These three goals are so impactful. It's succinct and clearly expresses the important ingredients for successful learning-focused relationships.

I love HyperPhysics!

eLife Assessment

This paper provides important insight into how early life experience shapes adult behavior in fruit bats. The authors raised juvenile bats either in an impoverished or enriched environment and studied their foraging behaviors. The evidence is convincing that bats raised in enriched environments are more active, bold, and exploratory. The work will be of interest to ethologists and developmental psychologists.

Reviewer #1 (Public review):

Summary:

The authors show that early life experience of juvenile bats shape their outdoor foraging behaviors. They achieve this by raising juvenile bats either in an impoverished or enriched environment. They subsequently test the behavior of bats indoors and outdoors. The authors show that behavioral measures outdoors were more reliable in delineating the effect of early life experiences as the bats raised in enriched environments were more bold, active and exhibit higher exploratory tendencies.

Strengths:

The major strength of the study is providing a quantitative study of animal "personality" and how it is likely shaped by innate and environmental conditions. The other major strength is the ability to do reliable long term recording of bats in the outdoors giving researchers the opportunity to study bats in their natural habitat. To this point, the study also shows that the behavioral variables measured indoors do not correlate to that measured outdoor, thus providing a key insight into the importance of test animal behaviors in their natural habitat.

Weaknesses were in the first round of review:

It is not clear from the analysis presented in the paper how persistent those environmentally induced changes, do they remain with the bats till end of their lives.

Comments on revisions:

The authors have addressed those weaknesses and the paper is much stronger.

eLife Assessment

This revised manuscript presents an important characterization of mouse auditory cortex receptive field organization, utilizing two-photon imaging of specific subpopulations. They demonstrate a degradation of tonotopic organization from the input to the output neurons. The strength of the evidence is convincing.

Reviewer #1 (Public review):

Summary:

In this study, Gu et al., employed novel viral strategies, combined with in vivo two-photon imaging, to map the tone response properties of two groups of cortical neurons in A1 - The thalamocortical recipient (TR neurons) and the corticothalamic (CT neurons). They observed a clear tonotopic gradient among TR neurons but not in CT neurons. Moreover, CT neurons exhibited high heterogeneity of their frequency tuning and broader bandwidth, suggesting increased synaptic integration in these neurons. By parsing out different projecting-specific neurons within A1, this study provides insight into how neurons with different connectivity can exhibit different frequency response-related topographic organization.

Strengths:

This study reveals the importance of studying neurons with projection specificity rather than layer specificity since neurons within the same layer have very diverse molecular, morphological, physiological, and connectional features. By utilizing a newly developed rabies virus CSN-N2c GCaMP-expressing vector, the authors can label and image specifically the neurons (CT neurons) in A1 that project to the MGB. To compare, they used an anterograde trans-synaptic tracing strategy to label and image neurons in A1 that receive input from MGB (TR neurons).

Weaknesses:

- Perhaps as cited in the introduction, it is well known that tonotopic gradient is well preserved across all layers within A1, but I feel if the authors want to highlight the specificity of their virus tracing strategy and the populations that they imaged in L2/3 (TR neurons) and L6 (CT neurons), they should perform control groups where they image general excitatory neurons in the two depths and compare to TR and CT neurons, respectively. This will show that it's not their imaging/analysis or behavioral paradigms that are different from other labs.  

- Fig 1D and G, the y-axis is Distance from pia (%). I'm not exactly sure what this means. How does % translate to real cortical thickness? 

- For Fig. 2G and H, is each circle a neuron or an animal? Why are they staggered on top of each other on the x-axis? If x-axis is thedistance from caudal to rostral, each neuron should have a different distance? Also,it seems like it's because Fig. 2H has more circles, that's why it has morevariation thus not significant (for example, at 600 or 900um, 2G seems to haveless circles than 2H).  

- Similar in Fig 2J and L, why are the circles staggered onthe y-axis now? And is each circle now a neuron or a trial? It seems they havemuch more circles than Fig 2G and 2H. Also I don't think doing a correlation isthe proper stats for this type of plot (this point applies to Fig. 3H and 3J)

- What does inter-quartile range of BF (IQRBF, in octaves) imply? What's the interpretation of this analysis? I am confused why TR neurons showhigh IQR in HF areas compared to LF areas mean homogeneity among TR neurons (line 213 - 216). On the same note, how is this different from the BF variability?  Isn't higher IQR = tohigher variability?

- Fig. 4A-B, there's no clear critieria on how the authors categorize V, I, and O Shape. The descriptions in the Methods (line 721 - 725) are also very vague.  

Comments on revisions:

The authors have addressed all my questions in the previous round.

Reviewer #2 (Public review):

Summary:

Gu and Liang et. al investigated how auditory information is mapped and transformed as it enters and exits a auditory cortex. They use anterograde transsynaptic tracers to label and perform calcium imaging of thalamorecipient neurons in A1 and retrograde tracers to label and perform calcium imaging of corticothalamic output neurons. They demonstrate a degradation of tonotopic organization from the input to output neurons.

Strengths:

The experiments appear well executed, well described, and analyzed.

Weaknesses:

(1) Given that the CT and TR neurons were imaged at different depths, the question as to whether not these differences could otherwise be explained by layer-specific differences is still not 100% resolved. Control measurements would be needed either by recording 1) CT neurons upper layers 2) TR in deeper layers 3) non-CT in deeper layers and/or 4) non-TR in upper layers.

(2) What percent of the neurons at the depths being are CT neurons? Similar questions for TR neurons?

(3) V-shaped, I-shaped, or O-shaped is not an intuitively understood nomenclature, consider changing. Further, the x/y axis for Figure 4a is not labeled, so it's not clear what the heat maps are supposed to represent.

(4). Many references about projection neurons and cortical circuits are based on studies from visual or somatosensory cortex. Auditory cortex organization is not necessarily the same as other sensory areas. Auditory cortex references should be used specifically, and not sources reporting on S1, V1.

Comments on revisions:

The authors have fully addressed my concerns.

Reviewer #3 (Public review):

Summary:

The authors performed wide-field and 2-photon imaging in vivo in awake head-fixed mice, to compare receptive fields and tonotopic organization in thalamocortical recipient (TR) neurons vs corticothalamic (CT) neurons of mouse auditory cortex. TR neurons were found in all cortical layers while CT neurons were restricted to layer 6. The TR neurons at nominal depths of 200-400 microns have a remarkable degree of tonotopy (as good if not better than tonotopic maps reported by multiunit recordings). In contrast, CT neurons were very heterogenous in terms of their best frequency (BF), even when focusing on the low vs high frequency regions of primary auditory cortex. CT neurons also had wider tuning.

Strengths:

This is a thorough examination using modern methods, helping to resolve a question in the field with projection-specific mapping.

Weaknesses:

There are some limitations due to the methods, and it's unclear what the importance of these responses are outside of behavioral context or measured at single timepoints given the plasticity, context-dependence, and receptive field 'drift' that can occur in cortex.

(1) Probably the biggest conceptual difficulty I have with the paper is comparing these results to past studies mapping auditory cortex topography, mainly due to differences in methods. Conventionally, tonotopic organization is observed for characteristic frequency maps (not best frequency maps), as tuning precision degrades and best frequency can shift as sound intensity increases. The authors used six attenuation levels (30-80 dB SPL) and report that the background noise of the 2-photon scope is <30 dB SPL, which seems very quiet. The authors should at least describe the sound-proofing they used to get the noise level that low, and some sense of noise across the 2-40 kHz frequency range would be nice as a supplementary figure. It also remains unclear just what the 2-photon dF/F response represents in terms of spikes. Classic mapping using single-unit or multi-unit electrodes might be sensitive to single spikes (as might be emitted at characteristic frequency), but this might not be as obvious for Ca2+ imaging. This isn't a concern for the internal comparison here between TR and CT cells as conditions are similar, but is a concern for relating the tonotopy or lack thereof reported here to other studies.

(2) It seems a bit peculiar that while 2721 CT neurons (N=10 mice) were imaged, less than half as many TR cells were imaged (n=1041 cells from N=5 mice). I would have expected there to be many more TR neurons even mouse for mouse (normalizing by number of neurons per mouse), but perhaps the authors were just interested in a comparison data set and not being as thorough or complete with the TR imaging?

(3) The authors definitions of neuronal response type in the methods needs more quantitative detail. The authors state: ""Irregular" neurons exhibited spontaneous activity with highly variable responses to sound stimulation. "Tuned" neurons were responsive neurons that demonstrated significant selectivity for certain stimuli. "Silent" neurons were defined as those that remained completely inactive during our recording period (> 30 min). For tuned neurons, the best frequency (BF) was defined as the sound frequency associated with the highest response averaged across all sound levels." The authors need to define what their thresholds are for 'highly variable', 'significant', and 'completely inactive'. Is best frequency the most significant response, the global max (even if another stimulus evokes a very close amplitude response), etc.

Comments on revisions:

I think the authors misunderstood my point about sound level and characteristic frequency vs best frequency tonotopic maps. Yes, many studies of cortical responses present stimuli at higher intensities than the characteristic frequencies, but as tuning curves widen with sound level, the macroscopic tonotopic organization of primary auditory cortex breaks down at higher intensities. This is why most of the classic studies of tonotopy e.g., from the Merzenich lab) generated maps of characteristic frequency. As I mentioned before, this isn't so much of an issue for the authors' comparisons of TR vs CT organization in their own study, but in general, this makes it difficult to compare aspects of spatially-organized tonotopy from imaging studies with the older electrophysiological 'truer' tonotopic maps. That said, this means that CT cells also might be tonotopically organized if the authors had been able to look at lower intensity tuning properties.

??? Opinion based YouTube video that provides no value to the article

Very passionate about the perspective of fish.

there is no way to tell who this author is or how credible they are on this topic

Hailey keeps trying to compare fish above water to humans underwater, but that is not a fair comparison. She doesn't back it up with any evidence. Fish are not the same as humans.

This is a broad, supposed to be factual statement that is in no way backed up by quoting what study said this or other sources

The name is just an informal first name, and the link it leads to is an error page

Did not have any actual relevance to the topic.

Where did the author find this information?

Do fish actually have feelings?

This is not directly Linked with Milestone Payment Schedule as if any changes made in the Milestone section, it should reflect it here as well.

eLife Assessment

This study presents a valuable assessment of and solid evidence for increased similarity in visual appearance combined with increased chemical differences between two butterfly species in sympatry compared with differences between three populations of one of the two species in allopatry. The similarity in visual appearance hints to an evolutionary response to shared predators (but alternative explanations are possible). Thus, the difference in chemical signaling likely helps to avoid between-species mating in sympatry.

Joint Public Review:

Summary:

Ledamoisel et al. examined the evolution of visual and chemical signals in closely related Morpho butterfly species to understand their role in species coexistence. Using an integrative, state-of-the-art approach combining spectrophotometry, visual modeling, and behavioral mate choice experiments, they quantified differences in wing iridescence and assessed its influence on mate preference in allopatry and sympatry. They also performed chemical analyses to determine whether sympatric species exhibit divergent chemical cues that may facilitate species recognition and mate discrimination. The authors found iridescent coloration to be similar in sympatric Morpho species. Furthermore, male mate choice experiments revealed that in sympatry, males fail to discriminate conspecific females based on coloration, reinforcing the idea that visual signal convergence is primarily driven by predation pressure. In contrast, the divergence of chemical signals among sympatric species suggests their potential role in facilitating species recognition and mate discrimination. The authors conclude that interactions between ecological pressures and signal evolution may shape species coexistence.

Strengths:

The study is well-designed and integrates multiple methodological approaches to provide a thorough assessment of signal evolution in the studied species. We appreciate the authors' careful consideration of multiple selective pressures and their combined influence on signal divergence and convergence. Additionally, the inclusion of both visual and chemical signals adds an interesting and valuable dimension to the study, enhancing its importance. Beyond butterflies, this research broadens our understanding of multimodal communication and signal evolution in the context of species coexistence.

Reviewing Editor comment:

The authors have improved their submission after revisions and responded to the previous concerns of the reviewers.

Author response:

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

Reviewer #1 (Public review):

Summary:

In this study, Ledamoisel et al. examined the evolution of visual and chemical signals in closely related Morpho butterfly species to understand their role in species coexistence. Using an integrative, state-of-the-art approach combining spectrophotometry, visual modeling, and behavioral mate choice experiments, they quantified differences in wing iridescence and assessed its influence on mate preference in allopatry and sympatry. They also performed chemical analyses to determine whether sympatric species exhibit divergent chemical cues that may facilitate species recognition and mate discrimination. The authors found iridescent coloration to be similar in sympatric Morpho species. Furthermore, male mate choice experiments revealed that in sympatry, males fail to discriminate conspecific females based on coloration, reinforcing the idea that visual signal convergence is primarily driven by predation pressure. In contrast, the divergence of chemical signals among sympatric species suggests their potential role in facilitating species recognition and mate discrimination. The authors conclude that interactions between ecological pressures and signal evolution may shape species coexistence.

Strengths:

The study is well-designed and integrates multiple methodological approaches to provide a thorough assessment of signal evolution in the studied species. I appreciate the authors' careful consideration of multiple selective pressures and their combined influence on signal divergence and convergence. Additionally, the inclusion of both visual and chemical signals adds an interesting and valuable dimension to the study, enhancing its importance. Beyond butterflies, this research broadens our understanding of multimodal communication and signal evolution in the context of species coexistence.

Weaknesses:

(1) The broader significance of the findings needs to be better articulated. While the authors emphasize that comparing adaptive traits in sympatry and allopatry provides insights into selective processes shaping reproductive isolation and coexistence, it is unclear what key conceptual or theoretical questions are being addressed. Are these patterns expected under certain evolutionary scenarios? Have they been empirically demonstrated in other systems? The authors should explicitly state the overarching research question, incorporate some predictions, and better contextualize their findings within the existing literature. If the results challenge or support previous work, that should be highlighted to strengthen the study's importance in a broader context.

We thank the reviewer for their valuable feedback. We understand that the framing of the results and the discussion may fail to convey the broader significance of our findings. In the first version of the manuscript, we framed our manuscript around the processes shaping reproductive isolation and co-existence in sympatry, but now realize that this question was too broad in regards to our results. We thus strictly focused on outlining the importance of ecological interactions in the evolution of traits in sympatric species. In the revised version of the manuscript, we rewrote the first paragraph of the introduction to introduce context regarding the effect of ecological interactions on trait evolution (lines 43-60). We then explicitly introduce the theoretical question investigated in our paper (i.e. “we investigate how ecological interactions in sympatry can constrain natural and sexual selection shaping trait evolution”, lines 62-63) and our predictions regarding the evolution of traits in sympatry vs. allopatry (lines 74-80). We also added predictions regarding our experiments on Morpho at the end of the introduction (lines 146-157). As a result, the discussion is now better aligned with the introduction, by discussing the putative effect of predation and mate choice on the evolution of wing iridescence in Morpho.

(2) The motivation for studying visual signals and mate choice in allopatric populations (i.e., at the intraspecific level) is not well articulated, leaving their role in the broader narrative unclear. In particular, the rationale behind experiments 1, 2, and 3 is not well defined, as the authors have not made a strong case for the need for these intraspecific comparisons in the introduction. This issue is further compounded by the authors' primary focus on signal evolution in sympatry throughout both the results and the discussion. For instance, the divergence of iridescence in allopatry is a potentially interesting result. But the authors have not discussed its implications.

We now clearly state in the introduction our motivation for studying visual signals and mate choice in allopatric populations (lines 74-80, lines 146-157). We argued that intraspecific comparisons help identify whether visual cues can be used in mate recognition between phylogenetically close subspecies, between whom visual resemblance is supposed to be higher than between closely-related species (tetrad experiment, and experiment 1). As M. h. bristowi and M. h. theodorus have different wing pattern, we also used this comparison to identify the traits involved in male mate preference within a species, testing the importance of iridescent color (experiment 2) or iridescent patterning (experiment 3). The results of those experiments can then be used to assess whether these traits are used in species recognition between sympatric species. See also our answers to recommendations 11 and 15 from reviewer #1.

