Why the brown ghost chirps at night

  1. Livio Oboti
  2. Federico Pedraja
  3. Marie Ritter
  4. Marlena Lohse
  5. Lennart Klette
  6. Rüdiger Krahe

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    This study addresses a question in sensory ethology and active sensing in particular. It links the production of a specific signal - electrosensory chirps - to various contexts and conditions to argue that the main function is to enhance conspecific localization rather than communication as previously believed. The study provides a lot of valuable data, but the methods section is incomplete making it difficult to evaluate the claims.

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Abstract

Since the pioneering work by Moeller, Szabo, and Bullock, weakly electric fish have served as a valuable model for investigating spatial and social cognitive abilities in a vertebrate taxon usually less accessible than mammals or other terrestrial vertebrates. These fish, through their electric organ, generate low-intensity electric fields to navigate and interact with conspecifics, even in complete darkness. The brown ghost knifefish is one of the most widely studied species due to its rich electric vocabulary, made by individually variable and sex-specific electric signals. These are mainly characterized by brief frequency modulations of the oscillating dipole moment emitted continuously by their electric organ and are known as chirps. Different types of chirps are believed to convey specific and behaviorally salient information, serving as behavioral readouts for different internal states during behavioral observations. Despite the success of this model in neuroethology over the past seven decades, the code to decipher their electric communication remains unknown.This study re-evaluates this view, aiming to offer an alternative, and possibly complementary, explanation for why these freshwater bottom dwellers emit electric chirps. By uncovering correlations among chirping, electric field geometry, and detectability in enriched environments, we present evidence for a previously unexplored role of chirps as specialized self-directed signals, enhancing conspecific electrolocation during social encounters.

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  1. Version published to 10.7554/elife.88287.3 on eLife
  2. Version published to 10.7554/elife.88287 on eLife
  3. eLife

    eLife assessment

    This study addresses a question in sensory ethology and active sensing in particular. It links the production of a specific signal - electrosensory chirps - to various contexts and conditions to argue that the main function is to enhance conspecific localization rather than communication as previously believed. The study provides a lot of valuable data, but the methods section is incomplete making it difficult to evaluate the claims.

  4. eLife

    Reviewer #1 (Public Review):

    The authors investigate the role of chirping in a species of weakly electric fish. They subject the fish to various scenarios and correlate the production of chirps with many different factors. They find major correlations between the background beat signals (continuously present during any social interactions) or some aspects of social and environmental conditions with the propensity to produce different types of chirps. By analyzing more specifically different aspects of these correlations they conclude that chirping patterns are related to navigation purposes and the need to localize the source of the beat signal (i.e. the location of the conspecific).

    The study provides a wealth of interesting observations of behavior and much of this data constitutes a useful dataset to document the patterns of social interactions in these fish. Some data, in particular the high propensity to chirp in cluttered environments, raises interesting questions. Their main hypothesis is a useful addition to the debate on the function of these chirps and is worth being considered and explored further.

    After the initial reviewers' comments, the authors performed a welcome revision of the way the results are presented. Overall the study has been improved by the revision. However, one piece of new data is perplexing to me. The new figure 7 presents the results of a model analysis of the strength of the EI caused by a second fish to localize when the focal fish is chirping. From my understanding of this type of model, EOD frequency is not a parameter in the model since it evaluates the strength of the field at a given point in time. Therefore the only thing that matters is the phase relationship and strength of the EOD. Assuming that the second fish's EOD is kept constant and the phase relationship is also the same, the only difference during a chirp that could affect the result of the calculation is the potential decrease in EOD amplitude during the chirp. It is indeed logical that if the focal fish decreased its EOD amplitude the target fish's EOD becomes relatively stronger. Where things are harder to understand is why the different types of chirps (e.g. type 1 vs type 2) lead to the same increase in signal even though they are typically associated with different levels of amplitude modulations. Also, it is hard to imagine that a type 2 chirp that is barely associated with any decrease in EOD amplitude (0-10% maybe), would cause a doubling of the EI strength. There might be something I don't understand but the authors should provide a lot more details on how this result is obtained and convince us that it makes sense.

    Finally, the reviewer is concerned about this sentence in the rebuttal - "The methods section has been edited to clarify the approach (not yet)". This section is unfinished, which suggests that it is difficult to explain the modeling results from a logical point of view. Thus the reviewer's major concern from the previous review remains unresolved. To summarize, the model calculates field strengths at an instant in time and integrates over time with a 500 ms window. This window is 10 times longer than the small chirps, while the longer chirps cover a much larger proportion of the window. Yet, the small chirps have a bigger impact on discriminability than the longer chirps. The authors should attempt to explain this seemingly contradictory result. This remains a major issue because this analysis was the most direct evidence that chirping could impact localization accuracy.

  5. eLife

    Reviewer #2 (Public Review):

    Studying Apteronotus leptorhynchus (the weakly electric brown ghost knifefish), the authors provide evidence that 'chirps' (brief modulations in the frequency and amplitude of the ongoing wave-like electric signal) function in active sensing (specifically homeoactive sensing) rather than communication. Chirping is a behavior that has been well studied, including numerous studies on the sensory coding of chirps and the neural mechanisms for chirp generation. Chirps are largely thought to function in communication behavior, so this alternative function is a very exciting possibility that should have a great impact on the field.

    The authors provide convincing evidence that chirps may function in homeoactive sensing. In particular, the evidence showing increased chirping in more cluttered environments and a relationship between chirping and movement are especially strong and suggestive. Their evidence arguing against a role for chirps in communication is not as strong. However, based on an extensive review of the literature, the authors conclude, I think fairly, that the evidence arguing in favor of a communication function is limited and inconclusive. Thus, the real strength of this study is not that it conclusively refutes the communication hypothesis, but that it calls this hypothesis into question while also providing compelling evidence in favor of an alternative function.

    In summary, although the evidence against a role for chirps in communication is not as strong as the evidence for a role in active sensing, this study presents very interesting data that is sure to stimulate discussion and follow-up studies. The authors acknowledge that chirps could function as both a communication and homeactive sensing signal, and the language arguing against a communication function is appropriately measured. A given electrical behavior could serve both communication and homeoactive sensing. I suspect this is quite common in electric fish (not just in gymnotiforms such as the species studied here, but also in the distantly related mormyrids), and perhaps in other actively sensing species such as echolocating animals.

  6. eLife

    Reviewer #3 (Public Review):

    Summary:
    This important paper provides the best-to-date characterization of chirping in weakly electric fish using a large number of variables. These include environment (free vs divided fish, with or without clutter), breeding state, gender, intruder vs resident, social status, locomotion state and social and environmental experience, without and with playback experiments. It applies state-of-the-art methods for reducing the dimensionality of the data and finding patterns of correlation between different kinds of variables (factor analysis, K-means). The strength of the evidence, collated from a large number of trials with many controls, leads to the conclusion that the traditionally assumed communication function of chirps may be secondary to its role in environmental assessment and exploration that takes social context into account. Based on their extensive analyses, the authors suggest that chirps are mainly used as probes that help detect beats caused by other fish as well as objects.

    Strengths:
    The work is based on completely novel recordings using interaction chambers. The amount of new data and associated analyses is simply staggering, and yet, well organized in presentation. The study further evaluates the electric field strength around a fish (via modelling with the boundary element method) and how its decay parallels the chirp rate, thereby relating the above variables to electric field geometry. The BEM modelling also convincingly predicts how the electric image of a receiver conspecific on a sending fish is enhanced by a chirp.

    The main conclusions are that the lack of any significant behavioural correlates for chirping, and the lack of temporal patterning in chirp time series, cast doubt on a primary communication goal for most chirps. Rather, the key determinants of chirping are the difference in frequency between two interacting conspecifics as well as individual subjects' environmental and social experience. The paper concludes that there is a lack of evidence for stereotyped temporal patterning of chirp time series, as well as of sender-receiver chirp transitions beyond the known increase in chirp frequency during an interaction. The authors carefully submit that the new putative echolocation function of chirps is not mutually exclusive with a possible communication function.

    These conclusions by themselves will be very useful to the field. They will also allow scientists working on other "communication" systems to perhaps reconsider and expand the goals of the probes used in those senses. A lot of data are summarized in this paper, with thorough referencing to past work.

    The alternative hypotheses that arise from the work are that chirps are mainly used as environmental probes for better beat detection and processing and object localization, and in this sense are self-directed signals. This led to their prediction that environmental complexity ("clutter") should increase chirp rate, which is fact was revealed by their new experiments. The authors also argue that waveform EODs have less power across high spatial frequencies compared to pulse-type fish, with a resulting relatively impoverished power of resolution. Chirping in wave-type fish could temporarily compensate for the lower frequency resolution while still being able to resolve EOD perturbations with a good temporal definition (which pulse-type fish lack due to low pulse rates).

    The authors also advance the interesting idea that the sinusoidal frequency modulations caused by chirps are the electric fish's solution to the minute (and undetectable by neural wetware) echo-delays available to it, due to the propagation of electric fields at the speed of light in water. The paper provides a number of experimental avenues to pursue in order to validate the non-communication role of chirps.

  7. eLife

    Author response:

    The following is the authors’ response to the previous reviews.

    Public Reviews:

    Reviewer #1 (Public Review):

    The authors investigate the role of chirping in a species of weakly electric fish. They subject the fish to various scenarios and correlate the production of chirps with many different factors. They find major correlations between the background beat signals (continuously present during any social interactions) or some aspects of social and environmental conditions with the propensity to produce different types of chirps. By analyzing more specifically different aspects of these correlations they conclude that chirping patterns are related to navigation purposes and the need to localize the source of the beat signal (i.e. the location of the conspecific).

    The study provides a wealth of interesting observations of behavior and much of this data constitutes a useful dataset to document the patterns of social interactions in these fish. Some data, in particular the high propensity to chirp in cluttered environments, raises interesting questions. Their main hypothesis is a useful addition to the debate on the function of these chirps and is worth considering and exploring further.

    After the initial reviewers' comments, the authors performed a welcome revision of the way the results are presented. Overall the study has been improved by the revision. However, one piece of new data is perplexing to me. The new Figure 7 presents the results of a model analysis of the strength of the EI caused by a second fish to localize when the focal fish is chirping. From my understanding of this type of model, EOD frequency is not a parameter in the model since it evaluates the strength of the field at a given point in time. Therefore the only thing that matters is the phase relationship and strength of the EOD. Assuming that the second fish's EOD is kept constant and the phases relationship is also the same, the only difference during a chirp that could affect the result of the calculation is the potential decrease in EOD amplitude during the chirp. It is indeed logical that if the focal fish decreased its EOD amplitude the target fish's EOD becomes relatively stronger. Where things are harder to understand is why the different types of chirps (e.g. type 1 vs type 2) lead to the same increase in signal even though they are typically associated with different levels of amplitude modulations. Also, it is hard to imagine that a type 2 chirps that is barely associated with any decrease in EOD amplitude (0-10% maybe), would cause doubling of the EI strength. There might be something I don't understand but the authors should provide a lot more details on how this result is obtained and convince us that it makes sense.

    We thank the author for the comments and we agree that the approach could have been better detailed. As anticipated by the Reviewer, the Boundary Element Method (BEM) model can be used simply to calculate the electric field and electric image at a specific point in time (instantaneously), regardless of EOD frequency. However, our model allows for the concatenation of consecutive instants and thus is able to render an entire sequence of electric fields - and resulting electric images - incorporating realistic EOD characteristics such as shape, duration, and frequencies (see Pedraja et al., 2014).

    Chirp-triggered EIs were modeled using real chirps produced by interacting fish. Each chirp was thus associated to its duration and peak parameters, as well as the fish positional information (distance and angle).

    However, since we did not know the beat phase at which chirps were produced, we computed electric images for each fish position and chirp scenario by simulating various phases (here referred to the initial offset of the two EODs, set at 4 phases, equally spaced). These are intended as phases of the sender EOD and simply refer to the initial OFFSET between the two interacting EODs. However, since our simulations were run over a time window of 500 msec, all phases are likely to be covered, with a different temporal order relative to the chirp (always centered within the 500 msec).

    The simulation was run maintaining consistent timing for both chirp and non-chirp conditions, across approximately 800 body nodes. At each node, the current flow was calculated from the peak-to-peak of the EOD sum (i.e. the point-to-point of the difference between the beat positive and negative envelopes). Analyzing the EIs over this fixed time window enables us to assess the unitary changes of current flow induced by chirps over units of time (ΔI/Δt). From this, we can calculate a cumulative sum of current flow changes - expressed as delta(EI) and use it to show the effect of the chirps on the spatiotemporal EI (Figure 7C).

    One can express this cumulative change mapped onto the fish body (keeping the 800 points separated, as in Figure 7C) or further sum the current changes to obtain a single total (as shown in Figure 7D).

    One can check this by considering that a sum for example of a set of 500/800 points - judging from the size of the blue areas in C not all 800 points have a detectable change - each valued 0.1-to-0.3 mA/s, one could get circa 100 mA/s, which is what is shown in D. (is this what is happening ?)

    We do not know why chirps of different types triggered similar effects. It is possible that, since EI measurements are pooled over several chirps produced at different angles and distances, in case of a lower amount of chirps considered for a given type (as in the case of rises, very low) these measurements may not highlight more marked differences among types. In a publication we are currently working on, we are considering a larger dataset to better assess these results.

    The methods section has been edited to clarify the approach (not yet).

