Author Response:
Public Reviews:
Reviewer #1 (Public review):
Summary:
Overall, this is an interesting and well-written manuscript on a fascinating question in a"charismatic" model system.
Strengths:
(1) The Introduction is concise, though it might be helpful to the non-specialist reader to learn a bit more about what is known about the social control of somatic growth across diverse species (including humans), which would help to make this work more generally interesting.
(2) The experiment is well-designed.
(3) The data collected are comprehensive.
(4) The complementary analysis of both feeding and aggression/submission data with and without known social roles is a neat idea and compelling!
Thank you for the positive feedback!
Here, we investigate phenotypic plasticity associated with the adoption of social roles in the clown anemonefish, with strategic growth being just one aspect of that plasticity. Strategic growth, also known as social control of growth, is a fascinating form of adaptive phenotypic plasticity, whereby individuals modify their growth and size in response to fine-scale changes in social conditions (Buston & Clutton-Brock, 2022). In cooperative breeding systems with high reproductive skew, particularly fishes and mammals (possibly including humans), individuals have been shown to i) increase growth/size on the acquisition of dominant status (Dengler-Crish & Catania, 2007; Johnston et al., 2021; Thorley et al., 2018; Van Schaik & Van Hooff, 1996; Walker & McCormick, 2009), ii) increase growth/size when paired with size matched reproductive rivals (Huchard et al., 2016; Reed et al., 2019; this study), and iii) decrease growth/size to avoid conflict (Buston, 2003; Heg et al., 2004; Wong et al., 2007). While strategic growth is fascinating and clearly occurring in this study, we show coordinated changes of multiple aspects of the phenotype as fish adopt social roles. Therefore, we deliberately framed the Introduction broadly to avoid biasing the reader toward viewing growth as the sole or main driver.
Weaknesses:
(1) I was surprised that the HPA/stress axis was not considered here at all. Wouldn't we expect that subordinates have increased stress axis activation, which in turn could inhibit their growth and aggressive behavior?
We also expected to see the HPA/stress axis activated in subordinates, which is why we carried out a targeted exploration of genes known to play a role in this axis. We did not find any genes that were significantly differentially expressed. We believe that there could be two explanations for this. First, from a methodological perspective, it could be due to our use of a whole-body RNA-seq, which may have masked this signal. Alternatively, the stress axis might play a more complex role than just acting as a simple on/off switch for reduced growth. Its activation may peak when competition over size is at its highest (during week one) or, conversely, it may peak later and help maintain reduced growth once hierarchies are firmly established (particularly after the dominant individual reaches its maximum size). To understand the role of the stress axis, future studies should observe how its activation varies over time. We acknowledge that the absence of a stress‑axis signal and its potential explanations were not clearly discussed in the original manuscript, in the revised version, we will address this issue.
(2) To what extent are growth, food intake, agonistic behavior, and/or gene expression patterns coordinated across P1 vs P2 pairs? The lack of such an analysis seems like a missed opportunity.
We had a similar thought. Specifically, we were interested in testing the hypothesis that the final size ratio of pairs, which is indicative of the amount of conflict remaining, would predict gene expression. We examined gene expression within pairs to test for coordinated changes and repeated the analysis, accounting for the pair size ratio. In both cases, we found no clear or consistent pattern within pairs. We will consider including these figures in the Supplementary Materials document.
(3) What was the rationale for using whole bodies for the transcriptome analysis? Given the hypotheses, the forebrain or hypothalamus and certain other organ systems (e.g.,liver, gonads, skin, etc.) would have been obvious candidate tissues here. I realize that cost is always a consideration, but maybe a focus on the fore-/midbrain could have been prioritized.
We decided to use whole-body samples for this initial transcriptomic analysis to capture a broad view of gene-expression differences while keeping sequencing costs and sample requirements manageable. We agree with the reviewer that future work should explore specific tissues sampled from individuals at multiple time points to disentangle transcriptomic differences across tissue types.
(4) Given the preceding point, why was a fold-change threshold used for assessing DEGs (supplementary Figure 3)? There is no biological justification to ever use a fold-change threshold, especially in bulk RNA-seq analysis. This is particularly true here, where wholebodies were used for RNA-seq analysis, which is a bit unusual. Relatively small cell populations (such as hypothalamic neurons that regulate growth or food intake) may show substantial gene expression variation across social types, yet will be masked by the masses of other cells in the whole body sample. However, gene expression may still vary significantly, albeit the fold-difference may be small. I therefore suggest a reanalysis that omits any fold-change threshold.
