Chirp type categorization based on FM parameters.

A: Schematic diagram of the recording setup: holding aquariums (N = 8) were divided by a plastic mesh divider to prevent physical contact between the interacting fish (N = 2 per aquarium) while allowing electric interactions. Brown ghost EODs were detected by electrodes placed in each tank compartment (2 input channels per tank), amplified, digitized (20 kHz sampling rate) and recorded using custom-written MATLAB scripts. B: Electric signals and chirps were analyzed using their fast-fourier transforms (FFT, 1) and assigned to different fish based on the signal intensity (2). Frequency modulations were detected using a MATLAB-based heuristic method for automatic peak detection (APD, 3). False-positives, false-negatives or wrongly assigned chirps were revised manually (4) and chirp FM and duration were then measured (5). C: Chirp distribution by frequency modulation (Hz) and duration (ms). K-means chirp clustering indicated an optimal value of 2-3 for clustering chirps based on these two parameters (cluster centroids are indicated with black crosses; silhouette values are shown in the bottom right panel). The red lines indicate the cut-off values used to classify the different chirp types (type 1 = 1, type 2 = 2, type 3 = 3, rises = 4): duration 50 ms and modulation 105 Hz. These values are based on a dataset acquired at the beginning of the study (red, in the scatter plot; N chirps = 11342; N chirping fish = 16, N fish pairs = 8) and on the distribution density (D) of the whole chirp population (gray, in the scatter plot; N chirps = 30486; N chirping fish = 130, N fish pairs = 78). E: Representative examples of the 4 different chirp categories (voltage data on the first row, instantaneous frequency on the second and spectrogram on the third). F: Scatter plot showing the distribution of different chirp types by DF (frequency difference between sender and receiver fish): note the gradual change in chirp type composition (color coded) at different DF values (especially visible for type 1-3). Due to the sex difference in the brown ghost EOD frequency, negative DF values correspond mainly to females, positive values to males.

Factor analysis of mixed data (FAMD) – social contexts and tank experience.

A: Scatter plot showing the contribution of all chirp-related variables to the overall variance of the whole chirp dataset: among these, EOD parameters such as amplitude (EODamp), frequency (EODs or EODr, based on sender or receiver identity), spectral power density (pow) were considered together with variables related to chirps, such as frequency modulation (freq. modulation), duration, sex of sender or receiver fish (sex_s, sex_r), the time of occurrence within a 1 hour trial (timestamp), the type (1 to 4), the DF. Variables related to the fish experience with either the tank environment (tank experience: resident = 1-week tank experience, intruder = new to the tank, equal = both new) and experience with the paired conspecific (context). This latter category refers to the reciprocal experience of each fish pair (novel or experienced), their hierarchical status (dominant or subordinate), the type of interaction (divided = behind a plastic mesh barrier, free = freely swimming) and the simulated breeding season (based on water conductivity levels: high conductivity = ca. 400 μS, no breeding; low conductivity = ca. 100 μS, breeding) at which the interaction takes place (see methods for details). Triangles indicate the coordinates of the variable centroids, their contribution (“contrib”) is coded by color intensity, whereas the quality of their representation on the transformed coordinates is coded by color hue (“cos2”). B: Estimates of the total variance explained indicate that tank experience, together with DF and context, are the most important factors explaining chirp variance. C: Representation of chirps in the transformed coordinates. The clustering is based on qualitative coordinates (tank experience, context and chirp type). Cluster distance represents the correlation among variables. The marginal plots show the kernel distribution of the chirp population color-coded according to chirp type (legend on the bottom right). Labeling chirps by DF shows how chirps can be meaningfully clustered based on this parameter (inset, top right).

Invariant chirping responses to playback chirps in freely swimming fish.

