Pathology findings and correlation with body condition index in stranded killer whales (Orcinus orca) in the northeastern Pacific and Hawaii from 2004 to 2013

Stephen Raverty; Judy St. Leger; Dawn P. Noren; Kathy Burek Huntington; David S. Rotstein; Frances M. D. Gulland; John K. B. Ford; M. Bradley Hanson; Dyanna M. Lambourn; Jessie Huggins; Martha A. Delaney; Lisa Spaven; Teri Rowles; Lynne Barre; Paul Cottrell; Graeme Ellis; Tracey Goldstein; Karen Terio; Debbie Duffield; Jim Rice; Joseph K. Gaydos

doi:10.1371/journal.pone.0242505

Peer Review History

Original SubmissionJuly 24, 2020
16 Sep 2020 Decision Letter - Susana Caballero, Editor PONE-D-20-23093 Pathology findings and correlation with body condition index in stranded killer whales (Orcinus orca) in the northeastern Pacific and Hawaii from 2004 and 2013 PLOS ONE Dear Dr. Gaydos, 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. Both reviewers were very positive regarding this manuscript and its results and they both agree this is an important contribution to the field and will help many researchers working in this and similar species. I think it is important that the authors re-evaluate the statistics and data analyses they included, since one of the reviewers points out that the statistical tests used for the data may not fit due to criteria regarding data normality, etc. For that reason I suggest that the authors double check this comments and consider alternative ways to analyze their data. Please submit your revised manuscript by 15th of October 2020. 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). 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For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Susana Caballero, PhD Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf Additional Editor Comments (if provided): Both reviewers were very positive regarding this manuscript and its results and they both agree this is an important contribution to the field and will help many researchers working in this and similar species. I think it is important that the authors re-evaluate the statistics and data analyses they included, since one of the reviewers points out that the statistical tests used for the data may not fit due to criteria regarding data normality, etc. For that reason I suggest that the authors double check this comments and consider alternative ways to analyze their data. [Note: HTML markup is below. Please do not edit.] 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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: No ****** 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 ****** 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: Yes ****** 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: I congratulate the authors on this piece of work. I found it interesting and easy to read, and a valuable addition to existing knowledge. I have only minor comments to make, mostly of an editorial rather than a content nature. These are detailed below. Line 162 (BCI equation) - 'enterior' should be 'anterior' Line 190-191 - this should either be 'association .... was evaluated' or 'associations.... were evaluated' Line 231 - add 'which' ("was an offshore calf which had a hiatal hernia...") Line 258 - should be bone remodelling rather than boney Line 392 - add 'with' or 'in' (was found with moderate...) Line 395. Meconium staining of what? Also please detail the signs of fetal distress that you screened for but were not present Line 677 - Please remove the comma after peritonitis LIne 678 - Bacteria should be bacterial Line 694 - I think this should be cytpolasmic rather than cytoplastic? Line 716 - 'concerns' is not a particularly scientific word. Is there a more precise subheading that could be used here? Line 718 and 719 - 'this' lesion is used in the first sentence and then 'these lesions' in the following one. Please be consistent Line 719 - 722. Please include references for pathogenesis of myocardial fibrosis. The sentence that begins with "The pathogenesis" requires some work. What do you mean by 'long past inflammation'. The word 'infectious' can be removed (pathogens is sufficient). 'Diving issues' is not a scientific term; please use more precise language here. Finally, the absence of cardiomyopathy, pulmonary congestion and hepatic congestion does not rule out the possibilty of non-congestive cardiac abnormalities such as arrhythmias, as has been described in association with myocardial fibrosis in humans and domestic species. A final general comment. I found the conclusions about blubber depth and BCI particularly interesting and relevant to researchers working on cetacean health and disease, and wondered if it would be worth re-emphasising these findings in the concluding paragraph. Reviewer #2: This study of causes of death of killer whales is an important addition to the marine mammal literature. However, I do have the following comments/questions about aspects of this study. This paper also discusses very important points relative to the significance, difficulty, and limitations of evaluation of post-mortem body condition in killer whales. The discussion about potential fisheries interaction with killer whales is also important. Based on how the data was collected, I am doubtful that the data fit the assumptions of the ANCOVA (normality, homogeneity of variance and randomized independent samples- samples were independent, not random) used to determine differences between body length and blubber thickness measurements relative to location. Therefore, I recommend an alternative procedure (e.g. generalized linear model, data transformations, nonlinear model) and for other results a non-parametric one-sided t-test alternative (wilcoxon signed rank test).Can you also explain figure 10 in a bit more detail? I did not see the figure title or legend. Also, it would be helpful, in addition to the large table of observations, to summarize some of the data in a graph or other type of data visualization(e.g. cause or causes of death and any significant associations with other factors). ****** 6. 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. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0242505.r001