    Reviewer #2 (Public Review):

    Studying Apteronotus leptorhynchus (the weakly electric brown ghost knifefish), the authors provide evidence that 'chirps' (brief modulations in the frequency and amplitude of the ongoing electric signal) function in active sensing (specifically homeoactive sensing) rather than communication. Chirping is a behavior that has been well studied, including numerous studies on the sensory coding of chirps and the neural mechanisms for chirp generation.

    Chirps are largely thought to function in communication behavior, so this alternative function is a very exciting possibility that could have a great impact on the field.

    We thank the Reviewer for the extensive and constructive comments. We would like to add that, while it is true that many detailed studies have been published on the anatomy and physiology of the circuits implicated in the production and modulation of “electric chirps”, most of this research assumed, and focused exclusively on, their possible role in communication. In addition, most behavioral studies did the same and a meta-analysis of the existing literature on chirping allows to trace back the communication idea mainly to two studies: Hagedorn and Heiligenberg, 1985 (“Court and spark: electric signals in the courtship and mating of gymnotoid fish”) and Hopkins, 1974 (“Electric Communication: Functions in the Social Behavior of Eigenmannia Virescens”), among the main sources. Importantly, in these studies only contextual observations have been made (no playback experiment or other attempts to analyze more quantitatively the correlation of chirping with other behaviors).

    The authors do provide convincing evidence that chirps may function in homeoactive sensing. However, their evidence arguing against a role for chirps in communication is not as strong, and fails to sufficiently consider the evidence from a large body of existing research. Ultimately, the manuscript presents very interesting data that is sure to stimulate discussion and follow-up studies, but it suffers from dismissing evidence in support of, or consistent with, a communicative function for chirps.

    Although the tone of some statements present in our earlier draft may suggest otherwise, through our revisions, we have made an effort to clarify that we do not intend to dismiss a function of chirps in communication, we only intend to debate and discuss valid alternative hypothesis, advanced from reasonable considerations.

    Before writing this manuscript, we have attempted to survey literally all the existing literature on chirps (including studies focused on behavior, peripheral sensory physiology as well as brain physiology). Although it is not unlikely that some studies have eluded our attention, an effort for a comprehensive review was made. Based on this survey we realized that none of the studies provided a clear and unambiguous piece of evidence to support the communication hypothesis (we refer here to the weak points highlighted in the discussion and mentioned in the previous comment). Which in fact does not come without its weak points and contradictions (see later comments).

    It follows a summary of the mentions made to the communication theory in the different section of the manuscript including several edits we have applied in response to the Reviewer’s concern:

    In the abstract we clearly state that we are considering an alternative that is only hypothetically complementary, not for sure. Nonetheless, we have identified a couple of instances that could sound dismissive of the “communication hypothesis” in the following section.

    In the introduction we write in fact about the possibility of interference between communication signals and conspecific electrolocation cues, as they are both detected as beat perturbations. We did not mean to use “Interference” here as “reciprocal canceling”, rather we intended it as “partial or more or less conspicuous overlap” in the responses triggered in electroreceptors.

    Hoping to convey a clearer message, we have edited the related statement and changed it to “both types of information are likely to overlap and interact in highly variable ways”.

    We have also removed the statement: “According to this idea, beats and chirps are not only detected through the same input channel, but also used for the same purpose.” as at this point in the manuscript it may be too strong.

    In the results section we do not include statements that might be seen as dismissive of the communication hypothesis but only statements in support of the “probing with chirps” idea (which is the central hypothesis of the study).

    In the discussion paragraphs we elaborate on why the current functional view is either flawed or incomplete (first paragraph “existing functional hypotheses''). Namely: 1) multiple triggering factors implied in chirp responses covary and need to be disentangled (example DF/ sex), 2) findings on brown ghosts and a few other gymnotiforms have been used to advance the hypothesis of “communication through chirps'' in all weakly electric fish (including pulse species). 3) social encounters - in which chirps are recorded - imply also other behaviors (such as probing) which have not been considered so far. This point is related to the first one on covariates. 4) most studies referring to big chirps as courtship chirps were not done in reproductive animals (added now) and 5) no causal evidence has been provided so far to justify a role of chirps in social communication.

    We are discussing these points as challenges to the communication hypothesis, not to dismiss the hypothesis, but rather to motivate future studies addressing these challenges.

    We do not want to appear dismissive of the communication hypothesis and had therefore previously edited the manuscript to avoid the impression of exclusivity of the probing hypothesis. We have now gone over the manuscript once more and edited several sentences. Nevertheless, we want to point out again that - despite the large consensus - the communication hypothesis has, until now, never been investigated with the kind of rigor applied here.

    The authors do acknowledge that chirps could function as both a communication and homeactive sensing signal, but it seems clear they wish to argue against the former and for the latter, and the evidence is not yet there to support this.

    In both rounds of revision we have made an effort to convey a more inclusive interpretation of our findings. We tried our best to express our ideas as hypothetical, not as proof that communication through chirps does not exist. The aim of this study is to propose an alternative view, and this cannot be done without underlining the weak points of an existing hypothesis while providing and supporting reasonable arguments in favor of the alternative we advance. The actual evidence for a role of chirping in communication is much less strong than appears from the pure number of articles that have discussed chirps in this context.

    Regarding the weak evidence against communication, here we can list a few additional important points related to the proposed interpretations of chirp function (more specific than those made earlier):

    (1) A formally sound assessment of signal value/meaning - as typically done in animal communication studies should involve:

    a) the isolation of a naturally occurring signal and determination of the context in which it is produced

    b) the artificial replication of the signal

    c) the observation that such mimic is capable of triggering reliable and stereotyped responses in a group of individuals (identified by sex and/or species) under the same conditions (conditioned, unconditioned, state-dependent, etc.). As discussed for instance in Bradbury and Vehrencamp, 2011; Laidre and Johnstone, 2013; Wyatt, 2015; Rutz et al., 2023.

    This approach has so far not been applied to weakly electric fish. The initial purpose of the present study was in fact to conduct this type of validation.

    (2) The hypothesis of chirps used for DF-sign discrimination - for “social purposes” - although plausible in the face of theoretical considerations, does not seem to be reasonable in practice, when one considers emission rates of 150 chirps per minute. We do find a strong correlation of chirp type with DF, which is often very abrupt and sudden (as if the fish were tracking beat frequency to guess its value) but the consideration made above on chirp rates seems to discourage this interpretation.

    (3) The hypothesis of chirp-patterning (i.e. chirping may have meaning based on the sequence of chirps of different types, a bit like syllables in birdsongs) - assessed by only one study conducted in our group - has not been enough substantiated by replication. We have surveyed all possible combinations of chirps produced by interacting pairs in different behavioral conditions using different value for chirp sequence size: 2, 3,... ,8 chirps (both considering the sender alone as well as sender+receiver together). In all cases we found no evidence for a context dependent “modulation” of chirp types (i.e. no specific chirp type sequence in specific contexts).

    (4) The hypothesized role of “large chirps” as courtship signals could be easily criticized by noting the symmetrical distribution of these events around a DF of 0 Hz . Although one could argue about a failure to discriminate DF-sign, to explain this well known pattern. However, we know from Walter Heiligenberg’s work and physiological considerations that such task can be solved easily through t-units and … in principle even just by motion (which would change the EOD phase in frequency dependent ways, thus potentially revealing the DF sign).

    Overall, these considerations made us think that certainly chirping occurs in a social context, but it is the meaning of this behavior that remains elusive. We noticed that environmental factors are also strongly implied … we then formulate an alternative hypothesis to explain chirping but we do so without dismissing the communication idea.

    All this seems to us just a careful way to critically discuss our results and those of other studies, without considering the issue resolved.

    In the introduction, the authors state, "Since both chirps and positional parameters (such as size, orientation or motion) can only be detected as perturbations of the beat, and via the same electroreceptors, the inputs relaying both types of information are inevitably interfering." I disagree with this statement, which seems to be a key assumption. Both of these features certainly modulate the activity of electroreceptors, but that does not mean those modulations are ambiguous as to their source. You do not know whether the two types of modulations can be unambiguously decoded from electroreceptor afferent population activity.

    We thank the Reviewer for noting this imprecision. We have addressed the Reviewer’s concern in another reply (see above).

    My biggest issue with this manuscript is that it is much too strong in dismissing evidence that chirping correlates with context. In your behavioral observations, you found sex differences in chirping as well as differences between freely interacting and physically separated fish. Chirps tended to occur in close proximity to another fish. Your model of chirp variability found that environmental experience, social experience, and beat frequency (DF) are the most important factors explaining chirp variability. Are these not all considered behavioral or social context? Beat frequency (DF) in particular is heavily downplayed as being a part of "context" but it is a crucial part of the context, as it provides information about the identity of the fish you're interacting with. The authors show quite convincingly that the types of chirps produced do not vary with these contexts, but chirp rates do.

    We believe the “perceived claim” may be an issue of unclear writing. We have now tried to better clarify that “context” affects chirp rates, but it does not affect chirp types as much (except when beat frequency is high).

    We have edited two statements possibly susceptible to misinterpretation:

    (1) In the results: “It also indicates that chirp parameters such as duration and FM do not seem to be associated with any particular context in a meaningful way, other than being affected by beat frequency.”

    (2) In the discussion: the statement

    “Recordings from interacting fish pairs confirmed the absence of any significant correlation between chirp type choice and behavioral context (Figure S2) although the variance of chirp parameters appears to be significantly affected by this factor (Figure 2). This may suggest that the effect of behavioral context is mainly detectable in the number of chirps produced (Figure S1), rather than the type (Figure S2).”

    has been changed to:

    “Recordings from interacting fish pairs confirmed the absence of any significant correlation between chirp type choice and behavioral context, except for those cases characterized by higher beat frequencies (Figure S2). This suggests that the effect of behavioral context highlighted in our factor analysis (Figure 2) is mainly due to the number of chirps produced (Figure S1), rather than their type (Figure S2).”

    Eventually, in the results we emphasize the relatively higher impact of previously unexplored factors on chirp variance: “The plot of individual chirps (Figure 2C) shows the presence of clustering around different categorical variables and it reveals that experience levels or swimming conditions are important factors affecting chirp distribution (note for instance the large central “breeding” cluster in which fish are divided and the smaller ones in which fish are free). Sender or receiver identity does not individuate any clear clustering relative to either sex (see the overlap of male_s/male_r and female_s/female_r) or social status (dominant/subordinate). Chirps labeled based on tank experience (i.e. resident vs intruder) are instead clearly separated.”.

    Further, in your playback experiments, fish responded differently to small vs. large DFs, males chirped more than females, type 2 chirps became more frequent throughout a playback, and rises tended to occur at the end of a playback. These are all examples of context-dependent behavior.

    We do note that male brown ghosts chirp more than females. But we do also say - and show in figure 8 - that males move more in proximity to and around conspecifics. We do acknowledge that chirp time-course may be different during playbacks in a type-dependent manner. But how this can support the communication hypothesis - or other alternatives - is unclear. This result could equally imply the use of different chirp types for different probing needs. Since we cannot be sure about either, we do not want to put too much emphasis to it. Eventually, the fact that “context” (here meant broadly to define different experimental situations in which social but also physical and environmental parameters are altered) affects chirping is undeniable: cluttered and non-cluttered environments do represent different contexts which differently affect chirping in conspicuous ways.

    In the results, the authors state, "Overall, the majority of chirps were produced by male subjects, in comparable amounts regardless of environmental experience (resident, intruder or equal; Figure S1A,C), social status (dominant or subordinate; Figure S1B) or social experience (novel or experienced; Figure S1D)." This is not what is shown in Figure S1. S1A shows clear differences between resident vs. intruder males, S1B shows clear differences between dominant vs. subordinate males, and S1D shows clear differences between naïve and experienced males. The analysis shown in Figure 2 would seem to support this. Indeed, the authors state, "Overall, this analysis indicated that environmental and social experience, together with beat frequency (DF) are the most important factors explaining chirp variability."

    The Reviewer is right in pointing at this imprecise reference and we are grateful for spotting this incongruence. The writing refers probably to an earlier version of the figure in which data were grouped and analyzed differently. We now edited the text and changed it to: “Overall, the majority of chirps were produced by male subjects, at rates that seemed affected by environmental experience (resident, intruder or equal; Figure S1A,C), social status (dominant or subordinate; Figure S1B) and social experience (novel or experienced; Figure S1D).”

    The choice of chirp type varied widely between individuals but was relatively consistent within individuals across trials of the same experiment. The authors interpret this to mean that chirping does not vary with internal state, but is it not likely that the internal states of individuals are stable under stable conditions, and that individuals may differ in these internal states across the same conditions? Stable differences in communication signals between individuals are frequently interpreted as reflecting differences between those individuals in certain characteristics, which are being communicated by these signals.

    It seems here we have been unclear in the writing: while it is true that behavioral states are stable and can imply stable chirp patterning (if the two are related), since chirp types vary abruptly and in a reliable DF-dependent manner, different types of chirps are unlikely to be matched to different internal states following the same temporal order in such a reliable way (similarly repeated through consecutive trials).

    This would imply the occurrence of different internal states in rapid sequence, reliably triggered by repeated EOD ramps, regardless of whether the playback is 20 sec long or 180 sec long.

    We have edited this paragraph to better explain this: “The reliability by which the chirping response adapts to both the rate and direction of beat frequency is variable across individuals but rather stable across trials (relative to a given subject), further suggesting that chirp type variations may not reflect changes in internal states or in the animal motivation to specific behavioral displays (which are presumably subject to less abrupt variations and stereotypical patterning based on DF).”

    I am not convinced of the conclusion drawn by the analysis of chirp transitions. The transition matrices show plenty of 1-2 and 2-1 transitions occurring.

