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 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 (i.e. the cumulative sum of the percentage of time spent in each ROI) and summed across different DFs and modes. 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 alter estimates of transcutaneous voltage in sender and 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 EOD (gray silhouette) and that of the receiver conspecific (white silhouette) are shown in black. B: Normalized heatmaps superimposed on the sender fish’s contour at the same instant as in A, representing the boundary element model (BEM, see methods) simulation of the electric image resulting from field interactions measured on the sender fish’s skin. C: Top: Schematic example of two fish, a chirp sender (black fish) and a chirp receiver (red contour), and the location of 3 body points used in D. Bottom left: all simulations include 8 different EOD phases (in 45° steps). Bottom right: the simulations used in E, include different body locations and are calculated across 835 nodes for each fish. In addition, different reciprocal body positions are included (i.e. distances and angles) for the calculation of the electric image data in E. D: Voltage measured at three points on the sender fish’s body as shown in C (upper panel) for a baseline 10% difference in fish EODf (i.e. normalized DF = 0.1). Top row: chirp condition, bottom row: beat with no chirp. The green area represents the integral of the voltage change over time (i.e. beat area under the curve, or AUC). The red signal indicates the chirp receiver’s EOD at the same three points on the chirp sender’s body. E: Net effect of chirps on electric images for different fish orientations and distances (insets in C, bottom right), represented as the sum of the voltage integral (AUC) over time (measured throughout the beat) due to chirps across the entire body (835 nodes), compared to the carrier beats alone, for seven baseline differences in fish EODf. Black asterisks indicate significant chirp-beat differences. Data for the sender fish (top) and receiver fish (bottom) are displayed separately. Significant differences between sender and receiver are indicated with red asterisks.
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
Chirp dataset (total = 67522 chirps)
Playback chirp parameters.
