females, although females with higher EOD frequencies tend to be larger and dominant (Dunlap & Oliveri 2002; Tallarovic & Zakon 2002). It is possible that fish with lower EOD frequencies raise their EOD frequencies during social encounters to appear more dominant, what we refer to as electrical puffing up by analogy with feather or hair erection, or elevations in posture that occur during aggressive interactions in many groups of animals. If this were the case, then during social encounters, we would expect fish with lower baseline EOD frequencies to increase their EOD frequencies beyond those of conspecifics, and fish with higher baseline EOD frequencies to raise theirs even higher. Since brown ghosts can raise their EOD frequency at least 30 Hz for many seconds or even minutes (Bastian et al. 2001; J. Oestreich, personal communication), the ability to raise EOD frequency higher than our stimulus signal is not a constraint. The electrical puffing up hypothesis predicts that the fish with the lower EOD frequency will try to surpass the EOD frequency of the other fish. Wave-type electric fish show a form of behaviour called phase coupling, where one fish actively moves its EOD frequency to lock onto the EOD frequency of a second fish. During phase coupling the EODs of the two fish are synchronized rather than moving into and out of phase, as occurs when the EOD frequencies are similar but not identical. This has been considered an alternative type of JAR since jamming is minimized when the two frequencies are completely in phase (Langner & Scheich 1978; Gottschalk & Scheich 1979). The phase-coupling hypothesis predicts that a fish with a lower EOD frequency will raise its EOD frequency to exactly match the EOD

frequency of the fish with the higher EOD frequency so that a cycle-by-cycle phase locking of both EODs occurs over tens of seconds. In our experiments, fish with low EOD frequencies raised their EOD frequencies up to or usually just below the frequency of the higher-frequency fish, and the higher-frequency fish subsequently showed only a slight increase in EOD frequency. Furthermore, in playback experiments with a constant frequency sine wave that was 15 Hz above the subject’s EOD frequency, responses of EOD elevations rarely surpassed the stimulus in frequency (Fig. 7a). This suggests that fish were not electrically puffing up. In contrast, we lack direct evidence to test whether phase coupling occurs during frequency elevations in brown ghosts because we did not measure the instantaneous phase relationship between the two signals. In most cases, however, the fish with the lower EOD frequency raised its EOD frequency to within a few hertz of the higher-frequency fish but seldom matched the EOD frequency of its partner. Maximum jamming occurs when EOD frequencies are within 4 Hz (Heiligenberg 1973; Bastian 1987a; Dye 1987). Inspection of the amplitude recordings of fish interacting with a playback electrode show large modulations of EOD amplitude due to the beating of the fish’s own EOD frequency and the sine wave stimulus (Fig. 7b). This beating of the amplitude envelope is a result of the interference caused by two waveforms overlapping imperfectly. This supports the jamming hypothesis and would seem to indicate that jamming behaviour is intentional. If so, this may represent the first documented description of a bioelectrical signal used for jamming in a navigation system.

EOD Rises Signal Aggression in Brown Ghosts Elevations in EOD frequency of long duration were overlain with upward modulations in EOD frequency of shorter duration, called EOD rises, which lasted hundreds of milliseconds to seconds (Fig. 8). In a previous study we distinguished three classes of EOD rises using cluster analysis: short, medium and long rises (Tallarovic & Zakon 2002). In the present study we did not attempt to differentiate among the different types of rises, mainly because the elevation of baseline frequency makes it very difficult to determine the duration, a key factor in identifying each type. We noted that fish made EOD rises more often in response to playback frequencies that were lower than their own EOD frequencies in both the resident–intruder and neutral arena studies, and this is consistent with our previous work in which fish in separate tanks were allowed to interact electrically by using pairs of wires in their respective tanks (Tallarovic & Zakon 2002). In that study we found that females, in particular, made short rises most often to neighbouring females whose EOD frequencies were lower than their own. Individuals with lower EOD frequencies are likely to be perceived by both males and females as more subordinate. Therefore, we suggest that EOD rises are associated with aggression, and that the slower frequency elevation may

TALLAROVIC & ZAKON: EOD JAMMING IN ELECTRIC FISH

Frequency (HZ)

(a) Generated sine wave

930

910

Fish’s EOD 890 2:34

2:38

2:42

2:46

2:50

2:54

2:50

2:54

Amplitude (arbitrary units)

Time elapsed (mm:ss)

400

(b)

200 0 –200 –400 2:34

2:38

2:42

2:46

Time elapsed (mm:ss) Figure 7. Recording of a fish interacting with a playback signal. (a) A spectrogram showing the computer-generated sine wave and the fish’s EOD frequency. (b) An oscillogram of the same recording above, showing beat patterns in the amplitude envelope as a result of interference.

also be associated with aggression. Studies of another gymnotiform electric fish, Eigenmannia virescens, suggest that long rises function as a submissive signal, whereas short rises may convey aggression (Hopkins 1974; Hagedorn & Heiligenberg 1985). A recent study in a closely related species, Apteronotus albifrons, also indicates that gradual frequency rises are submissive signals (SerranoFerna´ndez 2003). None of the behaviours observed in this study appear to function as a submissive signal, but

further studies are necessary to understand whether additional information is conveyed by different types of EOD rises.

