Brain size predicts learning abilities in bees

When it comes to the brain, bigger is generally considered better in terms of cognitive performance. While this notion is supported by studies of birds and primates showing that larger brains improve learning capacity, similar evidence is surprisingly lacking for invertebrates. Although the brain of invertebrates is smaller and simpler than that of vertebrates, recent work in insects has revealed enormous variation in size across species. Here, we ask whether bee species that have larger brains also have higher learning abilities. We conducted an experiment in which field-collected individuals had to associate an unconditioned stimulus (sucrose) with a conditioned stimulus (coloured strip). We found that most species can learn to associate a colour with a reward, yet some do so better than others. These differences in learning were related to brain size: species with larger brains—both absolute and relative to body size—exhibited enhanced performance to learn the reward-colour association. Our finding highlights the functional significance of brain size in insects, filling a major gap in our understanding of brain evolution and opening new opportunities for future research.

P5 L16: reads odd since you are making the case that a bigger brain might not have an advantage over a small brain and now you say the benefits of a small brain. And the whole paper tries to prove that bigger brains are better. P6 L12: Citation: Laloir vs Laloi on Page 23 P6 L12: This is only true for bumble bees and honey bees. What about the solitary bees in your study? P7 L2: I would recommend moving the relevant parts of table S2 into the main text and adding numbers of animals into figure legends. Reading the manuscript, it would be nice to know how many animals you have per species. P7 L13: How certain are you the different bee species have the same receptors and can distinguish the colors? This might also be one reason some of the solitary bees fail. See Briscoe and Chittka 2001. P7: Did you change the rewarded color between animals? P8 L22: Please discuss change in size due to fixation with PFA (see also Wehrl et al., 2014). Since you are weighing the brains it might not matter as much but still, different shrinkage might impact measurements. P10 L3 controlling for not by P11 Please make sure to add all figure numbers where you discuss data also plotted in the figures. P11 L12/13 It is unclear whether you discuss the training or test trials here. P11 L22/23 among vs across species. Please consider using a different wording here, it is not clear what you mean, I assume across species? P12 L8: 'but we found the opposite effect' please elaborate. This is only clear if you also study the figure. P12: Please consider adding figure numbers to the discussion as well. P12 L24: end of sentence missing? P12 L15: 'This supports the view that they learn faster' This sounds misleading. P14 L15: I am in general missing a discussion of the ecological background of the species in connection to their brain size as well as a mentioning of the discussed species by name. P16 L12: consider rewriting sentence, it is hard to understand. P17 Figure 5. Please add explanation for Boxplot P28: Images are too dark. It is hard to see the animal. P28 L46: Holes were done, use drilled? Extremes-use sides? P31: Please add in mg to labels.

Decision letter (RSOS-201940.R0)
We hope you are keeping well at this difficult and unusual time. We continue to value your support of the journal in these challenging circumstances. If Royal Society Open Science can assist you at all, please don't hesitate to let us know at the email address below.

