The evolution of cavefish odor perception shifts fish response from avoidance to approach when exposed to alarm and death odors

Reviewer #1 (Public review):

In their submitted manuscript, Harkinish-Murray and colleagues from the Kozol lab present convincing evidence for a genetically encoded shift in the odor perception of cavefish compared to their surface ancestors. Surface Astyanax, just as zebrafish, are attracted to food odors and are repelled by death odors and the alarm substance Schreckstoff (released from damaged skin by specialized club cells). Based on the experimental evidence in this manuscript, however, their cavefish counterparts are attracted to these odors as well. This would make sense, in an evolutionary framework, as predation is less likely in cave settings and decaying fish are a valuable source of nutrients for their living counterparts.

Using an F2 hybrid cross scheme between surface fish and cavefish, authors also provide compelling evidence that genetic factors are behind this behavioral shift. Furthermore, they also show that this behavior (i.e., attraction to skin and decay extracts) can be observed in surface fish given long enough food deprivation. This latter observation also makes sense in the light of evolution and is genuinely interesting as it also provides a plausible roadmap to the shift in behavior through Waddingtonian genetic assimilation.

The manuscript is generally well written and clear, we have identified only few weaknesses, some regarding the presentation of the data.

(1) For Figure 3, on the x-axis of panels b, e, and h, supposedly we see surface fish vs. different cavefish populations. This is currently missing and makes the figure harder to interpret. Also, two populations (panel e) show a bimodal distribution upon indirect white light exposure, suggesting that some fish still acted as if they were exposed to direct light, while others acted as if they were in darkness (infrared light). We believe this warrants more consideration as it could tell us something about the existing (and relevant) genetic variance within this population. It is also notable that the third cavefish population also showed increased odor indices under indirect white light and infrared light conditions, suggesting that increasing the number of observations could have yielded a statistically significant result.

(2) Some extra details about the methods could also be provided to enhance the reproducibility of the experiments.

(3) A more serious concern is about the anatomical designation of particular brain regions in Figure 7d and consequently Figure 7f. Whereas we would agree with the positioning of the medial pallium (Dm), we think the region depicting the thalamus is in fact still part of the telencephalon, and the real thalamus should be more posteriorly. On the other hand, we think that the preoptic areas should be under the pallium and not posterior to it (see PMID: 22586363 for corresponding zebrafish anatomy). We would suggest, therefore, that the authors revisit this issue (a minor one, considering the depth of the results presented in the manuscript), and provide a better anatomical annotation - e.g., the identity of particular brain regions could be backed up by Hybridization Chain Reaction experiments for region-specific transcripts. (Disclaimer: we do not consider ourselves experts in adult cavefish neuroanatomy; therefore, we consulted in this case a colleague with much more knowledge on this topic.)

(4) It would also be useful to expand the brain imaging data displaying results for similar tests in surface fish, to see if skin and decay extracts trigger different or similar brain activity in those fish.

Further work will surely be able to discern the more precise genetic changes that made the shift in behavior possible. Once these causative variants (or at least linked markers) are determined, it will be quite revealing to see if these variants are indeed already present in the surface population (as hinted by the authors), and also, if besides the Surface x Tinaja F2 hybrids, crosses between other cave populations and surface fish can be performed, we could also see how much evolutionary convergence happened in the parallel evolution of different cave morphs. Were there multiple possible pathways for similar behaviors in different cave populations, or - as in freshwater stickleback populations - do we see broadly the same genetic playbook repeated each time?

Another outstanding question, also demonstrated and discussed, albeit briefly, in this paper relates to the behavior-modulating effect of light in cavefish. What is the physiological relevance for a dark-dwelling animal to have this capacity? Is this just the chance result of occasional gene flow from surface populations, or does it have a genuine evolutionary significance?

Reviewer #2 (Public review):

Summary:

The authors tested whether the olfactory cues that drive attraction or avoidance behavior have diverged between surface‑dwelling and cave‑adapted strains of the Mexican cavefish Astyanax mexicanus. They use high‑throughput odor‑discrimination assays between known attractants and repellents by calculating an "odor index" per fish (=the difference in time spent in an odor zone versus a control zone). Further, hybrid crosses to probe heritability, starvation experiments to assess plasticity of odor perception, and whole‑brain pERK detection/mapping to link behavioral changes with known localized neural activity. The results support the hypothesis that the extreme cave environment has selected for an approach response to stimuli that are ancestrally aversive (like alarm or death odors) but in harsh environments can be used as guidance to the rare food sources in this ecosystem.

