Constitutive Innate Immunity of Migrant and Resident Long-Nosed Bats (Leptonycteris yerbabuenae) in the Drylands of Mexico

Round 1

Reviewer 1 Report

The presented manuscript seems to me interesting and worthy of being published. Perhaps other reviewers, more competent in immunology, will find some inconsistencies or flaws, but I did not see them. From my point of view, the methods and materials are quite adequate to the conclusions, which sound quite cautious and not categorical, which also speaks in favor of the authors. As another plus (from my point of view), it can be noted that the authors did not include in the analysis a number of categories of research objects due to insufficient samples sizes (although they are included into the Results, so that future readers can form their own opinion about the presence or absence of certain tendencies).

The research topic is quite specific; on the one hand, the work, as I have already said, is of interest to a problem that is not often discussed by the bat researchers, on the other hand, it will arouse interest primarily among those who are professionally involved in the chiropteran ecology and physiology. Therefore, I designated Scientific soundness and Interest for readers as "average", although the mentioned experts may not agree with me.

Thus, I see no grounds for any significant claims to the semantic part. The language of the manuscript, it seems to me, is quite understandable. So the manuscript requires only a minor revision, concerning some inaccuracies or places that remain unclear to me, which I write about below.

Lines 143 and further: “2.1. Study sites and sample collection” – Sampling was carried out in different seasons in two different places, one of which is included in, relatively speaking, the “winter” species range, and the second is in the breeding area of the species. However, as I understand it, there is no reason to believe that females from Don Panchito Island fly to Mariana cave to breed, or that the animals (adults and young) from Mariana cave return exactly to the Don Panchito Island. Thus, the animals included in the work, caught in these two places, may belong to different populations. Perhaps this does not affect the results obtained in any way, but this aspect at least requires an appropriate comment from the authors.

Line 154: “Lactating females have been reported” – It was previously said that the breeding area of the species lies in the deserts of northeastern Mexico and in the US, so it is not clear why lactating females suddenly appeared on Don Panchito Island. This point requires a brief explanation.

Lines 175: “in contrast with those with scrotal testicles but no dorsal patch” – It follows from the introduction that the testicles of adult males are located in the scrotum, regardless of reproductive activity. That is, the scrotal testicles simply mark adult males, while the dorsal spot marks breeding males. Maybe it would be better to initially characterize different age and reproductive categories, and then simply name them in the text?

Lines 253-254: Closing bracket is forgotten.

Figure 4: – A purely technical note: maybe for better understanding it might be better to change the order of the graphs in Figure 4b so that (as in other figures) it starts with the “Lactating” category?

Line 358: “non-reproductive and scrotal males,” – What I wrote about above. "Non-reproductive" - are they young or not reproductively active adults? But adult reproductively inactive males are also “scrotal”. And active ones (with a dorsal spot) are also “scrotal”. I repeat, so that there is no confusion, it is worth at the beginning to single out and describe the age and reproductive categories and then just mention them.

Line 368: “This discrepancy suggests that the effect of reproductive season on the immune system in bats is not widespread.” – This statement is generally true. However, Daubenton's bat has an extended mating period, males do not undergo significant visible changes, which indicates a relatively low level of hormonal fluctuations (especially compared to hibernation). I'm not sure that it is adequate to compare it with a species that does not hibernate, with a pronounced mating period, during which the hormonal background of males changes dramatically, causing anatomical changes visible from the outside?

I am sure that the responses to these comments and the introduction of appropriate corrections do not present any difficulties for the authors. After that, the manuscript could be recommended for publication in Diversity.

Author Response

We thank the reviewer for his/her insights. We addressed each comment, although we are certain that we fully understood the last request.

Lines 143 and further: “2.1. Study sites and sample collection” – Sampling was carried out in different seasons in two different places, one of which is included in, relatively speaking, the “winter” species range, and the second is in the breeding area of the species. However, as I understand it, there is no reason to believe that females from Don Panchito Island fly to Mariana cave to breed, or that the animals (adults and young) from Mariana cave return exactly to the Don Panchito Island. Thus, the animals included in the work, caught in these two places, may belong to different populations. Perhaps this does not affect the results obtained in any way, but this aspect at least requires an appropriate comment from the authors.

AUTH. We agree that this is an important issue. We added the following: “We cannot be certain that females migrate specifically between Don Panchito and Mariana cave, but we assumed that the study sites are representative of the general migratory range of L. yerbabuenae in this part of its distribution.”

Line 154: “Lactating females have been reported” – It was previously said that the breeding area of the species lies in the deserts of northeastern Mexico and in the US, so it is not clear why lactating females suddenly appeared on Don Panchito Island. This point requires a brief explanation.

AUTH. We pointed in the previous sentence that some females remain in Don Panchito, and we suspect that they are the origin of these lactating individuals. We added the following to clarify:

“The remaining population in west-central Mexico is nearly exclusively composed of males throughout August, although some females remain in the cave. Lactating females have been reported in the cave at Don Panchito Island in January-July [21] but is it uncertain if they are part of the resident population of this cave or if they originated from other caves in the region.”

Lines 175: “in contrast with those with scrotal testicles but no dorsal patch” – It follows from the introduction that the testicles of adult males are located in the scrotum, regardless of reproductive activity. That is, the scrotal testicles simply mark adult males, while the dorsal spot marks breeding males. Maybe it would be better to initially characterize different age and reproductive categories, and then simply name them in the text?