Dear Dr Collado
The Editors assigned to your paper RSOS-201940 "Brain size predicts learning abilities in bees" have now received comments from reviewers and would like you to revise the paper in accordance with the reviewer comments and any comments from the Editors. Please note this decision does not guarantee eventual acceptance.
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Reviewer comments to Author: Reviewer: 1 Comments to the Author(s) This manuscript addresses the longstanding hypothesis that bigger brains correlate with increased learning capabilities. This hypothesis has previously been supported in mammals and birds, and the experiments outlined here provide support for this hypothesis in bees. Overall, the authors present a very clear, well-thought out experiment that adds significantly to the field. In particular, I think the methods of this experiment nicely allow for the behavioral comparisons of many species, which has been a challenge in this area of study. The authors also discuss possible reasons for why some species were unable to be tested in this paradigm, allowing for further comparative studies. I have only a few small comments.
First of all, the choice to weigh brain tissue after fixation is an interesting one -why did the authors choose to do so? Fixation causes shrinkage of tissue and as far as I know, most brain weight measurements in the literature use fresh tissue (e.g. Seid, Castillo, andWcislo 2011, andSheehan et al. 2019). This being said, if all brains were treated the same way, the relative differences in weights are likely still valid, as long as all tissue was fixed the same amount. However, this assumes that all species' brains shrink the same amount. This is an assumption that is often made when staining tissue for immunohistochemistry for measure brain region volumes, although the authors should note these assumptions in their manuscript.
The authors divide up the bee species into two groups: bigger and smaller brains. What are the criteria for each group? I assume these designations are different for the absolute and relative mass measurements -this information should be mentioned in the methods and possibly incorporated into a table or figure.
Along these same lines, it also would be useful to have a table (even in the supplemental materials) listing species and the data associated with each (e.g. brain/body size and learning capability). This could be incorporated with the information noted above regarding bigger and smaller brain groups. Page 12, Line 24: It seems as though there is a line of text missing here.
Page 13, Line 7: A citation for the statement that "larger bees have larger visual organs and higher mobility" is needed.
Page 13, Lines 8-10: This sentence seems to contradict the authors' findings that the bees with larger relative brain size did not decrease their latency to choosing as quickly as those with smaller relative brain size.
Page 14, Lines 16-17: Which of the bees studied here are considered social? This information would be nice to have in this discussion.
Reviewer: 2 Comments to the Author(s) General comments: In this study Collado and colleagues investigate whether insects with bigger brains perform better in an associative learning task. They show that absolute and relative brain size relate to better performance in the task. Their data complements similar findings in birds and mammals. The experiments are done rigorously.
Enthusiasm is somewhat mitigated by the fact that except for the brain size analysis there is no further discussion of the studied species in relation to ecological niche and brain size. Two of the four analyzed families seem not to learn the task which should be further discussed. The learning task which they use is designed for honey bees. This makes honey bees by default good at performing. The families with the smaller sized brains however do not learn the task/perform worse. This might not be related to brain size but rather to their ecological niche that makes this kind of color learning less important. I wonder within honey bees and bumble bees, is the correlation bigger brains learn better still true? These points should be discussed.
However, this study provides sufficient data that add to the existing body of work in vertebrates, which makes the project of interest to the scientific community.
Specific points: Please discuss the huge variation in brain size within families. Is the variation similar within species? A figure would help here. Please consider revising your manuscript with respect to species/families. It seems like you are using the term species but give names of insect families (Apidae). Please discuss that 2 of the 4 species did not seem to learn the task (Andrenidae, Halictidae), figure 4a). See general comment. Please add number of animals per species to figure legends. P4 L21: Maybe add than insects at the end? Sentence reads incomplete. P4 L22/L23: A bit exaggerated. P5 L1: s missing (obtains) P5 L16: reads odd since you are making the case that a bigger brain might not have an advantage over a small brain and now you say the benefits of a small brain. And the whole paper tries to prove that bigger brains are better. P6 L12: Citation: Laloir vs Laloi on Page 23 P6 L12: This is only true for bumble bees and honey bees. What about the solitary bees in your study? P7 L2: I would recommend moving the relevant parts of table S2 into the main text and adding numbers of animals into figure legends. Reading the manuscript, it would be nice to know how many animals you have per species. P7 L13: How certain are you the different bee species have the same receptors and can distinguish the colors? This might also be one reason some of the solitary bees fail. See Briscoe and Chittka 2001. P7: Did you change the rewarded color between animals? P8 L22: Please discuss change in size due to fixation with PFA (see also Wehrl et al., 2014). Since you are weighing the brains it might not matter as much but still, different shrinkage might impact measurements. P10 L3 controlling for not by P11 Please make sure to add all figure numbers where you discuss data also plotted in the figures. P11 L12/13 It is unclear whether you discuss the training or test trials here. P11 L22/23 among vs across species. Please consider using a different wording here, it is not clear what you mean, I assume across species? P12 L8: 'but we found the opposite effect' please elaborate. This is only clear if you also study the figure. P12: Please consider adding figure numbers to the discussion as well. P12 L24: end of sentence missing? P12 L15: 'This supports the view that they learn faster' This sounds misleading. P14 L15: I am in general missing a discussion of the ecological background of the species in connection to their brain size as well as a mentioning of the discussed species by name. P16 L12: consider rewriting sentence, it is hard to understand. P17 Figure 5. Please add explanation for Boxplot P28: Images are too dark. It is hard to see the animal. P28 L46: Holes were done, use drilled? Extremes-use sides? P31: Please add in mg to labels.

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Author's Response to Decision Letter for (RSOS-201940.R0)
See Appendix A.

Recommendation?
Accept with minor revision (please list in comments)

Comments to the Author(s)
The authors have thoroughly revised their manuscript and have addressed all of my comments.
In particular, the addition of Table S2 is helpful for understanding the behavioral results more fully. However, a few minor issues remain.
As I mentioned previously, in Figure 5, the authors divide up the bee species into two groups: bigger and smaller brains. The authors clarified that brain size was used as a continuous variable in these analyses. If this is the case, however, I'm confused as to why there are two lines in this graph, one for bigger and one for smaller brains. This needs to be further clarified because the figure does not seem to match the text.
Page 11, Line 12: "Although" instead of "despite" Page 13, Line 9: How long after the training trials was the test done?
Page 18, Line 17: I think the phrase "solving the learning" should be replace by performing the learning task successfully or something similar.
Page 19, Line 21: Refer to supplemental table S2 here Figure S1 is not visible (I'm not sure if this is a problem solely on my end).

Comments to the Author(s) All concerns and comments have been addressed by the authors.
Decision letter (RSOS-201940.R1) We hope you are keeping well at this difficult and unusual time. We continue to value your support of the journal in these challenging circumstances. If Royal Society Open Science can assist you at all, please don't hesitate to let us know at the email address below.

