PONE-D-21-35366Morphological and quantitative analysis of hemocytes in free-living Australian black flying foxes (Pteropus alecto)PLOS ONE

Dear Dr. Hansen,

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.

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Both reviewers and myself agree that this is a well-written and helpful article for characterizing the hematological profiles of Australian flying foxes. Both reviewers provide a number of suggestions that will improve the manuscript. Most notably, reviewer 1 notes some discrepancies in how certain leukocytes are visualized/analyzed compared to other leukocytes as well as the need to explicitly analyze leukocyte data in relation to body condition and to identify the parasite gametocytes. Reviewer 2 also has also requested some additional analyses that would strengthen the paper. In relation to these comments, I had a few further comments for the authors: L80: Is there a reference the authors can cite for the ordinal body condition score? L124: Can the authors remind the reader what fraction of individual blood smears were from adults? L128: Note that the log transformation is not appropriate for proportion data, as proportion data are bound between 0-1 (see Warton & Hui 2011 Ecology for a full discussion). I suggest the authors consider logit transforming proportions, which also facilitates back-transforming the proportions to the true 0-1 boundary of the data. L159: Perhaps clarify here that “individuals” = “adult bats”. L160: Was a statistical test used (here and elsewhere) to compare leukocyte proportions between sexes and/or healthy/unhealthy bats? Or is this statement based only on data visualizations and raw summary statistics? I would suggest the authors consider the former as a more definitive way to make comparisons among groups. L275: Intraerythrocytic gametocytes of which parasites? It would help to identify the parasites to genus if possible, if not broader taxonomic level like order (e.g., haemosporidia).

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We acknowledge the Kabi Kabi and Yuggera Ugarapul people, who are the Traditional Custodians of the land upon which this work was conducted. This study was funded by the DARPA PREEMPT program Cooperative Agreement # D18AC00031, and the U.S. National Science Foundation (DEB-1716698). DH and BH were funded by the Montana State University Undergraduate Scholars Program. BH was also funded by the Montana State University McNairs Scholar Program (Grant #P217A130148). RKP was also funded by the USDA National Institute of Food and Agriculture (Hatch project 1015891). AJP was supported by an ARC DECRA fellowship (DE190100710). KR acknowledges the support of The University of Sydney Institute for Infectious Diseases. Special thanks to Dr. Charlotte Hollinger, Dr. Dee McAloose, Dr. Siobhan Egan, Lauren Warner, Amelia Graves, and Lindsay Lee. 

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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: Yes

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

Reviewer #1: Yes

Reviewer #2: 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

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

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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: This paper reports the morphological and descriptive statistics of leukocyte parameters from 134 Bat flying foxes (Pteropus alecto) from Australia between 2018-2020. Pteropus species are known to be hosts of many zoonotic viruses of interest and therefore a descriptive study of this kind is incredibly important for understanding baseline health of these animals. Overall, this paper does a fantastic job of summarizing key statistics, including differences in animals with injuries and those deemed healthy, and provides a jumping off point for future studies to analyze blood smears. Overall, the paper is written quite well, and I have very few minor comments to improve the paper.

Reviewer #2: I was happy to read the manuscript entitled “Morphological and quantitative analysis of hemocytes in free-living Australian black flying foxes (Pteropus alecto)” written by Hansen and colleagues.

The authors analyzed the differential white blood cell counts (and red blood cells and platelets) of 134 Australian black flying foxes from blood smears via light microscopy and provide quantitative and morphological information on these – including an atlas of images. Although this method is time consuming, in my opinion has a tremendous value in comparison to more automated methods, especially for wildlife species. I very much like this study; despite the use of classic methods and a more descriptive study design, I personally think this is a nice manuscript, especially considering the recent interest of infectious biologists in bats, but also has value from a conservation physiology point of view.

Especially the atlas of images will be used not only by researchers but also by veterinarians, vet nurses – in a recent textbox about wild and exotic animal haematology, I could not find any photo or information on Chiroptera, despite being the second largest mammalian order. This manuscript definitively helps to fill this gap.

I have few comments, which hope can be easily addressed after a minor revision.

1. through the entire manuscript please replace hemocytes with white blood cells or leukocytes, as hemocytes refers to the cells of invertebrates

2. in the abstract it is written “…leukocyte morphological traits that are relevant for future research on bat health, especially in context of viral spillover and emergence”. I could not find these in the discussion. Would it be possible to rephrase this sentence, present and focus more the specific results?

3. introduction – “bat-borne pathogens”: are all these serious threat to human health, or some of them only? Again I think being more specific on viruses and/or intracellular pathogens, would help also to decrease the negative distinction of bats received lately due to their reservoir competence.