AUTH. We agree that is confusing our use of the terms. We modified the terminology in different sections of the whole manuscript (abstract, methods, results, discussion, figures and table 1).

Lines 253-254: Closing bracket is forgotten.

AUTH. Fixed.

Figure 4: – A purely technical note: maybe for better understanding it might be better to change the order of the graphs in Figure 4b so that (as in other figures) it starts with the “Lactating” category?

AUTH. We agree that the order should be changed but following a chronological order (e.g.it would not make much sense to initiate with lactating females and then present pregnant females). Therefore, we modified Fig. 3b and Fig 4b to follow the chronological order in which female bats were collected.

Line 358: “non-reproductive and scrotal males,” – What I wrote about above. "Non-reproductive" - are they young or not reproductively active adults? But adult reproductively inactive males are also “scrotal”. And active ones (with a dorsal spot) are also “scrotal”. I repeat, so that there is no confusion, it is worth at the beginning to single out and describe the age and reproductive categories and then just mention them.

AUTH. Thank you for pointing this. To avoid the confusion that you mention (“"Non-reproductive" - are they young or not reproductively active adult s”?), we specified that this comparison concerns only to adult males.

Line 368: “This discrepancy suggests that the effect of reproductive season on the immune system in bats is not widespread.” – This statement is generally true. However, Daubenton's bat has an extended mating period, males do not undergo significant visible changes, which indicates a relatively low level of hormonal fluctuations (especially compared to hibernation). I'm not sure that it is adequate to compare it with a species that does not hibernate, with a pronounced mating period, during which the hormonal background of males changes dramatically, causing anatomical changes visible from the outside?

AUTH. We are not sure about this comment. Jorge A. Encarnacao appears to describe what seems to be a mating period that is not extended as indicated by the reviewer. Here is what his J. Ethol. 2012 paper states: ”Daubenton’s bats were tracked during three different periods. The definition of these periods was based on the identification of birth and fledging dates (Dietz and Kalko 2007), as well as seasonal variations in epididymal distension and evidence of mating (Encarnac¸a˜o et al. 2004, 2006, 2007). During late spring, females are pregnant and give birth in the first week of June, while males are reproductively inactive (13 May–17 June: nmale = 12, nfemale = 4). During early summer, young of the year get weaned from females, and in males gonadal development induces spermatogenesis (29 June–31 July: nmale = 15, nfemale = 4). During late summer, females are post-lactating, and in males spermatogenesis peaks (11–30 August: nmale = 15, nfemale = 4). In August, mating willingness should be high for both sexes.” We are sorry but we do not know how to proceed about this comment.

Reviewer 2 Report

The study presented in this ms. compares the innate immunity of bats of different sex during their reproductive and migration cycle. It is an exciting subject, particularly since zoonosis and animal immunity are of general interest.

I work on bats in arid landscapes, but with an ecological approach, so I have no competence to evaluate the lab procedure. I trust that it is okay. The numerical analysis also seems adequate for the data you gathered. Unfortunately, the sample size is very small, which proves most limiting in answering some of the questions proposed in the study.

The text needs to be revised thoroughly. I am not a native English speaker and found several typos (see the pdf attached). Namely, the common name of the studied species is often misspelt. The methodology section could also be written clearer and more concisely. There are several paragraphs where the text is repetitive, and I had to read it several times and take notes to grasp it all. The most obvious case was the description of where the body mass and the sex were not considered due to the low sample size.

The results are presented in terms of either body mass or BKA comparisons. If I understand correctly, the authors used separate models to make these comparisons. So, they show differences in the body mass of some groups and differences in the BKA of the plasma in some groups. However, the relation between the two variables is never presented clearly, a major drawback of this study. I suggest the authors give the GLM models fitted for each case, even as supplementary material.

The discussion provides an interesting read. It is appropriately supported and acknowledges some of the study’s limitations.

Overall, I find the study very interesting, and it may be a good contribution to an area that seems to be still understudied. Most studies would gain in having better quality data, which is flagrant in this case. However, I believe there is space to improve the ms. in other ways. Namely in how the results are presented and by improving the text.

Comments for author File: Comments.pdf

Author Response

We appreciate the suggestions to improve our manuscript. We addressed all the suggestions made by the reviewer in the PDF of the manuscript. 

We added an appendix (S1) with the models to address the following comment:

"The results are presented in terms of either body mass or BKA comparisons. If I understand correctly, the authors used separate models to make these comparisons. So, they show differences in the body mass of some groups and differences in the BKA of the plasma in some groups. However, the relation between the two variables is never presented clearly, a major drawback of this study. I suggest the authors give the GLM models fitted for each case, even as supplementary material."

Reviewer 3 Report

The manuscript presents well-rounded study, reporting innovative advances that further knowledge about a topic of importance. Moreover, bats have garnered attention as suspected reservoirs for viruses that can cause disease in humans, including the SARS-related coronaviruses. Recent studies suggest that 64 million years of adaptive evolution have shaped the host defence system; so, their uniquely adapted immune system limits inflammatory responses upon infectious challenge. It should be underlined that the Chiropteran immune system gathered momentum since early 2010s. The aim is clearly stated. However, Introduction does not provide sufficient background information to enable readers to better understand the problem (see some essential comments in the text).

Conclusions clearly support by the results. I suggest moderate changes

Comments for author File: Comments.pdf

Author Response

We thank the reviewer  for the suggestions made in the PDF of the manuscript, which we incorporated in the new version.