Strengths:

The odor index analysis is convincing, and the experiments for odor attraction/avoidance are robustly performed. The light-to-darkness shift reflected by avoidance to attraction in cavefish towards skin odors is compelling and carefully analyzed. The analysis of odor indices of three cave-dwelling populations in comparison to surface fish highlights a similar regime, yet with differences among the different populations, suggesting population-specific genetic variation.

Another strength of the paper is exactly this genetic inheritance study by generating F2 hybrids of cave-dwelling and surface-living individuals. The hybrids displayed a continuous range of odor indices for social, alarm, and death odors, indicating that these traits are heritable and likely based on additive genetic markers. Further, the authors uncovered a sexual dimorphism: only female cavefish exhibited approach behavior to social odors, whereas males remained neutral. This result aligns with known differences in olfactory organ morphology between sexes of other species from harsh environments.

Although limited in number, the neurophysiological correlation using whole‑brain pERK mapping after 10 min of odor exposure is convincing. The data revealed overlapping activation in the thalamus and pre‑optic region for food and decay odors, suggesting that these brain areas mediate the evolved attraction response to previously repellent stimuli.

Overall, the manuscript presents a concise story: cavefish have evolved attraction to alarm and death odors as a result of shifting from ancestral avoidance-driven to attraction by genetic changes and physiologically similar activation of specific neural circuits. The evidence is robust, with multiple independent experiments (behavioral assays, hybrid genetics, starvation experiments, and brain mapping) that collectively support the conclusions.

Furthermore, exposure to unpleasant odors can not only be tolerated but can even serve as a trigger for foraging. This plasticity demonstrates that genetic predispositions can be put into practice through active changes in physiology in species or organisms confronted with (drastically) changing environmental conditions.

Weaknesses:

I value that the authors are critical of their own data, indicating low numbers in the pERK/brain experiments. Yet this is a weak point as the statistical power is thus limited. However, their reasoning is careful, based on the results and not over-interpreting.

The layout/design of the ethograms (bout category plots) for both individual and population-wise are not easy to follow. Reworking these display items to convey the information is necessary.

Taken together, the manuscript uses odor perception and attraction/avoidance behavior studies to show that environmental changes (light-to-darkness) have an immediate impact on smell perception and behavior. Attraction to otherwise repellent odors is used by cavefish to likely adapt to harsh environments with low food sources. The manuscript convincingly demonstrates this plasticity, which is an interesting idea to follow up for other traits spreading among a population. This also underlines that a genome may be fixed and the blueprint for behavioral traits, but extrinsic cues can readily be adapted to change wired behavior even to the extreme as reported here: changing avoidance to attraction.

Author response:

The authors thank the reviewers for their thorough and fair assessment of our manuscript. We are currently working to edit the manuscript based on the critiques and guidance offered by the reviewers. This will consist of fixing grammar and typos, expanding the material and methods section to include more information on the behavioral assays, modifying graphs for clarity between visuals and interpretations, and correcting our mistakes in neuroanatomical labeling.

Public Reviews:

Reviewer #1 (Public review):

In their submitted manuscript, Harkinish-Murray and colleagues from the Kozol lab present convincing evidence for a genetically encoded shift in the odor perception of cavefish compared to their surface ancestors. Surface Astyanax, just as zebrafish, are attracted to food odors and are repelled by death odors and the alarm substance Schreckstoff (released from damaged skin by specialized club cells). Based on the experimental evidence in this manuscript, however, their cavefish counterparts are attracted to these odors as well. This would make sense, in an evolutionary framework, as predation is less likely in cave settings and decaying fish are a valuable source of nutrients for their living counterparts.

Using an F2 hybrid cross scheme between surface fish and cavefish, authors also provide compelling evidence that genetic factors are behind this behavioral shift. Furthermore, they also show that this behavior (i.e., attraction to skin and decay extracts) can be observed in surface fish given long enough food deprivation. This latter observation also makes sense in the light of evolution and is genuinely interesting as it also provides a plausible roadmap to the shift in behavior through Waddingtonian genetic assimilation.

The manuscript is generally well written and clear, we have identified only few weaknesses, some regarding the presentation of the data.

