PONE-D-23-42752Thermal limits of bumblebees and honeybees are modulated by different functional traits: predictions of a mechanistic modelPLOS ONE

Dear Dr. MacQueen,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================In particular, both reviewers raise issues with the lack of clarity about parts of the model. It would be important to address these issues and provide further explanations. It would also be good to take note  of the required qualifications to statements in the text with reference to previous work suggested.

==============================

Please submit your revised manuscript by May 25 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this manuscript, the authors present a thermodynamic model for insect thorax temperatures that includes metabolic processes and behavioural adaptations. The study is interesting, thorough and paves the way for species specific models on adaptation to changing climatic conditions while considering specific behavioural adaptations. I found the manuscript easy to read and understand for the most part but have some major and minor comments:

Major comments:

1. Questions on Qh: I found the explanation of how Qh is calculated difficult to understand. From Fig. 1, Qh is implied as heat taken away from thorax. In Eq. 1, Qh is added to (and not subtracted from) the thoracic temperature. In the text (L104-109) Qh is a combination of heat gain and loss due to environmental processes similar to what is described for the thorax. However, for honey bees the authors point out that the temperature differential between head and thorax is maintained at a constant value irrespective of this heat gain and loss. I think the authors should improve their description of Qh to make it’s definition and role clearer.

2. Several assumptions of parameter values have been made in the model without clear explanations. For example, one assumption in the model related to Qh is that the temperature differential between head and thorax is the same for bumblebees as it is for honey bees. This assumption is not present in the main text but is marked in the supplementary table 4. This and other assumptions should be explicitly stated and justified in the Methods section and should not be delegated only to the supplementary information as it is essential to understand the model.

3. Discussion regarding the effect of body mass: I found some of the inferences and discussion on this topic circular. In the model inputs, the body mass of bumblebees and honey bees are input with different levels of variation (for bumblebees the range is nearly 10 fold while the variation in honey bees is much less). While I understand that this corresponds to the extant variation seen, this makes it difficult to disentangle whether the effects of body mass on flight in the model is simply due to the reduced variation. One way would be to model similar levels of variation around the mean body mass in honey bees and bumblebees. While artificial, this will help strengthen the authors’ claim of body mass being an important factor for bumblebees but not honey bees.

4. Behavioural cooling mechanisms: This was an interesting addition to the model. However, I have some questions. Why is active cooling through evaporation of nectar only possible during flight in honey bees? The discussion states that it is strongest during flight (L220) but in the model it is taken to be completely absent (and not weaker) during rest. Can the authors clarify why they chose to not include it as a behavioural process in resting honey bees?

5. Fig.2: I think this figure could be expanded by adding panels from Supplementary figure 1. The results on temperature ranges for flight with and without cooling are an important part of the manuscript (it is highlighted in the first paragraph of the discussion, L189-194) and it is not clear why they are not presented in the main results.

6. L143-145: I had difficulty understanding how and why the parameter E was chosen for the restricted sensitivity analysis, given that it only explains very little amount of the variation (supplementary tables 3 and 4). E is only present when non-environmental variables are considered and even then, it only accounts for 1-3% of the remaining variation. The authors should justify their choice of using E more clearly.

7. In the parameters which require input of surface area values, only the surface area of the thorax or the head is provided in the various equations. Is heat gain or loss through the abdomen negligible? Can the authors elaborate on this.

Minor comments:

8. The introduction was well written and covered the major points succinctly. I enjoyed reading this section.

9. L64: It is not clear to me why certain subscripts in Eq 1 are in small letters (Qab) compared to others? What is the convention being used?

10. L96: How is the average thorax diameter calculated?

11. L108: the section number is missing.

12. L210: Bumblebees is wrongly spelt as 'bumblebeest'

13. L229-230: I’m not sure how cooling capacity benefits bumblebees more mid-foraging given that in both species the active cooling process is present during flight.

