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Protecting honey bees (Apis mellifera) from thermal stress: Probiotics and prebiotics buffer the survival and antioxidant enzyme activity

PLOS One

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Dear Dr. Bahreini,

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Additional Editor Comments:

The three reviewers acknowledge the value of this study and commend the authors for their work. However, they have also identified several issues that need to be addressed before the manuscript can be considered for publication. Minor typographical errors should be corrected throughout the manuscript. More importantly, several aspects of the methodology require further clarification, including the rearing conditions, experimental protocol and dietary treatments. In addition, the statistical analyses need to be described in greater detail to ensure clarity and reproducibility.

The reviewers have also provided useful suggestions to strengthen both the Introduction and the Discussion sections, which should be carefully considered. I encourage the authors to address all of these points thoroughly. I would be pleased to reconsider the manuscript for publication in PLOS ONE  once a revised version has been submitted.

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-->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

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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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: Yes

Reviewer #3: Yes

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-->4. 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 #2: No

Reviewer #3: 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: Comments for the Authors:

Minor corrections:

- Page 3, line 56: “Howevewr” please, spell it correctly.

- Page 3, line 59: “environmtal” please, spell it correctly.

- Page 3, line 61: “immunoe” please, spell it correctly.

- Page 3, line 62: “bahvious” please, spell it correctly.

- Page 3, line 76: compounds[15], a space is missing.

- Page 4, line 77: “agnist” please, spell it correctly.

- Page 4, line 92: The term “microflora” is no longer preferred. Instead, the community of live microorganisms associated with the host is referred to as “microbiota”.

- Page 5, line 105: “responces”, please, spell it correctly.

- Page 6, line 137: “Treatmnts” please, spell it correctly. The word “include” should be written in the past tense.

- Page 6, line 148: “Temperature stress treatments have been carried out”, please change it for: were carried out.

- Page 22, line 539: “requirments”, please spell it correctly.

Additional comments:

- The authors should consider addressing thermal stress and the importance of temperature maintenance in the Introduction. A brief paragraph would be sufficient.

- Lines 140-142: The paragraph “The efficacy of various combinations of Progen and Inulin was evaluated to determine the role of these diets in mitigating the detrimental effects of environmental heat stress on honey bee survival and antioxidant defense system.” should not be included in Material and Methods.

- Lines 145 – 147: “Newly emerged honey bees (1-day-old) were fed a combination of various concentrations of Progen probiotic and Inulin prebiotic over a 21-day period [29]. Following this period, samples were subjected to distinct thermal stress conditions”. For how long were they exposed to the thermal stress? Please, specify this.

- Lines 149 – 150: “Each treatment was repeated five times with 100 honey bee individuals (500 honey bees per treatment in total).” It is unclear whether the five repetitions represent independent experiments conducted at different times or simultaneous replicates within the same experiment. Please, clarify.

- Line 187: In the “Peroxidase assay”, it is not mentioned how much of honey bee tissue homogenate were added to the reaction mixture. Please, specify it.

- Figure 1: Please indicate statistically significant differences between treatments in the figure (using asterisks or letters).

- In the thermal stress results, as well as in the antioxidant activities results, there is repetitive information. The figures are sufficient to explain the results. Any additional comments should be included in the Discussion, not in the Results section. Authors should not discuss in the Results section.

- Line 362: The expression “adverse weather conditions” should be revised to “simulated weather conditions,” as the experiments were conducted under laboratory conditions.

- The authors suggest that the gut microbiota was improved and that this explains the observed effects (e.g., increased bee survival). However, no direct analysis of gut microbiota was performed to support this claim. Such conclusions should be revised or rephrased.

- Line 477: The study discussed in reference [64] is based on experiments in mice, not in honey bees. I therefore suggest removing this paragraph and restricting the discussion to literature relevant to bees.

- The authors discuss short-chain fatty acids (SCFAs) and attribute the antioxidant effects observed in the assays to their production. However, SCFAs were not measured in this study. I therefore suggest removing this part of the discussion.

Reviewer #2: I would like to commend the authors for conducting this study and for their dedication, as handling bees is inherently challenging. However, several key points require clarification to strengthen the scientific relevance and clarity of the work. It is unclear whether worker bees were reared from emergence (day 0) to 21 days under the different diets and temperature conditions. The timing of mortality assessment should be clarified, as it is not specified whether it was recorded from emergence or only after 21 days. The rationale for choosing a 21-day period for rearing and measurements should also be explained, and including a photograph of the experimental cage would improve reproducibility. Regarding diet composition, the study provided syrup and water but no pollen. Since pollen is the primary protein source for bees, its absence may affect the interpretation of the results. Furthermore, only a combined probiotic and prebiotic treatment was tested. Testing individual treatments could help determine whether the observed effects are due to a synergistic action or a single component. Concerning temperature treatments, the reported survival under extreme conditions of 4°C and 40°C from day 0 to 21 seems unlikely. Details on the initial number of bees, mortality rates, and any sampling losses should be provided to justify survival under these conditions. The statistical methods section also requires clarification. The software used, such as SPSS, should be stated at the beginning of the analysis section. Survival analysis should specify that the Kaplan–Meier estimator was applied, while other parameters should be analyzed using a two-way ANOVA with diet and temperature as the two factors. In summary, this study is valuable and the work is commendable, but clarification on the rearing protocol, diet, temperature conditions, and statistical analysis is essential to strengthen the manuscript, improve reproducibility, and increase its scientific impact.