(1) For Figure 3, on the x-axis of panels b, e, and h, supposedly we see surface fish vs. different cavefish populations. This is currently missing and makes the figure harder to interpret. Also, two populations (panel e) show a bimodal distribution upon indirect white light exposure, suggesting that some fish still acted as if they were exposed to direct light, while others acted as if they were in darkness (infrared light). We believe this warrants more consideration as it could tell us something about the existing (and relevant) genetic variance within this population. It is also notable that the third cavefish population also showed increased odor indices under indirect white light and infrared light conditions, suggesting that increasing the number of observations could have yielded a statistically significant result.

We agree with the reviewer that our light testing data suggests complexity in the response to indirect white light within certain cave populations. In addition, an expanded sample size would likely provide clarity on whether individuals fall within two groups, behavior that looks like direct light or infra-red light, that could relate to genetic variation within cavefish populations. We are currently working to reassess the current data and determining the best course of action for continued studies related to light exposure.

(2) Some extra details about the methods could also be provided to enhance the reproducibility of the experiments.

We agree with both reviewers that the methodological section on behavior needs to be expanded. We are currently editing our methods section to include more detail on water exchanges, odor preparation, timing, biological replicates, and binning.

(3) A more serious concern is about the anatomical designation of particular brain regions in Figure 7d and consequently Figure 7f. Whereas we would agree with the positioning of the medial pallium (Dm), we think the region depicting the thalamus is in fact still part of the telencephalon, and the real thalamus should be more posteriorly. On the other hand, we think that the preoptic areas should be under the pallium and not posterior to it (see PMID: 22586363 for corresponding zebrafish anatomy). We would suggest, therefore, that the authors revisit this issue (a minor one, considering the depth of the results presented in the manuscript), and provide a better anatomical annotation - e.g., the identity of particular brain regions could be backed up by Hybridization Chain Reaction experiments for region-specific transcripts. (Disclaimer: we do not consider ourselves experts in adult cavefish neuroanatomy; therefore, we consulted in this case a colleague with much more knowledge on this topic.)

We agree that our annotation was incorrect or more accurately mislabeled in our write-up of the preprint and submitted manuscript. Therefore, we have now re-assessed the regions using the tissue cleared and light sheet collected zebrafish atlas, Adult Zebrafish Brain Atlas (AZBA; doi: 10.7554/eLife.69988). We are now editing the resubmission in the following manner: our initial labeling of the ventromedial thalamus will be changed to the lateral olfactory tract (nLOT) of the pallium and the preoptic region to the ventromedial thalamus (VM). We will provide a comparable z-slice of the AZBA segmentation file to illustrate the similarity in position. This would also support a known continuous circuit of olfactory integration, with information flowing from the lateral olfactory tract-to the piriform cortex-to the thalamus. We also agree that a more accurate assessment in Astyanax would require HCR in situ hybridization of markers for those specific brain regions or a neurocomputational brain atlas for adult Astyanax populations. Finally, we assert that this small dataset is preliminary at best and only provides regions of shared activity that could explain anything from perception related processes to relay of odor signaling unrelated to perception. Further work with larger sample sizes and additional populations are currently underway for a follow-up study on the neurobiological basis of olfactory processing and perception in adult cavefish.

(4) It would also be useful to expand the brain imaging data displaying results for similar tests in surface fish, to see if skin and decay extracts trigger different or similar brain activity in those fish.

We agree with the reviewer that the brain mapping section lacks a sufficient sample size and no control group for comparison (surface fish). However, we found the variation in pERK intensity (notably the putative nLOT) to be informative and decided to include the dataset in the manuscript. We are currently working to fill in these data gaps by sampling all populations and increasing the Pachon cavefish sample size. This will be a follow-up study as mentioned above in the last rebuttal paragraph.

Further work will surely be able to discern the more precise genetic changes that made the shift in behavior possible. Once these causative variants (or at least linked markers) are determined, it will be quite revealing to see if these variants are indeed already present in the surface population (as hinted by the authors), and also, if besides the Surface x Tinaja F2 hybrids, crosses between other cave populations and surface fish can be performed, we could also see how much evolutionary convergence happened in the parallel evolution of different cave morphs. Were there multiple possible pathways for similar behaviors in different cave populations, or - as in freshwater stickleback populations - do we see broadly the same genetic playbook repeated each time?

We agree with the reviewer that the hybrid results setup a promising follow up project to map these traits genetically. We are continuing to test odor perception in other hybrid populations and have started Quantitative Trait Locus mapping experiments.