Is This a Novel Behaviour? Despite numerous observations of aggressive interactions in this species (Hagedorn & Heiligenberg 1985;

Frequency (Hz)

750 Fish enters tube

Fish backs out of tube Biting electrodes

730

710 EOD of female

690

Generated sine wave

3:35

3:45

3:55

4:05

4:15

4:25

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Time elapsed (mm:ss) Figure 8. Spectrogram of a female brown ghost knifefish interacting with a playback signal. When the female entered the playback tube and began biting the electrodes, she elevated her EOD frequency and produced rises as she attacked the electrodes.

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Dunlap 2002; Dunlap et al. 2002; Dunlap & Larkins-Ford 2003), EOD elevations in response to an opponent fish’s EOD frequency during aggressive interactions has not been reported before because most studies of social communication in brown ghosts have focused on the intense bouts of chirping behaviour made by male Apteronotus during agonistic interactions. Slow, relatively low-frequency (15–20 Hz) EOD elevations are less readily observable than high-frequency (up to 150 Hz), rapid (20–30 ms) elevations that characterize chirps. On the other hand, a behaviour similar to what we report here has been described in the studies using a chirp chamber. In these experiments fish are placed in long plastic tubes with electrodes at either end to detect their EODs and electrodes that are perpendicular to the long axis of the tube to provide EOD mimic stimuli. While this design has a number of experimental advantages, the fish is restrained in a stressful situation and presented with a stimulus playback from which it cannot escape. Under these circumstances brown ghosts make a so-called nonselective response (NSR), which is an elevation of EOD frequency to an EOD mimic higher in frequency than their own (Dye 1987; Bastian et al. 2001); the function of the NSR is unknown. Since the NSR has only been studied in a chirp chamber it is not surprising that the social function of the NSR is unknown. It is possible that NSRs are the same behaviour that we describe here. Interestingly, males show NSR in response to stimuli of higher frequency than do females (Bastian et al. 2001). In this regard EOD frequency elevations and NSR are similar. The magnitude of the response in a chirp chamber is less than that of playback stimuli. One explanation for this difference is that the artificial geometry of the EOD mimic in a chirp chamber is a poorer stimulus than the more natural geometry of a playback electrode in this study and therefore elicits a response of lower intensity. In addition to observing frequency elevations in interacting pairs of fish, and in playback experiments, these frequency-jamming interactions have been observed between females in established social tanks even 3 months after the fish were introduced (Tallarovic et al. 2002). Therefore, we have no reason to suspect that this behaviour is an artefact of social isolation or a novelty response to a new fish. Kramer (1987) proposed that the JAR may be socially mediated in a related species of wave-type electric fish, Eigenmannia virescens. He observed in this species that not all fish perform a JAR when presented with a jamming stimulus. He found that adult males gave poor or no responses; females performed a JAR if the stimulus frequency was higher than their own EOD frequency, but showed little or no response if the stimulus frequency was lower than their own; and juveniles gave the strongest response overall.

Examples of Jamming in Nature Examples of intentional jamming behaviour in social communication in nature are rare and usually confined to male–male interference behaviours in the context of

mating competition. Male Smilisca frogs (Ryan 1986) and male Magicada cicadas (Cooley & Marshall 2001) both alter their call rate so as to interfere with a rival calling male. In Apteronotus, the jamming occurs in a dual purpose communication system that potentially jams navigation systems. Apteronotus, like other wave-type electric fish, modulate their EOD for electrolocation and modulate it briefly for social communication, thus the effect of one fish raising its EOD frequency into the frequency range of a nearby fish may simultaneously jam the rival’s electrolocation abilities and serve as a communication signal. Jamming avoidance behaviours are not only well known in electric fish systems, but have also been studied in other animals with navigation systems such as bats. Bats can either alter their intercall intervals (Obrist 1995), or change the frequency of their echolocation calls to avoid being jammed (Habersetzer 1981; Surlykke & Moss 2000; Ulanovsky et al. 2004) very much like wave-type electric fish. Intentional jamming in bats has not been reported. Possible jamming behaviour may exist in one other electric fish species, Pollimyrus isidori (Kramer 1978): males of this pulse-type species of the family Mormyridae (Order Mormyriformes) display a shift in EOD pulse rate called the preferred latency response (PRL). Unlike other Mormyrids, however, the PRL in P. isidori appears to actually increase the probability of overlap between two nearby ¨ cker & Kramer 1981); thus, in males (Kramer 1978; Lu this species, PRL does not appear to be a form of jamming avoidance. In addition, because female P. isidori display a different behaviour than males called preferred latency ¨ cker & Kramer (1981) proposed that avoidance (PLA), Lu PLA and PLR function in sexual recognition rather than representing jamming avoidance.