Overall, given that the primary conclusions are based on results and analyses in sympatry, the role of allopatric populations in shaping these conclusions needs to be better integrated and justified. Without a stronger link between the comparative framework and the study's key takeaways, the use of allopatric populations feels somewhat peripheral rather than central to the study's aim. Since the primary conclusions remain valid even without the allopatric comparisons, their inclusion requires a clearer rationale.

To make a stronger case for the use of the allopatric population in our manuscript, we strengthened the justification behind the study of intraspecific allopatric populations vs. interspecific sympatric populations, as the iridescence measurements and the mate choice experiments in allopatric populations can serve as a baseline in studying how species interactions can shape the evolution of traits and mate recognition when compared to sympatric populations. Following your major comment #1, we rewrote the introduction to include a justification to the need for studying allopatric vs. sympatric populations (lines 74-80), and also further highlighted the need to study iridescence in sympatric species to fully understand the trait evolution of sympatric species in the discussion (339-343).

(3) While the authors demonstrate that iridescence is indistinguishable to predators in sympatry, they overstate the role of predation in driving convergence. The present study does not experimentally demonstrate that iridescence in this species has a confusion effect or contributes to evasive mimicry. Alternatively, convergence could result from other selective forces, such as signal efficacy due to environmental conditions, rather than being solely driven by predation.

We acknowledge that our study does not directly demonstrate that iridescence contributes to evasive mimicry. We did tone down the interpretation of the results in the discussion and state that predation is not the only selective pressure that could have promoted a convergent evolution of iridescence in sympatric species, as iridescence is a trait that could be involved in thermoregulation (lines 346-353) and camouflage (lines 363-369) for example. We made sure to mention that convergence in iridescent signals in sympatry is only an indirect support to the evasive mimicry hypothesis, and that further research is still needed, including direct predation experiments, to show that this convergence is indeed triggered by predation (lines 391-396).  

Reviewer #2 (Public review):

This study presents an investigation of the visual and chemical properties and mating behaviour in Morpho butterflies, aimed at addressing the nature of divergence between closely related species in sympatry. The study species consists of three subspecies of Morpho helenor (bristowi, theodorus, and helenor), and the conspecific Morpho achilles achilles. The authors postulate that whereas the iridescent blue signals of all (sub)species should function as a predator reduction signal (similar to aposematism) and therefore exhibit convergence, the same signals should indicate divergence if used as a mating signal, particularly in sympatric populations. They also assess chemical profiles among the species to assess the potential utility of scent in mediating species/sex discrimination.

The authors first used reflectance spectrometry to calculate hue, brightness, and chroma, plus two measures of "iridescence" (perhaps better phrased as angular dependence) in each (sub)species. This indicated the ubiquitous presence of sexual dimorphism in brightness (males brighter), which also appears to be the case for iridescence (Figure 3A-B). Analysis of these data also indicated that whereas there is evidence for divergence among subspecies in allopatry, the same evidence is lacking for species in sympatry (P = 0.084). This was supported further by visual modelling, which showed that both conspecifics and birds should be (theoretically) capable of perceiving the colour difference among allopatric populations of M. helenor, whereas the same is not true for the sympatric species.

The authors then conducted mate choice trials, first using live individuals and second using female dummies. The live experiments indicated the presence of assortative mating among the two subspecies of M. helenor (bristowi and theodorus). The dummy presentations indicated (a) bristowi males prefer conspecific wings, whereas theodorus have no preference, (b) bristowi males prefer the con(sub)specific colour pattern, (c) theodorus prefer the con(sub)specific iridescence when the pattern is manipulated to be similar among female dummies. A fourth experiment, using sympatric M. achilles and M. helenor, indicated no preference for conspecific female dummies. Finally, chemical analysis indicated substantial differences between these two species in putative pheromone compounds, and especially so in the males.

The authors conclude that the similarity of iridescence among species in sympatry is suggestive of convergence upon a common anti-predation signal. Despite some behavioural evidence in favourof colour (iridescence)-based mate discrimination, chemical differences between Achilles and Helenor are posed as more likely to function for species isolation than visual differences.

Overall, I enjoyed reading this manuscript, which presents a valiant attempt at studying visual, chemical and behavioural divergence in this iconic group of butterflies.

Major comments

My only major comment concerns the authors' favoured explanation for aposematism (or evasive mimicry) for convergence among species, which is based upon the you-can't-catch-me hypothesis first presented by Young 1971. Although there is supporting work showing that iridescent-like stimuli are more difficult to precisely localize by a range of viewers, most of the evidence as applied to the Morpho system is circumstantial, and I'm not certain that there is widespread acceptance of this hypothesis. Given that the present study deals with closely-related  (sub)species, one alternative explanation - a "null" hypothesis of sorts - is for a lack of divergence (from a common starting point) as opposed to evolutionary convergence per se. in other words, two subspecies are likely to retain ancestral character states unless there is selection that causes them to diverge. I feel that the manuscript would benefit from a discussion of this alternative, if not others. Signalling to predators could very well be involved in constraining the extent of convergence, but this seems a little premature to state as an up-front conclusion of this work. There is also the result of a *dorsal* wing manipulation by Vieira-Silva et al. 2024 which seems difficult to reconcile in light of this explanation. Whereas this paper is cited by the authors, a more nuanced discussion of their experimental results would seem appropriate here.

We thank the reviewer for their constructive comments on our manuscript. We appreciate the reviewer’s concern regarding the way iridescence convergence between sympatric species is discussed in our manuscript, which align with similar concerns raised by Reviewer 1. Indeed, the you-can't-catch-me hypothesis has not been yet empirically tested in Morpho, this is currently a working hypothesis only supported by indirect lines of evidence.

Among the 30 known Morpho species, iridescence is most likely the ancestral character, notably because iridescence is a trait shared by a majority of Morpho (we now mention this in the introduction lines 108-110). In this paper, we thus did not aim to identify the evolutionary forces involved in the appearance of iridescence in this group, but rather wanted to understand to what extent ecological interactions can impact the diversification (or not) of this trait. As such, the dorsal manipulations performed in Vieira-Silva et al 2024 showing that iridescence in Morpho may have a similar effect than crypsis does not impact our working hypothesis. Instead, we use VieraSilva et al 2024 to discuss the potential anti-predator effect of iridescence, that could potentially promote convergent evolution of iridescent patterns.

In the main text, we now clearly mention our null hypothesis: under a scenario of neutral evolution of iridescence, we would expect that the divergence in wing coloration between two M. helenor subspecies would be lower than between two different Morpho species (M. helenor and M. achilles) and showed that our results sharply differ from this null expectation.

We then improved the discussion by adding alternative hypotheses potentially explaining the convergent iridescent signal detected in sympatric species: we discussed the expected effect under neutral evolution (lines 339-343), but also added alternative hypotheses regarding the diversification of iridescence due to camouflage (lines 363-369), predator evasion (lines 373-377) and thermoregulation (lines 346-353).

Reviewer #3 (Public review):

The authors investigated differences in iridescence wing colouration of allopatric (geographically separated) and sympatric (coexisting) Morpho butterfly (sub)species. Their aim was to assess if iridescence wing colouration of Morpho (sub)species converged or diverged depending on coexistence and if iridescence wing colouration was involved in mating behaviour and reproductive isolation. The authors hypothesize that iridescence wing colouration of different (sub)species should converge in sympatry and diverge in allopatry. In sympatry, iridescence wing colouration can act as an effective antipredator defence with shared benefits if multiple (sub)species share the same colouration. However, shared wing colouration can have potential costs in terms of reproductive interference since wing colouration is often involved in mate recognition. If the benefits of a shared antipredator defence outweigh the costs of reproductive interference, iridescence wing colouration will show convergence and alternative mate recognition strategies might evolve, such as chemical mate recognition. In allopatry, iridescence wing colouration is expected to diverge due to adaptation to different local conditions and no alternative mate recognition is expected.

Strengths:

(1) Using allopatric and sympatric (sub)species that are closely related is a powerful way to test evolutionary hypotheses

(2) By clearly defining iridescence and measuring colour spectra from a variety of angles, applying different methods, a very comprehensive dataset of iridescence wing colouration is achieved.

(3) By experimentally manipulating wing coloration patterns, the authors show visual mate recognition for M. h. bristowi and could, in theory, separate different visual aspects of colouration (patterns VS iridescence strength).

(4) Measurements of chemical profiles to investigate alternative mate recognition strategies in case of convergence of visual signals.

Weaknesses:

In my opinion, studies should be judged on the methods and data included, and not on additional measurements that could have been taken or additional treatments/species that should be included, since in most ecological and evolutionary studies, more measurements or treatments/species can always be included. However, studies do need to ensure appropriate replication and appropriate measurements to test their hypothesis AND support their conclusions. The current study failed to ensure appropriate replication, and in various cases, the results do not support the conclusions.

First, when using allopatric and sympatric (sub)species pairs to test evolutionary hypotheses, replication is important. Ideally, multiple allopatric and sympatric (sub)species pairs are compared to avoid outlier (sub)species or pairs that lead to biased conclusions. Unfortunately, the current study compares 1 allopatric and 1 sympatric (sub)species pair, hence having poor (no) replication on the level of allopatric and sympatric (sub)species pairs,

We would like to thank the reviewer for their constructive feedback. We agree that replication is important to test evolutionary hypotheses and that our study lacks replication for allopatric and sympatric Morpho populations. Ideally, one would require several allopatric and sympatric replicates to conclude on the effect of species interaction in trait evolution. Our study is a preliminary attempt at answering this question, covering a few Morpho populations but proposing a broad assessment of iridescence and mate preference for those populations. We clearly mentioned in the discussion that investigating multiple populations is needed to test whether the trend we observed in this paper can be generalized (line 388-392).

Second, chemical profiles were only measured for sympatric species and not for allopatric (sub)species, which limits the interpretation of this data. The allopatric (sub)species could have been measured as non-coexistence "control". If coexistence and convergence in wing colouration drives the evolution of alternative mate recognition signals, such alternative signals should not evolve/diverge for allopatric (sub)species where wing colouration is still a reliable mate recognition cue. More importantly, no details are provided on the quantification of butterfly chemical profiles, which is essential to understand such data. It is unclear how the chemical profiles were quantified and what data (concentrations, ratios, proportions) were used to perform NDMS and generate Figure 5 and the associated statistical tests.

We recognize that having the chemical profiles of the genitalia of the Morpho from the allopatric populations would have made a stronger case in favor of reinforcement acting on the divergence of the chemical compounds found on the genitalia of the sympatric Morpho species. Due to limited access to the biological material needed at the time of the chromatography, we could not test for lower divergence in the chemical profiles of allopatric Morpho butterflies. We made sure to mention this limitation in the discussion (lines 457-461). 

We already stated in the methods that we compiled the area under the peak of each components found in the chromatograms of our samples and that we performed all the statistical analyses on this dataset. To make it clearer, we mention in the new version of the manuscript that the area under the peak of each component allows to measure the concentration of the components (in the methods lines 720, 723, 733). We also added some precisions in the legend of Figure 5.

Third, throughout the discussion, the authors mention that their results support natural selection by predators on iridescent wing colouration, without measuring natural selection by predators or any other measure related to predation. It is unclear by what predators any of the butterfly species are predated on at this point

We made sure to mention in the introduction (line 132-136) and in the discussion (line 373-377) that previous predation experiments performed on Morpho and other butterflies showed evidence that birds are likely predators for these species. These observations lead us to test for the putative effect of predation on the evolution of their color pattern, without directly testing predatory rates. We made sure this information is transparent in the revised manuscript, and now precise that assessing wing convergence is only an indirect way of testing the escape mimicry hypothesis (line 393-396).

To continue on the interpretation of the data related to selection on specific traits by specific selection agents: This study did not measure any form of selection or any selection agent. Hence, it is not known if iridescent wing colouration is actually under selection by predators and/or mates, if maybe other selection agents are involved or if these traits converge due to genetic correlations with other traits under selection. For example, Iridescent colouration in ground beetles has functions as antipredator defence but also thermo- and water regulation. None of these issues are recognized or discussed.

The lack of discussion of alternative selective pressures involved in the evolution of iridescence was pointed out by all reviewers. We thus modified the text to account for this comment, and no longer limit our discussion to the putative effects of predation. We now specifically discuss alternative hypotheses, including crypsis (362-369) and thermoregulation (line 346-353).

Finally, some of the results are weakly supported by statistics or questionable methodology.

Most notably, the perception of the iridescence coloration of allopatric subspecies by bird visual systems. Although for females, means and errors (not indicated what exactly, SD, SE or CI) are clearly above the 1 JND line, for males, means are only slightly above this line and errors or CIs clearly overlap with the 1 JND line. Since there is no additional statistical support, higher means but overlap of SD, SE or CI with the baseline provides weak statistical support for differences.

We thank the reviewer for bringing interpretation issues concerning the chromatic distances of allopatric Morpho species measured with a bird vision model. We made sure to be nuanced in the description of this graph in the results section (line 208-212). Note that this addition does not change our main conclusion stating that Morpho and predator visual models better discriminate iridescence differences between allopatric subspecies than between sympatric species.

We now also clearly mention in the figure’s legend that the error bars represent the confidence intervals obtained after performing a bootstrap analysis, in addition to the mention of the nature of the error bars already mentioned in the methods (line 580).

Regarding the assortative mating experiment, the results are clearly driven by M. bristowi. For M. theodorus, females mate equally often with conspecifics (6 times) as with M. bristowi (5 times). For males, the ratio is slightly better (6 vs 3), but with such low numbers, I doubt this is statistically testable. Overall low mating for M. bristowi could indicate suboptimal experimental conditions, and hence results should be interpreted with care.

We recognize that the tetrad experiment results are mainly driven by M. bristowi’s behavior as already mentioned in the results (line 231-232) but we now also mention it in the discussion (lines 401-402). This experiment would have benefited from more replicates, but the limited access to live males and virgin females for both subspecies was a limiting factor. Fisher’s exact test used to assess assortative mating is specifically appropriate to small sample sizes. We recognize that the sampling size is not ideal, however it is still statistically testable.

Regarding the wing manipulation experiment, M. theodorus does not show a preference when dummies with non-modified wings are presented and prefers non-modified dummies over modified dummies. This is acknowledged by the authors but not further discussed. Certainly, some control treatment for wing modification could have been added.

The use of controls to consider the effect of wing modification and odor by the permanent marker were already mentioned in the methods (lines 636-639). Following your recommendation and comments from the other reviewers, we now mention the use of this control in the results (lines 278283). We also address a potential issue that would have resulted in the rejection of these modified dummies by live males: we cannot be sure whether butterflies perceive these modifications as equivalent to natural coloration (lines 281-282). An additional control could have been used, adding black ink on the black dorsal parts of the pattern to assess its potential visual effect. The constraints on sampling unfortunately did not allow to add another treatment.

Overall, the fact that certain measurements only provide evidence for 1 of the 2 (sub)species (assortative mating, wing manipulation) or one sex of one of the species (bird visual systems) means overall interpretation and overgeneralization of the results to both allopatric or sympatric species should be done with care, and such nuances should ideally be discussed.

The aim of the authors, "to investigate the antagonistic effects of selective pressures generated by mate recognition and shared predation" has not been achieved, and the conclusions regarding this aim are not supported by the results. Nevertheless, the iridescence colour measurements are solid, and some of the behavioural experiments and chemical profile measurements seem to yield interesting results. The study would benefit from less overinterpretation of the results in the framework of predation and more careful consideration of methodological difficulties, statistical insecurities, and nuances in the results.

