Telomere length varies substantially between blood cell types in a reptile

Telomeres are repeat sequences of non-coding DNA-protein molecules that cap or intersperse metazoan chromosomes. Interest in telomeres has increased exponentially in recent years, to now include their ongoing dynamics and evolution within natural populations where individuals vary in telomere attributes. Phylogenetic analyses show profound differences in telomere length across non-model taxa. However, telomeres may also differ in length within individuals and between tissues. The latter becomes a potential source of error when researchers use different tissues for extracting DNA for telomere analysis and scientific inference. A commonly used tissue type for assessing telomere length is blood, a tissue that itself varies in terms of nuclear content among taxa, in particular to what degree their thrombocytes and red blood cells (RBCs) contain nuclei or not. Specifically, when RBCs lack nuclei, leucocytes become the main source of telomeric DNA. RBCs and leucocytes differ in lifespan and how long they have been exposed to ‘senescence' and erosion effects. We report on a study in which cells in whole blood from individual Australian painted dragon lizards (Ctenophorus pictus) were identified using flow cytometry and their telomere length simultaneously measured. Lymphocyte telomeres were on average 270% longer than RBC telomeres, and in azurophils (a reptilian monocyte), telomeres were more than 388% longer than those in RBCs. If this variation in telomere length among different blood cell types is a widespread phenomenon, and DNA for comparative telomere analyses are sourced from whole blood, evolutionary inference of telomere traits among taxa may be seriously complicated by the blood cell type comprising the main source of DNA.

I have enjoyed reading your manuscript. It discuss a very common issue in measuring telomere length from blood. At any given time point blood could vary substantially in promotion of different blood cells and how will this affect the average telomere length of blood need to be carefully investigated in different species. Manuscript is written well and I have a few comments that will help you to revise the manuscript.
1. why authors did not include the age in the model, which is the strongest predictor of telomere length in blood.
2. How telomere length of these three cell types correlates to blood telomere length. Why authors did not compare it.
3. Discussion need shorten and more focused. There is data available in mammals investigating the telomere length in different cell lines as well as it correlation. 7. Line 327-330. In recent study, it has been shown that although cell promotion change during infection but does not correlate with whole blood telomere length (Asghar et al. 2018).

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

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

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

Comments to the Author(s)
This is a really interesting study on average telomere length differences in three blood cells types in reptiles: red blood cells, lymphocytes and azurophils. The authors use a study system, the painted dragon, in which they have plenty of experience and information, in addition to having developed a robust methodology for their aim.
I generally agree on their interpretation of the results, though I feel the authors should take more careful consideration when stressing the implications of their findings in the evolutionary context. I agree on the importance of the extant differences in telomere length between cell types. However, I feel the discussion of the authors on this respect could be more focus as I find it too loose for the brief of the results, i.e. differences on average telomere length among three blood cell types.
Furthermore, as stated by the authors (L282-297), the proportion of each cell type should be critical to over/underestimate blood cell average telomere length. Indeed, in the study model, the longer average telomere length in the azurophils might not be relevant for the final telomere length of the individual. I wonder if the authors have information on the final telomere length estimate in the whole blood sample and in that case how the estimated within-cell telomere length influence this? I think adding this kind of information will provide a direct example for the point discussed in L282-297.
I understand that cell counts are not always easily normalised, but have the authors considered to analyse the telomere distribution of these three types in a single model instead (L184-187)? E.g. using the current DataBase in long-format with telomere length as a dependent variable, the cell type as a 3-level factor and the ID of the individual as a random effect to account for the nonindependence in the measurements.
Minor L104-126. I suggest to directly provide in the text the information about the individuals used in the study. It is nice to have information about the species ecology but sex, age or sample size should be easily accessible, so far only available in the DataBase. L190-193. Could the authors be slightly more specific about the model selection process used? Also, the authors state that they used mixed models, in that case which is the random structure? Please state it. On behalf of the Editors, I am pleased to inform you that your Manuscript RSOS-192136 entitled "Telomere length varies substantially between blood cell types in a reptile" has been accepted for publication in Royal Society Open Science subject to minor revision in accordance with the referee suggestions. Please find the referees' comments at the end of this email.
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Royal Society Open Science openscience@royalsociety.org on behalf of Kevin Padian (Subject Editor) openscience@royalsociety.org Associate Editor Comments to Author: Comments to the Author: Thank you for the submission, which has been reviewed by three referees. They all recommend that, after a number of comparatively minor modifications, your paper may be accepted. Please ensure you engage constructively with their comments, and provide a full point-by-point response in addition to a revised manuscript. We'll look forward to receiving the updated paper in due course.
Reviewer comments to Author: Reviewer: 1 Comments to the Author(s) Dear Dr. Olsson and coauthors, I have read and reviewed the manuscript: "Telomere length varies substantially between blood cell types in a reptile" (RSOS-192136).
I think your manuscript is interesting, well written and worth to publish it. This manuscript apports new and valuable information that should be taken into account in future studies done with telomeres, especially those done from blood samples. My only general concern is about methods which, from my point of view, are briefly explained and more details may be required. I want to point that there are references cited, but some more information may be useful for a general reader, not interested in review all information referent to telomeres already published. Moreover, a more detailed methodology about flow cytometry may be useful.
I made a few specific comments below.
Line 117: please, after blue heads close the " Line 158: please, provide a reference or data supporting the sentence: "The resulting fluorescence intensity of the cells is directly correlated with the length of the telomeres" Line 159: You provide information on the reproducibility of the techniques, but it may be interesting also provide a measurement of repeatability of your own data.
Line 176: please, delete one r of forr.