We thank the reviewer for this important point, and agree that an arbitrary fold‑change cutoff is inappropriate/unnecessary. It should be noted that this fold-change cut-off was only used in this single figure, and all other analyses used p-values from the entire dataset. We will remove the fold‑change threshold cutoff and correct Supplementary Figure 3, and any corresponding text.
(5) Why is the analysis of color (hue, saturation) buried in the supplementary materials?Based on the hypotheses that motivated the study, color seems just as relevant as food intake, growth, and agonistic behavior, so even if the results are negative, they should be presented in the main paper.
We agree that color can be an important social signal, so we included color measurements in our experimental design. However, after careful consideration of the color results, we decided that our experimental timing and husbandry changes introduced multiple confounding factors, preventing us from drawing confident conclusions. Specifically, our fish were ≈1 month old at the transfer from larval to experimental tanks and had already begun to deepen their orange hue, before our experiment. (In the wild, they would settle at two weeks of age, prior to the deepening of the orange hue). Once individuals attain a certain hue, it seems that color development can be halted, but not reversed. The transfer also involved changes in lighting, tank background, and diet, factors known to strongly affect coloration. Our results show a uniform shift in orange hue and saturation across social groups, suggesting that these confounding factors might have dominated changes in hue.
For transparency, we report the color data in the Supplementary Materials, but we caution against drawing any strong conclusions. In the revised manuscript, we will recommend that future work include a targeted experiment to robustly test for the effect of the adoption of social roles on coloration or the effect of coloration on the adoption of social roles.
(6) The Discussion is sometimes difficult to follow. The authors may want to consider including a conceptual graphic that integrates the different aspects of growth and satiety regulation, etc., into a work-in-progress model of sorts, which would also facilitate clearer hypotheses for future research.
Thank you for flagging that parts of the Discussion are a bit difficult to follow. In the revised manuscript, we will work to improve readability of the Discussion. We also appreciate the suggestion of including a conceptual schematic. We will consider whether adding such a graphic will add value to this manuscript or future manuscripts.
Reviewer #2 (Public review):
In this manuscript, the authors test growth, behavior, and gene expression in pairs of clownfish as they establish social dominance hierarchies, examining patterns of gene expression in these pairs after dominance has been established. The authors show solid evidence that emerging dominant clownfish show increased growth, aggression, and food consumption compared to their submissive or solitary counterparts, eventually adopting distinct gene expression profiles.
Major Comments:
(1) The Introduction is comprehensive, but it could be condensed. Likewise, the discussion could be condensed. There is considerable redundancy between the methods, the results,and the legend in Figure 1. The authors should consolidate and remove the redundancy.
Thank you for flagging that parts of the manuscript could be condensed, we will work on this as we revise the manuscript.
(2) For Figure 3, the authors are showing PC2 and PC3; why is PC1 not shown? There is so much overlap between the three groups in PC2 vs PC3; it seems unlikely that researchers could conclusively identify any individual as belonging to a group based on the expression profile. The ovals shown do not capture all the points within each of the groups, and particularly the grey S oval seems misaligned with the datapoints shown.
We understand the concern raised by the reviewer about the overlap among points in the PCA. We have explored PC1-PC3 and found that PC2 and PC3 showed the clearest, statistically significant clustering by social position, while PC1 did not capture any variation due to social position. We have explored whether other factors might be masking differences, such as genetic relatedness, tank effects, total read count per sample, and found that none of these factors explained sample clustering. Regarding the ellipses shown around the points, they were not intended to capture all points, but rather they show the estimated 95% multivariate t-distribution for that given social group. We will make sure this is clearly explained in the figure legend, and Methods section. In addition, in the revised version, we will show PC1 and PC2, and PC1 and PC3, in the Supplements for transparency.
(3) The authors indicate that the 15 replicates exhibiting the greatest size difference between P1 and P2 were selected for gene profiling. Does this mean that each of the P1and P2 were pairs with each other? Have the authors tried examining the gene expression patterns in a paired manner? E.g., for the pairs that showed the greatest size differences,do they also show the greatest differences in gene expression? Do the P1s show the most extreme differences from P2s that also show the most extreme P2 differences? Perhaps lines on Figure 3A connecting datapoints from the P1 and P2 pairs would be informative.
Yes, “15 replicates exhibiting the greatest size difference between P1 and P2 were selected for gene profiling” refers to pairs of P1 and P2, we will make sure this is clearly stated in the revised Methods. Yes, we have explored gene expression data considering the size difference between pairs, and found that it showed no clear differences in gene expression patterns (see earlier response to Reviewer #1). We will consider including these figures in the Supplementary Materials document, as well as adding a version of Figure 3A that clearly shows information on pairs, as suggested by the reviewer.