Overall, we would like to thank all reviewers for their thorough assessment of our work. We understand that the imbalance between mate choice data, visual model data and chemical data only gives us a partial assessment of species recognition in Morpho butterflies, thus requiring more precision in the interpretation and the discussion of our results. We made sure to add balanced interpretations in our discussion, by mentioning the lack of replicates for allopatric and sympatric populations (lines 391-392), and the lack of chemical characterization of allopatric species (lines 458361, see previous comments) and by being more transparent on methodological limitations that we failed to convey in the first version of our manuscript. We brought nuance to our discussion and also discussed alternative hypotheses to predation to explain the convergence of iridescence found in sympatry.

Reviewing Editor Comments:

While all reviewers acknowledge the value of your data, they converge in their recommendations to tone down the evolutionary interpretations. Ideally, to test your main hypothesis, you would need several species pairs, or if only one, as in your case, replicated sympatric and allopatric sites for both species. Furthermore, your more specific hypotheses about convergence (vs. nondivergence), response to predators (vs. other environmental variables), and avoiding interspecific mating in sympatry (vs. not avoiding it in allopatry) would require appropriate alternative treatments/controls. We therefore recommend that you focus on those statements that you can support with your experiments and data, and introduce these statements in the introduction with reference to the appropriate literature.

Reviewer #1 (Recommendations for the authors):

(1) Line 25: This stated aim seems a bit off. The authors did not sensu stricto quantify 'how shared adaptive traits may shape genetic divergence' in this study. I suggest rewriting or deleting this whole sentence altogether. The study's aim is already clear in lines 29-34.

We deleted the mention of the characterization of genetic divergence, since this study did not focus on any genetic analysis.

(2) Line 34: The authors here state that they compared allopatric vs sympatric populations. This is strictly not true for M. Achilles. Further, the results after this sentence focus solely ondivergence/convergence in sympatry, nothing at the intraspecific level and implications of the findings

We now mention that we tested allopatric vs. sympatric species of M. helenor only (lines 28-29). We also mention that the behavioral experiments were based on intraspecific comparisons, and discuss the implications of this result in the discussion.

(3) Line 35: 'convergence driven by predation': this is a strong statement and cannot be directly inferred from the present set of experiments. Consider toning it down.

We added nuance to this statement by rephrasing it “suggesting that predation may favors local resemblance” (lines 32-33)

(4) Line 36: Replace 'behavioral results' with 'behavioral experiments' or something similar.

Corrected

(5) Line 45-49: These opening statements need some citations.

We provided references for the first few lines, by citing terHorst et al 2018 (line 44) underlining the importance of species interactions in trait evolution, and Blomberg et al 2003 (line 45) showing that closely-related species tend to resemble each other by quantifying the phylogenetic signal of various traits.

(6) Line 83, 165: 'visual effect', not sure what the authors are referring to. Please rewrite.

We defined “visual effect” as the way wing color patterns could be perceived by predators or mates. We removed mentions of “visual effect” and directly used its definition instead.

(7) Line 105 onwards: This section of the introduction could benefit from more concise writing. The authors might consider reducing the number of specific examples and instead offering broader general statements, supported by citations from multiple studies.

We reduced the number of examples given in this paragraph and used general statements supported by multiple citations as examples. (lines 102-119).

(8) Line 108-110: This sentence seems to be redundant with the previous one.

We merged this sentence with the previous one to improve clarity. (lines 103-105)

(9) Line 140: 'with chemical defenses': include citations here.

We added citations of Joron et al 1999 and Merrill et al 2014, which document the evolution of convergent wing patterns (mimicry) in butterfly species with chemical-defenses.

(10) Line 149: This is a bit of a stretch. Note that genetic divergence could be influenced by many other things, not only the processes that the authors examined.

We agree with the reviewer that the study of the convergent vs. divergent evolution of visual cues is not enough to fully understand the mechanisms allowing genetic divergence between species. Because this paper does not focus on characterizing genetic divergence, we removed it from the manuscript to avoid oversimplification.

(11) Line 151: Again. Here, the author's primary focus seems to be at an interspecific level. One is left to wonder about the need for comparisons at the intraspecific level in M.helenor and the implications. Please clarify

In the end of the introduction (lines 146-157), we specifically highlighted the importance of intraspecific comparisons. While studying the effect of sympatry on the evolution of the iridescent color pattern, we use this intraspecific comparison as a baseline to account for convergence or divergence of iridescence in a sympatric interspecific pair of Morpho, because under neutral evolution two subspecies are expected to be more similar than two different species (this assumption has been clarified line 147-148). We also used intraspecific mate choice to test for the use of visual cues in mate recognition (experiment 1) and to test what type of signal could be perceived by Morphos (the iridescent coloration or the iridescent pattern, experiment 2 and 3). These results help contextualize the interspecific mate choice, focused on determining whether visual cues could also be used in species recognition. Since we show that iridescent coloration is important in mate recognition at the intraspecific scale, it helps understand why species recognition is low at the interspecific scale because of wing color convergence between M. helenor and M. achilles.

(12) Line 154: 'signals on mate preferences'.

Corrected.

(13) Line 189: 'At the intraspecific level', maybe in the brackets include 'allopatric populations' just so the results are in a similar format as in the color contrast section below.

We added details to make clearer that the intraspecific level is studied between allopatric Morpho populations (line 189).

(14) Line 189-192: Please rearrange the figure (current B as A and vice versa) or present the results in order as in the figure (interspecific first and then intraspecific level).

We rearranged Figure 3 so that the intraspecific comparison (allopatric population) appears as A and the interspecific level (sympatric population) appears as B, to follow the order of presentation in the main text.

(15) Line 232: The motivation behind experiments 1, 2, and 3 is unclear. The authors have not made a strong point in the introduction about the need for these comparisons at an intraspecific level. Given that the authors are focused on divergence/convergence at an interspecific level, this set of experiments seems to be irrelevant to the present study. The implications of these findings are also not discussed.

We added motivation to the use of experiment 1, 2, and 3 in the introduction (lines 151-154) by stating that those experiments were used to assess whether blue color could indeed be used as a mating cue in Morpho helenor (experiment 1) and to try to understand what part of the visual signal is important in mate choice in Morpho helenor: the wing pattern (experiment 2) or the iridescent coloration (experiment 3). Although motivation for these experiments was not detailed in our manuscript, we already discussed the implications of the results of experiments 1, 2 and 3 in the discussion by stating that visual cues can take many forms and that considering both color AND pattern is important in understanding visual cues (lines 408-416). We carefully reworked this new version to make it more straightforward.

(16) Line 260: Insert 'wild-type' before model to ensure similar wording as in the previous section.

Corrected.

(17) Line 286: Insert 'sympatric' after mimetic.

Corrected.

(18) Line 307: Include a reference to the figures or table where these results are presented.

We now mention in the main text that the different proportions of beta-ocimene found between males M. helenor and M. achilles are shown in Table S2.

(19) Line 343: These inferences are speculative. Add a line here, something like 'although this warrants further research in this species'.

We detailed what additional experiments are needed lines 388-396.

(20) Line 357: The authors have not discussed their results on iridescence divergence in allopatric populations (line 190) and its implications.

We now made clear in the beginning of the discussion that the divergence of iridescence in allopatric populations is used as a baseline to test for convergent iridescence between species (lines 339-343).

(21) Line 361 onwards: This first paragraph is a bit confusing, as the results mainly focus on allopatry, while the title refers to sympatry.

To avoid confusion between the title and the content of the discussion, we divided the last part of the discussion into two different parts. As the first paragraph mainly focus on allopatry, we isolated it and titled it “Iridescent color patterns can be used as mate recognition cues in M. helenor” (line 498). The next paragraph of the discussion, focusing on the sympatric Morpho populations, has been titled “Evolution of visual and olfactory cues in mimetic sister-species living in sympatry” (line 418).

(21)  Line 383: visual cues 'as' poor species.

Corrected.

(23) Line 405: Why females here and not males? This is again confusing since the authors tested for male mate choice in the main experiments. Some background information on sex-specific mate choice in the methods might help.

In this specific sentence, we talk about performing mate choice experiments to test for the discrimination of olfactory cues by females (and not males) because we found a high divergence in the chemical compounds found on male genitalia. Although female chemical compounds could also be used as a cue by males in mate recognition, olfactive mate choice is often driven by female choice in butterflies. We recognize that this perspective does not line up with the mate choice presented in our results section which focused on male mate choice based on visual cues, because of ecological reasons (Morpho males tend to be attracted to bright blue colorations but not females) and technical reasons (in cages, females tend to hide away from the males or male dummies, and this behavior is not compatible with experiments involving flying around false males). In the discussion, we made sure to precise that the perspective we cite here is about testing the implications of divergence in male olfactory cues (line 454). We also added motivation to why we chose to investigate male (and not female) mate choice based on visual cues in the methods (lines 613-618) and in the results (219-223).

(24) Line 417: This inference is speculative. Consider toning it down.

We rewrote the sentence: “We find evidence of converging iridescent patterns in sympatry suggesting that predation could play a major role in the evolution of iridescence. Further work is nevertheless needed to directly test this hypothesis and establish the important of evasive mimicry in Morpho” (lines 465-468).

(25) Line 429: 'Convergent trait evolution leads to mutualistic interactions enhancing coexistence'. Careful here. It is not very evident how convergent trait evolution (iridescence) is mutualistic in this case, as there is no experimental evidence for evasive mimicry yet. Consider rewording or toning this sentence down.

We agree with the reviewer and removed this statement, only keeping the end of the sentence: “Altogether, this study addresses how convergence in one trait as a result of biotic interactions may alter selection on traits in other sensory modalities, resulting in a complex mosaic of biodiversity. (lines 479-481).

(26) Line 442: Since the samples come from a breeding farm, I have a few questions. How are the authors sure about the location where the specimens were collected? How long have they been kept in captivity? Have they been subjected to any artificial selection? More details are needed here.

Since M. helenor bristowi and M. helenor theodorus are only found in the wild in West and East Ecuador respectively, those M. helenor subspecies can only be collected in those two allopatric populations. Their phenotype is directly linked to their geographic repartition, this is how we made sure about their collect location. M. h. theodorus we used in this study were caught in East Ecuador in Tena, and M. h. bristowi were caught in West Ecuador in Pedro Vincente Madonado. We received pupae from the breeding farm, meaning that the Morpho used for the experiments were raised in captivity since their date of emergence. Upon emergence, they were transferred into cages for 4 to 5 days to wait for sexual maturity before performing the tetrad and mate choice experiments. This information was added to the method (lines 490-496).

(27) Line 476: Include some citations supporting this statement.

We now cite Bennett and Théry (2007), reviewing avian color vision, and Briscoe (2008), characterizing the sensitivity of the photoreceptors found in the eyes of butterflies. Both citations show that the 300-700nm range is seen by avian and butterfly visual systems.

(28) Line 480 onwards: Please clarify if the analysis used only one value (mean?) per species, sex, angle of measurement, and locality or included data from multiple individuals.

The analyses of both colorimetric variables and global iridescence were performed using iridescence data from multiple individuals (10 males and 10 females from M. h. bristowi, M. h. theodorus, M. h. helenor and M. a. achilles), for which we measured iridescence at 21 angles of illumination. Sampling size are mentioned lines 507, 515, 540-542.

(29) Line 510: Is there a specific reason that authors did not investigate achromatic contrasts? Provide some justification here. Or include the results of achromatic contrasts in the supplement.

We added the achromatic results in the supplement and in the results (lines 200-204). For both the avian visual model and the Morpho visual model, the confidence intervals always overlapped with the JND threshold, showing that neither birds nor butterflies could theoretically discriminate the wing reflectance brightness in allopatric and sympatric populations.

(30) Line 552 onwards: I may have missed it. It is not entirely clear why the authors focused on male mate choice rather than female preference for visual cues. The authors should explicitly justify this choice and cite previous studies demonstrating that male mate choice, rather than female preference, is important in this species. This should be stated in the results section as well.

We added a paragraph in the method (lines 613-618) to describe the ecological and technical reasons leading to testing only male mate choice using visual cues (also see our response to recommendation #23).

(31) Line 537 onwards: What was the criterion used to score that mating had occurred? Why first mating and not how long they were mating? Please add these details.

We stopped the experiment as soon as a male/female pair was formed by joining their genitalia (we added this information in the method lines 599-600). Since the tetrad experiment involves the interaction of two males and two females from different subspecies, we considered that mate choice happened before the formation of any couple, and is not necessarily dependent on how long they mate by observing their mating behavior. For instance, we witnessed avoidance behaviors from females that systematically hide their genitalia and refused to join their abdomen to some males, while being very ‘open’ to others (but did not quantify it).  

(32) Line 571: The authors used a black permanent marker to modify wing patterns but did not validate whether butterflies perceive these modifications as equivalent to natural coloration. It is possible that the alterations introduced unintended visual cues and may explain why most males rejected the dummies (line 267). The authors should acknowledge this limitation here.

We now acknowledge this limitation in the method (lines 638-639) and in the results section (lines 278-283).

(33) Line 591: Insert 'above' after protocol.

Corrected.

(34) Line 605: If the authors included random effects in their model, then it should be generalized linear mixed model (GLMM) and not GLM as they wrote.

We indeed included a random effect in our model accounting for male ID and trial number, we thus replaced “GLM” by “GLMM” in the manuscript.

(35) Line 615: This set of analyses does not seem to account for pseudo-replication, as the data were recorded from the same male more than once (Line 583). Please clarify and redo the analysis with the GLMM framework

We run new analyses using the GLMM framework: we used a binomial GLMM to test whether individuals preferentially interacted with dummy 1 vs. dummy 2 while accounting for pseudoreplication. The previously detected tendencies hold true with these new analyses, except for the visual mate discrimination of M. achilles: we now find statistical evidence that M. achilles tend to approach more their conspecifics during the mate choice experiment, although the signal is weak (line 297-307). Indeed, while we previously concluded that both species in sympatry (M. helenor and M. achilles) could not discriminate their conspecific mates, we now emphasize that M. achilles is somewhat sensitive to some visual signals. However, its estimated probability of approaching a conspecific is only 0.54, which is low compared to the estimated probability of approaching (0.61) or touching (0.84) a con-subspecific for M. bristowi. We thus concluded that even though some visual cues could be relevant for mate recognition, they are less reliable for male choice in sympatric populations were color patterns are more convergent, compared to allopatric populations. We thus updated Figure 4 and Figure S8 and S9, which are now picturing the probability of approaching or touching a conspecific or con-subspecific with the updated pvalues retrieved from the GLMM analyses. We also updated the results (line 297-307) and the discussion (lines 430-438) to bring nuance to our previous results.  

(36) Line 963: Figure 3D. Is there a particular reason for comparing allopatric populations only within Ecuador rather than between Ecuador and French Guiana for M. helenor? Please clarify.

We aimed at comparing the putative discrimination of blue coloration using visual models vs. what the butterflies actually discriminate using mate choice experiments. Since we only performed mate choice experiments involving M. h. bristowi x M. h. theodorus (allopatric populations within Ecuador) and M. h. helenor x M. a. achilles (sympatric population from Ecuador), we only looked at those comparisons using visual models. We added this precision lines (559-560).

(37) Line 980: Are these predicted probabilities or just mean proportions as written in line 614? Then the label should be changed to 'Proportion of approaches' or something similar.

Following our answer to recommendation #35, the points now represent the probability of touching a conspecific in the graph for each male, for every trial of every male tested. We corrected the legend of the figure. 

Reviewer #2 (Recommendations for the authors):

(1) Line 25: "...therefore facilitating co-existence in sympathy".

Corrected.

(2) Line 28: "contrasting" instead of contrasted.

Corrected.

(3) Line 33: begin a new sentence at the colon.

Corrected.

(4) Line 49: the phrase "habitat filtering" is unclear and should perhaps be defined or qualified.

We replaced “habitat filtering” by its definition and cited Keddy (1992), describing the community assembly rules and defining habitat filtering (line 46)

(5) Line 52: remove "even".