    The only groups in which 1-2 and 2-1 transitions are as frequent as 1-1 and 2-2 (being 1 and 2 the numerical IDs of the two interacting fish) are F-F pairs. This is a result of the fact that in females chirp rates are so low that within-fish-correlations end up being as low as between-fish-correlations. We believe the impression of the Reviewer could be due to the fact that these are normalized maps (see legend of Figure 5A-B).

    Further, the cross-correlation analysis only shows that chirp timing between individuals is not phase-locked at these small timescales. It is entirely possible that chirp rates are correlated between interacting individuals, even if their precise timing is not.

    We agree with the Reviewer, this is a possibility. To address this point, we did edit the results section to acknowledge that what we see may be related to the time window chosen (i.e. 4 sec):

    “More importantly, they show that - at least in the social conditions analyzed here and within small-sized time windows - chirp time series produced by different fish during paired interactions are consistently independent of each other.”

    Further, it is not clear to me how "transitions" were defined. The methods do not make this clear, and it is not clear to me how you can have zero chirp transitions between two individuals when those two individuals are both generating chirps throughout an interaction.

    We thank the Reviewer for bringing up this unclear point. We have now clarified how transitions were calculated in the method section: “The number of chirp transitions present in each recording (dataset used for Figures 1, 2, 5) was measured by searching in a string array containing the 4 chirp types per fish pair, all their possible pairwise permutations (i.e. all possible permutations of 4+4=8 elements are: 1-1, 1-2, 1-3 … 7-6, 7-7, 7-8; considering the following legend 1 = fish1 type 1, 2 = fish 1 type 2, 3 = fish1 type 3 … 6 = fish2 type 2, 7 = fish2 type 3 and 8 = fish2 rise).”.

    Zero transitions are possible if two fish (or groups of fish) do not produce chirps of all types. Only transitions of produced types can be counted.

    In the results, "Although all chirp types were used during aggressive interactions, these seemed to be rather less frequent in the immediate surround of the chirps (Figure 6A)." A lack of precise temporal correlation on short timescales does not mean there is no association between the two behaviors. An increased rate of chirping during aggression is still a correlation between the two behaviors, even if chirps and specific aggressive behaviors are not tightly time-locked.

    The Reviewer is right in pointing out the limited temporal scaling of our observations/analysis. We have now edited the last paragraph of the results related to figure 6 to include the possibility mentioned by the Reviewer: “The significantly higher extent of chirping during swimming and locomotion, consistently confirmed by 4 different approaches (PSTH, TM, CN, MDS), suggests that - although chirp-behavior correlations may exist at time-scales larger than those here considered - chirping may be linked more strongly with scanning and environmental exploration than with a particular motivational state, thus confirming findings from our playback experiments.”

    The Reviewer here remarks an important point, yet, due to space limitations, we have considered only a sub-second scale. Most playback experiments in weakly electric fish implied the use of EOD mimics for a few tens of seconds - to avoid habituation in the fish behavioral responses - while inter-chirp intervals usually range between a few hundreds of milliseconds to seconds (depending on how often a fish would chirp). This suggested to us that a 4 second time window may not be a bad choice to start with.

    In summary, it is simply too strong to say that chirping does not correlate with context, or to claim that there is convincing evidence arguing against a communication function of chirps. Importantly, however, this does not detract from your exciting and well-supported hypothesis that chirping functions in homeoactive sensing. A given EOD behavior could serve both communication and homeoactive sensing. I actually suspect this is quite common in electric fish (both gymnotiforms and mormyrids), and perhaps in other actively sensing species such as echolocating animals. The two are not mutually exclusive.

    We agree with the Reviewer that context - broadly speaking - does affect chirping (as we mentioned above). We hope we have improved the writing and clarified that we do not dismiss communication functions of chirping, but we do lean towards electrolocation based on the considerations above made and our results.

    We do conclude the manuscript remarking that communication and electrolocation are not mutually exclusive: ”probing cues could function simultaneously as proximity signals to signal presence, deter approaches, or coordinate behaviors like spawning, if properly timed (Henninger et al., 2018).” (see the conclusion paragraph of the discussion) .

    Therein, we further add “These findings aim to stir the pot and initiate a discussion on possible alternative functions of chirps beyond their presumed communication role.”.

    With this, we hope we’ve made it clear how we intend our manuscript to be read.

    Reviewer #3 (Public Review):

    Summary:

    This important paper provides the best-to-date characterization of chirping in weakly electric fish using a large number of variables. These include environment (free vs divided fish, with or without clutter), breeding state, gender, intruder vs resident, social status, locomotion state and social and environmental experience, without and with playback experiments. It applies state-of-the-art methods for reducing the dimensionality of the data and finding patterns of correlation between different kinds of variables (factor analysis, K-means). The strength of the evidence, collated from a large number of trials with many controls, leads to the conclusion that the traditionally assumed communication function of chirps may be secondary to its role in environmental assessment and exploration that takes social context into account. Based on their extensive analyses, the authors suggest that chirps are mainly used as probes that help detect beats caused by other fish and as well as objects.

    Strengths:

    The work is based on completely novel recordings using interaction chambers. The amount of new data and associated analyses is simply staggering, and yet, well organized in presentation. The study further evaluates the electric field strength around a fish (via modelling with the boundary element method) and how its decay parallels the chirp rate, thereby relating the above variables to electric field geometry.

    The main conclusions are that the lack of any significant behavioural correlates for chirping, and the lack of temporal patterning in chirp time series, cast doubt on a primary communication goal for most chirps. Rather, the key determinants of chirping are the difference frequency between two interacting conspecifics as well as individual subjects' environmental and social experience. The paper concludes that there is a lack of evidence for stereotyped temporal patterning of chirp time series, as well as of sender-receiver chirp transitions beyond the known increase in chirp frequency during an interaction.

    These conclusions by themselves will be very useful to the field. They will also allow scientists working on other "communication" systems to perhaps reconsider and expand the goals of the probes used in those senses. A lot of data are summarized in this paper, with thorough referencing to past work.

    The alternative hypotheses that arise from the work are that chirps are mainly used as environmental probes for better beat detection and processing and object localization, and in this sense are self-directed signals. This led to their prediction that environmental complexity ("clutter") should increase chirp rate, which is fact was revealed by their new experiments. The authors also argue that waveform EODs have less power across high spatial frequencies compared to pulse-type fish, with a resulting relatively impoverished power of resolution. Chirping in wave-type fish could temporarily compensate for the lower frequency resolution while still being able to resolve EOD perturbations with a good temporal definition (which pulse-type fish lack due to low pulse rates).

    The authors also advance the interesting idea that the sinusoidal frequency modulations caused by chirps are the electric fish's solution to the minute (and undetectable by neural wetware) echo-delays available to it, due to the propagation of electric fields at the speed of light in water. The paper provides a number of experimental avenues to pursue in order to validate the non-communication role of chirps.

    We thank the reviewer for the kind assessment.

    Weaknesses:

    My main criticism is that the alternative putative role for chirps as probe signals that optimize beat detection could be better developed. The paper could be clearer as to what that means precisely, especially since beating - and therefore detection of some aspects of beating due to the proximity of a conspecific - most often precedes chirping. One meaning the authors suggest, tentatively, is that the chirps could enhance electrosensory responses to the beat, for example by causing beat phase shifts that remediate blind spots in the electric field of view.

    We agree with the Reviewer that a better and more detailed explanation of how beat processing for conspecific electrolocation may be positively affected by chirps would be important to provide. We are currently working on a follow-up manuscript in which we intend to include these aspects. For space limitations and readability we had to discard from the current manuscript a lot of results that could further clarify these issues.

    A second criticism is that the study links the beat detection to underwater object localization. The paper does not significantly develop that line of thought given their data - the authors tread carefully here given the speculative aspect of this link. It is certainly possible that the image on the fish's body of an object in the environment will be slightly modified by introducing a chirp on the waveform, as this may enhance certain heterogeneities of the object in relation to its environment. The thrust of this argument derives mainly from the notion of Fourier analysis with pulse type fish EOD waveforms (see above, and radar theory more generally), where higher temporal frequencies in the beat waveform induced by the chirp will enable a better spatial resolution of objects. It remains to be seen whether experiments can show this to be significant.

    Perhaps the Reviewer refers to the last discussion paragraph before the conclusions in which we mention the performance of pulse or wave-type EODs in electrolocation (referring here to ideas illustrated in a recent review by Crampton, 2019). We added to this paragraph a statement which could better clarify that we do not propose that chirping could enhance object electrolocation. What we mean is that, in a context in which object electrolocation occurs through wave-type EODs - given the generally lower performance of such narrow-band signals in resolving the spatial features of any object, even a 3D electric field - chirping could improve beat detection during social encounters by increasing the amount of information obtained by the fish.

    The edited paragraph now reads: “While broadband pulse signals may be useful to capture highly complex environments rich in foliage, roots and other structures common in vegetation featuring the more superficial habitats in which pulse-type fish live, wave-type EODs may be a better choice in the relatively simpler river-bed environments in which many wave-type fish live (e.g., the benthic zone of deep river channels; Crampton, 2019). In this case, achieving a good spatial resolution is critical during social encounters, especially considering the limited utility of visual cues in these low-light conditions. In such habitats, social encounters may “electrically” be less “abrupt”, but spatially less “conspicuous” or blurred (as a 3D electric field may be). In such a scenario, chirps could serve as a means to supplement the spatial information acquired via the beat, accentuating these cues during periods of reduced resolution.”

    Recommendations for the authors:

    Reviewer #3 (Recommendations For The Authors):

    None, my points in the original review have been properly addressed in this resubmission.

  8. Version published to 10.1101/2022.12.29.522225v7 on bioRxiv
  9. Version published to 10.1101/2022.12.29.522225v6 on bioRxiv
  10. Version published to 10.7554/elife.88287.2 on eLife
  11. eLife

    Author response:

    The following is the authors’ response to the original reviews.

    eLife assessment

    This is a valuable contribution to the electric fish community, and to studies of active sensing more generally, in that it provides evidence that a well-studied behavior (chirping) may serve in active sensing rather than communication. For the most part, the evidence is solid. In particular, the evidence showing increased chirping in more cluttered environments and the relationship between chirping and movement are convincing. Nevertheless, evidence to support the argument that chirps are mostly used for navigation rather than communication is incomplete.

    Thank you for the comment. In response to what seemed to be a generalized need for more evidence to support our hypothesis, we have extensively reviewed the manuscript, changed the existing figures and added new ones (3 new figures in the main text and 4 in the supplementary information section). Our edits include:

    (1) changes to the written text to remove categorical statements ruling out the possible communication function of chirps. When necessary, we have also added details on why we believe a social communication function of chirps could interfere with a role in electrolocation.

    (2) new experiments (and related figures) adding details on the behavioral correlates of chirping, on the effects of chirps on electric images (which are a way to represent current flow on the fish skin), and behavioral responses to ramp frequency playback EODs (used to test a continuous range of beat frequencies and fill the sampling gaps left by our experiments using real fish).

    Public Reviews:

    Reviewer #1 (Public Review):

    The authors investigate the role of chirping in a species of weakly electric fish. They subject the fish to various scenarios and correlate the production of chirps with many different factors. They find major correlations between the background beat signals (continuously present during any social interactions) or some aspects of social and environmental conditions with the propensity to produce different types of chirps. By analyzing more specifically different aspects of these correlations they conclude that chirping patterns are related to navigation purposes and the need to localize the source of the beat signal (i.e. the location of the conspecific).

    We thank the Reviewer for the extensive feedback received. Hereby we respond to each of the points raised.

    We have better clarified that our intention is not to propose chirps as tools for “conspecific localization” intended as the pinpointing of its particular location. Instead, based on our observation of chirps being employed at very close ranges, we suggest that chirps may serve to assess other parameters related to “conspecific positioning” (which in a wide sense, it is still “electrolocation”), and that could be derived from the beat. These parameters might include size, relative orientation, or subtle changes in position during movement. While the experiments discussed in the manuscript do not provide a conclusive answer in this regard, we prioritize here the presentation of broader evidence for a different use of chirping. We are actively working on another manuscript that explores this aspect more in detail, but, due to space limitations, additional results had to be excluded.

    In the abstract we mention a role of chirps in the enhancement of “electrolocation”, but - as above mentioned - it is here meant only in a broad sense. In the introduction (at the very end) we propose chirps as self-directed signals (homeoactive sensing). In the result paragraph dedicated to the novel environment exploration experiment the following lines were added “Most chirps (90%) in fact are produced within a distance corresponding to 1% of the maximum field intensity (i.e. roughly 30 cm; Figure S12B), indicating that chirping occurs way below the threshold range for beat detection (i.e. roughly in the range of 60-120 cm, depending on the study; see appendix 1: Detecting beats at a distance) and likely does not represent a way to improve it”. We conclude this paragraph mentioning “This further corroborates the hypothesized role of chirps in beat processing.”. The last result paragraph (on chirping in cluttered environments) ends with “This supports the notion of chirps as self-referenced probing cues, potentially employed to optimize short-range aspects of conspecific electrolocation, such as conspecific size, orientation, and swimming direction - a hypothesis that will certainly be explored in future studies.”. In the discussion paragraph entitled “probing with chirps”, we do provide hints to possible mechanisms implied in the role of chirps in beat processing. As mentioned, we have planned to add further details in another manuscript, currently in preparation.

    The study provides a wealth of interesting observation of behavior and much of this data constitute a useful dataset to document the patterns of social interactions in these fish. Some data, in particular the high propensity to chirp in cluttered environments, raises interesting questions. Their main hypothesis is a useful addition to the debate on the function of these chirps and is worth being considered and explored further. However, the data they provide does not support strong conclusion statements arguing that these chirps are used for localization purposes and is even less convincing at rejecting previously established hypotheses on the communication purpose of the chirps.