A: Schematic diagram of the setup used for the playback experiments: both the fish EOD and the fish swimming behavior are recorded during 60 s long playback trials. During each trial the fish locomotion is scored based on the % coverage of tank space (60 x 30 cm) in 4 regions of interest (ROI) at increasing distance from the playback electrodes (1 = close, 2-3 = intermediate, 4 = far). Playback trials are organized in 4 different modes (0-3), each including 15 DF levels, all shuffled and randomized. B: The box plots on the top row show the total number of chirps produced by either male or female brown ghosts produced in response to plain EOD mimics (mode 0), sinewaves containing type 3 chirps (red, mode 1), sine waves containing trains of type 2 chirps (blue, mode 2) or rises (grey, mode 3). Chirp counts relative to each individual fish are summed across different DFs. The boxplots on the bottom row show the trial scores relative to the same subjects and summed across different DFs. C: Heatmaps showing the DF-dependent but mode-invariant distribution of mean chirp types produced by male and female subjects in response to different playback regimes. D: Score heatmaps showing that fish of both sexes approach playback sources (i.e. higher scores in ROI 1) with equal probability regardless of playback types or DFs. Female fish may be more stationary in proximity of the electrodes, resulting in slightly higher ROI-1 scores.

Responses to EOD frequency ramps.

A-C) Examples of spectrograms from playback trials showing the responses to frequency ramps (increasing from −300 Hz to 300 Hz or decreasing within the same range) of different durations: 20, 60 and 180 sec respectively. D-F) Chirp raster plots relative to the three trial types. Chirp responses are grouped based on fish identity (unspaced rows represent responses by the same fish). Playback time is represented by the gray areas. Some fish produced chirps even in absence of a playback EOD. G-I) Histograms showing chirp type distributions by DF. Trials in which decreasing ramps were used were adjusted by flipping the time array to match the DF values. The pie plots show the relative amounts of the four types of chirp.

Chirp time-series correlations during social interactions.

A,B: Chirp transition maps representing the median transition probability (normalized) of all possible chirp type pairs calculated for female and male fish, considered together. For each type of social pairing, the identity of the two fish is indicated by different ID numbers in the different map quadrants: 1-1 = fish 1 to fish 1, 1-2 = fish 1 to fish 2, 2-1 = fish 2 to fish 1, 2-2 = fish 2 to fish 2. In mixed pairs, the order given in the sex tag of each plot follows the same order of the fish ID. The presence of higher chirp-transition frequencies in the 2nd and 4th quadrants of the matrices (labeled with 1-1 and 2-2) indicates a substantial independence between chirp time-series (i.e. lack of temporal correlation). Transitions from female to male fish (A) or male-to-female fish (B) are considered separately. For each case, chirps were selected based on the sex of the sender fish. The 1-2 numbering refers to fish identity. On the right side of each matrix, chirp totals are displayed in boxplots for sender and receiver fish (outliers are represented as dots lying beyond the boxplot whiskers; red bars = 100) and cross-correlation indices (cci, 50 ms binning) are provided for chirp time series relative to the same fish pairs (red dotted lines = confidence intervals corresponding to 3 cci standard deviations). C,D: Chirp transitions relative to same sex pairings. A higher level of interaction for F-F pairs (visible in the first and third quadrants in C) is probably due to the extremely low chirp rates in these pairs. Notably, higher chirp rates (as in M-M pairs, D,E) do not result in higher cross-correlation levels. E,F: Chirp transitions for resident-intruder pairs (M-M and F-F). Since most chirps are produced by M-M resident-intruder pairs in divided aquariums, plots in E resemble those in D, as they are relative to overlapping datasets. G,H: Chirp transitions for dominant-subordinate pairs (M-M and F-F). I,J: Chirp transitions for freely swimming (naive, I or experienced, J) opposite-sex pairs. Note the reversed sexual dimorphism in chirp rates, in both cases.

Chirping is mainly correlated with locomotion and active sensing.