Corrected.

(6) Line 53: divergent suites may also result because traits are often constrained by genetic architecture (multivariate genetic covariances). This is discussed at length and specifically in relation to ornamental coloration by Kemp et al. 2023

We rewrote the introduction and focused on only reviewing the ecological interactions promoting trait divergence in sympatric species, and did not mention genetics in this paper.

(7) Line 87: (and throughout) refer to "colouration" or "colour pattern" rather than "colourations".

Corrected.

(8) Line 151: Remove "To do so,".

Corrected.

(9) Line 191: I would like to see the degrees of freedom for this test.

We added the F-statistic=2.09 and the degrees of freedom df=1 of this test, and for all the following tests.

(10) Line 201: (and throughout) replace "on" with "of".

Corrected.

(11) Line 205: modelling the visual properties of the wings allows one to infer what is theoretically visible/distinguishable. The modelling is useful but not necessarily definitive of vision/behaviour per se under different conditions in the wild. I therefore think it is appropriate to phrase the wording around the modelling approach more carefully. Perhaps refer to "theoretical" or "inferred" discriminability, or state (e.g.) that species should/should not be capable of perceiving differences based on the modelling data. You do this well in your wording of lines 207-209. This need not apply in the discussion because you're then dealing with the combination of modelling results and behaviour (mating trials).

We agree with the reviewer that visual modelling only allows to infer what is theoretically discriminated by the butterflies, and that the wording of our sentence is confusing. We therefore modified the sentence to account for those precisions: “Morpho butterflies and predators can theoretically visually perceive the difference in the blue coloration between different subspecies of M. helenor…… using both bird and Morpho visual models” (line 206-209).

(12) Line 222: Either the chi-square test or Fisher's exact test should be sufficient (why report both?)

Chi-square test relies on large-sample assumptions (expected counts>5) whereas Fischer’s exact test does not and is valid even with small or unbalanced sample sizes. Since the M. bristowi female/M. h. theodorus male paring only occurred 3 times, we do not meet the primary assumptions to apply a Chi-square test, although it is significant. We used a Fischer’s test to confirm the results. Using both and finding that both tests are significant shows that the results are robust, although they may appear redundant. To simplify, we remove the results of the Chisquare test and only keep the Fisher’s test in the methodology and the results.

(13) Line 224 (and throughout): Degrees of freedom should be provided for statistical tests.

We reported the statistic value and the degrees of freedom for all mentions of the statistical tests in the main text, except for the Fischer test which does not rely on an asymptotic distribution like the Chi-squared distribution as it is an exact test.

(14) Lines 266-267: This sentence has interest, but it is rather vague at present. Wouldn't your controls account for the effect of manipulation? This could be explained further.

During our mate choice experiments, all Morpho female dummies used for the experiments were painted with black markers, either on their dorsal blue band to modify their blue iridescent phenotype, or on their ventral side, thus controlling for the effect of manipulation. However, we cannot rule out that the modification of the dorsal blue iridescence could have had a “repulsive” effect for males for several reasons. For example, depending on the visual discrimination of darker colors by Morphos, the painted black band could have a slightly different color compared to the dark “brown” usually surrounding their blue iridescent patterns. We now explain this in the results (lines 278-283) and in the methodology (lines 638-639)  

(15) Line 316: I'm not certain that the similarity is best described as "striking", given a P-value of 0.084 for this contrast

We agree with the reviewer and removed this adjective for this line.

(16) Lines 387-390: This sentence is puzzling because, theoretically speaking, we should expect selection on visual preference to be heightened (not relaxed) in sympatry if colouration isincluded among the traits used in mate selection. I'm not certain I have understood the meaning here.

We would like to thank the reviewer for pointing out this typo. If shared predatory pressures favors convergent evolution of color pattern, then the visual signals become less reliable for species recognition. As a result, sexual selection on visual preference is heightened and becomes stronger, favoring the evolution of alternative cues used to discriminate conspecific mates. We changed the sentence and now write “the convergent evolution of iridescent wing patterns… may have negatively impact visual discrimination and favored the evolution of divergent olfactory cues” (lines 457-458).

(17) Line 529: Mating experiments. Given that these are quite large butterflies, I wondered whether a 3x3x2m cage would be sufficient in size to allow the expression of male courtship. A brief description of the courtship behaviour in these species or Morphos generally would be a useful addition to the paper.

A cage this size was enough for the males to express a flight behavior similar to what can be seen in nature, while also being able to see the females (live females or dummies). We tried to perform mate experiments in a larger cage (7m x 5m x 3m) but the trials were not conclusive because male did not find the dummies depending on where they were flying in the cage. A 3mx3mx2m cage is a good compromise maximizing interactions while still allowing enough space to fly. We now describe Morpho male behavior and female behavior in the methods (lines 613-618).

(18) Line 546: Why are both tests needed (chi-square AND Fisher's exact)?

Similarly to our answer on recommendations #12, were used both tests to show robustness in the statistical results. We only kept the Fisher’s test results to simplify the results.

semiotics/semiology

he provides a critique of treating language as a "naming process"

signifier (sound image) x signified (the concept) = sign (naming)

example;

• dog = is the sign/naming
• "a four-legged animal that barks" is signified concept (picture) in our minds
• ‘/dɒɡ/’ is the sound image/ signifier

the concept of "arbitrariness"

Line 266 says, "The river sweats / Oil and tar." This line is contradictory in obvious ways; for one, a healthy river should not contain industrial waste like "oil and tar." The polluted river is another example of how nature is disrupted by modern life and industrialization. Also, a river shouldn't "sweat;" it's supposed to flow. The following line, as well as line 273, describes how "the barges drift" and "wash" rather than sail with purpose. There are hints of rhythm in this section, but they are inconsistent and sluggish, mimicking "the turning tide." Further down, lines 277-78 state "Weialala leia / Wallala leialala," which, in the context of Wagner, refers to the Rhinemaidens' song, a sorrowful song of lost gold and a pure, natural world despoiled by greed. By placing it here, Eliot transplants this lament to the polluted Thames. The modern world has its own stolen gold (industrial wealth), which has similarly cursed it, leading to spiritual decay. The song becomes a ghostly echo of a lost purity, now meaningless in its new context, just a sound the river makes. The scene of the poem then shifts to a ghostly memory: "Elizabeth and Leicester / Beating oars / The stern was formed / A gilded shell / Red and gold." This seems like a moment of romance and pageantry. However, Froude reveals the reality beneath the "gilded shell." Froude's history details the political intrigue, the suspected murder of Leicester's wife, and Elizabeth's calculated use of marriage as a political tool. Their relationship was not pure passion but a "fatal affection" entangled with power, suspicion, and death. The "brisk swell" in line 284 that they create is not just water, but the ripple of historical consequence and personal sin. The same Rhinemaidens' lament follows them, subtly linking their political greed to Alberich's curse on the ring.

A social democratic with the three branches of government executive, judicial, and legislative branches and also has a national parliament.

That’s amazing. Vikings influenced the English language and engineering principles that made society more advanced.

I always wonder what Valhalla truly meant. I'm glad I do now!

I wonder what fashion trends developed over time?

Not just resourceful but cautious of not wasting money or resources.

More and more I read, the more and more i'm intrigued by the culture to learn more behind the scenes especially when art is involved

This is very amazing. Vikings were ahead of their time. By almost 1,220 years

I always wondered if they had a soft side! I knew deep down the Vikings had a soft side. No one is truly that violent or the whole group, especially when children are involved.

Sounds like my kind of people, minus the violence

complexity, not simplistic

From what I can gather from this, the Vikings were the "bad apples" of the bunch, setting out to make their own lives by traveling to find a new home and stealing resources to sustain themselves.

I wonder the same. What was the main agenda?

Wow, this is interesting — even though Rome was destroyed by the Gauls in 390 BCE, it didn’t give up and actually came back stronger! It’s kind of like a comeback story after a major setback. The Latin League was like a team-up for protection, but once Rome got powerful enough, it defeated its own allies and took full control — kind of like when a smaller group grows so strong that it doesn’t need help anymore and decides to lead on its own.

Wow, this is so cool — Zeno saw the whole universe as divine, and believed everyone shares a bit of its rational spark! It’s kind of like saying we’re all connected through the same energy or wisdom, like how people today talk about being “one with the universe.” His idea of a stateless community is also wild — it’s almost like an ancient version of trying to imagine a world with no governments or borders, just people living by shared reason and virtue.

Ska kortas ned men får stå fn.

Har inte sett supplement men jag tror generellt att denna studie är värd att submitta rätt högt. Den är bra! Men då krävs ofta lite lull-lull i supplement. Tydligt vad som är inkluderat i SAH, kanske nån sensitivitetsanalys. Kanske kolla det du resonerar om i discussion som jag nämnde förut. Är det bara så att SAH-patienter följs upp så sjukt bra och får SSRI insatt därför. Kolla kanske om de får annat insatt i mkt högra utsträckning som INTE har koppling till depression som jag snackade om där uppe nånstans. Vi kan snacka mer om det vb /JE

Kan ha missat i Methods men VET vi att SA/DP kommer efter insättning av antidepp? Kan det vara så att temporalt kommer först SA/DP, det blir man deprimerad av och får därför AD insatt? Alltså är det kodat så att SA/DP är tvingat att hända efter uthämtade AD? Också competing risks /JE --> <!--# Jag tycker att ett tillägg i diskussionen bör ha med SSRI som neuromodulator per se. Det kommer data från djur och humanstudier kring hjärnplasticitet och SSRI-effekten av detta. Kan vi sen ökning av SSRI senaste åren dvs 2020-2024 jmf 2014-2019? De senaste 5 åren har mer data talat för SSRI som en stroke-regenerator.../PA

Saknar en tabell, med antal patienter, som visar matchade SAH mot icke SAH. Denna matchning ligger till grund för pekets huvudbudskap och är därför jätteviktig. /AO

Skulle vara kanske mer intressant att se tabell 2 för de kontroller som de facto jämfördes. Alltså de matchade kontrollerna. Åtminstone båda varianterna (239,570 kontroller samt de 4*3637 kontrollerna som användes i analysen). /JE Man kanske kan lägga till en tredje kolumn som visar matchade populationen? / RFJ-

Kan man tänka sig att SAH-patienterna följs upp mycket bättre än en generell IVA-kohort. Dvs att det finns en diagnostik-bias eller vad det nu kan heta. SAH får oftare uppföljning hos dr Alpqvist, Lundberg, Svensson som är inkännande och sätter in AD? Gubben med dille får uppföljning vid behov? Om ja så kanske man kan gräva i det och jämföra med en annan omhuldad grupp. Alternativt testa med “negativ kontroll”. Välj ett LM som INTE är kopplat till depression. Typ Omeprazol. Eller nåt vanligt blodtrycksläkemedel. Om SAH också har en ökad förskrivning av detta icke-depressionskopplade läkemedel så indikerar det på att de bara följs upp mer och bättre och inte givet har mer depressioner. /JE Fundera och regionala skillnader??? /LH

Jag vet inte hur generella IVA-pat följs upp men SAH-pat har en enormt standard uppföljning och kontroll-schema sedan 2005. Samtliga inkluderas i neurorehab-kedja, samtliga har åt 3 mån + 1år hos ansv neurokir. Samtliga har en uppföljning via neurorehab i öppen-vård, efter utskrivning från inneliggande neurorehab och därefter går remissen till husläkare. ALLA aSAH pat som är yrkesföra (dvs inte pensionärer) får en sjuksrivningsperiod om minst 100% i 3 mån /PA

Här vill jag att studiepopulationen beskrivs bättre. Är siffror samma som aSAH? Ålder? Incidens? Är det rimligt att ha med 2024 års kohort, läkemedel för depression sätts ofta in senare i förloppet är min erfarenhet./PA

Fixa suppl

Kolla, fler frågor

Här kanske man får en fråga om vad denna exklusion innebär för resultaten. Ev göra en sensitivitetsanalys med non-random dropouts due to death. /JE Kolla Dufoil et al Statist Med 2004 el Scharfstein et al Am Stat Assoc 1999 samt Eriks opioidpek i CCM

Här tycker jag det finns utrymme för att betona selektionen. I60-I60.9 inkluderar även blödning pga fistlar, AVM med blödning utan parenkymblödning samt dissektionsorsakad blödning. I text tycker jag det ska beskrivas även och inte bara ref till supplementary. Räknar jag grovt blir detta incidens om 7/100000/år, vilket är i överkant mtp att vi idag snarare har sjukhusbehandlad incidens omkring 3/100000/år sedan 2010.

The nature imagery here is showing the beauty in nature as things grow, and is also a symbol for death and life, because in nature plants have to die at some point in the winter to come back to life in the spring. This connects to lincolns death because although witman is mourning his loss, he tries to remember his life in a good and positive light.

The speaker feels that their voice can only be heard through feeling over logical terms. Personification is used as the star is called a comrade and the bird is a singer, signaling that they have a more powerful meaning to their connection.

the star represents the loss of something important along with the grief and powerless feelings that come along loosing something important

This stanza shows romanticization of death through having rich love for their loss. Describing ways of connection with their lost one.

The descriptions show the beauty in the song the speaker hears that will eventually leave, symbolizing the loss they already are mourning. Whitman romanticizes death through hearing their lost ones' voice leaving them.

In a mourning of death, roses and lilies and lilacs were shown to express the deep ways of love and grief. Covering death with flowers that connect into beauty, turning grief into a deep expression and feeling of love. Whitman shows Romanticism through nature that mirrors human emotions and peace or beauty even in loss.

There are no differences at all — both species look and act the same everywhere.

What it means: Either they’re responding identically to the environment, or competition is so weak that no differentiation happens.

Both species’ traits change in the same direction across zones — for example, both are larger or smaller in certain areas — but they stay distinct overall.

Instead of diverging due to competition, both species adapt to the same local conditions — for instance, both get larger in cooler or drier habitats.

Very expansive and thoughtful. This report was to recommend an implementation of social insurance right after the war ended. It classifed a broad range of people that would recieve these benefits. I believe it touched on where the money would come from.

This article is a great example of the downsides when it comes to the simplification of data. To briefly summarize, it basically explains that during the 2024 Superbowl, 75.85% of traffic from X's advertising was fake. Advertising companies learned that while their viewership seemed high, their website clicks/content engagement was staggeringly low in comparison. This revealed that X had a huge bot problem. A real example talked about in the article was how a small business owner, despite having 29,000 views on his ad posted to X, Google Analytics reported that X wasn't the source of any of the traffic during that time. This shows that simplifying certain data, such as social media traffic, can blur the line between what is human vs. bot. However, bots cannot be entirely to blame, as the article talks about how this could be an Elon Musk problem as well. The 2023 Superbowl was only a few months after Musk had acquired twitter and had 72% less fake traffic. So, it’s possible that money hungry billionaires like him could be purposely exploiting data simplification, and allowing more bots onto his site in order to increase profits made from advertisers.

The image from this tweet shows the gender selection options from what is assumed to be an online tax form. Hilariously though, besides "Male" and "Female", it also displays the options "N/A", "Unknown", and "Tax Entity". Aside from this being a funny yet really bad mistake on the website's part, it also ironically displays the difficulties people within the LGBTQ community may face when it comes to data collection. Since gender can be considered a spectrum, it can become extremely complicated to accurately create a way to gather data on ALL the genders, besides the main two (male and female).

This article is about Elon's query of the percentage of fake accounts and his plan of buying Twitter. Currently, twitter is already under the name of Elon Musk, but the question about the fake accounts on social media is still a worth-considering question. Data on the internet is simplified and it comes with some bad impacts, so when using internet, we should be able to realize what is real and what is fake. And when we post data on the internet, we should try to use the most helpful way to represent the reality we want to show to others.