Mar Comas
Reviewer: 2 Comments to the Author(s) Dear Authors, I have enjoyed reading your manuscript. It discuss a very common issue in measuring telomere length from blood. At any given time point blood could vary substantially in promotion of different blood cells and how will this affect the average telomere length of blood need to be carefully investigated in different species. Manuscript is written well and I have a few comments that will help you to revise the manuscript.
1. why authors did not include the age in the model, which is the strongest predictor of telomere length in blood.
2. How telomere length of these three cell types correlates to blood telomere length. Why authors did not compare it.
3. Discussion need shorten and more focused. There is data available in mammals investigating the telomere length in different cell lines as well as it correlation. 7. Line 327-330. In recent study, it has been shown that although cell promotion change during infection but does not correlate with whole blood telomere length (Asghar et al. 2018).

Reviewer: 3
Comments to the Author(s) This is a really interesting study on average telomere length differences in three blood cells types in reptiles: red blood cells, lymphocytes and azurophils. The authors use a study system, the painted dragon, in which they have plenty of experience and information, in addition to having developed a robust methodology for their aim. I generally agree on their interpretation of the results, though I feel the authors should take more careful consideration when stressing the implications of their findings in the evolutionary context. I agree on the importance of the extant differences in telomere length between cell types. However, I feel the discussion of the authors on this respect could be more focus as I find it too loose for the brief of the results, i.e. differences on average telomere length among three blood cell types.
Furthermore, as stated by the authors (L282-297), the proportion of each cell type should be critical to over/underestimate blood cell average telomere length. Indeed, in the study model, the longer average telomere length in the azurophils might not be relevant for the final telomere length of the individual. I wonder if the authors have information on the final telomere length estimate in the whole blood sample and in that case how the estimated within-cell telomere length influence this? I think adding this kind of information will provide a direct example for the point discussed in L282-297.
I understand that cell counts are not always easily normalised, but have the authors considered to analyse the telomere distribution of these three types in a single model instead (L184-187)? E.g. using the current DataBase in long-format with telomere length as a dependent variable, the cell type as a 3-level factor and the ID of the individual as a random effect to account for the nonindependence in the measurements.
Minor L104-126. I suggest to directly provide in the text the information about the individuals used in the study. It is nice to have information about the species ecology but sex, age or sample size should be easily accessible, so far only available in the DataBase. L190-193. Could the authors be slightly more specific about the model selection process used? Also, the authors state that they used mixed models, in that case which is the random structure? Please state it. Decision letter (RSOS-192136.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 Olsson,
It is a pleasure to accept your manuscript entitled "Telomere length varies substantially between blood cell types in a reptile" in its current form for publication in Royal Society Open Science. The comments of the reviewer(s) who reviewed your manuscript are included at the foot of this letter.
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Thank you for your fine contribution. On behalf of the Editors of Royal Society Open Science, we look forward to your continued contributions to the Journal. Thank you for accepting our MS for publication in RSOS. Please, find below our responses (following **) to the few comments provided by the reviewers: Reviewer comments to Author: Reviewer: 1 Comments to the Author(s) Dear Dr. Olsson and coauthors, I have read and reviewed the manuscript: "Telomere length varies substantially between blood cell types in a reptile" (RSOS-192136).
I think your manuscript is interesting, well written and worth to publish it. This manuscript apports new and valuable information that should be taken into account in future studies done with telomeres, especially those done from blood samples. My only general concern is about methods which, from my point of view, are briefly explained and more details may be required. I want to point that there are references cited, but some more information may be useful for a general reader, not interested in review all information referent to telomeres already published. Moreover, a more detailed methodology about flow cytometry may be useful. ** Some of these comments (and the one referring to l. 158 below) seem to suggest that the reviewer is not familiar with Flow Cytometry as a method in general and I don't think this is the right forum for such a review. Cells are incubated with a dye of choice targeting the specific trait to estimate and this results in fluorescence that can be 'counted'. Our methods description on Flow Cytometry (FC) of telomere length, specifically, is already two pages and contains four specific references and the web site from Dako manufacturing the kit. The sentence 'general reader, not interested in review all information referent to telomeres already published' demonstrates further inconsistency between the request for Flow Cytometry methods per se, and the huge literature and numerous reviews on telomere methods. We can add additional information if the editor finds this necessary but since this is not a request, but more of a comment, we have left the FC methods as is while adding a reference to line 158 (see below).
I made a few specific comments below.
Line 117: please, after blue heads close the " ** Many thanks, this has been done.
Line 158: please, provide a reference or data supporting the sentence: "The resulting fluorescence intensity of the cells is directly correlated with the length of the telomeres"