Revision 1
12 Oct 2020 Author Response Dear Dr. Caballero, Thank you for reviewing our manuscript PONE-D-20-23093, “Pathology findings and correlation with body condition index in stranded killer whales (Orcinus orca) in the northeastern Pacific and Hawaii from 2004 and 2013.” We were encouraged to hear that both reviewers were very positive regarding this manuscript and its results and they both agree this is an important contribution to the field and will help many researchers working on this and similar species. All of the comments were very helpful and improved the quality of the manuscript. As requested, we have addressed all concerns raised by both Reviewers. We have uploaded a marked-up copy of the manuscript that highlights changes made to the original version and labeled it “Revised Manuscript with Track Changes.” Also, we have uploaded an unmarked version without tracked changes as a separate file labeled “Manuscript.” We have proofread the manuscript closely for mistakes and I hope you will find this revised manuscript suitable for publication. Here is a detailed description of each point brought up during the review and what we did to address it: Reviewer #1: Line 162 (BCI equation, now line 165) - 'enterior' should be 'anterior' - corrected Line 190-191 (now line 201)- this should either be 'association .... was evaluated' or 'associations.... were evaluated' - corrected Line 231(now line 267)- add 'which' ("was an offshore calf which had a hiatal hernia...") - done Line 258 (now line 297) - should be bone remodeling rather than boney - corrected Line 392 (now line 436) - add 'with' or 'in' (was found with moderate...) - done Line 395 (now line 439). Meconium staining of what? Also please detail the signs of fetal distress that you screened for but were not present - we removed this sentence from the manuscript “There was no indication of meconium staining or in utero fetal distress.” Line 677 - Please remove the comma after peritonitis - done Line 678 (now line 763)- Bacteria should be bacterial - fixed Line 694 (now line 780) - I think this should be cytoplasmic rather than cytoplastic? - fixed Line 716 (now line 804) - 'concerns' is not a particularly scientific word. Is there a more precise subheading that could be used here? - changed “concerns” to say “pathology” Line 718 and 719 (now line 807) - 'this' lesion is used in the first sentence and then 'these lesions' in the following one. Please be consistent - corrected all to “this lesion” Line 719 – 722 (now lines 808-814) - Please include references for pathogenesis of myocardial fibrosis. The sentence that begins with "The pathogenesis" requires some work. What do you mean by 'long past inflammation'. The word 'infectious' can be removed (pathogens is sufficient). 'Diving issues' is not a scientific term; please use more precise language here. Finally, the absence of cardiomyopathy, pulmonary congestion and hepatic congestion does not rule out the possibility of non-congestive cardiac abnormalities such as arrhythmias, as has been described in association with myocardial fibrosis in humans and domestic species. - we included two citations for pathogenesis of myocardial fibrosis (citations 54 and 55) - we edited “long-past” to say “resolving and possibly progressive fibrosis” - we removed the word “infectious” before pathogens - “diving issues such as” were replaced with “gas bubble disease” - as suggested, we added the final concluding comment, “however, the lack of these lesions does not preclude the possibility of an arrythmia or other non-congestive cardiac abnormalities.” A final general comment. I found the conclusions about blubber depth and BCI particularly interesting and relevant to researchers working on cetacean health and disease and wondered if it would be worth re-emphasizing these findings in the concluding paragraph. - as suggested, we added several sentences to re-emphasize the findings related to blubber depth and BCI in the concluding paragraph (lines 922-931). Reviewer #2: Based on how the data was collected, I am doubtful that the data fit the assumptions of the ANCOVA (normality, homogeneity of variance and randomized independent samples- samples were independent, not random) used to determine differences between body length and blubber thickness measurements relative to location. Therefore, I recommend an alternative procedure (e.g. generalized linear model, data transformations, nonlinear model) and for other results a non-parametric one-sided t-test alternative (Wilcoxon signed rank test). Note: edits discussed below include additions to materials and methods (lines 190-199; 203-207; 223-237), results (lines 478-491; 520-536), Fig. 9 description (lines 511-513), Fig. 10 description (line 564), Discussion (line 605; line 626; line 640; line 646; and lines 922-931). - As suggested, we used a generalized linear model to assess relationships between body length and blubber thickness measurements for all animals combined as well as for older animals and young animals separately. All data incorporated into ANCOVA passed both normality and homogeneity of variance tests, but we had previously failed to include those details in the methods. Because of the results