[d28] Hoffman, "Data Violence" (2018): Harm arises not only from biased outcomes but also from categorization/interface choices that force people to "adapt." Key takeaway: If the affected community doesn't shape the architecture, technical fairness improvements can still perpetuate violence. This reframes the formal failure in §4.3: a flawed drop-down menu isn't a failure; it's a governance failure.

It’s interesting that Elon Musk is so concerned about bots on Twitter. Not all bots are harmful, and it’s hard to know the exact number of bots on the platform. Claiming there are less than 5% might oversimplify a complex situation. Even if some accounts are fake or spam, that doesn’t change the fact that bots exist and can have different impacts.

This article introduces a book that generally talks about the connection between ignorance and racism. The gap for knowledge is not the only reason causes the racism, and the main reason is the accidental result of epistemological oversight.

In July 2022, Tesla CEO Elon Musk announced the termination of the $44 billion agreement to acquire Twitter. This decision stemmed from the company's misrepresentation of fake account data and refusal to provide critical information. According to the merger agreement, if Twitter prevails in court, it can compel Musk to complete the transaction; otherwise, he must pay a $1 billion termination fee. Legal experts believe Twitter has a high probability of winning the case.

The URL isn't working. Here I am offering a new source! https://thehappybeavers.com/blog/2025-06-27-word-count-differences-explained The passage is basically saying about differennt editors (Microsoft google etc.) use different algorithms to count words, which is why the same text yields different word-counts in each program. How they handle numbers, puncutations, URLs are different. Bad news for the PhD who are struggling with essay length!

I checked out the W3Schools page on Python Lists ([d7]) and it actually helped me understand lists way better than before. It showed that lists can hold different data types at the same time, like strings, numbers, and even other lists. I didn’t realize you could mix them like that! It also explained how you can use list methods like append() and remove(), which I had seen before in examples but never fully got. The examples were simple but made it easier to connect what the book said about indexing and list operations.

This article made me reflect back to a question in one of our assignments in regards to how would the perspective be changed if someone were to figure out that a comment were from a bot rather than a human. Reading that the Super Bowl advertisements were reflected on engagement being over 75% is shocking to hear and the controversy that can come from this is very significant especially towards the topic of X as this is the social media platform that applied the "biggest" topic and relevance. This also makes me wonder how these types of situations will deal with the criticism when events turn out in the wrong. Does their reputation continue to go down in the long-run? Or do people eventually forget and move on from the topic.

This article explains that during the super bowl in 2024, 75% of X's site visits were bots. This is wild to me. It makes me wonder, are social media sites like X even worth it anymore? If the vast majority of users are bots, we aren't even interacting with other humans anymore and the purpose of social media (to connect and share) is lost.

This wikipedia article covers the social media app X formally known as Twitter. Created by billionaire Elon Musk to have a platform to promote free speech, live engagment, and more. The app allows users to post,share, and like a variety of social media style of content. Over the years the app has undergoed multiple different policy changes to moderate content and add verification. The app wants to reduce its spread of harassment, misinformation, and more.

Version 4 of this preprint has been peer-reviewed and recommended by Peer Community in Infections.<br /> See the peer reviews and the recommendation.

I agree with this idea because I believe everyone can be creative in their own way. Creativity doesn’t have to mean inventing something completely new, but it can come from noticing problems or ways things could be improved. I realize that I’ve sometimes doubted my ideas because they felt small or “obvious,” but this reading helped me see that even those ideas have value.

This made me think of the phrase "with age comes wisdom". I think most people can agree that nowadays, people don't really use this phrase because they don't believe in it. But I think the phrase still holds some truth, as this passage describes. With age (and time), you can gain experience through trial and error in whatever you are doing. Experience cannot be taught; one must go through it themselves to gain the wisdom/knowledge from it.

Who was the recruiting agent?

eLife Assessment

This study presents important information about the role of mu opioid receptors in neurotransmission between the medial habenula and the interpeduncular nucleus. The authors provide convincing evidence that mu opioid receptor activation has differential effects on transmission from substance P neurons and cholinergic neurons, and that blockade of potassium channels can unmask a nicotinic cholinergic synaptic response. This work will be of high interest to those studying this brain region, and potentially to the larger neuroscience community studying motivated behavior.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors demonstrate for the first time that opioid signaling has opposing effects on the same target neuron depending on the source of the input. Further, the authors provide evidence to support the role of potassium channels in regulating a brake on glutamatergic and cholinergic signaling, with the latter finding being developmentally regulated and responsive to opioid treatment. This evidence solves a conundrum regarding cholinergic signaling in the interpeduncular nucleus that evaded elucidation for many years.

Strengths:

This manuscript provides 3 novel and important findings that significantly advance our understanding of the medial habenula-interpeduncular circuitry:

(1) Mu opioid receptor activation (mOR) reduces postsynaptic glutamatergic currents elicited from substance P neurons while simultaneously enhancing postsynaptic glutamatergic currents from cholinergic neurons, with the latter being developmentally regulated.

(2) Substance P neurons from the Mhb provide functional input to the rostral nucleus of the IPN, in addition to the previously characterized lateral nuclei.

(3) Potassium channels (Kv1.2) provide a break on neurotransmission in the IPN,

The findings here suggest that the authors have identified a novel mechanism for the normal function of neurotransmission in the IPN, so it would be expected to be observable in almost any animal. In the revised manuscript, the authors put forth significant effort to increase the n, thus increasing the confidence in the observations.

There are also significant sex differences in nAChR expression in the IPN that might not be functionally apparent using the low n presented here. In the revised manuscript, the authors increased the n, and provided data to the reviewers that no significant sex differences were apparent, although there was a trend. Future studies should examine sex differences in detail.

There are also some particularly novel observations that are presented but not followed up on, and this creates a somewhat disjointed story. For example, in Figure 2, the authors identify neurons in which no response is elicited by light stimulation of ChAT-neurons, but application of DAMGO (mOR agonist) un-silences these neurons. Are there baseline differences in the electrophysiological or morphological properties of these "silent" neurons compared to the responsive neurons? In the revised manuscript, the authors directly tested this with new experiments in SST+ neurons in the IPN, demonstrating convincingly that mOR activation unsilences these neurons.

With the revisions, the authors have addressed the reviewers' concerns and significantly improved the manuscript. I find no further weaknesses.

Reviewer #2 (Public review):

Summary:

In this paper, Chittajallu and colleagues present compelling evidence that mu opioid receptor (MOR) activation can potentiate synaptic neurotransmission in a medial habenula to interpeduncular nucleus (mHb-IPN) subcircuit. While, projections from mHb tachykinin 1 (Tac1) neurons onto lateral IPN neurons show a canonical opioid-induced synaptic depression in glutamate release, excitatory neurotransmission in mHb choline acetyltransferase (ChAT) projections to the rostral IPN is potentiated by opioids. This function emerges around age P27 in mice, when MOR expression in the IPN peaks.

Strengths:

Carefully executed electrophysiological experiments with appropriate controls. Interesting description of a neurodevelopmental change in the effects of opioids on mHb-IPN signaling.

Weaknesses:

A minor concern is that the genetic strategy used to target the mHb-IPN pathway (constitutive ChR2 expression in all ChAT+ and Tac1+ neurons) might not specifically target this projection. Future studies are needed to examine the precise mechanism whereby MOR signaling can potentiate glutamatergic neurotransmission in ChAT+ MHb-IPN projections."

Reviewer #3 (Public review):

Summary:

Here the authors describe the role of mORs in synaptic glutamate release from substance P and cholinergic neurons in the medial habenula to interpeduncular nucleus (IPN) circuit in adult mice. They show that mOR activation reduces evoked glutamate release from substance P neurons yet increases evoked glutamate release and Ach release from cholinergic neurons. Unlike glutamate release, Ach release is only detected when potassium channels are blocked with 4-AP or dendrotoxin. The authors also report a previously unidentified glutamatergic input to IPR from SP neurons and describe the developmental timing of mOR- facilitation in adolescent mice.

Strengths:

- The experiments provide new insight into the role of mORs in controlling evoked glutamate release in a circuit with high levels of mORs and established roles in relevant behaviors.

- The experiments are rigorous, and the results are clear cut. The conclusions are supported by the data.

- The findings will be of interest to those working in the field of synaptic transmission and those interested in the function of the medial habenula or interpeduncular nucleus, as well as those seeking to understand the role of opioids on normal and pathological behaviors.

Weaknesses:

- The mechanistic underpinnings of these interesting and novel results are not pursued.

I totally agree with this. All data is a simplification of reality, but in the process of simplification, some information is lost. So when we are choosing the way of simplification, we should consider the pros and cons, then decide which simplification would be the best to satisfy the need of different users.

While reading §4.2 and the debate over Twitter bots, I was struck by how definitions can quietly gain influence. For a class project, I modified a spam heuristic (number of URLs + account age), and my bot estimate went from 3% to 14%—the same data, just simplified. This convinced me that the "<5%" number wasn't a fact, but a governance decision about what's considered criticaI. Platforms should publish ranges based on uncertain scenarios and disclose the underlying assumptions. Question: Why don't we require confidence intervals and other definitions for platform metrics, as we do in epidemioIogy?

Feels like this can be explained by the Linear Direction Hypothesis, each direction encodes a "latent" or some form of "concept". Thus, when we use LoRa its like doing PCA and identifying the most relevant directions for this dataset and encoding them in the model, making the model more "sensitive" to them. When the dataset size is larger than the rank then we cant quite encode all necessary information.

repetition of 'dry' as a motif -> arid/barren --> nothing to supporrt the 'nutrrition' of humanity

collective: the speaker is one of them -> link to spirituality individuality is reduced

eLife Assessment

This important study elucidates the role of the exocyst component EXOC6A at distinct stages of ciliogenesis, which advances our understanding of ciliary membrane remodeling and cilium formation. The authors provide solid evidence that EXOC6A interacts with myosin-Va and is dynamically recruited via dynein-, microtubule-, and actin-dependent mechanisms, to support proper formation of the ciliary membrane. The study will be of interest to cell biologists and other researchers interested in vesicular trafficking, organellar membrane dynamics, and ciliogenesis.

Reviewer #1 (Public review):

Summary:

The study by Lin et al. studies the role of EXOC6A in ciliogenesis and its relationship with the interactor myosin-Va using a range of approaches based on the RPE1 cell line model. They establish its spatio-temporal organization at centrioles, the forming ciliary vesicle and ciliary sheath using ExM, various super-resolution techniques, and EM, including correlative light and electron microscopy. They also perform live imaging analyses and functional studies using RNAi and knockout. They establish a role of EXOC6A together with myosin-Va in Golgi-derived, microtubule- and actin-based vesicle trafficking to and from the ciliary vesicle and sheath membranes. Defects in these functions impair robust ciliary shaft and axoneme formation due to defective transition zone assembly.

Strengths:

The study provides very high-quality data that support the conclusions. In particular, the imaging data is compelling. It also integrates all findings in a model that shows how EXOC6A participates in multiple stages of ciliogenesis and how it cooperates with other factors.

Weaknesses:

The precise role of EXOC6A remains somewhat unclear. While it is described as a component of the exocyst, the authors do not address its molecular functions and whether it indeed works as part of the exocyst complex during ciliogenesis.

Reviewer #2 (Public review):

Summary:

The molecular mechanisms underlying ciliogenesis are not well understood. Previously, work from the same group (Wu et al., 2018) identified myosin-Va as an important protein in transporting preciliary vesicles to the mother vesicles, allowing for initiation of ciliogenesis. The exocyst complex has previously been implicated in ciliogenesis and protein trafficking to cilia. Here, Lin et al. investigate the role of exocyst complex protein EXOC6A in cilia formation. The authors find that EXOC6A localizes to preciliary vesicles, ciliary vesicles, and the ciliary sheath. EXOC6A colocalizes with Myo-Va in the ciliary vesicle and the ciliary sheath, and both proteins are removed from fully assembled cilia. EXOC6A is not required for Myo-Va localization, but Myo-VA and EHD1 are required for EXOC6A to localize in ciliary vesicles. The authors propose that EXOC6A vesicles continually remodel the cilium: FRAP analysis demonstrates that EXOC6A is a dynamic protein, and live imaging shows that EXOC6A fuses with and buds off from the ciliary membrane. Loss of EXOC6A reduces, but does not eliminate, the number of cilia formed in cells. Any cilia that are still present are structurally abnormal, with either bent morphologies or the absence of some transition zone proteins. Overall, the analyses and imaging are well done, and the conclusions are well supported by the data. The work will be of interest to cell biologists, especially those interested in centrosomes and cilia.

Strengths:

The TEM micrographs are of excellent quality. The quality of the imaging overall is very good, especially considering that these are dynamic processes occurring in a small region of the cell. The data analysis is well done and the quantifications are very helpful. The manuscript is well-written and the final figure is especially helpful in understanding the model.

Weaknesses:

Additional information about the functional and mechanistic roles of EXOC6A would improve the manuscript greatly.

Reviewer #3 (Public review):

Summary:

Lin et al report on the dynamic localization of EXOC6A and Myo-Va at pre-ciliary vesicles, ciliary vesicles, and ciliary sheath membrane during ciliogenesis using three-dimensional structured illumination microscopy and ultrastructure expansion microscopy. The authors further confirm the interaction of EXOC6A and Myo-Va by co-immunoprecipitation experiments and demonstrated the requirement of EHD1 for the EXOC6A-labeled ciliary vesicles formation. Additional experiments using gene-silencing by siRNA and pharmacological tools identified the involvement of dynein-, microtubule-, and actin in the transport mechanism of EXOC6A-labeled vesicles to the centriole, as they have previously reported for Myo-Va. Notably, loss of EXOC6A severely disrupts ciliogenesis, with the majority of cells becoming arrested at the ciliary vesicle (CV) stage, highlighting the involvement of EXOC6A at later stages of ciliogenesis. As the authors observe dynamic EXOC6A-positive vesicle release and fusion with the ciliary sheath, this suggests a role in membrane and potentially membrane protein delivery to the growing cilium past the ciliary vesicle stage. While CEP290 localization at the forming cilium appears normal, the recruitment of other transition zone components, exemplified by several MKS and NPHP module components, was also impaired in EXOC6A-deficient cells.

Strengths:

(1) By applying different microscopy approaches, the study provides deeper insight into the spatial and temporal localization of EXOC6A and Myo-Va during ciliogenesis.

(2) The combination of complementary siRNA and pharmacological tools targeting different components strengthens the conclusions.

(3) This study reveals a new function of EXOC6A in delivering membrane and membrane proteins during ciliogenesis, both to the ciliary vesicle as well as to the ciliary sheath.

(4) The overall data quality is high. The investigation of EXOC6A at different time points during ciliogenesis is well schematized and explained.

Weaknesses:

(1) Since many conclusions are based on EXOC6A immunostaining, it would strengthen the study to validate antibody specificity by demonstrating the absence of staining in EXOC6A-deficient cells.

(2) While the authors generated an EXOC6A-deficient cell line, off-target effects can be clone-specific. Validating key experiments in a second independent knockout clone or rescuing the phenotype of the existing clone by re-expressing EXOC6A would ensure that the observed phenotypes are due to EXOC6A loss rather than unintended off-target effects.

(3) Some experimental details are lacking from the materials and methods section. No information on how the co-immunoprecipitation experiments have been performed can be found. The concentrations of pharmacological agents should be provided to allow proper interpretation of the results, as higher or lower doses can produce nonspecific effects. For example, the concentrations of ciliobrevin and nocodazole used to treat RPE1 cells are not specified and should be included. More precise settings for the FRAP experiments would help others reproduce the presented data. Some details for the siRNA-based knockdowns, such as incubation times, can only be found in the figure legends.