    We intentionally framed our aims a bit provocatively to underscore that, to date, the role of chirps in social communication has been supported solely by correlative evidence. While the evidence we provide to support the role of chirps as probes is also correlative, it opens at the same time critical questions on the long assumed role of chirps in social communication. In fact, chirping is strongly dependent on fish reciprocal positioning, highly constrained by beat frequency, and patterned in such ways that - in our opinion - makes the existence of links between chirp types and internal states less likely, as suggested instead by the current view. Moreover, the use of different chirp types does not appear specific to any of the social contexts analyzed but is primarily explained by DF (beat frequency). This observation, coupled with the analysis of chirp transitions (more self-referenced than reflecting an actual exchange between subjects), leads us to hypothesize with greater confidence that chirp production may be more related to sensing the environment, rather than transmitting information about a specific behavioral state.

    Nevertheless, the Reviewer's comment is valid. We've tempered the study's conclusions by introducing the possibility of chirps serving both communication and electrolocation functions, as stated in the conclusion paragraph: "While our results do not completely dismiss the possibility of chirps serving a role in electrocommunication—probing cues could, for instance, function as proximity signals to signal presence, deter approaches, or coordinate behaviors like spawning (Henninger et al., 2018).". Nonetheless, we do emphasize that our hypothesis is more likely to apply - based on our data. We refrain from categorically excluding a communicative function for chirps (between subjects), but we hypothesize that this communication - if occurring - may contain the same type of information as the self-directed signaling implied by the “chirps as probes” idea (i.e. spatial information).

    In response to the Reviewer's feedback, we've revised the end of the introduction, removing suggestions of conclusiveness: "Finally, by recording fish in different conditions of electrical 'visibility,' we provide evidence supporting a previously neglected role of chirps: homeoactive sensing." (edit: the word “validating” has been removed to give a less “conclusive” answer to the open functional questions about chirping).

    I would suggest thoroughly revising the manuscript to provide a neutral description of the results and leaving any speculations and interpretations for the discussion where the authors should be careful to separate strongly supported hypotheses from more preliminary speculations. I detail below several instances where the argumentation and/or the analysis are flawed.

    Following to the reviewer’s comment, we have revised the manuscript to emphasize the following points: 1) the need for a revision of the current view on chirping, 2) our proposal of an alternative hypothesis based on correlations between chirping and behavior, which were previously unexplored, and 3) our acknowledgment that while we offer evidence supporting a probing role of chirps (e.g., lack of behavioral correlation, DF-dependency, stereotypy in repeated trials, modulation by clutter and distance), we do not present here conclusive evidence for chirps detecting specific details of conspecific positioning. Neither do we exclude categorically a role of chirps in social communication.

    They analyze chirp patterning and show that, most likely, a chirp by an individual is followed by a chirp in the same individual. They argue that it is rare that a chirp elicits a "response" in the other fish. Even if there are clearly stronger correlations between chirps in the same individual, they provide no statistical analysis that discards the existence of occasional "response" patterns. The fact that these are rare, and that the authors don't do an appropriate analysis of probabilities, leads to this unsupported conclusion.

    We employed cross-correlation indices, calculated and assessed with a 3 standard deviation symmetrical boundary (which is a statistically sound and strict criterion). Median values were utilized to depict trends in each group/pair. To support our findings, we added new experiments and new figures: 1) a correlation analysis between chirps and behaviors, providing more convincing evidence of how chirps are employed during "scanning" swimming activity (backward swimming); 2) a text mining approach to underscore chirp-behavior correlations, employing alternative and statistically more robust methods.

    One of the main pieces of evidence that chirps can be used to enhance conspecific localization is based on their "interference" measure. The measure is based on an analysis of "inter-peak-intervales". This in itself is a questionable choice. The nervous system encodes all parts of the stimulus, not just the peak, and disruption occurring at other phases of the beat might be as relevant. The interference will be mostly affected by the summed duration of intervals between peaks in the chirp AM. They do not explain why this varies with beat frequency. It is likely that the changes they see are simply an artifact of the simplistic measure. A clear demonstration that this measure is not adequate comes from the observation in Fig7E-H. They show that the interference value changes as the signal is weaker. This measure should be independent of the strength of the signal. The method is based on detecting peaks and quantifying the time between peaks. The only reason this measure could be affected by signal strength is if noisy recordings affect how the peak detection occurs. There is no way to argue that this phenomenon would happen the same way in the nervous system. Furthermore, they qualitatively argue that patterns of chirp production follow patterns of interference strength. No statistical demonstration is done. Even the qualitative appraisal is questionable. For example, they argue that there are relatively few chirps being produced for DFs of 60 or -60 Hz. But these are DF where they have only a very small sample size. The single pair of fish that they recorded at some of these frequencies might not have chirped by chance and a rigorous statistical analysis is necessary. Similarly, in Fig 5C they argue that the position of the chirps fall on areas of the graph where the interferences are strongest (darker blue) but this is far from obvious and, again, not proven.

    We would like to clarify that the estimation of the effects of chirps on the beat (referred to as “beat interference”) was not intended to serve as the primary evidence supporting a different use of chirping. In fact, all the experiments conducted prior to that calculation already provide substantial evidence supporting the hypothesis we have proposed. In an attempt to address the Reviewer’s concern and to avoid misleading interpretations, we moved this part now to the Supplementary Information (see now Figures S8 and S9), in agreement with the non crucial relevance of this approach. We also added the following statement to the result paragraph entitled “Chirps significantly interfere with the beat and enhance electric image contrast”: “Obviously, measuring chirp-triggered beat interferences by using an elementary outlier detection algorithm on the distribution of beat cycles does not reflect any physiological process carried out by the electrosensory system and can be therefore used only as an oversimplified estimate.”.

    Regarding the meaning of “beat interference” (as here estimated) from a perspective of brain physiology: chirp interference was calculated using the beat cycles as a reference. Beat peaks were used only to estimate beat cycle duration. Regardless of whether or not a beat peak is represented in the brain, beat cycle duration (estimated using the peaks) is the main determinant of p-unit rhythmic response to a beat. Regarding the effect of signal amplitude, this is also not very relevant. It is obvious that a chirp creates more - or less - interference based on the chirp FM and its duration (but also the sign of the DF and the magnitude of the amplitude modulation). If electroreceptor responses are entrained in waves of beat AMs and if “interference” is a measure of how such waves are scrambled, then “interference” is a measure of how chirps scramble waves of electroreceptor activity by affecting beat AMs.

    The reason why the interference fades with the signal (previous figure 7, now Figure S12) is because it is weighted on the signal strength (the signals used as carrier for chirps are recalculated based on real measurements of signal strength at different distances). Nonetheless, the Reviewer is right: mathematically speaking interference would not change at all because it is just the result of an outlier detection algorithm. This outlier detection is actually set to have a 1% threshold (percent of beat contrast).

    Regarding the comparison “chirps vs interference”, we did not make a statistical analysis because we wanted to just show a qualitative observation. Similar results can be obtained for slightly shorter or longer time windows, within certain limits of course (see added Figure S9, in the Supplementary Information). We hope that moving this analysis to the supplementary information makes it clear that this approach is not central to make our point.

    The Reviewer’s point on the DF sampling is correct, we have reconsidered the low chirping at 60Hz as potentially the result of sampling bias and edited the respective result paragraph.

    They relate the angle at which one fish produces chirps relative to the orientation of the mesh enclosing. They argue that this is related to the orientation of electric field lines by doing a qualitative comparison with a simplified estimate of field lines. To be convincing this analysis should include a quantitative comparison using the exact same body position of the two fish when the chirps are emitted.

    We agree with the Reviewer, this type of experiment would be much better suited to illustrate the correlations between chirping and reciprocal positioning in fish. What we can see is that chirping occurs at certain orientations more often than others. This could have something to do with either field geometry or with locomotion in the particular test environment we have used. As mentioned earlier, we are currently editing a second manuscript which will include the type of analysis/experiment the Reviewer is thinking of. We preferred to focus in this first study on the broader behavioral correlates of chirping. We removed the mention to the field current lines because - we agree - the argument is vague as presented here.

    They show that the very vast majority of chirps in Fig 6 occur when the fish are within a few centimeters (e.g. very large first bin in Fig6E-Type2). This is a situation when the other fish signal will be strongest and localization will be the easiest. It is hard to understand why the fish would need a mechanism to enhance localization in these conditions (this is the opposite of difficult conditions e.g. the "cluttered" environment).

    Agreed, in fact we do not explicitly propose chirps as means to improve “electrolocation” (this word is used only broadly in the abstract) but instead as probes to extract spatial information (e.g. shape, motion, orientation) from a beat source. In a broader sense, all these spatial parameters contribute to any given instance of "localization." Because we were unable to explore all these aspects in greater detail, we chose to maintain a broader perspective. If chirps contribute to a better resolution of fine spatial attributes of conspecific locations, it is reasonable to expect higher chirping rates in proximity to the target fish.

    The argumentation aimed at rejecting the well-established role of chirp in communication is weak at best. First, they ignored some existing data when they argue that there is no correlation between chirping and behavioral interactions. Particularly, Hupe and Lewis (2008) showed a clear temporal correlation between chirps and a decrease in bites during aggressive encounters. It could be argued that this is "causal evidence" (to reuse their wording) that chirps cause a decrease in attacks by the receiver fish (see Fig 8B of the Hupe paper and associated significant statistics). Also, Oboti et al. argue that social interactions involve "higher levels of locomotion" which would explain the use of chirps since they are used to localize. But chirps are frequent in "chirp chamber" paradigms where no movement is involved. They also point out that social context covaries with beat frequency and thus that it is hard to distinguish which one is linked to chirping propensity and then say that it is hard to disentangle this from "biophysical features of EOD fields affecting detection and localization of conspecific fish". But they don't provide any proof that beat frequency affects detection and localization so their argument is not clear. Last, they argue that tests in one species shouldn't be extrapolated to other species. But many of the studies arguing for the role of chirps in communication was done on brown ghost. In conclusion of this point, they do not provide any strong argument that rejects the role of chirps as a communication signal. A perspective that would be better supported by their data and consistent with past research would be to argue that, in addition to a role in communication, chirps could sometimes be used to help localize conspecifics.

    We did not intend to disregard the extensive body of literature supporting a role of chirps in social communication. Rather, the primary goal of this study was to present a valid alternative perspective to this prevailing view. The existence of a well-established hypothesis does not imply that new evidence cannot change it; it simply indicates that changing it may be challenging either because it's genuinely difficult or because the idea has not been thoroughly explored. Whatever the case may be, proposing new hypotheses, whether complementary or alternative to established theories, is a challenging undertaking for a single study. We judged that starting from broad correlations would be the most desirable approach.

    We did not ignore data from Hupé and Lewis 2008. We cited this study repeatedly and compared their findings to those of others, not only for the correlation chirp-behaviors but also for chirping distance considerations. However, following the Reviewer’s comment, we now cite this study in the context of the behavioral analysis recently added (data from the PSTH plots could possibly confirm the observation of lower chirps during attacks). We also cited the study by Triefenbach and Zakon 2008, which reports something along the same lines. See the statement: “Overall, these results provided mutually reinforcing evidence indicating that chirps are produced more often during locomotion or scanning-related motor activity and confirm previous reports of a lower occurrence of chirping during more direct aggressive contact (as shown also by Triefenbach and Zakon, 2008; Hupé and Lewis, 2008).”, in the result paragraph related to the behavioral correlates of chirping.

    In our study we make it clear how we distinguish causal evidence (i.e. providing evidence that A is required for B) from correlation (i.e. evidence for A simply occurring together with B). We also make it clear that we are not going to provide causal evidence but we are going to provide new evidence for correlations that were so far not considered, in order to propose a new unexplored function of chirps.

    The Reviewer's point on chirping during motion and while caged in a chirp chamber is valid. Indeed at first we were also puzzled by this finding. However, under the “chirps as probes” paradigm, chirping in a chirp-chamber can be explained by the need to obtain spatial information from an otherwise unreachable beat source (brown ghosts are typically exploring new environmental objects or conspecifics by actively swimming around them - something caged fish can’t do). So, eventually the observation of chirping under conditions of limited movement (such as in a chirp chamber experiment) is not in contradiction with our hypothesis, rather it can be used to support it. Further experiments are required - as rightfully pointed out - to evaluate the effects of beat frequency on beat detection. We added a note about this in the “probing with chirps” discussion paragraph.

    The Reviewer's comment regarding generalization is unclear. We acknowledge that most studies are conducted in brown ghosts, as stated in the abstract. Our intention was to highlight that insights gained from this species have been applied to broaden the understanding of chirps in other species. Specifically, the "behavioral meaning idea" of chirping has been extended to other gymnotiform species producing EOD frequency modulations .

    Our study's aim is not to dismiss the idea of chirps being used for communication but to present an alternative hypothesis and to provide supporting evidence. While our results may not align well with the communication theory, our intention is not dismissal but rather engaging in a discussion and exploration of alternative perspectives.

    The discussion they provide on the possible mechanism by which chirps could help with localization of the conspecific is problematic. They imply that chirps cause a stronger response in the receptors. For most chirps considered here, this is not true. For a large portion of the beat frequencies shown in this paper, chirps will cause a de-synchronization of the receptors with no increase in firing rate. They cannot argue that this represents an enhanced response. They also discuss a role for having a broader frequency spectrum -during the chirp- in localization by making a parallel with pulse fish. There is no evidence that a similar mechanism could even work in wave-type fish.