A: Peri-stimulus time histograms (PSTH) centered around different chirp types (window = 4 sec) indicating that chirps are reliably emitted during locomotion-related behaviors (“knife”, “loc”, and “tail1”) but also during resting states (“rest”). Chirps do not seem to trigger any defensive or aggressive behavior directly (i.e. no evident left/right bias in the PSTH). A more detailed description of each behavior is provided in the methods section (see also panel C). B: Transition matrix showing the total number of behavioral transitions (data pooled from all pairs). Note that all behaviors are considered both individually as well as together with those co-occurring (example: knife-type2 indicates a type 2 chirp produced during backward swimming). The color threshold has been lowered to visualize the most significant transitions. The marginal bar plots represent the sum of the number of transitions along each axis. C: To extract meaningful behavioral correlations, a text-mining algorithm (see methods) was used to perform a co-occurrence analysis on the whole set of annotations in chronological order (data pooled from 12 fish pairs). In the graph, words closely associated with each other are linked with darker lines (according to a 0-1 coefficient scale). This analysis emphasizes the presence of modularity in a group of strings (i.e. chirps and behaviors), which represents the degree of partitioning of the whole word dataset (N = 5). Each cluster delineates strings sharing similar co-occurrence patterns (see color code). D: The multi-dimensional scaling of the same word-database (2D in this case) shows behavioral combinations having similar patterns of occurrence, regardless of their co-occurrence with other behavioral instances. For this representation, each word is considered individually and the color code represents the clustering of objects based on their proximity (method: Kruskal, distance: Jaccard, N of clusters = 8).

Chirps enhance the contrast of electric images in the sender but not in the receiver fish.

A: Heatmaps representing the electric field (V/cm) generated by a sender fish alone (left) and by two interacting fish (right, fish length = 15 cm, distance = 10 cm). The electric field lines induced by the sender fish’s own EOD (gray) and that of the receiver conspecific (white) are shown in black. B: Normalized heatmaps superimposed on the sender fish contour representing the boundary element model (BEM) simulation of the electric image as a result of the field interactions measured on the sender fish skin. The electric images are calculated by subtracting the transcutaneous current in the presence and absence of the receiver fish. C: Sum of the electric images modeled on the skin of the sender fish and generated by different chirp types produced by the same subject (N = 297 chirps). These effects are compared to the respective beats without chirps (beat range ca. 40-300 Hz). D: Net effect of chirps on electric images represented as absolute current changes induced by chirps and compared to their carrier beats alone. Data for sender (top) and receiver fish (bottom) are displayed separately. Note that chirps do not elicit significant changes in the electric images calculated for the receiver fish.

Chirping during novel environment exploration.

A: Diagram of the recording arena showing the criteria used to define the different regions of interest (ROIs): 1) the presence of shelters (PVC tubes), 2) the proximity to the tank walls (distance < 5 cm), 3) the presence of a fish (caged in a mesh tube), 4) the presence of an “unknown/novel” conductive object (a 3 cm piece of graphite). B: Proportion of time spent in the different ROIs (N = 14 females, Friedman X2 22,9 p < 0.001 wall vs open p < 0.001, social vs object p = 0.02, open vs shelter/social/object < 0.01; N = 15 males, Friedman X2 19,8 p < 0.001 wall vs open p = 0.049, wall vs shelter/object p < 0.05, open vs shelter/social/object p < 0.001). C: Chirp locations (red) overlaid to the heatmaps showing the average swimming activity of females and males (for the specific locations of different types of chirps see Figure S10). D: Polar histograms showing the angles between the two fish during chirping. Angles are referred to the X-axis and are sorted based on chirp type and sex (male = blue, female = red). E: Histograms of the chirping distances in males (blue) and females (red) relative to different types of chirps.

Effect of environmental clutter on interacting fish pairs.