Reviewer #3: This study is very timely in the era of global climate change, as winters are becoming increasingly harsh and honey bee health is declining due to cold-season nutritional deficiencies. Supplementation with pre- and probiotics is therefore important. The study is well written and suitable for minor revision.

1. Lines 63–64: The claim that stress leads to bee population losses is overstated. Stressors can threaten colony health, but they do not necessarily cause population decline, particularly in managed honey bees, where resilience, husbandry practices, and nutrition mitigate impacts. Please temper this statement and, throughout the manuscript, avoid overgeneralizations by distinguishing colony-level stress from population-level trends. Support any population-level claims with appropriate, recent citations and revise the wording to reflect the current evidence base.

2. Hemagglutinins- check spelling

3. Line 68: Please insert a paragraph break to separate the oxidative stress discussion from the preceding text, improving readability and logical flow.

4. In the final paragraph, please add a dedicated, well-structured section on honeybee–thermal–microbiome interactions. Summarize how the gut microbiome responds under thermal stress (both heat waves and cold spells), and how these shifts may influence resilience, colony health, and pathogen susceptibility. Highlight mechanisms by which bees maintain their microbiome and how the microbiome may, in turn, bolster bee resilience. After this synthesis, transition to probiotics and prebiotics, explaining how they may directly or indirectly support the gut microbiome. To create a clear, “funnel-shaped” narrative: start with thermal stress impacts, then discuss bee–microbiome resilience mechanisms, and conclude with interventions (probiotics/prebiotics). Please incorporate and appropriately cite relevant studies, for example:

• High temperatures: “Hot and Bothered: Bees’ Gut Microbiome Shifts Under Thermal Stress and Pathogen Infection” (J.I. Van Wyk et al., 2025); “Thermal niches of specialized gut symbionts: the case of social bees” (T.J. Hammer, E. Le, N.A. Moran).

• Low temperatures: “High abundance of lactobacilli in the gut microbiome of honey bees during winter” (Brar et al., 2025); “Overwintering Honey Bee Colonies: Effect of Worker Age and Climate on the Hindgut Microbiota” (P.W. Maes, 2021); “Gut microbiota structure differs between honeybees in winter and summer” (L. Kešnerová, 2020)

5. Material and methods: Please specify the exact months that constitute the summer and autumn seasons for 2023–2024 in your study.

6. Paragraph 2: Please include a clear photograph/figure of your experimental setup to improve readability and understanding. Add this image to the supplementary materials.

7. Commercial probiotic Progen and prebiotic inulin: please specify their composition. This is very important.

8. Please clarify the timeline: “Following this period” is ambiguous. For how many days was the reference control maintained? Was feeding conducted for 21 days and then samples moved to thermal treatment, or did these occur concurrently? The current wording is confusing; please specify the exact durations and sequence for each group.

9. Newly emerged bees do not have an intact gut microbiota because most bacterial species are acquired through trophallaxis, a key component of bee physiology. Bees with an intact microbiota behave and perform physiologically better than newly emerged bees lacking it. Why did the authors not include a treatment with bees having an intact microbiota? For example, newly emerged bees could be marked and co-housed with mature workers to inoculate their microbiome, serving as a control group. In practical applications, probiotics are fed to whole colonies comprising workers of different ages and castes, not only to newly emerged bees. Please provide a clear justification for the chosen treatments and controls and explain why a control group with an intact microbiota was not included.

10. Lines 338–340: This is an interesting observation. Please discuss how these temperatures may affect the gut microbiota, particularly lactobacilli (Lactobacillus spp.), which are often highly abundant under such conditions.

11. Line 366: Please refer back to the suggested citations and include them in this section as appropriate.

12. Please add a dedicated paragraph on the study’s limitations (e.g., the absence of gut microbial analysis and limited assessment of antimicrobial peptides), and outline future studies that could be conducted based on these data to strengthen the conclusions.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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<p>Protecting honey bees (Apis mellifera) from thermal stress: Probiotics and prebiotics buffer the survival and antioxidant enzyme activity

PONE-D-26-03238R1

Dear Dr. Bahreini,

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,

Vicente Martínez López

Academic Editor

PLOS One

Additional Editor Comments (optional):

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: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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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 #2: Yes

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: 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 #2: 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 #2: 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 revised the manuscript according to the Reviewr´s suggestions. They have done a a good work.

Reviewer #2: The authors have carefully and thoroughly addressed all the comments and concerns raised during the previous review round. Their responses are clear, detailed, and scientifically sound. The requested clarifications and revisions have been appropriately incorporated into the revised manuscript, which has significantly improved its clarity, quality, and scientific rigor. I appreciate the effort made by the authors to address the reviewers' suggestions and to strengthen the manuscript accordingly. In my opinion, the revised version satisfactorily addresses all major concerns, and I therefore recommend its acceptance for publication.

Reviewer #3: The manuscript has been significantly improved. No further comments.

The manuscript has been significantly improved. No further comments.

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-->7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: Yes: Maria Agustina Rodriguez

Reviewer #2: Yes: KHEDIDJI Hassiba

Reviewer #3: No

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