    We have already commented on the “localization” idea in our previous responses. The Reviewer is right in saying that we have provided only vague descriptions of the potential mechanisms implied by our hypothesis. The studies by Benda and others (2005, 2006) demonstrate a clear synchronizing effect of chirps on p-unit firing rates, especially at low DFs (at ranges similar to those considered in this study). This synchronization could lead to an enhanced response at the electroreceptor level, as described in these very studies, which in turn would result in a higher probability of firing in downstream neurons (E-cells in the ELL).

    As also reported within the same works, chirps may also exert an opposite effect on p-units (i.e. desynchronization). This is what happens for large chirps at high DFs. Desynchronization may cause temporary lapses of p-unit firing, which in turn may lead to increased activity of I-cells in the ELL (which are indeed specifically tuned to p-unit lack of activity).

    So, in general, if we consider both ON and OFF pyramidal cells (in the ELL) and small and large chirps, we could state that chirps can be potentially used to enhance the activity of peripheral electrosensory circuits through different mechanisms, contingent on the chirp type and beat frequency. Unfortunately, space constraints limited our ability to dig into these details in the present study.

    However, to address the Reviewer’s rightful point, we now mention this in the manuscript: Since the beat AMs generated by the chirps always trigger reliable responses in primary electrosensory circuits (pyramidal cells in the ELL respond to both increases and decreases in beat AM), any chirp-triggered AM causing a sudden change in p-unit firing could potentially amplify the downstream signal (Marsat and Maler, 2010) and thus enhance EI contrast.” (see result paragraph on beat interference and electric images).

    They write the whole paper as if males and females had been identified in their experiments. Although EOD frequency can provide some guess of the sex the method is unreliable. We can expect a non-negligible percentage of error in assigning sex.

    We agree and in fact, in the method section we state:

    “The limitation of this approach is that females cannot be distinguished from immature males with absolute certainty, since no post-mortem gonadal inspection was carried out.”

    to this we added:

    “Although a more accurate way to determine the sex of brown ghosts would be to consider other morphological features such as the shape of the snout, the body size, the occurrence of developing eggs, EOD frequency has been extensively used for this purpose.”

    Moreover, the consistent behavioral differences observed in low frequency fish, measured with those behavioral experiments aimed at assessing responses to playback stimuli and swimming behavior in novel environments, could also be caused by a younger age (as opposed to femaleness). However, the size ranges of our fish (an admittedly unreliable proxy of age) were all comparable, making this possibility perhaps less likely.

    Reviewer #2 (Public Review):

    Studying the weakly electric brown ghost knifefish, the authors provide evidence that 'chirps' (brief modulations in the frequency and amplitude of the ongoing electric signal) function in active sensing (specifically homeoactive sensing) rather than communication. This is a behavior that has been very well studied, including numerous studies on the sensory coding of chirps and the neural mechanisms for chirp generation. Chirps are largely thought to function in communication behavior, so this alternative function is a very exciting possibility that could have a great impact on the field. The authors do provide convincing evidence that chirps may function in homeoactive sensing. However, their evidence arguing against a role for chirps in communication is not as strong, and neglects a large body of research. Ultimately, the manuscript has great potential but suffers from framing these two possibilities as mutually exclusive and dismissing evidence in favor of a communicative function.

    We thank the Reviewer for the comment. Overall, we have edited the manuscript to soften our conclusions and avoid any strong categorical statement excluding the widely accepted role of chirps in social communication. We have added some new experiments with the aim to add more detail to the behavioral correlates of chirping and to the DF dependency of the production of different types of chirps. Nonetheless, based on our results, we are prone to conclude that the communication idea - although widely accepted - is not as well substantiated as it should be.

    Although we do not dismiss the bulk of literature supporting a role of chirps in social communication, we think that our hypothesis (i.e. decoding of spatial parameters from the beat) may be not fully compatible with the social communication hypothesis for the following reasons:

    (1) Chirp type dependency on DF makes chirps likely to be adaptive responses to beat frequency. While this idea is compatible with a role of chirps in the detection of beat parameters, their concurrent role in social communication would imply that chirps interacting at given beat frequencies (DFs) would communicate only (or mainly) by delivering a very limited range of “messages”. For instance, assuming type 2 chirps are related to aggression (as widely suggested), are female-male pairs - with larger DFs - interacting less aggressively than same sex pairs? Our experiments often suggested this is not the case. In addition, large DFs are not always indicative of opposite sex interactions, while they are very often characterized by the emission of large chirps. Not to mention that, despite the fact that opposite sex interactions in absence of breeding-like conditions, cannot be considered truly courtship-related, large chirps are often considered courtship signals, regardless of the reproductive state of the emitting fish.

    (2) Chirping is highly affected by locomotion (consider female/male pairs with or without mesh divider) and distance (as shown in the novel environment exploration experiment). While the involvement of both parameters is compatible with a role of chirps in active sensing, a role of chirps in social communication implies that such signaling would occur only when fish are in very close proximity to each other. In this case, the beat is therefore heavily distorted not only by fish position/locomotion but also by chirps. Which means that when fish are close to each other, the 2 different types of information relayed by the beat (electrolocation and electrocommunication) would certainly interfere (this idea has been better phrased in the Introduction paragraph).

    (3) In our playback experiments we could not see any meaningful matching (e.g. angry-chirp → angry-chirp or sexy-chirp → approach) between playback chirps and evoked chirps, raising doubts on the meaning associated so far with the different types. Considering that playback experiments are typically used to assess signal meaning based on how animals respond to them, this result is suggesting quite strongly that such meaning cannot be assigned to chirps.

    (4) In playback experiments in which the same stimulus is provided multiple times, chirp type transitions (i.e. emission of a different chirp type after a given chirp) become predictable (as shown in the added playback experiments using ramping signals). This confirms that the choice to emit a given chirp type has something to do with beat frequency (or a change in this parameter) and not a communication of internal states. It would be otherwise unclear how a fish could change its internal state so quickly - and so reliably - even in the span of a few seconds.

    Despite this evidence against a semantic content of chirps in the context of social communication, we conclude the manuscript reminding that we are not providing strong evidence dismissing the communication hypothesis, and that both could coexist (see the example of “proximity signals” in the mating context given in the concluding paragraph).

    (1) The specific underlying question of this study is not made clear in the abstract or introduction. It becomes apparent in reading through the manuscript that the authors seek to test the hypothesis that chirps function in active sensing (specifically homeoactive sensing). This should be made explicitly clear in both the abstract and introduction, along with the rationale for this hypothesis.

    In the abstract we state “Despite the success of this model in neuroethology over the past seven decades, the underlying logic of their electric communication remains unclear. This study re-evaluates this view, aiming to offer an alternative, and possibly complementary, explanation for why these freshwater bottom dwellers emit electric chirps.”. This statement is meant as a summary of our aims. However, in order to convey a clearer message, we have revised the whole manuscript to more explicitly articulate our objectives. In particular we stress that with our experiments we intend to provide correlative evidence for a different role of chirps (previously unexplored) with the idea to stimulate a discussion and possibly a revision of the current theory about the functional role of chirps.

    In the introduction we have added a paragraph explaining our aim and also why we think that communicating through chirps could potentially interfere with efficient electrolocation: “Since both chirps and positional parameters (such as size, orientation or motion) can only be detected as perturbations of the beat (Petzold et al., 2016; Yu et al., 2012; Fotowat et al., 2013), and via the same electroreceptors, the inputs relaying both types of information are inevitably interfering. Moreover, as the majority of chirps are produced within a short range (< 50 cm; Zupanc et al., 2006; Hupé and Lewis 2008; Henninger et al., 2018; see appendix 1) this interference is likely to occur consistently during social interactions.

    Under the communication-hypothesis, the assumption that chirps and beats are conveying different types of information (i.e. semantic value as opposed to position and related geometrical parameters) is therefore leaving this issue unresolved.”.

    (2) My biggest issue with this manuscript is that it is much too strong in dismissing evidence that chirping correlates with context. This is captured in this sentence in the introduction, "We first show that the choice of different chirp types does not significantly correlate with any particular behavioral or social context." This very strong conclusion comes up repeatedly, and I disagree with it, for the following reasons:

    In your behavioral observations, you found sex differences in chirping as well as differences between freely interacting and physically separated fish. Your model of chirp variability found that environmental experience, social experience, and beat frequency (DF) are the most important factors explaining chirp variability. Are these not all considered "behavioral or social context"? Beat frequency (DF) in particular is heavily downplayed as being a part of "context" but it is a crucial part of the context, as it provides information about the identity of the fish you're interacting with.

    In your playback experiments, fish responded differently to small vs. large DFs, males chirped more than females, type 2 chirps became more frequent throughout a playback, and rises tended to occur at the end of a playback. These are all examples of context-dependent behavior.

    We agree with the Reviewer’s comment and we think that probably we have been unclear in what the meaning of that statement was. We also agree with the Reviewer about what is defined as “context”, and that a given beat frequency (DF) can in the end represent a “behavioral context” as well. In order to make it clearer, we have rephrased this statement and changed it to: “We first show that the relative number of different chirp types in a given recording does not significantly correlate with any particular behavioral or social context.”. This new form refers specifically to the observation that - in all different social conditions examined - the relative amounts of different types of chirps is unchanged (see Figure S2). We thought the Reviewer maybe interpreted our statement as if we suggested that chirp type choice is random or unaffected by any social variable. We agree with the Reviewer that this is not the case. We also reported that sex differences in chirping are present, but we have emphasized they may have something to do with the propensity of the brown ghosts of either sex to swim/explore as opposed to seek refuge and wait (as suggested by our experiments in which FM pairs were either divided or freely interacting and our novel environment exploration experiments).

    We agree DF is important, in fact it is the 3rd most important factor explaining chirp variance in our model. In our fish pair recordings, we see a strong correlation of chirp total variance with tank experience (one naïve, one experienced, both fish equally experienced) and social context (novel to each other/familiar to each other, subordinate/dominant, breeding/non breeding, accessible/not accessible) although data clustering seems to better distinguish “divided” vs “freely moving” conditions (and sex may also play a role as well because of the reversal of sexual dimorphism in chirp rates in precisely this case) more than other variables. However, we do not see a specific effect of these variables on the proportion of different types of chirps in any recording (see Figure S2).

    We also edited the beginning of the first result paragraph and changed it to “Thus, if behavioral meaning can be attributed to different types of chirps, as posed by the prevailing view (e.g., Hagedorn and Heiligenberg, 1985; Larimer and MacDonald, 1968; Rose, 2004), one should be able to identify clear correlations between behavioral contexts characterizing different internal states and the relative amounts of different types of chirp”, to emphasize we are here assessing the meaning of different types of chirps (not of the total amount of chirping in general).

    Further, you only considered the identity of interacting fish or stimulated fish, not their behavior during the interaction or during playback. Such an analysis is likely beyond the scope of this study, but several other studies have shown correlations between social behavior and chirping. In the absence of such data here, it is too strong to claim that chirping is unrelated to context.

    We agree with the Reviewer, in fact this analysis was previously carried out but purposely left out in an attempt to limit the manuscript length. We have now made space for this experimental work which is now added (see the new Figure 6).

    In summary, it is simply too strong to say that chirping does not correlate with context. Importantly, however, this does not detract from your hypothesis that chirping functions in homeoactive sensing. A given EOD behavior could serve both communication and homeoactive sensing. I actually suspect that this is quite common in electric fish. The two are not mutually exclusive, and there is no reason for you to present them as such. I recommend focusing more on the positive evidence for a homeoactive function and less on the negative evidence against a communication function.

    We aimed to clarify that our reference was to the lack of correlation between "chirp type relative numbers" and the analyzed context. Regarding the communication function, we tempered negative statements. However, as this study stems from evidence within the established paradigm of "chirps as communication signals", and aims at proposing an alternative hypothesis, eliminating all references to it could undermine the study's purpose.

    (3) The results were generally challenging to follow. In the first 4 sections, it is not made clear what the specific question is, what the approach to addressing that question is, and what specific experiment was carried out (the last two sections of the results were much clearer). The independent variables (contexts) are not clearly established before presenting the results. Instead they are often mentioned in passing when describing the results. They come across as an unbalanced hodgepodge of multiple factors, and it is not made clear why they were chosen. This makes it challenging to understand why you did what you did, the results, and their implications. For each set of major results, I recommend: First, pose a clear question. Then, describe the general approach to answering that question. Next, describe the specifics of the experimental design, with a rationale that appeals to the general approach described. Finally, describe the specific results.

    The introductory sentences of the first result paragraphs have been edited, rendering the aim of the experiments more explicit.

    (4) Results: "We thus predicted that, if behavioral meaning can be attributed to different types of chirps, as posed by the prevailing view (e.g., Hagedorn and Heiligenberg, 1985; Larimer and MacDonald, 1968; Rose, 2004)..." It should be made clear why this is the prevailing view, and this description should likely be moved to the introduction. There is a large body of evidence supporting this view and it is important to be complete in describing it, especially since the authors seem to seek to refute it.

    We understand the Reviewer’s question and we tried to express in the introduction the main reasons for why this is the current view. We state “Different types of chirps are thought to carry different semantic content based on their occurrence during either affiliative or agonistic encounters (Larimer and MacDonald 1968; Bullock 1969; Hopkins 1974; Hagedorn and Heiligenberg 1985; Zupanc and Maler 1993; Engler et al. 2000; Engler and Zupanc 2001; Bastian et al., 2001).”. To this we added: “Although supported mainly by correlative evidence, this idea gained popularity because it is intuitive and because it matches well enough with the numerous behavioral observations of interacting brown ghosts.”.