14. L262 ‘improce’ should be replaced with ‘improve’.

Reviewer #2: This paper presents a mechanistic model for predicting the thermal limits of activity in honeybees and bumblebees to facilitate predictions of how future climate changes will affect crop pollination. The model is based primarily on empirical data but is designed to be applied beyond these limits. In general, the text is well-written but is lacking important clarifications and includes generalisations and implicit assumptions that make the results of the model difficult to interpret and would make it problematic for other research groups to apply. As such, the work is not suitable for publication in its present form but could be if the concerns below are adequately addressed.

General comments

While the authors’ attempt to develop a general model by including two groups of bees is commendable, the work is limited by the lack of empirical data from more than a few species (and only one for honeybees). The general oversight regarding potential differences between honeybee and bumblebee species that inhabit different environments is the greatest weakness of the work. Although it is understandable that the authors are necessarily limited because the empirically measured parameters required for the model are only available for a limited number of species, it is important for this limitation to be made explicit. For example, bumblebee species (of which there are approximately 250) occupy a broad range of habitats, including the Arctic, the Mediterranean, the deserts of North America and the Amazon rainforest. Honeybee species also vary in both their habitat and their body size, which would also potentially affect the generalisability of the results. Because of this, it is unlikely that the parameters used for the model apply to species that have evolved in these more extreme environments and it is therefore important that the authors are aware of this and discuss this limitation in the text. The other problem associated with the limited number of species used in the model is that the conclusion that honeybee body size does not affect their thermal limits may be incorrect. This is because the honeybee species from which all empirical data was made tends to have workers that do not vary in body size. However, different honeybee species have very different body sizes and this could result in differences in thermal limits. As the authors do not clarify that the honeybee species they refer to is Apis mellifera, this leads to the impression that these results would apply to other honeybee species with different body sizes, but this may not be the case.

Line 31, 55 and line 181: the model apparently only accounts for temperature changes and not changes in other weather factors, such as cloud cover, wind or humidity. If this is not the case, then please specify what other weather variables were used, if this is the case then clarify by replacing ‘weather’ here with ’temperature’.

Equation 1: define the terms dt and dTth in the text before presenting the model

Equation 1: please also explain why QH is being added rather than subtracted here. Potential heat transfer from the thorax to the head would reduce the thorax temperature rather than increase it as suggested in this equation. Please clarify the reasoning behind this in the text.

Line 68: This statement should be supported with an explanation or a citation “and head transfer from the thorax to the head are necessary for bees”. There appears to be only two published studies that explore heat transfer to the head in bees and it appears as though they can regulate this. These works should be cited and the statement revised to reflect the state of our knowledge on this:

Meredith G. Johnson, Jordan R. Glass, Jon F. Harrison; A desert bee thermoregulates with an abdominal convector during flight. J Exp Biol 1 October 2022; 225 (19): jeb244147. doi: https://doi.org/10.1242/jeb.244147

Guadalupe Sepúlveda-Rodríguez, Kevin T. Roberts, Priscila Araújo, Philipp Lehmann, Emily Baird, Bumblebee thermoregulation at increasing temperatures is affected by behavioral state, Journal of Thermal Biology, 2024, 103830,

https://doi.org/10.1016/j.jtherbio.2024.103830.

Line 74: Isn’t the reflection of shortwave radiation from the Earth dependent on the substrate (i.e. if it is long grass, short grass, mud, water etc.)? If so, please explain the assumption of reflectance being made for this equation and note any potential caveats about substrate in the text.

Equation 2: define �P in the text

Equations 2 and 3: These equations are quite confusing. Why is the surface area of the bee’s thorax specifically important for the calculations of Qs and QL and why is it treated separately from the body surface calculations? Please clarify in the text.

Line 84: Where does this measure of ground temperature come from? Please explain in the text.

Line 103: This is quite confusing. The resting metabolic rate was used for the model but then applied to flying as well but these are two very different values in most insects. Furthermore, the metabolic rate for flight varies with air temperature. Please clarify how using the resting metabolic rate value is valid.