A: Recording of fish pairs (N = 6) in environments of different sensory complexity: lights ON = clear tank environment and direct illumination, lights OFF = clear tank environment, no illumination, lights OFF + clutter = no illumination and cluttered environment. B: Total amount of chirps produced in each condition and normalized on the lights ON session (green bars; Friedman’s p = 0.053; lights ON vs lights OFF + clutter = 0.024, lights OFF vs lights OFF + clutter = 0.051). Chirp counts relative to trials sorted in chronological order (1-3) are shown in gray. C: The box plots show the normalized chirp type counts relative to each session (lights ON, Friedman’s X2 = 21.9 p < 0.001, pairwise comparisons 1 vs 2 p = 0.006, type 1 vs 3, type 1 vs 4, type 2 vs 3, type 2 vs 4 p < 0.001; lights OFF Friedman’s X2 = 21.8 p < 0.001 pairwise comparisons type 1 vs 2, type 1 vs 3, type 1 vs 4, type 2 vs 3, type 2 vs 4 p < 0.001; clutter Friedman’s X2 = 19.5 p < 0.001 pairwise comparisons type 1 vs 2, type 1 vs 4, type 2 vs 3, type 2 vs 4 p < 0.001). D: Results of playback experiments in which EOD mimics were either directly detectable through a fine mesh barrier (clear) or more indirectly due to a barrier of plastic plants interposed between the mesh and the EOD source (cluttered). Clear and cluttered trials were presented in random succession (N fish = 6, 10 trials each, 60 sec ITI). E: Total chirp counts in the 2 conditions are normalized on the total amount of chirps produced by each subject (Wilcoxon, p = 0.025). F: Boxplots showing the chirp type composition of each condition (clear Friedman’s X2 = 17.4 p < 0.001 pairwise comparisons type 1 vs 3 p = 0.048, type 1 vs 4, type 2 vs 3, type 2 vs 4 p < 0.001; cluttered Friedman’s X2 = 29.9 p < 0.001 pairwise comparisons type 1 vs 3, type 1 vs 4, type 2 vs 3, type 2 vs 4 p < 0.001; Wilcoxon type 1 clear vs type 1 clutter p = 0.034). Tank sizes: A-C: 30 x 80 cm; D-F: 160 x 50 cm.

Chirp categories

Playback chirp parameters.

Chirp categorization used for chirp detection

Chirping in different social contexts and different tank experience conditions.

A: Total chirp counts obtained from resident-intruder assays. Resident fish were housed for 1 week in the test-aquarium while intruders were introduced only at the moment of testing (males, Mann-Whitney U = 0.032, females U < 0.001). B: Total chirp counts recorded during dominant-subordinate interactions (males, Mann-Whitney U = 0.015). C: Total chirp counts obtained from dyadic interactions in which both fish were novel to the test environment. D: Chirp counts evaluated during first time pairing (novel) and after 1 week of pairing (exp, experienced; pooled data, Mann-Whitney U = 0.016; males, Mann-Whitney U = 0.002). E: Chirp counts obtained from opposite sex pairs at the beginning (novel) and at the end of a 4 week-long water conductivity decrease protocol (used to simulate the reproductive season; novel, Mann-Whitney U = 0.034). Fish pairs were interacting only electrically, across a plastic mesh barrier (divided). F: Chirp counts relative to female-male interactions in absence of any mesh barrier (freely interacting). Chirps produced by opposite-sex pairs at the end of the water conductivity changes (experienced) are compared with chirp rates of female-male pairs recorded in absence of prior experience (novel). All chirp counts refer to 1 hour-long recordings except those of dominant-subordinate pairings, which lasted 30 minutes; see methods for details on the behavioral experiments.

Effect of social context and environmental experience on chirping behavior.

A: Histograms showing the normalized chirp type distribution relative to female senders and receivers of both sexes (F>F or F>M) in different behavioral contexts. Note the almost identical relative abundance (normalized) of different chirp types. B: Chirp type transitions displayed by female chirpers during different kinds of encounters. In all cases the most common sequence is “type 2-type 2” (the numbers on the X-axis represent the 4 different types, see Figure 1). C: Histograms showing the normalized chirp type distribution for male chirpers. The only difference is observed during male-female interactions in conditions of equal tank experience (“equal”). D: Occurrence of chirp transitions in males is similar to those observed in females with the exception of male-female pairs with equal tank experience or longer-term experience with each other. This is likely due to the lower amount of type 2 chirps produced at lower DFs which in turn results in a relatively higher number of larger chirps (chirp type dependency on DF can be better visualized in Figure 1F and Figure 5C,D).