    We believe the prevailing view is based on intuition and a series of basic observed correlations repeated throughout the years. The crystallization of this idea is not due to negligence but mainly to technical limitations existing at the time of the first recordings. In order to assess the role of chirps in behaving fish a tight and precise temporal control over synched video-EOD recordings is most likely necessary, and this is a technical feature probably available only much later than the 50-60ies, when electric communication was first described.

    (5) I am not convinced of the conclusion drawn by the analysis of chirp transitions. The transition matrices show plenty of 1-2 and 2-1 transitions occurring. Further, the cross-correlation analysis only shows that chirp timing between individuals is not phase-locked at these small timescales. It is entirely possible that chirp rates are correlated between interacting individuals, even if their precise timing is not.

    We agree with the Reviewer: chirp repertoires recorded in different social contexts are not devoid of reciprocal chirp transitions (i.e. fish 1 chirp - to - fish 2 chirp, or vice versa). Yet our point is to emphasize that their abundance is way more limited when compared to the self-referenced ones (i.e. 1-1 and 2-2). This is a fair concern and in order to further address this point, we have added a whole new set of analyses and new experiments (see chirp-behavior correlations, PSTHs and more analysis based on more solid statistical methods; see Figure 6).

    Reviewer #3 (Public Review):

    Summary:

    This important paper provides the best-to-date characterization of chirping in weakly electric fish using a large number of variables. These include environment (free vs divided fish, with or without clutter), breeding state, gender, intruder vs resident, social status, locomotion state and social and environmental experience, as well as with playback experiments. It applies state-of-the-art methods for reducing dimensionality and finding patterns of correlation between different kinds of variables (factor analysis, K-means). The exceptional strength of the evidence, collated from a large number of trials with many controls, leads to the conclusion that a number of commonly accepted truths about which variable affects chirping must be carefully rewritten or nuanced. Based on their extensive analyses, the authors suggest that chirps are mainly used as probes that help detect beats and objects.

    Strengths:

    The work is based on completely novel recordings using interaction chambers. The amount of new data and associated analyses is simply staggering, and yet, well organized in presentation. The study further evaluates the electric field strength around a fish (via modelling with the boundary element method) and how its decay parallels the chirp rate, thereby relating the above variables to electric field geometry.

    The main conclusions are that the lack of any significant behavioural correlates for chirping, and the lack of temporal patterning in chirp time series, cast doubt on a communication goal for most chirps. Rather, the key determinants of chirping are the difference frequency between two interacting conspecifics as well as individual subjects' environmental and social experience. These conclusions by themselves will be hugely useful to the field. They will also allow scientists working on other "communication" systems to at least reconsider, and perhaps expand the precise goal of the probes used in those senses. There are a lot of data summarized in this paper, and thorough referencing to past work. For example, the paper concludes that there is a lack of evidence for stereotyped temporal patterning of chirp time series, as well as of sender-received chirp transitions beyond the known increase in chirp frequency during an interaction.

    The alternative hypotheses that arise from the work are that chirps are mainly used as environmental probes for better beat detection and processing and object localization.

    The authors also advance the interesting idea that the sinusoidal frequency modulations caused by chirps are the electric fish's solution to the minute (and undetectable by neural wetware) echo-delays available to it, due to the propagation of electric fields at the speed of light in water.

    Weaknesses:

    My main criticism is that the alternative putative role for chirps as probe signals that optimize beat detection could be better developed. The paper could be clearer as to what that means precisely.

    We appreciate the Reviewer's kind comments. While we acknowledge that our exploration of chirp function in this study may be limited and not entirely satisfying, we made this decision due to space constraints, opting for a broader and diversified approach. We hope that future studies will build on these data and start filling the gaps. We are also working on another manuscript which is addressing this point more in detail.

    Nonetheless, we considered the Reviewer’s criticism and added not only a new figure (to show more explicitly what chirps can do to the perceived electric fields, as simulated by electric images) but also more descriptive parts explaining how we think chirps may act to improve the spatial resolution of beat processing (see the discussion paragraph “probing with chirps”). In this paragraph we rendered more clearly how chirps could improve beat processing by phase shifting EODs and recovering eventual blind-spots on the fish skin caused by disruptive EOD interferences (resulting in lower beat contrast). We also mention that enhancement of electrosensory input triggered by chirps, could be localized not only at the level of electroreceptors (consider the synchronizing effects small chirps have on p-units at low frequency beats) but also at the level of ON and OFF pyramidal cells in the ELL. Looked at from the perspective of these neurons, any chirp would enhance the activity of these input lines, yet in opposite ways.

    And there is an egg-and-chicken type issue as well, namely, that one needs a beat in order to "chirp" the beating pattern, but then how does chirping optimize the detection of the said beat? Perhaps the authors mean (as they wrote elsewhere in the paper) that the chirps could enhance electrosensory responses to the beat.

    According to the Reviewer’s comment, we have now revised several instances of the misleading phrasing identified.

    In the results on novel environment exploration: “If chirps enhance beat processing, for instance, chirping should occur within beat detection range but at a certain distance.”.

    “This, in turn, could be used to validate our beat-interference estimates as meaningfully related to beat processing.” and “In all this, rises may represent an exception as their locations are spread over larger distances and even in presence of obstacles potentially occluding the beat source (such as shelters, plants, or walls), all of which are conditions in which beat detection or beat processing could be more difficult (this, could be coherent with the production of rises right at the end of EOD playbacks; Figure S5).”

    Last result paragraph (clutter experiment): “Overall, these results indicate that chirping is significantly affected by the presence of environmental clutter partially disrupting - or simply obstructing - the processing of beat related information during locomotion”.

    In the probing with chirps discussion paragraph “In theory, chirps could also be used to improve electrolocation of objects as well (as opposed to the processing of the beat).”.

    In the conclusions: “optimizing the otherwise passive responses to the beat”.

    A second criticism is that the study links the beat detection to underwater object localization. I did not see a sufficiently developed argument in this direction, nor how the data provided support for this argument. It is certainly possible that the image on the fish's body of an object in the environment will be slightly modified by introducing a chirp on the waveform, as this may enhance certain heterogeneities of the object in relation to its environment. The thrust of this argument seems to derive more from the notion of Fourier analysis with pulse type fish (and radar theory more generally) that the higher temporal frequencies in the beat waveform induced by the chirp will enable a better spatial resolution of objects. It remains to be seen whether this is significant.

    The Reviewer is correct in noting that this point is not addressed in the manuscript. We introduced it as a speculative discussion point to mention alternative possibilities. These could be subject to further testing in future studies.

    I would also have liked to see a proposal for new experiments that could test these possible new roles.

    We have added clearer suggestions for future experiments throughout the discussion: these may be aimed at 1) improving playback experiments using more realistic copies of the brown ghost’s EODs (including harmonics), 2) assess fish reciprocal positioning during chirping in better detail and 3) test the use of chirping during target-reaching tasks in order to better assess the probing function of chirps.

    The authors should recall for the readers the gist of Bastian's 2001 argument that the chirp "can adjust the beat frequency to levels that are better detectable" in the light of their current. Further, at the beginning of the "Probing with chirps" section, the 3rd way in which chirps could improve conspecific localization mentions the phase-shifting of the EOD. The authors should clarify whether they mean that the tuberous receptors and associated ELL/toral circuitry could deal with that cue, or that the T_unit pathway would be needed?

    We thank the Reviewer for identifying this unclear point. We added reference to the p-units “Yet, this does not exclude the possibility that chirps could be used to briefly shift the EOD phase in order to avoid disruptive interferences caused by phase opposition (at the level of p-units)” in the above mentioned paragraph. We would prefer to omit a more detailed reference to t-units in order to avoid lengthy descriptions required to discuss the different electroreceptor types.

    On p.17 I don't understand what is meant by most chirps being produced, possibly aligned with the field lines, since field lines are everywhere. And what is one to conclude from the comparison of Fig.6D and 7A? Likewise it was not clear what is meant by chirps having a detectable effect on randomly generated beats.

    We agree on the valid point raised by the Reviewer and we have removed reference to current lines from the text.

    In the section on Inconsistencies between behaviour and hypothesized signal meaning, the authors could perhaps nuance the interpretation of the results further in the context of the unrealistic copy of natural stimuli using EOD mimics. In particular, Kelly et al. 2008 argued that electrode placement mattered in terms of representation of a mimic fish onto the body of a real fish, and thus, if I properly understand the set up here, the movement would cause the mimic to vary in quality. This may nevertheless be a small confounding issue.

    We agree with the Reviewer and added a comment at the beginning of the paragraph mentioned. “Nonetheless, it's plausible that playback stimuli, as employed in our study and others, may not faithfully replicate natural signals, thus potentially influencing the reliability of the observed behaviors. Future studies might consider replicating these findings using either natural signals or improved mimics, which could include harmonic components (excluded in this study).”

    Recommendations for the authors:

    8Reviewer #2 (Recommendations For The Authors):*

    (1) Abstract: "...is probably the most intensely studied species..." is a weak, unsupported, and unnecessary statement. Just state that it has been heavily studied, or is one of the most well-studied,...

    rephrased

    (2) Abstract: "...are thus used as references to specific internal states during recordings - of either the brain or the electric organ..." This was not clear to me.

    rephrased

    (3) Abstract: "...the logic underlying this electric communication..." It is not clear to me what the authors mean here by "logic".

    rephrased

    (4) I strongly recommend clearly defining homeoactive sensing and distinguishing it from allocative sensing when this term is first introduced in the introduction. This is not a commonly used term. Most readers likely think they understand what is meant by the term active sensing, however I recommend first defining it, and then distinguishing amongst these two different types of active sensing.

    rephrased

    (5) Introduction: "Together with a few other species (Rose, 2004),..." More than a few. There are hundreds of species with electric organs. It is certainly not a "unique" capability.

    rephrased

    (6) Introduction: "But the real advantage of active electrolocation can be appreciated in the context of social interaction." This is unclear. Why is this the "real advantage" of active electrolocation when an electrically silent fish could detect an electrically communicating fish just fine without interference? Active electrolocation is needed to detect objects that are not actively emitting an electric field. It is not needed to detect signaling individuals.

    rephrased

    (7) Introduction: why is active sensing using EODs limited to distances of 6-12 cm? Why does it not work at closer range?

    Here we meant to give a range based on published data. We rephrased it to “up to 12”.

    (8) Introduction: electric fields decay with the cubed of distance, as you show in appendix 1.

    rephrased

    (9) Introduction: it is not clear what is meant by "blurred EOD amplitude".

    rephrased (“noisy”)

    (10) Figure 2C is very challenging to interpret. I recommend spending more time in the manuscript walking the reader through this analysis and its presentation.

    We are grateful for the comment as we probably overlooked this point. We now added a small paragraph to explain these data in better detail.

    (11) Results: "This was done by calculating the ratio between the duration of the beat cycles affected by the chirp (beat interpeak intervals) and the total duration of the beat cycles detected within a fixed time window (roughly double the size of the maximum chirp duration, 700 ms)." This was not clear to me.

    We now rephrased to “Estimates of beat interference were made by calculating the ratio between the cumulative duration of the beat cycles affected by a given chirp (1 beat cycle corresponding to the beat comprised by two consecutive beat peaks, or - more simply - the beat inter-peak interval) over the cumulative duration of all the beat cycles within the time window used as a reference (700 ms; other analysis windows were tested Figure S9)” to clarify this method.

    (12) Results: "For each chirp, the interference values obtained for 4 different phases (90{degree sign} steps) were averaged." Why was this done?

    To consider an average effect across phases. Although it is true that chirp parameters may have a different impact on the beat, depending on EOD phase, including this parameter in our figure/s would have considerably increased the volume of data reported giving too much emphasis to an analysis we judged not crucially important. In addition, since we did not consider EOD phase in our recordings, we opted for an average estimate encompassing different phase values.

    (13) Discussion: "Third, observations in a few species are generalized to all other gymnotiforms without testing for species differences (Turner et al., 2007; Smith et al., 2013; Petzold et al., 2016)." I strongly disagree with this statement. First, the studies referenced here do explicitly compare chirps across species. Second, you only studied one species here, so it is not clear to me how this is a relevant concern in interpreting your findings.

    Here we have probably been unclear in the writing: the point we wanted to make is that the idea of chirps having semantic content has been generalized to other species without investigating the nature of their chirping with as much detail as done for brown ghosts.

    We have now rephrased the statement and changed it to: “Second, observations in a few species are generalized to all other gymnotiforms without testing whether chirping may have similar functions in other species (Turner et al., 2007; Smith et al., 2013; Petzold et al., 2016)”

    (14) Discussion: "The two beats could be indistinguishable (assuming that the mechanism underlying the discrimination of the sign of DF at low DFs, and thought to be the basis of the so called jamming avoidance response (JAR; Metzner, 1999), is not functional at higher DFs)." Why would you assume this?

    What we meant here is that it is unlikely that the two DFs are not discriminated by the same mechanisms implied in the JAR, even if the DF is higher than the levels at which usually JARs are detected (i.e. DF = 1-10 Hz?). To improve clarity, we rephrased this statement. “The two beats could be indistinguishable (assuming - perhaps not realistically - that the same mechanism involved in DF discrimination at lower DF values would not work in this case; Metzner, 1999)”.

    (15) Discussion: "...an idea which seems congruent with published electrophysiological studies..." How so?

    Rephrased to “Based on our beat interference estimates, we propose that the occurrence of the different types of chirps at more positive DFs (such as in male-to-female chirping) may be explained by their different effect on the beat (Figure 5D; Benda et al., 2006; Walz et al., 2013).”