Taken together, the authors achieved their goal of elucidating the role of EXOC6A in ciliogenesis, demonstrating its involvement in vesicle trafficking and membrane remodeling in both early and late stages of ciliogenesis. Their findings are supported by experimental evidence. This work is likely to have an impact on the field by expanding our understanding of the molecular machinery underlying cilia biogenesis, particularly the coordination between the exocyst complex and cytoskeletal transport systems. The methods and data presented offer valuable tools for dissecting vesicle dynamics and cilium formation, providing a foundation for future research into ciliary dysfunction and related diseases. By connecting vesicle trafficking to structural maturation of an organelle, the study adds important context to the broader description of cellular architecture and organelle biogenesis.

eLife Assessment

This valuable study investigates the role of HIF1a signaling in epicardial activation and neonatal heart regeneration in mice. Using a combination of genetic and pharmacological approaches, the authors demonstrate that stabilization of HIF1a enhances epicardial activation and extends the regenerative capacity of the heart beyond the typical neonatal window following myocardial infarction. The main conclusion is well supported by solid data, although some minor concerns regarding experimental interpretation require further clarification to ensure accuracy.

Reviewer #1 (Public review):

Summary:

The manuscript by Gamen et al. analyzed the functional role of HIF signaling in the epicardium providing evidence that stabilization of the hypoxia signaling pathway might contribute to neonatal heart regeneration. By generating different conditionally mouse mutants and performing pharmacological interventions, the authors demonstrate that stabilizing HIF signaling enhances cardiac regeneration after MI in P7 neonatal hearts.

Strengths:

The study presents convincing genetic and pharmacological approaches on the role of hypoxia signaling enhance the regenerative potential of the epicardium

Weaknesses:

The major weakness remains the lack of convincing evidence demonstrating the role of hypoxia signaling in EMT modulation in the epicardial cells. The authors claimed that EMT assays adopted in this study are based on similar previous studies. Surprisingly, two of the references provided correspond to their own research group (PMID: 17108969, PMID: 19235142), limiting the credit for such claims, and the other two (PMID: 27023710, PMID: 12297106) assessment of cell migration but not EMT is reported. Thus, EMT remains to be convincingly demonstrated.

Reviewer #2 (Public review):

Summary:

In this study, Gamen et al. investigated the roles of hypoxia and HIF1a signaling in regulating epicardial function during cardiac development and neonatal heart regeneration. The authors identified hypoxic regions in the epicardium during development and demonstrated that genetic and pharmacological stabilization of HIF1a during neonatal heart injury prolonged epicardial activation, preserved myocardium, enhanced infarct resolution, and maintained cardiac function beyond the normal postnatal regenerative window.

Strengths:

HIF1a signaling was manipulated in an epicardium-specific manner using appropriate genetic tools.

Weaknesses:

Some conclusions still need clarification.

Comments on revisions:

(1) The authors' comment on the partial overlap of HP1 and HIF1a IF signals (HIF1a is highly unstable ... broader regions of hypoxia) is reasonable and would help readers interpret the data if included in the text describing Fig. 1.

(2) The conclusion regarding WT1+ cells in Fig. 2a and b remains unclear. Both panels display larger and smaller magenta cells, and when all are taken into account, the overall number does not appear substantially different. Additional clarification is needed on how the quantification was performed.

(3) Regarding Figure 6-figure supplement 1c, it seems difficult to conclude the endothelial identity of WT1+ cells based on EMCN staining, as the markers do not overlap. The authors note that WT1 is upregulated in endothelial cells, but this has been reported in the context of injury, which differs from the context of the present study involving Molidustat.

Reviewer #3 (Public review):

Summary:

The author's research here was to understand the role of hypoxia and hypoxia-induced transcription factors Hif-1a in the epicardium. The authors noted that hypoxia was prevalent in the embryonic heart and this persisted into neonatal stages until post natal day 7 (P7). Hypoxic regions in the heart were noted in the outer layer of the heart and expression of Hif-1a coincided with the epicardial gene WT1. It has been documented that at P7, the mouse heart cannot regenerate after myocardial infarction and the authors speculated that the change in epicardial hypoxic conditions could play a role in regeneration. The authors then used genetic and pharmacological tools to increase the activity of Hif genes in the heart and noted that there was a significant improvement in cardiac function when Hif-1a was active in the epicardium. The authors speculated that the presence of Hif-1a improved cell survival.

Strengths:

A focus on hypoxia and its effects on the epicardium in development and after myocardial infraction. This study outlines a potential to extend the regenerative time window in neonatal mammalian hearts.

Weaknesses:

While the observations of improved cardiac function is clear, the exact mechanism of how increased Hif-1a activity causes these effects is not completely revealed. The authors mention improved myocardium survival, but do not include studies to demonstrate this.

There is an indication that fibrosis is decreased in hearts where Hif activity is prolonged, but there are no studies to link hypoxia and fibrosis.

Comments on revisions:

In the manuscript revision, the authors address my comments. They outline differences between genetic disruption of Phd2 and chemical inactivation could be due to dosing and drug half-life of Molidustat. The other comments are addressed by explaining that they have analyzed enough heart sections and hearts to come to their conclusions. The authors also state they cannot generate more numbers for this study, therefore I accept their conclusions as stated.

Author response:

The following is the authors’ response to the original reviews

eLife Assessment

This valuable study investigates the role of HIF1a signalling in epicardial activation and neonatal heart regeneration in mice. Through a combination of genetic and pharmacological approaches, the authors show that stabilization of HIF1a enhances epicardial activation and extends the regenerative capacity of the heart beyond the typical neonatal window following myocardial infarction (MI). However, several aspects of the study remain incomplete and would benefit from further clarification and additional experimental support to solidify the conclusions.

We reveal herein prolonged epicardial activation following myocardial infarction (MI) beyond post-natal days 1-7 (P1-P7) by genetic or pharmacological stabilisation of HIF-signalling. This extends the so-called “regenerative window” during an adult-like response to injury, leading to enhanced survived myocardium and functional improvement of the heart, even against a backdrop of persistent, albeit reduced, fibrosis. The epicardium is known to enhance cardiomyocyte proliferation and myocardial growth during heart development via trophic growth factor (for example, IGF-1, FGF, VEGF, TGFβ and BMP) signalling (reviewed in PMID:29592950) and epicardium-derived cell-conditioned medium reduces infarct size and improves heart function (PMID: 21505261). Further experiments, outside of the scope of the current study, are required to determine whether activated neonatal epicardium elicits similar paracrine support to sustain the myocardium and heart function after injury beyond P7 into adulthood.

Public Reviews:

Reviewer #1 (Public review):

Summary:

The manuscript by Gamen et al. analyzed the functional role of HIF signaling in the epicardium, providing evidence that stabilization of the hypoxia signaling pathway might contribute to neonatal heart regeneration. By generating different conditionally mouse mutants and performing pharmacological interventions, the authors demonstrate that stabilizing HIF signaling enhances cardiac regeneration after MI in P7 neonatal hearts.

Strengths:

The study presents convincing genetic and pharmacological approaches to the role of hypoxia signaling in enhancing the regenerative potential of the epicardium.

Weaknesses:

The major weakness is the lack of convincing evidence demonstrating the role of hypoxia signaling in EMT modulation in epicardial cells. Additionally, novel experimental approaches should be performed to allow for the translation of these findings to the clinical arena.

We respectfully disagree that we have not convincingly demonstrated a role for HIF-signalling in promoting epicardial EMT. We adopt epicardial explant assays utilising a well characterised ex vivo protocol previously described for studying EMT in embryonic, neonatal and adult epicardium (PMID: 27023710, PMID: 12297106; PMID: 17108969, PMID: 19235142). These assays demonstrate in WT1^CreERT2;Phd2^fl/fl explants enhanced cobblestone to spindle-like change in cell morphology, increased cell migration, appearance of stress fibres and an up-regulation of the mesenchymal marker alpha-smooth muscle actin (αSMA); all parameters associated with EMT. In addition, our in vivo analyses of Wt1^CreERT2;Phd2^fl/fl hearts, in response to neonatal injury, reveal elevated numbers of WT1+ epicardial cells within the sub-epicardial region and underlying myocardium as is associated with active EMT and subsequent migration from the epicardium.

Reviewer #2 (Public review):

Summary:

In this study, Gamen et al. investigated the roles of hypoxia and HIF1a signaling in regulating epicardial function during cardiac development and neonatal heart regeneration. They found that WT1⁺ epicardial cells become hypoxic and begin expressing HIF1a from mid-gestation onward. During development, epicardial HIF1a signaling regulates WT1 expression and promotes coronary vasculature formation. In the postnatal heart, genetic and pharmacological upregulation of HIF1a sustained epicardial activation and improved regenerative outcomes.

Strengths:

HIF1a signaling was manipulated in an epicardium-specific manner using appropriate genetic tools.

Weaknesses:

There appears to be a discrepancy between some of the conclusions and the provided histological data. Additionally, the study does not offer mechanistic insight into the functional recovery observed.

We respectfully disagree with the comment that our histological data does not support our conclusions and expand on this in the response to specific reviewer comments. We agree that further mechanistic experiments outside of the scope of the current study are required to identify precisely how activated neonatal epicardium results in increased healthy myocardium after injury beyond post-natal day 7 (P7).

Reviewer #3 (Public review):

Summary:

The authors' research here was to understand the role of hypoxia and hypoxia-induced transcription factor Hif-1a in the epicardium. The authors noted that hypoxia was prevalent in the embryonic heart, and this persisted into neonatal stages until postnatal day 7 (P7). Hypoxic regions in the heart were noted in the outer layer of the heart, and expression of Hif-1a coincided with the epicardial gene WT1. It has been documented that at P7, the mouse heart cannot regenerate after myocardial infarction, and the authors speculated that the change in epicardial hypoxic conditions could play a role in regeneration. The authors then used genetic and pharmacological tools to increase the activity of Hif genes in the heart and noted that there was a significant improvement in cardiac function when Hif-1a was active in the epicardium. The authors speculated that the presence of Hif-1a improved cell survival.

Strengths:

A focus on hypoxia and its effects on the epicardium in development and after myocardial infarction. This study outlines the potential to extend the regenerative time window in neonatal mammalian hearts.

We thank the reviewer for this positive endorsement and recognition of the importance of mechanistic insight into how to extend the window of neonatal heart regeneration.

Weaknesses:

While the observations of improved cardiac function are clear, the exact mechanism of how increased Hif-1a activity causes these effects is not completely revealed. The authors mention improved myocardium survival, but do not include studies to demonstrate this.

We report an increase in healthy myocardium arising from prolonged activation of the epicardium during the neonatal window and following injury at post-natal day 7 (P7). We speculate this recapitulates the role of the epicardium during heart development which is known to be a source of trophic growth factors that can enhance myocardial growth. Further experiments are required, out-of-scope of this study, to define a mechanistic link between HIF-signalling, epicardial activation and myocardial survival in the setting of prolonged neonatal heart regeneration.

There is an indication that fibrosis is decreased in hearts where Hif activity is prolonged, but there are no studies to link hypoxia and fibrosis.

We believe the decreased fibrosis is a natural consequence of the increase in survived myocardium arising from the activated epicardium. There is strong precedent here following injury at post-natal day 1 (P1) in which fibrosis is evident early-on but is resolved over time with growth of the myocardium in the regenerating heart (PMID: 23248315).

Recommendations for the authors:

Reviewing Editor Comments:

(1) Address issues related to image quality, colocalization, sample labeling, appropriate controls, and quantification - particularly in Figures 1, 2, 6, and Supplementary Figure 9. Increase sample size as noted by reviewers.

The issues of co-localisation and sample labelling have been addressed under response to reviewers. We are unable to increase sample numbers but have clarified the number of regions per section and numbers of sections per heart analysed where appropriate.

(2) Clarify the effects of epicardial HIF1a activation on neovascularization.

We have removed reference in the abstract to an effect on neovascularisation.

(3) Extend assessments of epicardial hypoxia and HIF1a expression to earlier embryonic stages, when epicardial EMT is more active.

Our earliest timepoint of E12.5 marks the onset of epicardial EMT and E13.5 is the stage with the most significant mobilisation of epicardium-derived cells (EPDCs) into the sub-epicardial region and underlying myocardium (PMID: 32359445). In the same study, E11.5 lineage tracing of epicardial cells is restricted to outer layer of the heart; thus, our timepoints are representative in capturing both the onset and progression of in vivo EMT.

(4) Strengthen EMT assays and mechanistic modeling. Provide evidence from physiologically relevant models, as current 2D culture assays do not adequately support conclusions about EMT. Include additional EMT markers and quantification where appropriate.

We respectfully disagree that epicardial explants are not a valid assay for assessing EMT. As noted under responses to reviewers, such primary explants have been widely described elsewhere (PMID: 27023710, PMID: 12297106; PMID: 17108969, PMID: 19235142) and enable documentation of multiple parameters that are associated with active EMT, including an assessment of the extent of cell migration, cobblestone (epithelial) to spindle-like (mesenchymal) cell morphologies, stress fibre formation and expression of alpha-smooth muscle actin as a mesenchymal marker. We support our findings in explants by revealing reduced WT1+ epicardium-derived cells (EPDCs) in the sub-epicardial region and underlying myocardium of WT1^CreERT2/+;Hif1a^fl/fl embryonic hearts (data in Figure 2) indicative of impaired epicardial EMT and migration of EPDCs and in vivo following neonatal MI with pharmacological inhibition of PHD2, where we observe the reciprocal phenotype of increased numbers of epicardium-derived cells emerging from the outer epicardial layer (data in Figure 6).

(5) Strengthen mechanistic insights into the role of epicardial cells in the functional recovery observed in MI hearts.

We agree that further experiments are required, out-of-scope of this study, to define a mechanistic link between HIF-signalling, epicardial activation and myocardial survival in the setting of prolonged neonatal heart regeneration.

Reviewer #1 (Recommendations for the authors):

The manuscript by Gamen et al. analyzed the functional role of HIF signaling in the epicardium, providing evidence that stabilization of the hypoxia signaling pathway might contribute to neonatal heart regeneration. By generating different conditionally mouse mutants and performing pharmacological interventions, the authors demonstrate that stabilizing HIF signaling enhances cardiac regeneration after MI in P7 neonatal hearts. The study is potentially interesting, but it presents several major caveats.

(1) One of the critical points reported in the early stages of this study is the early co-localization of Wt1, the hypoxic report (HP1), and HIF signaling pathways master regulators (i.e., HIF1a and HIF1b) during embryonic development. Figure 1 is meant to report such findings. However, unfortunately, I hardly see any co-localization at all in the Wt1+ epicardial cells for HP1, with some colocalization is seen for HIF1 and 2 alpha, although none of these data are quantified. Thus, it is hard to believe such co-localization.

We respectfully disagree with this comment. We highlight cells in Figure 1 that are co-stained for WT1+ and HP1. In addition, we identify HIF1-α and HIF2- α positive cells which either reside within the epicardium, as the outer cell layer, or within the underlying sub-epicardial region, respectfully.

(2) The authors claimed that they have analyzed the expression of the hypoxic report, as well as Wt1 and the HIF signaling pathways master regulators (i.e., HIF1a and HIF1b) in the AV groove, as compared to the apex, in embryonic heart ranging from E12.5 to E18.5 (Figure 1). Unfortunately, all images provided that are tagged as AV groove are rather misleading. They do not represent the AV groove but part of the right ventricular free wall. If the authors want to refer to the AV groove, AV cushions should be visible underneath.

We have removed specific reference to the AV groove and refer to the highlighted regions as the “Base” of the heart.

(3) The authors analyzed the hypoxic condition of the developing heart from E12.5 to E18.5. However, it remains unclear why the authors only explored the hypoxic conditions from E12.5 onwards, since epicardial EMT mainly occurs earlier than this time point, i.e., E10.5 onwards. Therefore, it would be needed to explore it already at this earlier time point.

We respectfully disagree with the reviewer and refer to the comment above regarding the fact that E12.5 marks the onset of epicardial EMT and E13.5 is the stage with the most significant mobilisation of epicardium-derived cells (EPDCs) into the sub-epicardial region and underlying myocardium (PMID: 32359445).