(4) For the specific target pathways that are up- and downregulated in the different backgrounds, I recommend that the authors include boxplots (or heatmaps) showing the actual expression values for these targets. Figure 6 shows a heatmap for appetite-related genes, and it would be great to see a similar graph for the metabolism and glycolytic genes; it would also be informative to see similar graphs for hormonal and sexual maturation pathways as well.
We have explored genes across a broad set of metabolic pathways (glycolysis, TCA cycle, lactic fermentation, PDH complex, cholesterol biosynthesis, fatty-acid synthesis, and beta-oxidation) and show all metabolic genes that showed significant differential expression between P1, P2, and S in Figure 6. Overall, very few metabolism-associated genes were significantly differentially expressed, which is why we decided to combine appetite-regulation and metabolism-associated genes into a single figure (Figure 6). In the revised version, we will ensure that Figure 6 clearly shows the gene sets associated with appetite and metabolism.
We also examined hormonal pathways (glucocorticoid and thyroid signaling), but did not find genes in these pathways that were significantly differentially expressed. Finally, we would like to clarify that our samples consist of two-month-old juvenile individuals that are sexually immature —under ideal conditions, clown anemonefish can mature in one to two years, but they can also remain sexually immature for a decade or more (Buston & García, 2007) — which is why we did not observe distinct molecular signatures of sexual maturation. We recognize that the sentence at line 520 may be misleading, as we did not identify any gene expression signature that we could confidently associate with signs of sexual maturation. We will make sure that these are clearly stated in the revised version of the manuscript.
(5) Particularly given that there is a relatively small number of genes enriched in the different rank conditions, I did not understand the need to do the WGCNA module analysis. I thought that an analysis of GO terms across the dataset would have been more meaningful than the GO term analysis shown in Figure 4, which considers only genes assigned to the "brown WGCNA module". This should be simplified or clarified.
To clarify, GO enrichment analysis does not establish correlations with traits, it only describes which functions or pathways are over-represented in a given gene set. That is why we began by using WGCNA to define gene sets (modules) that are correlated to phenotypes. Our primary rationale for WGCNA was to identify modules of co-expressed genes that show significant statistical correlation with the phenotypes of interest (social role: P1, P2, S; growth; and food intake). Pairwise differential expression analysis (Figure 3B) identified a few hundred significantly differentially expressed genes, but those tests treat genes independently and are not able to help us link coordinated changes of co-expressed genes to phenotypes of interest. Because WGCNA is blind to traits, it first identifies groups of co-expressed genes, which can help resolve gene expression patterns.
We therefore ran WGCNA on the rlog-transformed dataset to identify modules of co-expressed genes that show significant correlation with phenotypes of interests. For every module that showed such a correlation, we performed GO enrichment and carefully evaluated the resulting GO enrichment trees (see Supplementary Figs. 4–5). The brown module was highlighted in the main text because it was one of the modules with a significant correlation to growth, and its associated GO enrichment showed clear growth-related signals that were not identified in the pairwise differential expression analysis results.
(6) The authors say that they have identified coordinated changes in behaviors and the"underlying gene expression, leading to the emergence" of social roles. This is a little bit misleading, since the gene expression analysis occurred well after the behavioral and phenotypic differences emerged. Presumably, the hormonal and genetic shifts that actually caused the behavioral and phenotypic difference occurred during the weeks during which the experiment was underway, and earlier capture of the transcriptome would presumably reveal different patterns, and ones that would be considered more causative.The authors acknowledge this in 434-435, but it could be emphasized further.
We appreciate the reviewer raising this point. In the updated version of the manuscript, we will revise wording to convey that food intake, agonistic behavior, size and growth, and gene expression are all changing continuously, in response to each other and in response to social feedback. An underappreciated aspect of this system (and likely many other systems) is that phenotype (including transcriptome) influences the outcome of social interactions, and the outcome of social interactions influences the phenotype (including the transcriptome). Earlier capture of the transcriptome would reveal different levels of gene expression, reflecting the state of the system at that moment in time.
(7) The authors have measured a number of differences between the different dominance classes of fish. All these differences were measured relative to the other classes, but in my view, the Solitary group was the closest to a baseline control. So I'm not sure that it is fair to say that "P2 and S individuals showed consistent downregulation of these genes and pathways" (line 401). I encourage the authors to emphasize the differences in gene expression from the "perspective" of the P1 individuals compared to the baseline of P2and S individuals. Line 474 says that "P2 fish showed significant upregulation" of a number of pathways. It should be very clear what that is compared to (compared to P1, presumably?)
We agree with the reviewer that solitary individuals are the most intuitive baseline. Indeed, the experimental design included solitary fish because we expected they would serve as a useful control. Without social restraint, we anticipated they would show unrestricted growth, feeding, behavior, and associated gene‑expression patterns, similar to dominants.