    Reviewer #3 (Recommendations For The Authors):

    On p.2 there is a discrepancy between the quoted ranges for active sensing of objects, first 10-12 cm, and then 6-12 cm further down. And in the following paragraph right below this passage, electric fields are said to decay with the squared distance (appendix 1). That expression has a cos(theta) which is inversely proportional to the distance, and so one is really dealing, as expected for dipolar fields, with a drop-off that decays with the distance cubed.

    We thank the Reviewer for the comment, we have now corrected the mistake and added “cubed”. We also removed the imprecise reference to the range 6-12 cm, rephrased to “up to 12 cm”.

    At the end of the section on Inconsistencies..., it is not clear what "activity levels" refers to. It should also be made clearer at the outset, and reminded in this section too, that for the authors, behavioural context does not include social experience, which is somewhat counter-intuitive.

    We now specified we meant “locomotor activity levels”. Regarding the social experience we included it as “behavioral context”, we now made it clearer in the first result paragraph. We hope we resolved the confusion.

    The caption of Fig.8 could use more clarity in terms of what is being compared in (C) (and is "1*2p" a typo?)

    We corrected the typo and edited the figure to make the references more clear.

    The concept of "high self-correlation of chirp time series" is presented only in the Conclusion using those words. The word self-correlation is not used beforehand. This needs to be fixed so the reader knows clearly what is being referred to.

    Thank you for noting this. We have now changed the wording using the term “auto-correlation” and changed a statement at the beginning of the “interference” result paragraph accordingly, removing references to self-correlation.

  12. eLife

    eLife assessment

    This is a valuable contribution to the electric fish community, and to studies of active sensing, in that it provides evidence that a well-studied behavior (chirping) may serve in active sensing rather than communication. This is likely to stimulate follow-up behavioral and physiological studies to determine whether the active sensing component of the behavior is pre-eminent, or whether their major function is communication. For the most part, the evidence for increased chirping in more cluttered environments and the relationship between chirping and movement are convincing. However, the evidence used to argue that chirping does not vary with behavioral context is less so, and the arguments against a communicative function of chirps are not strong. The main conclusions are only supported by correlations and remain for now at the level of an interesting hypothesis to explore.

  13. eLife

    Reviewer #1 (Public Review):

    The authors investigate the role of chirping in a species of weakly electric fish. They subject the fish to various scenarios and correlate the production of chirps with many different factors. They find major correlations between the background beat signals (continuously present during any social interactions) or some aspects of social and environmental conditions with the propensity to produce different types of chirps. By analyzing more specifically different aspects of these correlations they conclude that chirping patterns are related to navigation purposes and the need to localize the source of the beat signal (i.e. the location of the conspecific).

    The study provides a wealth of interesting observations of behavior and much of this data constitutes a useful dataset to document the patterns of social interactions in these fish. Some data, in particular the high propensity to chirp in cluttered environments, raises interesting questions. Their main hypothesis is a useful addition to the debate on the function of these chirps and is worth considering and exploring further.

    After the initial reviewers' comments, the authors performed a welcome revision of the way the results are presented. Overall the study has been improved by the revision. However, one piece of new data is perplexing to me. The new Figure 7 presents the results of a model analysis of the strength of the EI caused by a second fish to localize when the focal fish is chirping. From my understanding of this type of model, EOD frequency is not a parameter in the model since it evaluates the strength of the field at a given point in time. Therefore the only thing that matters is the phase relationship and strength of the EOD. Assuming that the second fish's EOD is kept constant and the phases relationship is also the same, the only difference during a chirp that could affect the result of the calculation is the potential decrease in EOD amplitude during the chirp. It is indeed logical that if the focal fish decreased its EOD amplitude the target fish's EOD becomes relatively stronger. Where things are harder to understand is why the different types of chirps (e.g. type 1 vs type 2) lead to the same increase in signal even though they are typically associated with different levels of amplitude modulations. Also, it is hard to imagine that a type 2 chirps that is barely associated with any decrease in EOD amplitude (0-10% maybe), would cause doubling of the EI strength. There might be something I don't understand but the authors should provide a lot more details on how this result is obtained and convince us that it makes sense.

  14. eLife

    Reviewer #2 (Public Review):

    Studying Apteronotus leptorhynchus (the weakly electric brown ghost knifefish), the authors provide evidence that 'chirps' (brief modulations in the frequency and amplitude of the ongoing electric signal) function in active sensing (specifically homeoactive sensing) rather than communication. Chirping is a behavior that has been well studied, including numerous studies on the sensory coding of chirps and the neural mechanisms for chirp generation. Chirps are largely thought to function in communication behavior, so this alternative function is a very exciting possibility that could have a great impact on the field. The authors do provide convincing evidence that chirps may function in homeoactive sensing. However, their evidence arguing against a role for chirps in communication is not as strong, and fails to sufficiently consider the evidence from a large body of existing research. Ultimately, the manuscript presents very interesting data that is sure to stimulate discussion and follow-up studies, but it suffers from dismissing evidence in support of, or consistent with, a communicative function for chirps. The authors do acknowledge that chirps could function as both a communication and homeactive sensing signal, but it seems clear they wish to argue against the former and for the latter, and the evidence is not yet there to support this.

    In the introduction, the authors state, "Since both chirps and positional parameters (such as size, orientation or motion) can only be detected as perturbations of the beat, and via the same electroreceptors, the inputs relaying both types of information are inevitably interfering." I disagree with this statement, which seems to be a key assumption. Both of these features certainly modulate the activity of electroreceptors, but that does not mean those modulations are ambiguous as to their source. You do not know whether the two types of modulations can be unambiguously decoded from electroreceptor afferent population activity.

    My biggest issue with this manuscript is that it is much too strong in dismissing evidence that chirping correlates with context. In your behavioral observations, you found sex differences in chirping as well as differences between freely interacting and physically separated fish. Chirps tended to occur in close proximity to another fish. Your model of chirp variability found that environmental experience, social experience, and beat frequency (DF) are the most important factors explaining chirp variability. Are these not all considered behavioral or social context? Beat frequency (DF) in particular is heavily downplayed as being a part of "context" but it is a crucial part of the context, as it provides information about the identity of the fish you're interacting with. The authors show quite convincingly that the types of chirps produced do not vary with these contexts, but chirp rates do.

    Further, in your playback experiments, fish responded differently to small vs. large DFs, males chirped more than females, type 2 chirps became more frequent throughout a playback, and rises tended to occur at the end of a playback. These are all examples of context-dependent behavior.

    In the results, the authors state, "Overall, the majority of chirps were produced by male subjects, in comparable amounts regardless of environmental experience (resident, intruder or equal; Figure S1A,C), social status (dominant or subordinate; Figure S1B) or social experience (novel or experienced; Figure S1D)." This is not what is shown in Figure S1. S1A shows clear differences between resident vs. intruder males, S1B shows clear differences between dominant vs. subordinate males, and S1D shows clear differences between naïve and experienced males. The analysis shown in Figure 2 would seem to support this. Indeed, the authors state, "Overall, this analysis indicated that environmental and social experience, together with beat frequency (DF) are the most important factors explaining chirp variability."

    The choice of chirp type varied widely between individuals but was relatively consistent within individuals across trials of the same experiment. The authors interpret this to mean that chirping does not vary with internal state, but is it not likely that the internal states of individuals are stable under stable conditions, and that individuals may differ in these internal states across the same conditions? Stable differences in communication signals between individuals are frequently interpreted as reflecting differences between those individuals in certain characteristics, which are being communicated by these signals.

    I am not convinced of the conclusion drawn by the analysis of chirp transitions. The transition matrices show plenty of 1-2 and 2-1 transitions occurring. Further, the cross-correlation analysis only shows that chirp timing between individuals is not phase-locked at these small timescales. It is entirely possible that chirp rates are correlated between interacting individuals, even if their precise timing is not. Further, it is not clear to me how "transitions" were defined. The methods do not make this clear, and it is not clear to me how you can have zero chirp transitions between two individuals when those two individuals are both generating chirps throughout an interaction.

    In the results, "Although all chirp types were used during aggressive interactions, these seemed to be rather less frequent in the immediate surround of the chirps (Figure 6A)." A lack of precise temporal correlation on short timescales does not mean there is no association between the two behaviors. An increased rate of chirping during aggression is still a correlation between the two behaviors, even if chirps and specific aggressive behaviors are not tightly time-locked.

    In summary, it is simply too strong to say that chirping does not correlate with context, or to claim that there is convincing evidence arguing against a communication function of chirps. Importantly, however, this does not detract from your exciting and well-supported hypothesis that chirping functions in homeoactive sensing. A given EOD behavior could serve both communication and homeoactive sensing. I actually suspect this is quite common in electric fish (both gymnotiforms and mormyrids), and perhaps in other actively sensing species such as echolocating animals. The two are not mutually exclusive.

  15. eLife

    Reviewer #3 (Public Review):

    Summary:

    This important paper provides the best-to-date characterization of chirping in weakly electric fish using a large number of variables. These include environment (free vs divided fish, with or without clutter), breeding state, gender, intruder vs resident, social status, locomotion state and social and environmental experience, without and with playback experiments. It applies state-of-the-art methods for reducing the dimensionality of the data and finding patterns of correlation between different kinds of variables (factor analysis, K-means). The strength of the evidence, collated from a large number of trials with many controls, leads to the conclusion that the traditionally assumed communication function of chirps may be secondary to its role in environmental assessment and exploration that takes social context into account. Based on their extensive analyses, the authors suggest that chirps are mainly used as probes that help detect beats caused by other fish and as well as objects.

    Strengths:

    The work is based on completely novel recordings using interaction chambers. The amount of new data and associated analyses is simply staggering, and yet, well organized in presentation. The study further evaluates the electric field strength around a fish (via modelling with the boundary element method) and how its decay parallels the chirp rate, thereby relating the above variables to electric field geometry.

    The main conclusions are that the lack of any significant behavioural correlates for chirping, and the lack of temporal patterning in chirp time series, cast doubt on a primary communication goal for most chirps. Rather, the key determinants of chirping are the difference frequency between two interacting conspecifics as well as individual subjects' environmental and social experience. The paper concludes that there is a lack of evidence for stereotyped temporal patterning of chirp time series, as well as of sender-receiver chirp transitions beyond the known increase in chirp frequency during an interaction.

    These conclusions by themselves will be very useful to the field. They will also allow scientists working on other "communication" systems to perhaps reconsider and expand the goals of the probes used in those senses. A lot of data are summarized in this paper, with thorough referencing to past work.

    The alternative hypotheses that arise from the work are that chirps are mainly used as environmental probes for better beat detection and processing and object localization, and in this sense are self-directed signals. This led to their prediction that environmental complexity ("clutter") should increase chirp rate, which is fact was revealed by their new experiments. The authors also argue that waveform EODs have less power across high spatial frequencies compared to pulse-type fish, with a resulting relatively impoverished power of resolution. Chirping in wave-type fish could temporarily compensate for the lower frequency resolution while still being able to resolve EOD perturbations with a good temporal definition (which pulse-type fish lack due to low pulse rates).

    The authors also advance the interesting idea that the sinusoidal frequency modulations caused by chirps are the electric fish's solution to the minute (and undetectable by neural wetware) echo-delays available to it, due to the propagation of electric fields at the speed of light in water. The paper provides a number of experimental avenues to pursue in order to validate the non-communication role of chirps.

    Weaknesses:

    My main criticism is that the alternative putative role for chirps as probe signals that optimize beat detection could be better developed. The paper could be clearer as to what that means precisely, especially since beating - and therefore detection of some aspects of beating due to the proximity of a conspecific - most often precedes chirping. One meaning the authors suggest, tentatively, is that the chirps could enhance electrosensory responses to the beat, for example by causing beat phase shifts that remediate blind spots in the electric field of view.

    A second criticism is that the study links the beat detection to underwater object localization. The paper does not significantly develop that line of thought given their data - the authors tread carefully here given the speculative aspect of this link. It is certainly possible that the image on the fish's body of an object in the environment will be slightly modified by introducing a chirp on the waveform, as this may enhance certain heterogeneities of the object in relation to its environment. The thrust of this argument derives mainly from the notion of Fourier analysis with pulse type fish EOD waveforms (see above, and radar theory more generally), where higher temporal frequencies in the beat waveform induced by the chirp will enable a better spatial resolution of objects. It remains to be seen whether experiments can show this to be significant.

  16. Version published to 10.1101/2022.12.29.522225v5 on bioRxiv
  17. Version published to 10.7554/elife.88287.1 on eLife
  18. eLife

    eLife assessment

    This is a valuable contribution to the electric fish community, and to studies of active sensing more generally, in that it provides evidence that a well-studied behavior (chirping) may serve in active sensing rather than communication. For the most part, the evidence is solid. In particular, the evidence showing increased chirping in more cluttered environments and the relationship between chirping and movement are convincing. Nevertheless, evidence to support the argument that chirps are mostly used for navigation rather than communication is incomplete.

  19. eLife

    Reviewer #1 (Public Review):

    The authors investigate the role of chirping in a species of weakly electric fish. They subject the fish to various scenarios and correlate the production of chirps with many different factors. They find major correlations between the background beat signals (continuously present during any social interactions) or some aspects of social and environmental conditions with the propensity to produce different types of chirps. By analyzing more specifically different aspects of these correlations they conclude that chirping patterns are related to navigation purposes and the need to localize the source of the beat signal (i.e. the location of the conspecific).