Dear Dr Collado
On behalf of the Editors, we are pleased to inform you that your Manuscript RSOS-201940.R1 "Brain size predicts learning abilities in bees" has been accepted for publication in Royal Society Open Science subject to minor revision in accordance with the referees' reports. Please find the referees' comments along with any feedback from the Editors below my signature.
We invite you to respond to the comments and revise your manuscript. Below the referees' and Editors' comments (where applicable) we provide additional requirements. Final acceptance of your manuscript is dependent on these requirements being met. We provide guidance below to help you prepare your revision.
Please submit your revised manuscript and required files (see below) no later than 7 days from today's (ie 23-Mar-2021) date. Note: the ScholarOne system will 'lock' if submission of the revision is attempted 7 or more days after the deadline. If you do not think you will be able to meet this deadline please contact the editorial office immediately.
At this stage, we ask that you please archive your GitHub code within the Zenodo repository: https://guides.github.com/activities/citable-code/. By doing this, a formal, citable DOI will be associated with your data record, and an open license (CC-BY preferred) can be applied to your data. We would then ask that you please update your data availability statement to read as: Please note article processing charges apply to papers accepted for publication in Royal Society Open Science (https://royalsocietypublishing.org/rsos/charges). Charges will also apply to papers transferred to the journal from other Royal Society Publishing journals, as well as papers submitted as part of our collaboration with the Royal Society of Chemistry (https://royalsocietypublishing.org/rsos/chemistry). Fee waivers are available but must be requested when you submit your revision (https://royalsocietypublishing.org/rsos/waivers). Comments to the Author(s) The authors have thoroughly revised their manuscript and have addressed all of my comments.
In particular, the addition of Table S2 is helpful for understanding the behavioral results more fully. However, a few minor issues remain.
As I mentioned previously, in Figure 5, the authors divide up the bee species into two groups: bigger and smaller brains. The authors clarified that brain size was used as a continuous variable in these analyses. If this is the case, however, I'm confused as to why there are two lines in this graph, one for bigger and one for smaller brains. This needs to be further clarified because the figure does not seem to match the text.
Page 11, Line 12: "Although" instead of "despite" Page 13, Line 9: How long after the training trials was the test done?
Page 18, Line 17: I think the phrase "solving the learning" should be replace by performing the learning task successfully or something similar.
Page 19, Line 21: Refer to supplemental table S2 here Figure S1 is not visible (I'm not sure if this is a problem solely on my end).

Reviewer: 2
Comments to the Author(s) All concerns and comments have been addressed by the authors.

===PREPARING YOUR MANUSCRIPT===
Your revised paper should include the changes requested by the referees and Editors of your manuscript. You should provide two versions of this manuscript and both versions must be provided in an editable format: one version identifying all the changes that have been made (for instance, in coloured highlight, in bold text, or tracked changes); a 'clean' version of the new manuscript that incorporates the changes made, but does not highlight them. This version will be used for typesetting.
Please ensure that any equations included in the paper are editable text and not embedded images.
Please ensure that you include an acknowledgements' section before your reference list/bibliography. This should acknowledge anyone who assisted with your work, but does not qualify as an author per the guidelines at https://royalsociety.org/journals/ethicspolicies/openness/.
While not essential, it will speed up the preparation of your manuscript proof if you format your references/bibliography in Vancouver style (please see https://royalsociety.org/journals/authors/author-guidelines/#formatting). You should include DOIs for as many of the references as possible.
If you have been asked to revise the written English in your submission as a condition of publication, you must do so, and you are expected to provide evidence that you have received language editing support. The journal would prefer that you use a professional language editing service and provide a certificate of editing, but a signed letter from a colleague who is a native speaker of English is acceptable. Note the journal has arranged a number of discounts for authors using professional language editing services (https://royalsociety.org/journals/authors/benefits/language-editing/).

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Dear Dr Collado,
It is a pleasure to accept your manuscript entitled "Brain size predicts learning abilities in bees" in its current form for publication in Royal Society Open Science. The comments of the reviewer(s) who reviewed your manuscript are included at the foot of this letter.
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Thank you for your fine contribution. On behalf of the Editors of Royal Society Open Science, we look forward to your continued contributions to the Journal. Thank you for the swift handling of our manuscript. We have incorporated your insightful comments and added modifications to our article when relevant. We hope that this new manuscript suits the quality standards to be published in Royal Society Open Science. Find below a detailed response point by point to the reviewers comments. We also marked in blue font the main changes done to the main text.

Associate Editor Comments to Author (Dr Ryan Y Wong):
Your manuscript has now been reviewed by two reviewers. Both reviewers agree that the experiment is interesting and provides much needed data in the field. However, several concerns were raised that need to be addressed prior to further consideration. In particular, there were concerns about clarity of methodological procedures and applicability of data interpretation given the experimental design.
Answer: Thank you for your positive assessment. In this new version we clarified the methodological protocol used and clarified what we can interpret and what not.

Reviewer 1 comments to Author:
This manuscript addresses the longstanding hypothesis that bigger brains correlate with increased learning capabilities. This hypothesis has previously been supported in mammals and birds, and the experiments outlined here provide support for this hypothesis in bees. Overall, the authors present a very clear, well-thought out experiment that adds significantly to the field. In particular, I think the methods of this experiment nicely allow for the behavioral comparisons of many species, which has been a challenge in this area of study. The authors also discuss possible reasons for why some species were unable to be tested in this paradigm, allowing for further comparative studies. I have only a few small comments.