4. line 34 – “medical” history

5. lines 50 – 60: other drawbacks of automated methods are logistics (e.g. remote field conditions) and volume requirements (e.g. smaller sized species). These could be included in this section too.

6. The authors report and discuss the size of each cell type relative to others, which is indeed helpful when analyzing blood smears. However, I was wondering whether it would be possible to measure the size and thickness of each cell type and report these too?

7. figure 1 – please rearrange the order as differs between the figure and the order described in the legend

8. figure 1 and figure 2 – I would suggest to keep the logical order from the manuscript: neutrophils, lymphocytes, eosinophils and monocytes.

9. references - through the manuscript there were few sentences where I missed references supporting the statements. Could you please include these, as this would help the reader? See lines 320 (on neutrophil dominance in mammals); lines 356-364 (reference 19 is not the right one, please change it; LPS induced systemic inflammation causes neutrophilia in various bat species, including in Rousettus aegyptiacus; maybe some of these papers can be included here); lines 377-379 (indeed differential counts can allow comparison across larger scales. Reference missing – see Becker et al. 2019 Integr Comp Biol on common vampires).

10. I was surprised that the authors did not perform total white blood cell counts using standardized method with hemocytometer…It requires only small amount of blood and a field microscope. Why was this omitted? Although the differential counts alone have their values as discussed in this paper, still would be great if more studies would aim for total leukocyte counts too and not only via automated methods.

11. Besides the value of such study for disease research, I think also has importance for conservation (e.g. identifying anthropogenic stressors, species of conservation interest) – this aspect should be mentioned in the conclusion section.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-21-35366R1Morphological and quantitative analysis of leukocytes in free-living Australian black flying foxes (Pteropus alecto)PLOS ONE

Dear Dr. Hansen,

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.

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

The manuscript has now been seen by both original reviewers, who each agree that their prior concerns were addressed satisfactorily and that the manuscript will make a nice contribution to the literature on bat hematology and immunology more broadly. I agree with these assessments.

One of the conditions of publication in PLoS ONE is that conclusions are supported by data, and I would therefore ask a final set of edits by the authors. In the response letter, the authors note that the focus of the work was to present a reference of images and values for hematology of wild black flying foxes; because of this descriptive focus, the authors do not perform statistical analyses and instead present ranges (IQR) and medians of cell types. I don’t quite agree with the authors that you would need data on absolute numbers of cells to make such conclusions (you can analyze differences in the proportion of each leukocyte type), but descriptive analyses are perfectly fine for this journal. However, throughout the Results text, the authors do make inferential statements about how cell proportions differ between males and females and between apparently healthy and injured individuals (e.g., L174, “The interquartile ranges for neutrophils showed no major differences between males and females in apparently healthy individuals; however, in the group of bats with observed injuries, the interquartile ranges and medians were lower for females than males”). These statements are inferential, in that you are claiming a difference between the medians of two groups (this is the point of inferential statistics; e.g., a t-test or a non-parametric equivalent). I think these statements could mislead readers to infer comparisons that weren’t actually tested. Therefore, I would ask the authors to take one of two routes in this paper. On the one hand, you could clarify early in the Methods why you don’t perform formal statistical analyses, and then remove any of the inferential text from the Results. You could leave the tables and figures as they are, but not make any claims about median percent neutrophils, lymphocytes, eosinophils, etc being different between sexes or health status. On the other hand, you could retain these statements but support them with simple yet formal statistical tests (e.g., t-tests would be perfectly fine). Relevant parts of the Discussion (e.g., L380, 389) should then be modified or left as is, according to what the authors decide. Either option would fit well within PLoS ONE, but the authors should decide if they wish to infer sex- and health-related differences or leave the manuscript in a descriptive form.

Lastly, the authors must upload their raw data (i.e., individual-level data on leukocyte profiles) as a condition of acceptance.

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

Please submit your revised manuscript by Apr 28 2022 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.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• 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,

Daniel Becker

Academic Editor

PLOS ONE

Journal Requirements:

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: All comments have been addressed

Reviewer #2: 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

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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: (No Response)

Reviewer #2: 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

**********

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: I believe that the authors did a good job at addressing all the reviewer's concerns and I believe it will make a great contribution to the field.

Reviewer #2: I would like to congratulate the authors for the revision they did on this manuscript. All my concerns and questions have been dealt with, I have no more suggestions.

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Reviewer #1: No

Reviewer #2: No

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Morphological and quantitative analysis of leukocytes in free-living Australian black flying foxes (Pteropus alecto)

PONE-D-21-35366R2

Dear Dr. Hansen,

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.

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Kind regards,

Daniel Becker

Academic Editor

PLOS ONE

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