    The study provides a wealth of interesting observation of behavior and much of this data constitute a useful dataset to document the patterns of social interactions in these fish. Some data, in particular the high propensity to chirp in cluttered environments, raises interesting questions. Their main hypothesis is a useful addition to the debate on the function of these chirps and is worth being considered and explored further. However, the data they provide does not support strong conclusion statements arguing that these chirps are used for localization purposes and is even less convincing at rejecting previously established hypotheses on the communication purpose of the chirps. I would suggest thoroughly revising the manuscript to provide a neutral description of the results and leaving any speculations and interpretations for the discussion where the authors should be careful to separate strongly supported hypotheses from more preliminary speculations. I detail below several instances where the argumentation and/or the analysis are flawed.

    - They analyze chirp patterning and show that, most likely, a chirp by an individual is followed by a chirp in the same individual. They argue that it is rare that a chirp elicits a "response" in the other fish. Even if there are clearly stronger correlations between chirps in the same individual, they provide no statistical analysis that discards the existence of occasional "response" patterns. The fact that these are rare, and that the authors don't do an appropriate analysis of probabilities, leads to this unsupported conclusion.
    - One of the main pieces of evidence that chirps can be used to enhance conspecific localization is based on their "interference" measure. The measure is based on an analysis of "inter-peak-intervales". This in itself is a questionable choice. The nervous system encodes all parts of the stimulus, not just the peak, and disruption occurring at other phases of the beat might be as relevant. The interference will be mostly affected by the summed duration of intervals between peaks in the chirp AM. They do not explain why this varies with beat frequency. It is likely that the changes they see are simply an artifact of the simplistic measure. A clear demonstration that this measure is not adequate comes from the observation in Fig7E-H. They show that the interference value changes as the signal is weaker. This measure should be independent of the strength of the signal. The method is based on detecting peaks and quantifying the time between peaks. The only reason this measure could be affected by signal strength is if noisy recordings affect how the peak detection occurs. There is no way to argue that this phenomenon would happen the same way in the nervous system. Furthermore, they qualitatively argue that patterns of chirp production follow patterns of interference strength. No statistical demonstration is done. Even the qualitative appraisal is questionable. For example, they argue that there are relatively few chirps being produced for DFs of 60 or -60 Hz. But these are DF where they have only a very small sample size. The single pair of fish that they recorded at some of these frequencies might not have chirped by chance and a rigorous statistical analysis is necessary. Similarly, in Fig 5C they argue that the position of the chirps fall on areas of the graph where the interferences are strongest (darker blue) but this is far from obvious and, again, not proven.
    - They relate the angle at which one fish produces chirps relative to the orientation of the mesh enclosing. They argue that this is related to the orientation of electric field lines by doing a qualitative comparison with a simplified estimate of field lines. To be convincing this analysis should include a quantitative comparison using the exact same body position of the two fish when the chirps are emitted.
    -They show that the very vast majority of chirps in Fig 6 occur when the fish are within a few centimeters (e.g. very large first bin in Fig6E-Type2). This is a situation when the other fish signal will be strongest and localization will be the easiest. It is hard to understand why the fish would need a mechanism to enhance localization in these conditions (this is the opposite of difficult conditions e.g. the "cluttered" environment).
    - The argumentation aimed at rejecting the well-established role of chirp in communication is weak at best. First, they ignored some existing data when they argue that there is no correlation between chirping and behavioral interactions. Particularly, Hupe and Lewis (2008) showed a clear temporal correlation between chirps and a decrease in bites during aggressive encounters. It could be argued that this is "causal evidence" (to reuse their wording) that chirps cause a decrease in attacks by the receiver fish (see Fig 8B of the Hupe paper and associated significant statistics). Also, Oboti et al. argue that social interactions involve "higher levels of locomotion" which would explain the use of chirps since they are used to localize. But chirps are frequent in "chirp chamber" paradigms where no movement is involved. They also point out that social context covaries with beat frequency and thus that it is hard to distinguish which one is linked to chirping propensity and then say that it is hard to disentangle this from "biophysical features of EOD fields affecting detection and localization of conspecific fish". But they don't provide any proof that beat frequency affects detection and localization so their argument is not clear. Last, they argue that tests in one species shouldn't be extrapolated to other species. But many of the studies arguing for the role of chirps in communication was done on brown ghost. In conclusion of this point, they do not provide any strong argument that rejects the role of chirps as a communication signal. A perspective that would be better supported by their data and consistent with past research would be to argue that, in addition to a role in communication, chirps could sometimes be used to help localize conspecifics.
    -The discussion they provide on the possible mechanism by which chirps could help with localization of the conspecific is problematic. They imply that chirps cause a stronger response in the receptors. For most chirps considered here, this is not true. For a large portion of the beat frequencies shown in this paper, chirps will cause a de-synchronization of the receptors with no increase in firing rate. They cannot argue that this represents an enhanced response. They also discuss a role for having a broader frequency spectrum -during the chirp- in localization by making a parallel with pulse fish. There is no evidence that a similar mechanism could even work in wave-type fish.
    -They write the whole paper as if males and females had been identified in their experiments. Although EOD frequency can provide some guess of the sex the method is unreliable. We can expect a non-negligible percentage of error in assigning sex.

  20. eLife

    Reviewer #2 (Public Review):

    Studying the weakly electric brown ghost knifefish, the authors provide evidence that 'chirps' (brief modulations in the frequency and amplitude of the ongoing electric signal) function in active sensing (specifically homeoactive sensing) rather than communication. This is a behavior that has been very well studied, including numerous studies on the sensory coding of chirps and the neural mechanisms for chirp generation. Chirps are largely thought to function in communication behavior, so this alternative function is a very exciting possibility that could have a great impact on the field. The authors do provide convincing evidence that chirps may function in homeoactive sensing. However, their evidence arguing against a role for chirps in communication is not as strong, and neglects a large body of research. Ultimately, the manuscript has great potential but suffers from framing these two possibilities as mutually exclusive and dismissing evidence in favor of a communicative function.

    (1) The specific underlying question of this study is not made clear in the abstract or introduction. It becomes apparent in reading through the manuscript that the authors seek to test the hypothesis that chirps function in active sensing (specifically homeoactive sensing). This should be made explicitly clear in both the abstract and introduction, along with the rationale for this hypothesis.

    (2) My biggest issue with this manuscript is that it is much too strong in dismissing evidence that chirping correlates with context. This is captured in this sentence in the introduction, "We first show that the choice of different chirp types does not significantly correlate with any particular behavioral or social context." This very strong conclusion comes up repeatedly, and I disagree with it, for the following reasons:

    In your behavioral observations, you found sex differences in chirping as well as differences between freely interacting and physically separated fish. Your model of chirp variability found that environmental experience, social experience, and beat frequency (DF) are the most important factors explaining chirp variability. Are these not all considered "behavioral or social context"? Beat frequency (DF) in particular is heavily downplayed as being a part of "context" but it is a crucial part of the context, as it provides information about the identity of the fish you're interacting with.

    In your playback experiments, fish responded differently to small vs. large DFs, males chirped more than females, type 2 chirps became more frequent throughout a playback, and rises tended to occur at the end of a playback. These are all examples of context-dependent behavior.

    Further, you only considered the identity of interacting fish or stimulated fish, not their behavior during the interaction or during playback. Such an analysis is likely beyond the scope of this study, but several other studies have shown correlations between social behavior and chirping. In the absence of such data here, it is too strong to claim that chirping is unrelated to context.

    In summary, it is simply too strong to say that chirping does not correlate with context. Importantly, however, this does not detract from your hypothesis that chirping functions in homeoactive sensing. A given EOD behavior could serve both communication and homeoactive sensing. I actually suspect that this is quite common in electric fish. The two are not mutually exclusive, and there is no reason for you to present them as such. I recommend focusing more on the positive evidence for a homeoactive function and less on the negative evidence against a communication function.

    (3) The results were generally challenging to follow. In the first 4 sections, it is not made clear what the specific question is, what the approach to addressing that question is, and what specific experiment was carried out (the last two sections of the results were much clearer). The independent variables (contexts) are not clearly established before presenting the results. Instead they are often mentioned in passing when describing the results. They come across as an unbalanced hodgepodge of multiple factors, and it is not made clear why they were chosen. This makes it challenging to understand why you did what you did, the results, and their implications. For each set of major results, I recommend: First, pose a clear question. Then, describe the general approach to answering that question. Next, describe the specifics of the experimental design, with a rationale that appeals to the general approach described. Finally, describe the specific results.

    (4) Results: "We thus predicted that, if behavioral meaning can be attributed to different types of chirps, as posed by the prevailing view (e.g., Hagedorn and Heiligenberg, 1985; Larimer and MacDonald, 1968; Rose, 2004)..." It should be made clear why this is the prevailing view, and this description should likely be moved to the introduction. There is a large body of evidence supporting this view and it is important to be complete in describing it, especially since the authors seem to seek to refute it.

    (5) I am not convinced of the conclusion drawn by the analysis of chirp transitions. The transition matrices show plenty of 1-2 and 2-1 transitions occurring. Further, the cross-correlation analysis only shows that chirp timing between individuals is not phase-locked at these small timescales. It is entirely possible that chirp rates are correlated between interacting individuals, even if their precise timing is not.

  21. eLife

    Reviewer #3 (Public Review):

    Summary:
    This important paper provides the best-to-date characterization of chirping in weakly electric fish using a large number of variables. These include environment (free vs divided fish, with or without clutter), breeding state, gender, intruder vs resident, social status, locomotion state and social and environmental experience, as well as with playback experiments. It applies state-of-the-art methods for reducing dimensionality and finding patterns of correlation between different kinds of variables (factor analysis, K-means). The exceptional strength of the evidence, collated from a large number of trials with many controls, leads to the conclusion that a number of commonly accepted truths about which variable affects chirping must be carefully rewritten or nuanced. Based on their extensive analyses, the authors suggest that chirps are mainly used as probes that help detect beats and objects.

    Strengths:
    The work is based on completely novel recordings using interaction chambers. The amount of new data and associated analyses is simply staggering, and yet, well organized in presentation. The study further evaluates the electric field strength around a fish (via modelling with the boundary element method) and how its decay parallels the chirp rate, thereby relating the above variables to electric field geometry.

    The main conclusions are that the lack of any significant behavioural correlates for chirping, and the lack of temporal patterning in chirp time series, cast doubt on a communication goal for most chirps. Rather, the key determinants of chirping are the difference frequency between two interacting conspecifics as well as individual subjects' environmental and social experience. These conclusions by themselves will be hugely useful to the field. They will also allow scientists working on other "communication" systems to at least reconsider, and perhaps expand the precise goal of the probes used in those senses. There are a lot of data summarized in this paper, and thorough referencing to past work. For example, the paper concludes that there is a lack of evidence for stereotyped temporal patterning of chirp time series, as well as of sender-received chirp transitions beyond the known increase in chirp frequency during an interaction.

    The alternative hypotheses that arise from the work are that chirps are mainly used as environmental probes for better beat detection and processing and object localization.

    The authors also advance the interesting idea that the sinusoidal frequency modulations caused by chirps are the electric fish's solution to the minute (and undetectable by neural wetware) echo-delays available to it, due to the propagation of electric fields at the speed of light in water.

    Weaknesses:
    My main criticism is that the alternative putative role for chirps as probe signals that optimize beat detection could be better developed. The paper could be clearer as to what that means precisely. And there is an egg-and-chicken type issue as well, namely, that one needs a beat in order to "chirp" the beating pattern, but then how does chirping optimize the detection of the said beat? Perhaps the authors mean (as they wrote elsewhere in the paper) that the chirps could enhance electrosensory responses to the beat.

    A second criticism is that the study links the beat detection to underwater object localization. I did not see a sufficiently developed argument in this direction, nor how the data provided support for this argument. It is certainly possible that the image on the fish's body of an object in the environment will be slightly modified by introducing a chirp on the waveform, as this may enhance certain heterogeneities of the object in relation to its environment. The thrust of this argument seems to derive more from the notion of Fourier analysis with pulse type fish (and radar theory more generally) that the higher temporal frequencies in the beat waveform induced by the chirp will enable a better spatial resolution of objects. It remains to be seen whether this is significant.

    I would also have liked to see a proposal for new experiments that could test these possible new roles.

    The authors should recall for the readers the gist of Bastian's 2001 argument that the chirp "can adjust the beat frequency to levels that are better detectable" in the light of their current. Further, at the beginning of the "Probing with chirps" section, the 3rd way in which chirps could improve conspecific localization mentions the phase-shifting of the EOD. The authors should clarify whether they mean that the tuberous receptors and associated ELL/toral circuitry could deal with that cue, or that the T_unit pathway would be needed?

    On p.17 I don't understand what is meant by most chirps being produced possibly aligned with the field lines, since field lines are everywhere. And what is one to conclude from the comparison of Fig.6D and 7A? Likewise it was not clear what is meant by chirps having a detectable effect on randomly generated beats.

    In the section on Inconsistencies between behaviour and hypothesized signal meaning, the authors could perhaps nuance the interpretation of the results further in the context of the unrealistic copy of natural stimuli using EOD mimics. In particular, Kelly et al. 2008 argued that electrode placement mattered in terms of representation of a mimic fish onto the body of a real fish, and thus, if I properly understand the set up here, the movement would cause the mimic to vary in quality. This may nevertheless be a small confounding issue.

  22. Version published to 10.1101/2022.12.29.522225v4 on bioRxiv
  23. Version published to 10.1101/2022.12.29.522225v3 on bioRxiv
  24. Version published to 10.1101/2022.12.29.522225v2 on bioRxiv
  25. Version published to 10.1101/2022.12.29.522225v1 on bioRxiv