We initially ran analyses using solitaries as the baseline, but after examining the results, which showed subordinate‑like characteristics for the solitary individuals, we concluded that solitary individuals are not an ecologically appropriate control for this context. Removing juveniles from a social context and housing them in isolation may be stressful and can affect physiology and behavior in ways that do not reflect a natural baseline. From a life‑history standpoint, solitary living is not the typical state for A. percula.
For these reasons, we reanalysed the dataset using the dominant (P1) as the reference to enable more ecologically meaningful comparisons (this choice was somewhat arbitrary, subordinates could also have been used as the reference). Given that gene expression is relative, we interpret results from both the dominant (P1) and subordinate (P2) perspectives in the Discussion to provide a complete view. We will clarify wording throughout the manuscript to make it clear that everything is relative (e.g., revising Line 474).
(8) Along the same lines, the authors say in line 514 that subordinates and solitaries strategically downregulate their growth. I'm not convinced that this is the case: I would consider this growth trajectory to be the default and the baseline. I would interpret that under certain social conditions, a P1 dominant pattern of growth, behavior, and gene expression is allowed to emerge.
We respectfully disagree with the idea that a single baseline/reference growth trajectory exists for any individual of this species. Growth of individuals is entirely social context-dependent: neither fast nor slow growth represents an inherent baseline. When two size‑matched juveniles meet and compete to establish dominance, accelerated growth is the expected trajectory. By contrast, juveniles joining an existing hierarchy are expected to exhibit reduced growth, which minimizes conflict and facilitates their social integration. Unlike species that show non socially mediated growth trajectories, clown anemonefish do not have a context‑independent growth rate, rather, individuals constantly readjust their growth according to their immediate social environment.
Therefore, growth trajectories must be considered from the perspective of all group members, because they emerge from interactions among individuals rather than reflecting an intrinsic baseline. In this study, we were interested in the establishment of dominance hierarchy and how individuals adjust their phenotypes during this process. By experimentally pairing size‑matched rivals, both individuals are initially expected to pursue the dominant trajectory, and thus neither individual represents a default state. Instead, the outcome reflects a social decision, after which both individuals reinforce their emerging social roles through coordinated changes.
Reviewer #3 (Public review):
Summary:
The authors tested the hypothesis that interactions among size- and age-matched rivals will lead to the emergence of social roles, accompanied by divergence in four aspects of individual phenotypes: growth, feeding behavior, fighting behaviors, and gene expression in clownfish.
Strengths:
The data on growth, feeding rate, and fighting behaviors support the authors' claims.
Thank you for the positive feedback!
Weaknesses:
Gene analysis conducted in this study is not sufficient to clarify how the relevant genes actually regulate growth and behavior.
The information obtained from whole-body gene expression analysis is very limited.Various gene expression is associated with the regulation of fighting behaviors, food intake, growth, and metabolism, and these genes are regulated differently across tissues,even within a single individual. Gene expression analysis should be performed separately for each tissue.
We understand the reviewer’s concern about whole‑body transcriptomes and agree that tissue‑specific sampling would provide greater resolution of the mechanisms linking gene expression to growth, agonistic behaviors, and food intake. For this initial study, however, we deliberately chose whole‑body samples to capture a broad, unbiased view of gene expression differences while keeping sequencing costs and sample requirements manageable. We explicitly acknowledge the resulting interpretational limits in the Discussion (lines 464; 529–533), and suggest in the last paragraph that the patterns reported here should be used to build on in future studies exploring targeted, tissue‑specific hypotheses.
Clownfish undergo sex change depending on social status and body size, as the authors mention in the manuscript. Numerous gene expressions are affected by sex change. It is unclear how this issue was addressed.
We thank the reviewer for raising this point. Sex change and sexual maturation can indeed drive major transcriptional shifts in clown anemonefish, but our experiment did not encompass such a life‑history transition. All individuals in this experiment were juveniles (≈1 month old at the start, ≈2 months old at the end) and were sexually immature at these ages. Clown anemonefish reach sexual maturation around one to two years under ideal conditions, can delay sexual maturation for years under normal conditions (Buston & García, 2007), and sex change in the genus Amphiprion is known to take over ~5 months (Moyer & Nakazono, 1978). Accordingly, individuals in this study were not sexually mature, and sex change was not biologically plausible over the five-week experimental period of our study. We recognize that the sentence at line 520 may be misleading, as we did not identify any gene expression signature that we could confidently associate with signs of sexual maturation. We will make sure that it is clearly stated that the fish in this study were sexually immature in the revised version.
References:
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