Conclusions Our study is the first to show active jamming behaviour in brown ghosts in response to both conspecific rivals and to sine wave playbacks that mimic another fish’s EOD frequency. By allowing fish to interact under more natural circumstances, and allowing them to swim freely in a tank during signal presentations, we have gained more insight into their social behaviours. These findings underscore the importance of studying behaviour in naturalistic situations and using relevant and controllable social stimuli. Future studies will examine the neural basis of these behaviours and the hormonal mechanisms that underlie aggression (particularly in females) in this species. Acknowledgments We thank F. Triefenbach and D. Lim for assistance in the laboratory. We also thank F. Triefenbach, P. Narins, J. Oestreich and R. Steinberg for helpful discussions and encouragement on this project. Funding for this study was provided by National Institute of Child Health and Human Development in the form of a National Research Service Award Postdoctoral Fellowship HD08647-03 to

TALLAROVIC & ZAKON: EOD JAMMING IN ELECTRIC FISH

S. K. Tallarovic, and National Institute of Mental Health grant MH56535 to H. H. Zakon. The experiments reported comply with the Principles of animal care (publication No. 86-23, revised 1985) of the National Institutes of Health and also with current laws of the U.S.A. References Bastian, J. 1987a. Electrolocation in the presence of jamming signals: behaviour. Journal of Comparative Physiology A, 161, 811– 824. Bastian, J. 1987b. Electrolocation in the presence of jamming signals: electroreceptor physiology. Journal of Comparative Physiology A, 161, 825–836. Bastian, J., Schniederjan, S. & Nguyenkim, J. 2001. Arginine vasotocin modulates a sexually dimorphic communication behaviour in the weakly electric fish Apteronotus leptorhynchus. Journal of Experimental Biology, 204, 1909–1923. Bullock, T. H. 1970. Reliability of neurons. Journal of General Physiology, 55, 565–584. Bullock, T. H., Hamstra, R. H., Jr & Scheich, H. 1972. The jamming avoidance response of high frequency electric fish. I. General features. Journal of Comparative Physiology, 77, 1–22. Cooley, J. R. & Marshall, D. C. 2001. Sexual signaling in periodical cicadas, Magicicada spp. Behaviour, 138, 827–855. Dulka, J. & Maler, L. 1994. Testerone modulates female chirping behaviour in the weakly electric fish (Apteronotus leptorhynchus). Journal of Comparative Physiology A, 174, 331–343. Dunlap, K. D. 2002. Hormonal and body size correlates of electrocommunication behaviour during dyadic interactions in a weakly electric fish, Apteronotus leptorhynchus. Hormones and Behaviour, 41, 187–194. Dunlap, K. D. & Larkins-Ford, J. 2003. Production of aggressive electrocommunication signals to progressively realistic social stimuli in male Apteronotus leptorhynchus. Ethology, 109, 243–258. Dunlap, K. D. & Oliveri, L. M. 2002. Retreat site selection and social organization in captive electric fish, Apteronotus leptorhynchus. Journal of Comparative Physiology A, 188, 469–477. Dunlap, K. D., Thomas, P. & Zakon, H. H. 1998. Diversity of sexual dimorphism in electrocommunication signals and its androgen regulation in a genus of electric fish, Apteronotus. Journal of Comparative Physiology A, 183, 77–86. Dunlap, K. D., Pesczar, P. L. & Knapp, R. 2002. Social interactions and cortisol treatment increase the production of aggressive electrocommunication signals in male electric fish, Apteronotus leptorhynchus. Hormones and Behaviour, 42, 97–108. Dye, J. C. 1987. Dynamics and behavioural contexts distinguishing modes of pacemaker modulations in the weakly electric fish Apteronotus. Journal of Comparative Physiology A, 161, 175–185. Engler, G. & Zupanc, G. K. H. 2001. Differential production of chirping behaviour evoked by electrical stimulation of the weakly electric fish, Apteronotus leptorhynchus. Journal of Comparative Physiology A, 187, 747–756. Engler, G., Fogarty, C. M., Banks, J. R. & Zupanc, G. K. H. 2000. Spontaneous modulations of the electric organ discharge in the weakly electric fish, Apteronotus leptorhynchus. Journal of Comparative Physiology A, 186, 645–660. Gottschalk, B. & Scheich, H. 1979. Phase sensitivity and phase coupling: common mechanisms for communication behaviours in gymnotid wave and pulse species. Behavioral Ecology and Sociobiology, 4, 395–408. Habersetzer, J. 1981. Adaptive echolocation sounds in the bat Rhinopoma hardwickei. Journal of Comparative Physiology A, 144, 559– 566.

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