Sex comparison of fish responses to playback chirps by DF.

A: Heatmaps of responses to different playback signals sorted by DF (X-axis) and type of playback chirps (Y-axis, sine = no chirps, type 1, type 2 and rises). The chirps produced by female and male fish are sorted by type and shown in 4 different maps. Chirp counts are normalized on the max value per type. For a description of playback chirps see methods.

Playback chirps temporarily suppress chirping in freely swimming fish.

A-D: Peristimulus time-histograms (PSTH) of chirps produced by 16 fish (8 females and 8 males) during playback experiments. Fish responses to different DFs (±240 Hz, ±160 Hz, ±80 Hz, ±40 Hz, ±20 Hz, ±10 Hz, ±5 Hz, 0 Hz) are pooled together. Results for different types of chirps are displayed in different columns while each row is related to chirps produced in response to a given playback mode (mode 0 = plain sinewave, mode 1 = type 1 chirps at 0.2 Hz, mode 2 = type 2 chirps at 3 Hz, mode 3 = rises at 0.2 Hz; see methods for details). The PSTHs show that the main effect of playback chirps (black dots) on the chirps produced by the fish is a brief temporal suppression (accentuated when chirps are repeated in trains, C) but no sign of any significant temporal correlation, except for a transient suppression.

Chirp responses to playback EODs containing chirps.

A: Average number of chirps of different types (normalized) produced over the course of 1-minute long playback trials (mode 0, sinewave frequency range −240 Hz to +240 Hz). The timing of playback stimuli is represented by the thicker line on the X axis. Vertical ticks on the same axis represent playback chirps. B: Responses to the same set of sine wave EODs to which type 1 chirps were added (mode 1). C: Responses to EODs containing 3Hz trains of type 2 chirps (mode 2). Note the stronger inhibition of fish chirping exerted by trains of type 2 chirps. D: Responses to EODs containing playback rises (mode 3). See method section for details on the playback experiments.

Linear correlation of chirp counts and playback duration (playback frequency ramps).

A: Correlation coefficient of the mean chirp counts (normalized) recorded as a response to playback frequency ramps. B: Line plots related to the individual subjects. The outlier fish emitted 1 chirp during the 60 sec trial and no chirps in the other 2 trials. Fish never producing any chirp were excluded (N=2).

Frequency ramp playback experiments, inter-chirp intervals (ICI) of different chirp types.

A-D) The inter-chirp latencies following each type of chirp are reported for each of the 3 different frequency ramps (see values on the top). These represent the time passing after a given chirp, before a chirp of any given type would follow. Low ICI values are found more often around the DF= 0 Hz (more markedly for type 2 chirps). Low values are also found at trial onset, due to the often higher chirp rates observed at the beginning of a playback trial (see type 1 and type 3 chirps in Figure S5 for instance).

Chirp interference with beat regularity.

A: Artificial chirps were generated on an 862 Hz EOD baseline covering a 0-400 ms duration range and a 0-400 Hz peak amplitude. An additional EOD signal was added to the chirping signal (−300-300 Hz DF range) to simulate a wide range of beat frequencies. The sign of the DF is referred to the baseline reference EOD. The beat interference induced by chirps was calculated as the ratio of the cumulative duration of beat interpeak intervals (IPI) affected by a chirp (i.e. outliers in the IPI population relative to each EOD pair) on the total cumulative beat IPI duration (including outliers) within a 700 ms time window (see methods for details). B: Duration and frequency modulation (FM) histograms of recorded chirps (red, N = 30486) sorted by DF and matched to their estimated beat interference (blue). The gray histograms on the Y-axis represent the beat frequencies sampled. C: Normalized heatmaps showing examples of chirp-induced beat interferences (color coded in blue) calculated at different DF values. Real chirps produced at the same DF are overlaid in red. Only overlapping FM and duration ranges are shown. In the plots on the extreme right: beat interference values are summed over all DFs (top) and are shown next to the corresponding chirps (bottom). D: Comparison of average chirp-induced beat interferences sorted by type (left map, blue) and the actual observed chirps (log N, right map in red).