of the GLM, however, we assessed differences in thickness across the three blubber measurement sites separately for older animals (via ANCOVA) and young animals (via ANOVA) in this revised manuscript. Before conducting these analyses, we confirmed that data passed normality and homogeneity of variance tests. This is now stated in the methods. Finally, because both tests passed, including the test for normality, the data were not transformed. - As the reviewer pointed out, some data are nonlinear. This is the case for the relationship between body length and BCI. It was incorrect to evaluate the data via linear regression analysis. We now evaluate the relationship between body length and BCI via Spearman Rank Order Correlation. - Regarding the t-tests, it was an oversight to use t-tests for some of the comparisons. Data for a few of the t-tests did not pass the normality test. For these specific comparisons, the Mann-Whitney Rank Sum Test is now used. The methods were modified to specify this approach, and the results were modified to include the results of the Mann-Whitney Rank Sum Test, when appropriate. The Wilcoxon signed rank test was suggested by the reviewer, but this is actually not an appropriate test. The Wilcoxon signed-rank test is used to compare two related samples, matched samples, or repeated measurements on a single sample, which doesn’t apply in this situation. We used Mann-Whitney Rank Sum Tests to determine whether there were differences in lengths, BCI and blubber thickness measurements between animals that died from trauma and animals that died from disease or malnutrition (combined). T-tests are still used for the specific cases when the data are normal and other assumptions of t-tests are met. This is now explicitly stated in the methods and results. We reiterate that one-tailed tests were chosen because, based on previous publications, we assumed that animals that died from trauma would have thicker blubber and greater BCI values than those that died from disease and malnutrition. We selected one-tailed T-tests prior to conducting the statistical analyses. However, regardless of whether one-tailed or two-tailed tests are used for the comparisons, the results are still highly significant. Can you also explain figure 10 in a bit more detail? I did not see the figure title or legend. - Figure 10 has a lengthily description in the figure legend, however it appears on the page prior to the figure so I imagine Reviewer #2 just missed it. Also, it would be helpful, in addition to the large table of observations, to summarize some of the data in a graph or other type of data visualization(e.g. cause or causes of death and any significant associations with other factors). - Good idea. We added new summary text (lines 237-242) and Table 3 (line 247) to summarize the 62 significant pathologic findings by age class and help the reader better visualize them in a small table. - Additionally, we added an additional reference to Table 1 in the materials and methods (line 124) and alphabetized and corrected the terms used for cause of death in Table 1 (lines 127-129). Note: in reviewing the manuscript one final time, we noted that the pathogenesis described on lines 274-275 was not clear, so we altered the text slightly to clarify. Also, • we added an acknowledgment line of thanks to G. Anchor for help with the GLM (line 960) • we corrected the spelling of cholangiohepatitis in Table 1 for ID 20040503 • we added “cm” after the measurement 501 on line 159 All co-authors have approved these revisions. Again, I would like to reiterate that the peer-review process at PLOS ONE has improved the quality of this manuscript and I appreciate the thoughtful and helpful comments provided. I hope you will find this manuscript acceptable for publication. Please do not hesitate to contact me by email or by phone if you have any questions or need additional information. Attachments Attachment Submitted filename: Response to Reviewers PONE-D-20-23093R1.docx https://doi.org/10.1371/journal.pone.0242505.r002
4 Nov 2020 Decision Letter - Susana Caballero, Editor Pathology findings and correlation with body condition index in stranded killer whales (Orcinus orca) in the northeastern Pacific and Hawaii from 2004 and 2013 PONE-D-20-23093R1 Dear Dr. Gaydos, 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. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Susana Caballero, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Well done, excellent paper, will be a very good addition to marine mammal literature 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 ******** 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: (No Response) Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: (No Response) 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 ****** 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: (No Response) 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: (No Response) Reviewer #2: All comments have been adequately addressed. This article is an important addition to the marine mammal disease literature. ****** 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. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No https://doi.org/10.1371/journal.pone.0242505.r003
Formally Accepted
9 Nov 2020 Acceptance Letter - Susana Caballero, Editor PONE-D-20-23093R1 Pathology findings and correlation with body condition index in stranded killer whales (Orcinus orca) in the northeastern Pacific and Hawaii from 2004 and 2013 Dear Dr. Gaydos: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Susana Caballero Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0242505.r004

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