(4) The authors reported a conditional mouse model of HIF1alpha deletion by using the Wt1CreERT2 driver. Curiously, Wt1 is dependent on hypoxia signaling (i.e., HIF1a). Therefore, it is unclear whether there is a negative feedback loop between the deletion of Hif1alpha and the activation of the Cre driver might have functional consequences. Convincing evidence should be provided that such crosstalk does not interfere with Hif1alpha inactivation, and therefore, appropriate controls should be run in parallel.

We discount a negative feedback loop in this instance based on the fact we have utilised heterozygous mice for the WT1^CreERT2/+ line and observe a consistent and reproducible phenotype for the developing hearts on a Wt1^CreERT2/+;Hif1a^fl/fl background and following injury in Wt1^CreERT2/+;Phd2^fl/fl mice. Collectively this indicates that the WT1-CreERT2 driver is active in the context of diminishing HIF-1α and Phd2, respectively. In addition, have carried out parallel experiments using epicardial explants derived from R26R-CreERT2;Phd2^fl/fl (Figure 3) to circumvent any potential confounding issues; the results of which are consistent with increased epicardial EMT in support of our overall hypothesis.

(5) On Figure 2a-f the authors reported that epicardial cells are diminished in Wt1CreERT2Hif1alpha mice as compared to controls. I am very sorry, but I do not see any difference. Furthermore, it is unclear to me how the authors quantified such differences, i.e., what marker signal did they use and how it was performed (Figure 2c and d)?

We respectfully disagree with the reviewer and draw attention to the single channel panels of WT1+ staining in Figure 2, which show clear differences between numbers of epicardial cells in the mutant mice compared to controls (comparing magenta cells in panels a) versus b). Quantification was carried out for numbers of WT1+ cells residing within the PDPN-positive epicardium (and underlying PDPN-negative myocardium) across multiple images from multiple sections and multiple hearts.

(6) On Figure 2g, the authors reported differences in total vessel length. Are they referring to impaired microvasculature development? Or is this analysis also including major coronary vessels? What about the major coronary vessels and trees, is there any affection?

This analysis refers to the microvasculature and not the major coronary arteries or coronary trees.

(7) The authors reported that there might be some differences in EMT markers, but unfortunately, all of them are analyzed on 2D cultures, where no substrate for EMT is present, i.e., an underlying ECM bed. Thus, the authors cannot claim that EMT is altered. Additional experiments using either collagen substrate and/or Matrigel are required to fully demonstrate that EMT is impaired. Furthermore, quantitative analyses of such differences should be provided.

The 2D cultures are epicardial explants from mutant versus wild type hearts and represent a widely adopted previously published ex-vivo assay for investigating epicardial EMT across embryonic to adult stages (PMID: 27023710, PMID: 12297106; PMID: 17108969, PMID: 19235142); including an assessment of the extent of migration and cobblestone (epithelial) to spindle-like (mesenchymal) cell morphologies, stress fibre formation and expression of alpha-smooth muscle actin as a mesenchymal marker. We do not understand the comment regarding an “underlying ECM bed” as the cells exhibit EMT routinely on tissue culture plastic and will deposit their own ECM during the culture time course and in response to EMT/cell migration. In terms of quantification this was carried out for scratch assay experiments, as a proxy for EMT and emergent mesenchymal cell migration, as presented in Figure 3i, j with significant enhanced scratch closure and cell migration following Molidustat treatment.

(8) The description of data provided on Supplementary Figure 5 is spurious and should be removed. A note in the discussion might be sufficient.

We respectfully disagree. The ChIP-seq data, in what is now Figure 2- figure supplement 3, highlights a HIF-1 α binding site within the Wt1 locus suggesting putative upstream regulation of WT1 by HIF-1α. Thus this provides a potential explanation as to how HIF-1α may activate the epicardium through up-regulation of Wt1/WT1.

(9) On Figure 3, the authors further illustrate the change of EMT markers using ex vivo cardiac explants. They reported increased expression of Snai2 that, although statistically significant, is most likely of no biological relevance (increase of only 20% at transcript level). What about Snai1, Prrx1, and other EMT promoters? Are they also induced? As previously stated, these 2D cultures do not provide supporting evidence that EMT is occurring, thus 3D gel assays should be performed in which Z-axis analyses will provide evidence on the different migratory behaviour of those cells.

We respectfully suggest that a 20% change in snai2 expression is biologically meaningful with respect to EMT. This in-turn is supported by associated cell migration, reduced ZO-1 expression, increased stress fibres and increased alpha-SMA as a mesenchymal marker; all properties associated with active EMT. Other suggested markers have not been validated as formally required for EMT, for example Snai1 (PMID: 23097346). The migratory capacity of targeted versus epicardial cells was assessed by combined explant and scratch assay experiments.

(10) The description of single-cell analyses is very incomplete. Which mice were used for these analyses, wildtype control, or hypoxic mice? Please provide a clearer description of the samples used. Additionally, the entire rationale of these analyses is dubious. Doing single-cell analyses to analyze a couple or three markers in a very small cell population is rather ridiculous. qPCR might be far more appropriate and convincing, or a bulk RNAseq analysis of isolated epicardial cells.

The single-cell analyses represent an unbiased assessment of different pathways in epicardial cells (identified bioinformatically) between intact P1 and P7 stages in wild type (control) hearts, with a focus on hypoxia-related gene expression and HIF-dependent pathways. It was not designed to analyse a small number of genes, rather global differences in the hypoxic states between P1 and P7 hearts. Selected genes (Vegfa, Pdk3, Egln 1 (Phd2)) were analysed to highlight the key differences in hypoxic signalling across the regenerative window. The fact the hearts were uninjured/intact is clarified in the text and legends for Figure 4 and now Figure 4-figure supplement 1.

(11) The analyses provided in Figure 5 are very interesting and their findings are very relevant. However, I would think that the complementary experimental approach should also be done, i.e, MI followed by activation with tamoxifen, since that situation would be more realistic in the clinical setting.

Tamoxifen causes respiratory failure in neonates with MI, so the two cannot be combined at the same time or soon after surgery. Moreover, tamoxifen takes significant time to take effect on targeted gene down-regulation which may negate sufficient activation of the epicardium following injury.

The experiments in Figure 5 were designed to demonstrate that prolonged heart regeneration could be elicited in a cell-specific (epicardial-specific) manner via a genetic approach. The pharmacological experiments in Figure 6 are complementary in this regard by demonstrating equivalent effects with drug (Molidustat) delivery to reduce PHD2 and stabilise HIF post-MI.

(12) In Figure 6, expression of Wt1 is highly prominent in P7 controls, mainly restricted to the epicardial lining while in the experimental setting, such Wt1 expression is broadly distributed on the subepicardial space, nicely demonstrating epicardial activation. However, it is very surprising to see such Wt1 expression in controls, something that is not expected, as compared to the data reported in Figure 4g. Could the authors please reconcile these findings?

Figure 6 represents the injury setting and Figure 4g the intact setting (as clarified above, in the text and revised figure legends). Hence in the latter WT1 expression is significantly reduced in the P7 heart, as anticipated. With injury at P7 we anticipate activation of WT1 in control hearts, albeit restricted to the epicardial layer (as occurs in adult hearts, PMID: 21505261). In contrast, following Molidustat-treatment of P7 hearts post-MI we observe extensive epicardial expansion into the sub-epicardial region and EPDC migration into the underlying myocardium (Figure 6b).

Reviewer #2 (Recommendations for the authors):

The role of hypoxia and HIF1a signaling in epicardial activation is an important topic, and the genetic approaches employed in this study are appropriate. However, several aspects of the study remain unclear and would benefit from further clarification or explanation by the authors:

(1) The authors detected hypoxic regions using an anti-pimonidazole fluorescence-conjugated monoclonal antibody (HP1). The data would become more compelling if negative and positive controls were provided.

We believe the HP1 staining is compelling in the images shown and is consistent with hypoxic regions of the developing heart. We reveal HP1 staining at cellular resolution with neighbouring cells positive and negative for the HP1 signal in the apex of the heart and within the epicardium and sub-epicardial regions at E12.5 (Figure 1a) and diminished/altered hypoxic/HP1 regional signal through subsequent developmental stages at E14.5-18.5 (Figure 1a-d).

(2) Many HIF1a-positive cells in the AV groove region do not appear to overlap with HP1 staining (Figure 1a). Providing a low-magnification image of HIF1α expression would be helpful to better assess the extent of overlap with HP1 staining

HIF-1 is highly unstable and hence detection of HIF-1+ cells will likely only sample of cells compared to HP1 which is a surrogate for broader regions of hypoxia.

(3) Although the authors conclude that epicardial HIF1a deletion results in a significant reduction of WT1⁺ cells in both the epicardium and myocardium (Figure 2a-d), the provided images are not sufficiently clear to fully support this interpretation. Providing additional evidence to support this conclusion would be helpful.

We respectfully disagree with the reviewer and draw attention to the single channel panels of WT1+ staining which show clear differences between numbers of epicardial cells in the mutant mice compared to controls (Figure 2a versus 2b; magenta WT1+ staining).

(4) Similar to the point raised above, the authors' conclusion regarding the increased expression of WT1 following Molidustat treatment does not appear to be fully supported by the provided images (Figure 6b-f). Immunofluorescence staining for WT1 does not clearly demonstrate epicardial expression in the remote zone of either the control or Molidustat-treated hearts. In addition, while an increase of WT1⁺ cells is observed in the infarct zone of the Molidustat-treated heart, it is somewhat unexpected that such expansion is not evident in the corresponding region of the control heart, given that epicardial cells typically expand near the infarct area. Clarification on these points would be helpful.

Figure 6b reveals WT1 expression in controls (upper panel set) that is reactivated proximal to the infarct region, given WT1 is not expressed in adult epicardium but restricted to the epicardial layer (as occurs in injured adult mouse hearts PMID: 21505261). This contrasts with what is observed in the Molidustat-treated P7 hearts post-MI, where we observe epicardial expansion and migration of WT1+ cells into the underlying myocardium (Figure 6b, lower panel set, infarct zone).

(5) The authors conclude that WT1⁺ cells in the myocardial tissue exhibit endothelial identity based on the colocalization of WT1 and EMCN signals (Supplementary Figure 9c). However, this interpretation is difficult to assess, as WT1 is a nuclear marker and EMCN is a membrane protein, which makes precise colocalization challenging to confirm with confidence. Additional supporting evidence may be necessary to substantiate this conclusion.

WT1 is known to be up regulated in endothelial cells in response to injury as shown previously in several studies (for example, PMID: 25681586). Here we show clear co-localisation of nuclear WT1 and cytoplasmic Endomucin (EMCN) in what is now Figure 6- figure supplement 1c and would encourage the reviewer and readers to magnify the image by zooming-in on the relevant co-stained panel.

(6) The authors conclude that activation of epicardial HIF1a signaling has no effect on neovascularization in postnatal MI hearts (Figure 5c). However, the abstract states: "Finally, a combination of genetic and pharmacological stabilisation of HIF ... increased vascularisation, augmented infarct resolution and preserved function beyond the 7-day regenerative window" (Lines 38-41). Clarification regarding this apparent discrepancy would be appreciated.

The abstract has been altered to remove the statement of increased vascularisation.

(7) The study appears somewhat incomplete, as it lacks mechanistic insight into the functional recovery observed following epicardial Phd2 deletion and Molidustat treatment in postnatal MI hearts. Although the authors suggest a potential paracrine role of the epicardium in protecting cardiomyocytes from apoptosis, this hypothesis has not been experimentally addressed. Incorporating such analysis would help to reinforce the study's conclusions.

Further experiments are required, which are out-of-scope of this study, to define a mechanistic link between the genetic or pharmacological stabilisation of HIF-signalling, epicardial activation and myocardial survival in the setting of prolonged neonatal heart regeneration.

Other points:

(1) Providing single-channel images for Figures 1a-d and 6g would be helpful for clarity and interpretation.

We believe the combined channel views of co-staining for two markers on a background of DAPI staining to pin-point cell nuclei, are informative and support our conclusions.

(2) Have the authors considered using AngioTool to quantify the number of vessels in Figure 5b-c?

AngioToolTM was used to quantify the vessels, as we have used previously (PMID: 33462113) and this is now added to the methods and legend of Figure 2.

Reviewer #3 (Recommendations for the authors):

There are several areas where the manuscript can be improved, such that its conclusions can be solidified.

(1) The authors highlight a point where blocking Phd2 can enhance survival of cardiac tissue, but did not report on survival markers. They surmised that apoptosis could be decreased in Phd2 mutant or Molidustat treatment but did not show this. The authors should determine if apoptosis is decreased in the myocardium and epicardium.

We show evidence of increased levels of healthy myocardium in the genetic and pharmacological models of stabilised HIF-signalling. We exclude increased cardiac hypertrophy or increased cardiomyocyte proliferation as causative, so suggest as a reasonable alternative enhanced survival, albeit this need not necessarily be via an apoptotic pathway given the incidence of necrotic cell death during MI. We are unable to generate new surgeries and mutant/treated heart samples to analyse for apoptotic markers at this stage.

(2) There appears to be no difference in cardiomyocyte proliferation in Molidustat-treated animals, but the experiment was only performed on 2 to 3 animals. This is too small a sample size to conclude from these results. The authors should increase the sample size to make this assertion.

We respectfully disagree that we are unable to conclude no effect on cardiomyocyte proliferation. We analysed multiple heart regions per section, for EdU+/cTnT+ colocalised signals across several sections per heart, set against a consistency of effect on other parameters in hearts treated with Molidustat. We are unable to generate more P7 heart surgeries +/- Molidustat and +/- EdU at this stage.

(3) It is curious as to how, after myocardial infarction, the fibrotic scar tissue is decreased in the Phd2 deletion but not as profound in Molidustat-treated mice at d21. Can the authors speculate why the difference exists and how this decrease arises? For example, are there decreased pro-inflammatory signals in Phd2 deleted mice? Is there decreased collagen deposition and ECM gene expression? Do macrophage recruitment into the infarct zone differ between mutant/treated vs WT?

The representative images in Figure 6k reveal a trend towards reduced fibrosis with Molidistat treatment (Figure 6l), but across all hearts analysed this was not as significant as observed in the epicardial-specific deletion injured hearts (Figure 5g, h). This may be due to the relatively short half-life of Molidustat (approximately 4-10 hours, PMID: 32248614), the dosing regimen for the drug and/or the fact that it was not specifically delivered/targeted to the epicardium.

(4) The magnified images in Figure 1 do not match the boxes in the whole heart images. It is unclear what the white boxes signify.

The white boxes have been removed from Figure 1. The magnified image panels are from serial heart sections and this is now clarified in the Figure 1 legend.

Need to fix the logo on navigation bar

not doing anything french related is an attack on french values

seems a criminizalition and marginalization of the other

hypocritical to allow masks for coronovrius but not for religious reasons

orientalist, treats islam as a monolith of hate

same idea of why jews get protections but not muslims

muslims were not offended because it insulted muhammed but because it betittled their beliefs in muhammed

should not be branded muslims are religious freaks especiallt when trying to assimilate said muslims

the attackers are not a representation of muslims

by taking the claim that free speech attacking religion is fine because its not real makes the complexity of blasphemy in islam to not be accounted for

the reason why we allow free speech that makes fun of religion but not race is because of western belief that religion is fake

if we wouldn't make fun of jews then why would we do so for muslims

the idea that we must respect an individuals commitment to break the norms, but also recognize that we must not support it in order to mantain the guise of respecting free speech

supporting the contents of Hebedo as universal values is not supporting free speech but enforcing several kinds of speech

religion is just an excuse, not the cause

it hypocritical to blame islam for the attacks, when attacks by those carrying the american flag arent used as examples of nationalism being the cause

strict secularism is a problem because it makes jihadist groups look more presentable when they claim the west is out to get them

they are an another

if jews are protected under hate speech laws, why not muslisms

can fit within the orientalist context of trying to modernize those who are stuck with traditions and not with the modern west of secularism

strict secularism has been used when it pertains to muslims by treating them as others that need to be assimilated

should not champion the magazine because it would only heighten tension between groups

the killings were not only a response to the caricturates, but also a sentiment burned from a history of french and muslims relations

arguing that people only showed support to this obscure magazine because it was an attack commited by muslims

the magazine is very provacative with its liberal use of Muhammed

The minimum wage in the US was last raised on July 24, 2009.

