Figures and data
Chirp type categorization based on FM parameters.
A: Schematic diagram of the recording setup: holding aquariums (N = 8) were divided by a plastic mesh 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 for clustering chirps based on these two parameters (cluster centroids are indicated with black crosses). 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 (specially 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 (f. 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”). Note how “tank experience”, “context” and “DF” stand out of the variable group. 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.
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 (1 = sender, 2 = receiver). On the right side of each matrix, chirp totals are displayed in boxplots for sender and receiver fish (outliers are represented as dots laying beyond the boxplot whiskers; red bars = 100) and cross-correlation indexes (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 level of cross-correlation. 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.
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).
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. 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.
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.
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*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*type 1 clutter p = 0.034).
Chirp categories
Playback chirp parameters.
Chirp categorization used for chirp detection