Beat interference calculated using different time-windows.

The effect of the size of the analysis window on the beat interference estimates for the different types of chirps can be evaluated by comparing the heatmaps above. Since the estimate depends on the number of outliers among the beat cycles included within a fixed time interval, the interference value will decrease/change depending on the number of beat cycles considered (i.e. the size of the analysis window): a small window size will have more outliers for fewer beat cycles but a single chirp will affect a larger number of cycles (F). When a larger analysis window is used (A-C), the effect at low DFs is diluted as fewer cycles are affected overall. Other factors potentially affecting the interference estimate include: the algorithm used to detect outliers (here, the outliers are detected in the upper and lower quartiles in the distribution of peak durations) and the phase at which the chirp occurs (here the average of 4 phases is used).

Chirp type locations during novel environment explorations.

A: Sender fish locations during chirps of different types. Most chirps are produced while brown ghosts are swimming in close proximity to a caged conspecific (see Figure 8). This is particularly evident for freely swimming “sender” fish. Chirps produced by caged fish (“receiver”) are more widely distributed. Rises are produced when fish are perpendicularly oriented, along the wall (right) or half hidden behind shelters or plastic barriers. B: Sender fish locations during chirps produced by the caged conspecific (receiver). Receiver chirps are produced at similar locations, and in similar percentage (C).

Factor analysis of mixed data (FAMD) – novel environment exploration.

A: The scatter plot represents the contribution of chirp-related variables to the 2 main components of the transformed space. The contribution of each variable is indicated by both the position and the color of the corresponding marker on the plot. The variables closer to the origin contribute less to the overall sample variance. Variable contribution (contrib) is coded by color intensity and the quality of the representation by color hue (cos2). B: Bar plot showing the total variance (i.e. sum of each variable loading on all the 3 dimensions) explained by each variable in the transformed space. C: Representation of all 7894 chirps in the transformed coordinates. Triangles indicate the coordinates of the qualitative variable centroids (Fs = female sender, Fr = female receiver, Ms = male sender, Mr = male receiver). The contribution of individual chirps is color coded as in B. The clustering is based on both quantitative and qualitative coordinates (fish ID, sex sender, sex receiver, chirp type, average swimming speed, distance and angle between fishes, time spent in the tank ROIs, EOD frequencies of the interacting fish and DF, estimated beat interference and maximum interference possible). The marginal histograms show the kernel density distributions of different chirp types. In the insets, chirps are color-coded according to DF, distance and interference.

Chirps interfere with the beat within the electric field range.

A: Electric field generated by a 16 cm 3D dipole modeling an electric fish of the same size using BEM (boundary element methods). Iso-potential lines are shown for the near-field range in different colors, based on field polarity. Current is represented by the dashed lines, perpendicular to them. B: Electric field intensity mapped around the same modeled fish. The level lines represent the 1% and 5% intensity of the electric field generated by the ideal fish. C: Scatter plot of chirp locations. The overlay is centered at the origin and corresponds to 90% and 85% of all chirps produced, respectively. D: Plot showing the intensity range of an EOD mimic calculated at 22-25 °C and 200 μS (red) and the distribution range of chirps emitted by real fish (blue), for comparison. E-H: 3D plots showing the ideal beats calculated for different sinewave pairs during different chirp types and plotted over distance. The 2D plots on the side represent the beat interference (calculated using a threshold of 1% of maximum beat amplitude) caused by each chirp type over distance. Scale bars in the spectrograms are 100 ms and 100 Hz.