Line 104: The basis for this assumption is unclear. Why would a connection between the head and thorax affect the heat flux from the head to the environment? Would active transfer of warm haemolymph from the thorax to the head (as appears to be the case in bumblebees) affect this assumption? If so, how?

Line 105: This statement about a constant difference in head temperature was based on studies from honeybees and does not appear to be true for bumblebees or desert bees. The model should be revised to reflect this

Line 108: the reference to a section (in parentheses) is incomplete.

Line 116: The paper cited here is a study on honeybees and not on bumblebees, this value would also depend on whether the bees are resting or flying. Please clarify for the reader what conditions this statement is specific for.

Line 117: the conclusion that “flight speed thus plays no role in abdomen cooling” should be more clearly supported, as convective cooling will increase with flight speed and should therefore affect the abdomen cooling.

Table 1: Please specify the species for which these values were taken as it is important for the model and for the reader to know how specific they are. Particularly for bumblebees, there are around 250 different species that inhabit a broad range of habitats and that have different body sizes and hairiness. They likely have differences in their ability to thermoregulate and in their responses to temperature. The model likely uses data from very few, if more than one, species of honeybee and bumblebee but the authors do not highlight this as a limitation. Please address this in the Table and in the text, discussing how parameters taken from one, or just a few species might skew the results of the model.

Line 163: It is not clear what other environmental parameters the authors are referring to. Please clarify what they are here.

Line 174: when referring to ‘…ones experiencing greater wind speeds’, I assume that the authors mean head winds or would winds from other directions also have this effect? Please clarify in the text.

Line 189: The term ‘indefinitely’ is used incorrectly here and is misleading. Please revise.

Line 191: This conclusion does not make sense as how could bumblebees fly at 15°C if they cannot warm up their flight muscles? It is also an irrelevant statement as bumblebees do have the ability to warm up their flight muscles and to transfer heat to the abdomen during flight.

Line 201: correct the spelling of bumblebees

Line 205: Please provide a further clarification of why bumblebees would have a higher thermal limit.

Line 209: this statement is incorrect. Some bumblebee species do inhabit tropical environments, Bombus transversalis lives in the tropical lowlands of the Amazon rainforest, for example. Other bumblebee species live in deserts so they are not limited only to cold climates as implied here. Also, the honeybee Apis mellifera evolved in the tropical rainforests of India, so it is of course going to be adapted to flight in tropical environments. Please modify this statement to make it valid.

Line 223: This statement is incorrect as the primary behavioural cooling mechanism is dependent on flight speed as noted above (unless the authors can provide empirical data to show otherwise). Please modify.

Line 232: the use of the term ‘species’ here is incorrect as the terms honeybee and bumblebee are ambiguous and could refer to genera or to specific species. Please be more explicit with the terminology and what is meant by it in the text to avoid confusion.

Line 235 (and lines 244-255): As the empirical data used for honeybees in the model comes from one species, Apis mellifera, this statement is not correct without specification. Honeybee workers are not size polymorphic as bumblebee workers are, so the empirical data is likely only taken from bees that vary little in body size. This lack of size variation for honeybees and not bumblebees could have a strong effect on the conclusions made in the model and the authors should be explicit about this in the text and revise this statement to clarify.

Line 236: The information in this sentence is not supported by evolution. As mentioned above, bumblebee species do live in tropical environments and they do not have smaller body sizes than those that occupy temperate climates. The authors either need to specify what species they are referring to or to include more correct information that accounts for species differences.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-23-42752R1Thermal limits of bumblebees and honeybees are modulated by different functional traits: predictions of a mechanistic modelPLOS ONE

Dear Dr. MacQueen,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================There are a few suggestions for additions to the discussion which should be easily incorporated. The paper would be in good shape for acceptance then.