Another outstanding question, also demonstrated and discussed, albeit briefly, in this paper relates to the behavior-modulating effect of light in cavefish. What is the physiological relevance for a dark-dwelling animal to have this capacity? Is this just the chance result of occasional gene flow from surface populations, or does it have a genuine evolutionary significance?

Reviewer #2 (Public review):

Summary:

The authors tested whether the olfactory cues that drive attraction or avoidance behavior have diverged between surface‑dwelling and cave‑adapted strains of the Mexican cavefish Astyanax mexicanus. They use high‑throughput odor‑discrimination assays between known attractants and repellents by calculating an "odor index" per fish (=the difference in time spent in an odor zone versus a control zone). Further, hybrid crosses to probe heritability, starvation experiments to assess plasticity of odor perception, and whole‑brain pERK detection/mapping to link behavioral changes with known localized neural activity. The results support the hypothesis that the extreme cave environment has selected for an approach response to stimuli that are ancestrally aversive (like alarm or death odors) but in harsh environments can be used as guidance to the rare food sources in this ecosystem.

Strengths:

The odor index analysis is convincing, and the experiments for odor attraction/avoidance are robustly performed. The light-to-darkness shift reflected by avoidance to attraction in cavefish towards skin odors is compelling and carefully analyzed. The analysis of odor indices of three cave-dwelling populations in comparison to surface fish highlights a similar regime, yet with differences among the different populations, suggesting population-specific genetic variation.

Another strength of the paper is exactly this genetic inheritance study by generating F2 hybrids of cave-dwelling and surface-living individuals. The hybrids displayed a continuous range of odor indices for social, alarm, and death odors, indicating that these traits are heritable and likely based on additive genetic markers. Further, the authors uncovered a sexual dimorphism: only female cavefish exhibited approach behavior to social odors, whereas males remained neutral. This result aligns with known differences in olfactory organ morphology between sexes of other species from harsh environments.

Although limited in number, the neurophysiological correlation using whole‑brain pERK mapping after 10 min of odor exposure is convincing. The data revealed overlapping activation in the thalamus and pre‑optic region for food and decay odors, suggesting that these brain areas mediate the evolved attraction response to previously repellent stimuli.

Overall, the manuscript presents a concise story: cavefish have evolved attraction to alarm and death odors as a result of shifting from ancestral avoidance-driven to attraction by genetic changes and physiologically similar activation of specific neural circuits. The evidence is robust, with multiple independent experiments (behavioral assays, hybrid genetics, starvation experiments, and brain mapping) that collectively support the conclusions.

Furthermore, exposure to unpleasant odors can not only be tolerated but can even serve as a trigger for foraging. This plasticity demonstrates that genetic predispositions can be put into practice through active changes in physiology in species or organisms confronted with (drastically) changing environmental conditions.

Weaknesses:

I value that the authors are critical of their own data, indicating low numbers in the pERK/brain experiments. Yet this is a weak point as the statistical power is thus limited. However, their reasoning is careful, based on the results and not over-interpreting.

We agree with the reviewer and direct their attention to the same critique by reviewer 1. We believe this is predominantly preliminary data that was included due to the conspicuous increase in pERK signal from the putative lateral olfactory tract (nLOT) for food and decay exposed cavefish. We are continuing to work on odor stimulated brain mapping and look forward to publishing a comprehensive dataset across wildtype and hybrid populations.

The layout/design of the ethograms (bout category plots) for both individual and population-wise are not easy to follow. Reworking these display items to convey the information is necessary.

We agree with the reviewer that the ethograms are challenging to read, especially due to our use of different colors for odor categories. We are currently preparing alternative graphs for displaying ethograms that reduce confusion and make following bout transitions for individual traces and bout probabilities for populations easier on the eyes.

Taken together, the manuscript uses odor perception and attraction/avoidance behavior studies to show that environmental changes (light-to-darkness) have an immediate impact on smell perception and behavior. Attraction to otherwise repellent odors is used by cavefish to likely adapt to harsh environments with low food sources. The manuscript convincingly demonstrates this plasticity, which is an interesting idea to follow up for other traits spreading among a population. This also underlines that a genome may be fixed and the blueprint for behavioral traits, but extrinsic cues can readily be adapted to change wired behavior even to the extreme as reported here: changing avoidance to attraction.