I really like the idea of on-going assessment and modifying learning strategies for students. Assessments are not necessarily quizzes or tests, but a mere tool for measuring where a student is struggling. But this can be a time burden for teachers to grade.

I really like teachers assess their students and then modify their instructional approaches based on the needs and learning readiness.

Academics could be seen as part of the establishment.

the teacher embodies the strict sense of assimilation that placed on immigrant communities

strict sense of secularism in French society

could have been a source of his religious radicalism that clashed with the French ideals of secularism and freedom of expression

goal of the French model of education was assimilating different groups of people under French identity

this is a case related to blasphemy in Islam

I find it interesting how much work goes into programming a computer to know the steps necessary to do a task. Its fascinating how a complicated technical process comes down to minimal details, and how simply a small error can cause many issues.

I find this very practical. For instance, when saving videos, I deliberately choose a lower-quality mode to save storage space. And if there's a file size limit when uploading, I'll intentionally reduce the quality to minimize the file size.

My friends and I often love taking photos, and I've noticed that when we try sending photos to each other on apps such as Instagram..etc., the image quality does become fuzzy. Sending images through iMessage/email on the other hand, often doesn't distort the photo too much, or can even keep the same quality. Now relating back to our reading, this I found is because for such a big app like Instagram, compressing the photo makes it much faster to upload and download images. IMessage however keeps the quality because it is sent between apple servers (mainly relating to iPhones), and email just attaches the full file, which often takes longer to download but will retain much of it's resolution.

There are a lot of pre-existing opinions, advice and strategies for health care and promotion that are deep-rooted into many layers of current policies and understanding of health across all different groups of people. Many of these messages and strategies are actually harmful to health promotion and health which has been redefined through the Ottawa Charter and WHO among others. I believe that generational differences in opinions about all aspects of life also contribute to the lack of people actually trying to understand new concepts and the importance of health promotion on a population level. People can become "stuck in their old ways and understandings", perhaps.

It is arguably more important to target disadvantage groups when discussing health promotion strategies, as currently, they are much less likely to receive and understand information about the underlying causes of health, as more of their basic prerequisites for health are unlikely to be met

A reminder to readers that equity should be at the forefront of health promotion and action

This highlights that it is crucial for governments and policy makers to make change and adjust systems that affect health, as although the determinants are listed in academic literature - they are incredibly relevant in our everyday lives and contribute to ease and dis-ease for all groups of people

When I use to go to used bookstores to buy books I could sometimes find books with annotations in them it was interesting reading what people were thinking as I was thinking the same thing!

I had never even thought about that! Like you're writing an article about said disabilities but the article itself may not even cater to the very disability its speaking on and bring awareness to. Like even I had a hard time processing that text.

I've been using that site for years! I love Genuis! Sometimes I genuinely can't figure out what the meaning behind a song is or I make my own interpretations and see if I hit the name on what the song was about straight from the artist themself!

This has been probably my favorite part about English as a whole is that everything is fluid and nothing is set in stone. How I feel on the subject may not be the same way someone else is feeling on the subject. How I view a sentence may be the polar opposite of how my peer sees the sentence. Theres no one way to see something!

List is very convenient for the programers. We can store as many number we want into the list. When we want to use the specific number inside the list, we can just state the location of that number in the list. Also, if you want to add more into the list, it is also flexible for programmers.

I used to think it was weird that Python starts counting from 0 instead of 1. Like, why not just start with 1 like normal people? But after reading that it’s because of how programming languages were developed, it actually makes a bit more sense now. I also didn’t realize strings are kind of like lists too—that’s pretty cool. The example with the authors and the word “ethics” really helped me see how indexing works in real code.

Impressive and is telling of her potential and rigour

I will have to visit that national park

This quote is great way to represent the clarification of thinking through writing. I like that idea of turning vague notions in well thought out ideas I think that's very accurate.

The idea that when we know other people are going to see our thoughts, we make sure to fully evaluate and understand what we are saying so that it is the best it can be.

I like the opening sentence a lot. I connect with it because that is how people connect with one another through thought. It shows the importance of being able to make educated connections and connect with others to enhance knowledge.

This could be interesting to compare to AI use because the struggle to write is what led him to his ideas (which other authors have contended before)

The idea of an obligation not only to write to a loyal audience, but also to maintain your own credibility as you write is an interesting motivation to want to continue writing. Usually I assume people write because they have something they want to say.

In my teaching social studies for elementary class, we learn that ew should use literacy skills in social studies, and to use social studies in reading and writing. I think this is important for all content areas to see how the skills are all interconnected and to really make sure students are understanding the content.

In special education, we look at assessments similar to this for students in special education. I think that it is very interesting to see the similarities in some of the teaching instructions and learning supports for ELL and SPED.

While AI can be a helpful tool in some cases, I don’t think that students should rely on it to get their work done. By doing the work themselves, they are increasing their own language and critical thinking skills that are imperative to enhance your learning. Even just generating your own ideas can increase your problem solving, creativity, and promotes divergent thinking.

when ending a paper make sure to end with a proper ending and not a starter to a new idea

make thesis statement intriguing and eye-catching to attract the readers attention to keep them reading your work.

a good way to revise your work Is to have someone reading it out loud to you, it helps you go over your work slowly.

Revising a writing piece is vital step in making a good piece.

What are some good ways to end off a paragraph without it feeling like it's unfinished?

This one is hard for me because what might be important and interesting to me might not be to everyone else. This might be easier to do when writing for an audience that you KNOW.

I did this for my learning narrative and it was nice to get reminders of things I've overcome from people who know me personally. It made for a story that I am proud of, and when I read it back I even get emotional.

We post data, but we also need documentation on the data we posted, which means ore data. Administration is always acceleratable, praised and detested by people at the same time. Just find that amusing because it is another hard proof on the double sided world and its "one theory for all" theory.

AI can help doctors find out what is wrong with a patient faster and with fewer mistakes. This helps patients get answers and treatment sooner. It also makes visits easier and less stressful for patients. AI helps doctors do their jobs better and gives patients better care.

This would be super helpful because Al can make charts or graphs seeing where the hospital lacks and ways it can improve. Not only that, but when a patient makes a complaint, it can be resolved, and surveys can be given for feedback.

AI can help do many of the hard and slow jobs in making and grading exams. This saves teachers time and work and can also lower costs for schools. It helps make grading fair because AI checks all tests the same way. But teachers still need to look over the results to make sure everything is right. Overall, AI makes testing faster and gives teachers more time to help students learn.

One concern is that students may start to become overly reliant on AI and fail to develop independent critical thinking skills. AI may not always be reliable or correct which could result bad. Although AI has it uses, there are setbacks as well that must be properly addressed in settings related to healthcare and education.

AI are making a big difference in how scientific articles are published. AI can help make the peer-review process faster and more organized by checking papers and helping reviewers find mistakes. It also improves the quality of reviews and helps make sure research can be repeated and trusted. This means that scientists can share their work more quickly and readers can have more confidence that the information. AI helps make publishing more fair, accurate, and efficient for everyone i

The integration of AI into education can cause new problems like cheating. Some students might use AI to get answers or finish work without really learning the material. This can give them an unfair advantage and make the education system less fair and honest. It also shows why teachers need to find new ways to test students and make sure everyone learns on their own.

I didn't know that AI had become advanced enough to the point where radiology was included; however, I think this is a great thing because not only is radiation being less exposed, but it also doesn't harm the patients and healthcare workers. Since X-rays and CT scans are always needed, it would help, but if a code blue is called, the AI wouldn't be able to do anything other than the healthcare workers.

These sentences explain how AI can help enhance medical treatment by identifying and avoiding errors. Medical records can be examined by AI to find any mistakes such as an incorrect diagnosis or treatments. It can also help in detecting potential hazards before they develop into significant issues. AI also assists in making better choices and avoiding reoccurring errors. In order to improve healthcare accuracy, AI is supposed to operate with physicians rather than in place of them.

Here it explains how AI can analyze and read medical records to help see any errors or risks which will help all medical professionals. I think this is very helpful and beneficial. Technology continues to grow and it is exciting to see how this will continue to help the medical field.

The author highlights how the implementation of artifical intelligence and its use in radiology to improve patient outcomes. This is very important as humans are imperfect, and having a tool such as a ai algorithm to scan diagnosic images can help pick up discrepencies that were maybe overlooked. This still raises concerns opn growing dependencies on AI or how accurate this technology will be.

As AI is a growing tool in today's environment, the application methods are still being determined. In healthcare, this can be benefical for individuals as they can us AI to generate personalized information for their benefit. From generating personalized meal plans to workout plans this only scratches the surface of the uses of AI in healthcare.

Using the beginning sentence the author explains how AI can be used to respond to patients complaints and also analyze that feedback to identify any patterns and trends in the complaints. The author says that AI has a predictive function which lets healthcare providers see which patients are dissatisfied and helps on how to intervene to resolve any issues.

AI has made a big difference in both healthcare and education. It helps doctors look at medical information faster and find what is wrong with patients more easily. It can even suggest treatment plans that fit the person better. In schools it helps teachers and students by creating lessons that match how each student learns. I think AI is really helpful because it saves time and makes things more organized but only to a certain point. People still need to be involved because computers do not understand emotions or small details the way humans do.

This is a really good tool because many times patients especially those who are elderly have difficulty finding ways to go to the doctor’s office because they might not be able to drive. Having AI as a pre-screener and promoting remote medical can can save time and reduce costs for the patients as well.

These sentences acknowledge that although AI is a good resource it is still uses human assistance to answer questions. AI can be incorrect as the formatting of words may not be correct where it shows that human common sense and knowledge is usually important than just to get an answer. This emphasizes the need of the still developing ChatGPT/AI can be.

I believe this section truly shows how much artificial intelligence has helped healthcare, especially for people who cannot easily see a doctor. Remote monitoring allows patients to stay connected with their providers from home, which makes a big difference for those with mobility issues or no access to transportation. During times like the COVID-19 pandemic, telemedicine became an important option that kept people safe while still getting the care they needed. AI technology makes it easier for doctors to track patients’ health, notice changes early, and prevent emergencies. It also helps patients feel more supported because they know someone is watching out for them even from a distance. This innovation has made healthcare more accessible to everyone, not just those who can physically go to the clinic.

I really enjoyed reading this section. It shows how AI not only helped when it comes to patient safety but improves communication between patients and healthcare. In healthcare a common issue is patients and their families not always being heard. And with new AI technology it allows feedback so hospitals can then address any concerns patients feel and improve the healthcare experience. It allows patients’ voices to be heard and hopefully acted on. This section reminded me an app called Leap Frog which is healthcare which tells individuals how the hospital is (like a yelp in healthcare). It collected all the data about the current hospital and then gives a grade on the current hospital and how well it is prepared in areas.

This quote is showing how AI is moving forward with patient safety by improving healthcare workers’ more accurate technology that can be able to detect any illness early. I find this important because by detecting any illness early, may be the cause a patient can be treated early and can potentially save their life. I also wanted to add how AI does reduce the chances of errors which is vital in healthcare. Dave, M., Patel, N. Artificial intelligence in healthcare and education. Br Dent J 234, 761–764 (2023). https://doi.org/10.1038/s41415-023-5845-2

This is a great way to use AI in healthcare. If you tell AI to give you ways to be healthier or improve your overall well-being, it will give you personalized advice. If your goal is to lose weight, it can give you workout plans and even meal plans. This is very beneficial for an individual who does not have access to a doctor or a nutritionist.

This paragraph highlights the importance of AI and its potential to enhance patient care. AI can be faster, convenient, and accessible. It can make the diagnosis faster and easier by just analyzing the medical records and other things so patients won't get misdiagnosed, and they can get the best treatment they need.

The help from artificial intelligence can be used in so many ways, and seeing that it is being used for laboratory results and to support clinical decision-making is good. Not all the time, doctors have the time to do things that take up time. If they have the ability to use AI, then by all means, go for it.

I agree with this because I think people got too used to AI and at the end of the day you have to use your thinking process right at the time you need it. AI cannot help you in everyday use.

The definition of artificial intelligence (AI) is disclosed, and some of it's abilities are as well in terms of its usage abilities in healthcare environments. It's usage is extremely beneficial to both patients and healthcare professionals. These sentences stand as a thorough background on artificial intelligence (AI).

The article 'Artificial intelligence in healthcare and education,' discusses the usage of artificial intelligence within education and healthcare environments. The benefits and negatives of the usage of artificial intelligence are disclosed within the article as well. Furthermore, the usage itself is heavily discussed and analyzed in terms of its relevance to healthcare and education.

I think this is true because for our discussion I talked about this as well and AI will continue to advance. With these changes you would expect it to make education more effective.

In this section, and particularly in this sentence, it shows how advanced technology and AI are. AI is becoming something so big and is helping doctors in many ways. They are able to process the image that is being used and help doctors with diagnosis. AI has a multi-step role when it comes to supporting doctors. With AI, medical professionals will be able to have more information on what is going on and better imaging with better quality.

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the main idea

My question is: since the integrand is already separated into terms that depend only on (x), only on (y), and both variables, how do you quickly decide whether to integrate with respect to (y) first or (x) first? Is there a general rule of thumb for spotting when one order will make the work noticeably easier?

1: The letter D stands for the discriminant of the Hessian matrix. 4: The equation f(x,y) = abs(x) looks like a 3d V. It has a minimum at the point x=0 but no maximum.

This makes a lot of sense to me, It will seem a lot shorter if you end up enjoying it. I love Mexican culture and Spanish language and I feel left out every time my friends talk in Spanish so I very much enjoy learning it.

This statement shows how ones lifestyle and activity is important and can decrease the risk of dementia in the future. It shows that these are effective of each other. It is also shows that cognition is correlated to disabilities as well.

Canavan sees the launch of Sputnik as a break in human self-awareness, or known as the cosmic point of view that severs us from Earth. This abstracted look turns humans into astronimized spectators of there world but a preview of the posthuman condition in our current Anthropocene.

Before reading this, I had never thought of this situation (the woman posting with kids from a different country) in this way before. However, it's so true. The woman should not be posting a child without their consent. Additionally, posting about the trip and taking advantage of the kids in that way almost devalues the original purpose of the trip. It makes me question the woman's motives. Did she really go abroad to raise money and help out? Or did she just want to take photos so the world would know she was a good person who goes abroad and volunteers. It's important to remember that social media is only one side of the picture. We see only what the person is showing us, not their intention behind it.

An example of pernicious ignorance in social media interaction is when people ignore the fact that social media's algorithm is curated to what they want to see. Not realizing that social media platforms intentionally utilize their gathered data to curate content for that individual to maximize their engagement. A lot of information, videos, and posts on social media aren't a true representation of reality, but many viewers assume so. This leads to accepting their own mental reality or changing their true perception of reality. This ignorance is truly harmful and is overlooked l as it can spread misinformation, polarization, insecurities, and more.

I feel like in all cases the problem is the main topic that way it gets readers hook to read the article and also stating solutions to fix the problem

Its really sad that in most cases children suffer the most

I personally feel sad reading this quote because it literally shows that how much people suffered when they are forced to leave everything behind

Sounds like fun, but I doubt I’ll understand Old English, but it would still be fun to watch.

Folklore deep in the castle, also kinda creepy, but awesome that its deep within the castle

Do different diets affect the bats' social systems? And if so, why?