==============================

Please submit your revised manuscript by Jan 30 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Vivek Nityananda

Academic Editor

PLOS ONE

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #3: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have thoroughly responded to the previous reviews and the manuscript is now much improved. I am happy to recommend this for publication. However the authors should have a look over to ensure that minor grammatical and other issues are resolved.

A few that I noticed include:

1. L26 – 29: This sentence is too long with four commas bringing together 5 subsections. I would suggest to break down this sentence and other like this into 2 or 3 to make it easier to read.

2. L49: ‘our’ should be capitalised as it is the start of the sentence.

3. L326: Please use the author names to refer to the study at the start of the sentence instead of using the reference number as it makes it harder to read.

4. L339: ‘could be allow’ should be replaced with ‘could allow’

Reviewer #3: This study makes a valuable contribution to understanding the effects of temperature on bees, particularly by presenting a model to measure thorax temperature. Bumblebee thorax temperature is a critical parameter influencing their ability to fly, forage, and survive under varying environmental conditions. The model developed here is an invaluable tool for investigating how bees respond to thermal stress, offering key insights into their adaptability to climate change and other stressors. By defining these physiological limits, we can better predict the resilience of bumblebee populations amidst the increasing frequency and intensity of extreme weather events, such as heatwaves.

Main Comments:

1. Include species names in methods:

While the study includes one honeybee species and several bumblebee species, the names of these species are only mentioned in the Supplementary Material. Including these species in the Methods section would enhance the manuscript's clarity and accessibility. This change would be particularly valuable for researchers focused on the variability in thermal tolerance among bees.

2. Address high CTmax in some bumblebee species:

In L345–348, the discussion suggests that a resting bumblebee would reach lethal thorax temperatures at air temperatures of 42°C. However, some studies indicate that certain bumblebee species (including those from temperate regions) can tolerate air temperatures exceeding 45°C (CTmax) (e.g. https://doi.org/10.1242/jeb.165589; https://doi.org/10.1016/j.jtherbio.2023.103672). Incorporating these findings into the discussion would provide a more comprehensive view of thermal limits.

3. Consider the role of nutritional status:

It may be worth addressing whether nutritional status could influence an individual bee's flight activity and ability to thermoregulate under different weather conditions, particularly high temperature. This could add a valuable dimension to the study and highlight a potential avenue for future research.

General Feedback:

The authors have done an excellent job addressing reviewers’ concerns with appropriate revisions, thoroughly explaining the model, and discussing the results in depth. I particularly appreciated the discussion on how the model can be applied to other insect species and the thoughtful consideration of its limitations.

Minor Suggestions:

• L20–22: Include the range or minimum thorax temperature required for flight, if available. This would underscore the bees’ remarkable ability to elevate thorax temperature despite ambient conditions.

• L86: Clarify whether "ground temperature" refers specifically to soil temperature or includes other surfaces, such as impervious materials, which are present in urban areas.

• L241: Specify the approximate time required for the thorax to reach equilibrium temperature, as “after some time” is a bit vague.

Additional Minor Comments:

• L55: Capitalise “Our parameters.”

• Figure 1 caption: Correct “icoming” to “incoming.”

• L76: Change “tounge” to “tongue.”

• L245: Correct “activiating” to “activating.”

• Figure 3 caption: Correct “rigth” to “right” and revise the third sentence to: “Pink curves indicate flight, and green curves indicate resting.”

• L319–325: Clarify when referring to body mass or thorax mass.

• L338: Specify whether “higher thermal limit” refers to upper or lower thermal limits.

• L366: Change to “It is dependent.”

• L392: Revise to: “Could allow bees.”

Overall, this manuscript is well-written, thoroughly researched, and presents a model with significant implications for understanding insect physiology under climate change.

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Reviewer #1: No

Reviewer #3: No

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Thermal limits of bumblebees and honeybees are modulated by different functional traits: predictions of a mechanistic model

PONE-D-23-42752R2

Dear Dr. MacQueen,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Vivek Nityananda

Academic Editor

PLOS ONE

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Reviewers' comments: