Fertility and mortality impacts of thermal stress from experimental heatwaves on different life stages and their recovery in a model insect

With climate change creating a more volatile atmosphere, heatwaves that create thermal stress for living systems will become stronger and more frequent. Using the flour beetle Tribolium castaneum, we measure the impacts of thermal stress from experimental heatwaves in the laboratory on reproduction and survival across different insect life stages, and the extent and pace of any recovery. We exposed larvae, pupae, juvenile and mature adult male beetles to 5-day periods of heat stress where temperatures were maintained at either 40°C or 42°C, a few degrees above the 35°C optimum for this species' population productivity, and then measured survival and reproduction compared with controls at 30°C. Mortality due to thermal stress was greatest among juvenile life stages. Male reproductive function was specifically damaged by high temperatures, especially if experienced through pupal or immature life stages when complete sterility was shown at reproductive maturity; larval exposure did not damage adult male fertility. High temperatures impaired testis development and the production of viable sperm, with damage being strongest when experienced during pupal or juvenile adult stages. Despite this disruption, males recovered from heat stress and, depending on the stage of exposure, testis size, sperm production and fertility returned to normal 15–28 days after exposure. Our experiments reveal how thermal stress from heatwave conditions could impact on insect survival and reproduction across different life stages, and the potential and timescales of recovery.

Comments to the Author(s) I have read this paper a couple of times through and find the writing, analyses and interpretation are all clear. The work is well-justified, comprehensive and makes an excellent contribution to the growing literature, partially by this group, on better quantifying the potential role of climateinduced heat stress on insect reproduction. My only minor criticism is that the Discussion sometimes felt more like a review and if there are space considerations then I would recommend cutting down on line 779-852. The information in those sections are relevant but go somewhat beyond interpretation of the current results and future directions.

Review form: Reviewer 3
Is the manuscript scientifically sound in its present form? Yes

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

Comments to the Author(s)
This is a beautifully written manuscript on the consequences of heatwaves on the mortality of different life stages and, among the survivors, on the downstream effects on reproductive traits and function, using red flour beetles as a model organism. The authors found severe effects of heat stress on post-larval stages, with virtually all pupae dying or, in early imagoes, becoming sterile (including disrupted testicular development and reduced sperm viability) -at least temporarily.
This paper describes a carefully designed and executed experiment testing well-motivated hypotheses on a timely and relevant topic. It must be the most clearly and eloquently written manuscript of the many I have already peer-reviewed this year and probably one of my first to accept almost as is. My comments, therefore, are mostly of editorial nature. Chapeau to the authors! Here a few minor comments for further clarification: Lines 77-78: What do you mean by "increases in hotter environmental temperatures"? Simply increases in environmental temperature, increases in temperature particularly in hot climates (i.e. no change in more polar regions), or increases in average temperatures particularly of the hot season (i.e. no change in winter)?
Line 270: Is this error around mean post-eclosion time the SD or SE? Please define at first mention. And sample size = 49? Line 505 and throughout: It would be helpful to remind the reader of the sample sizes (or degrees of freedom) associated with all these z-statistics as it seems cumbersome to go find them in the Methods or supplementary material.
Lines 505-507: Is this really such a coincidence that 3 p-values in a row are identical to three decimal places (p = 0.671) despite vastly different z-values, or were these results copy-pasted and accidentally not updated?
Line 601: What do you mean by "immature, mature males and controls"? Immature and mature males relative to control males or immature, mature and control males with one another? Or what are immature, mature males if not two different sets of males?
Line 863: sable -> stable Decision letter (RSOS-201717.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 Professor Gage
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Reviewer comments to Author: Reviewer: 1 Comments to the Author(s) This study investigates on the impacts of exposure to high temperature on the biology of different life stage in Tribolium: survival, offspring production, testis size and spermatozoal recovery. One interesting aspects is about the time necessary for reproduction rate to recover following exposure to high temperature depending on the life stage that experienced the heat treatment. In general, the paper is well written, the methods are very clear and easily reproducible from the information provided.
The main problem I have is that the study applied treatments with constant temperature, at either 40 or 42°C, for 5 consecutive days. In the field, during heat waves, the temperature is never constant at so high temperature for so long (daily fluctuations still operate). Therefore, the study cannot conclude on the impacts of heatwave conditions. Instead, this study provides effects of high temperature on the biology of this species (which is still of interest). But this is thermal biology study and not anymore a study on the effect of heatwaves. The word heatwave should be suppress almost everywhere in the main text (the same error can be found in previous studies published by same and other authors).
In addition, the control treatment consisted in an exposure to 30°C (constant, again). However, the optimal temperature is said to be around 35°C. Therefore, I wonder why the control was not fixed at 35°C ? That would increase the deviation between control and high temperature treatments, reflecting the extent to which a moderate increase in temperature is sufficient to generate high impacts (although this is not new). Indeed, at some point lower on the thermal performance curve, there should be a lower temperature at which the biological traits will show similar values than during the high temperature treatments, and one can wonder to what extent the 30°C is near this temperature equivalence. The section on the statistical methodology is unusually long (4 pages) and should certainly be written in a more concise way by summarizing the first part on the general approach (the specific paragraphs after that provide the necessary details).
Reviewer: 2 Comments to the Author(s) I have read this paper a couple of times through and find the writing, analyses and interpretation are all clear. The work is well-justified, comprehensive and makes an excellent contribution to the growing literature, partially by this group, on better quantifying the potential role of climateinduced heat stress on insect reproduction. My only minor criticism is that the Discussion sometimes felt more like a review and if there are space considerations then I would recommend cutting down on line 779-852. The information in those sections are relevant but go somewhat beyond interpretation of the current results and future directions.
Reviewer: 3 Comments to the Author(s) This is a beautifully written manuscript on the consequences of heatwaves on the mortality of different life stages and, among the survivors, on the downstream effects on reproductive traits and function, using red flour beetles as a model organism. The authors found severe effects of heat stress on post-larval stages, with virtually all pupae dying or, in early imagoes, becoming sterile (including disrupted testicular development and reduced sperm viability) -at least temporarily.
This paper describes a carefully designed and executed experiment testing well-motivated hypotheses on a timely and relevant topic. It must be the most clearly and eloquently written manuscript of the many I have already peer-reviewed this year and probably one of my first to accept almost as is. My comments, therefore, are mostly of editorial nature. Chapeau to the authors! Here a few minor comments for further clarification: Lines 77-78: What do you mean by "increases in hotter environmental temperatures"? Simply increases in environmental temperature, increases in temperature particularly in hot climates (i.e. no change in more polar regions), or increases in average temperatures particularly of the hot season (i.e. no change in winter)?
Line 270: Is this error around mean post-eclosion time the SD or SE? Please define at first mention. And sample size = 49?
Line 505 and throughout: It would be helpful to remind the reader of the sample sizes (or degrees of freedom) associated with all these z-statistics as it seems cumbersome to go find them in the Methods or supplementary material.
Lines 505-507: Is this really such a coincidence that 3 p-values in a row are identical to three decimal places (p = 0.671) despite vastly different z-values, or were these results copy-pasted and accidentally not updated?
Line 601: What do you mean by "immature, mature males and controls"? Immature and mature males relative to control males or immature, mature and control males with one another? Or what are immature, mature males if not two different sets of males?
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Author's Response to Decision Letter for (RSOS-201717.R0)
See Appendix A.

RSOS-201717.R1 (Revision)
Review form: Reviewer 1 Is the manuscript scientifically sound in its present form? Yes

Are the interpretations and conclusions justified by the results? Yes
Is the language acceptable? Yes

Recommendation? Accept as is
Comments to the Author(s) All my comments have been considered with care -I thank the authors for that. I have absolutely nothing to add. This is an excellent paper. I still kind of disagree with the interpretation of the term heatwave, but the interpretation of the authors is quite clear such that the readers will have the opportunity to make their own idea. This will add to the hot debate about heatwave characteristics!

Review form: Reviewer 3
Is the manuscript scientifically sound in its present form? Yes

Are the interpretations and conclusions justified by the results? Yes
Is the language acceptable? Yes

Do you have any ethical concerns with this paper? No
Have you any concerns about statistical analyses in this paper? No

Recommendation?
Accept as is

Comments to the Author(s)
The authors have addressed my comments adequately. I have no further comments except that the green stain of the LIVE/DEAD viability kit is called "SYBR", not "SYBER". Please correct this in the proofs.
Decision letter (RSOS-201717.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 Professor Gage, It is a pleasure to accept your manuscript entitled "Fertility and mortality impacts of thermal stress from experimental heatwaves on different life stages and their recovery in a model insect" in its current form for publication in Royal Society Open Science.
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Associate Editor Comments to Author (Dr Kristina Sefc):
Dear authors, Your manuscript has been seen by three reviewers, who applaud your experiment and offer some advice for further improvement of your manuscript. Reviewer 1 comments on an important point, namely the simulation of natural heat waves, which involve fluctuations rather than stable temperature. This is a matter of what you claim to demonstrate in the manuscript, and can be resolved by revising your interpretation. When shortening the methods, please be careful to still include all information. Reviewer 1 also suggests that a 'read me first' text file should be added to the supplementary data, as the terms and headers in the tables are not comprehensible.
Reviewer 2 recommends cutting down the text of the discussion. Although there are no space limits, please keep in mind that readers will appreciate a concise text. Please attend to all of the reviewers' comments, and I look forward to seeing your revision.

RESPONSE:
We have attended to all reviewers' comments and altered the manuscript accordingly. In particular we have suppressed the use of the term heatwave throughout, reduced the statistical analysis methods section, provided a read me first' text file for the supplementary data, and reduced the length of the discussion. We also have removed the figures and supplementary material and resubmit as separate files.

Reviewer comments to Author:
Reviewer: 1 Comments to the Author(s) A. This study investigates on the impacts of exposure to high temperature on the biology of different life stage in Tribolium: survival, offspring production, testis size and spermatozoal recovery. One interesting aspects is about the time necessary for reproduction rate to recover following exposure to high temperature depending on the life stage that experienced the heat treatment. In general, the paper is well written, the methods are very clear and easily reproducible from the information provided.
The main problem I have is that the study applied treatments with constant temperature, at either 40 or 42°C, for 5 consecutive days. In the field, during heat waves, the temperature is never constant at so high temperature for so long (daily fluctuations still operate). Therefore, the study cannot conclude on the impacts of heatwave conditions. Instead, this study provides effects of high temperature on the biology of this species (which is still of interest). But this is thermal biology study and not anymore a study on the effect of heatwaves. The word heatwave should be suppress almost everywhere in the main text (the same error can be found in previous studies published by same and other authors).
RESPONSE: We agree of course that natural heatwaves will show thermal variation, so were initially careful throughout (and in other publications) to assert that this was an experimental study, and that our treatments exposed individuals to 'experimental heatwaves' or 'heatwave conditions'. We could not mimic exactly a natural heatwave, as many experts disagree on what even defines the conditions for a heatwave, and natural heatwaves will all be different. Our primary aim was to understand basic reproductive sensitivities at different life stages and their potential recovery in the extreme conditions that might be experienced through the increasing incidence of heatwaves. As the reviewer requests, we have suppressed the term 'heatwave' almost everywhere in the text, changing it to 'thermal stress', 'heat stress' or 'high temperature conditions'.