PONE-D-21-10790

Exceptional long-term sperm storage by a female vertebrate

PLOS ONE

Dear Dr. Booth,

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This is an interesting observation on an exceptionally long storage period of sperm in a snake. I agree with the two reviewers that the data are interesting and that the manuscript is well written. I do support their recommendations and comments and request minor revision of the paper.

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PLOS ONE

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Reviewers' comments:

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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: Yes

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

Reviewer #1: Yes

Reviewer #2: N/A

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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?

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

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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: The authors report on a wild-collected female Crotalus atrox, maintained in isolation from 1999, that produced two healthy litters approximately one and six years post capture. Genetic analysis of the second litter identified paternal contribution in all offspring, thus rejecting facultative parthenogenesis. They suggest the long-term sperm storage to last approximately 71 months, making it the longest period over which a female vertebrate has been shown to store sperm that resulted in the production of healthy offspring from LTSS.

This is an observational study reproting an interesting finding with implications for sperm competition, mating and reproductive strategies in a snake. The methods applied and the data obtained appear valid. I have only a very few minor points that have tob e clarified during a revision oft he paper.

Material and methods:

I am missing a note on the approval for the collection of the animal from the wild as well as a licence for keeping it in captivity.

Whole genomic DNA was obtained from offspring: Please explain how this was done: From which tissues? Were the animals killed? By which means? And, again, under which licence?

Discussion:

Appears rather long. Please shorten if possible.

Reviewer #2: This is manuscript is well written and provides unprecedented data on exceptional long-term sperm storage for 71 months in a female Western Diamond-backed Rattlesnake (Crotalus atrox). The methods are soundly carried out. Descriptions are detailed and well documented. The combination of data obtained in captivity, genetic screening and the observation of paternal alleles in all offspring showed a very accurate result, confirming the differentiation between long-term sperm storage (LTSS) and facultative parthenogenesis (FP). Both males and females were born, all in good condition and no anomalies present, reinforcing the results presented in the paper. Some inttriguing questions about long-term sperm storage remain uncertain: we still need to understand how spermatozoa are able to survive during this period and how females may produce multiple clutches using these stored sperm. The authors of this study are experts on this subject and this manuscript is a huge contribution to the knowledge on the reproductive biology of vertebrates. This is a good paper and I look forward to see this paper published. Minor suggestions are included below:

INTRODUCTION

Line 27-29: The authors mention in these lines that ectothermic vertebrates (non-avian reptiles) may show sperm storage for several years, however they do not mention where sperm is stored. A brief anatomical description of sperm storage sites in the oviducts of female reptiles, mainly for snakes is important. Later, the authors mention in the discussion section, sperm storage in UMT (uterine muscular twisting) in the útero-vaginal junction and in the infundibular portion. The inclusion of this information in the introduction will contribute to a general view on sperm storage in the oviduct, contributing to a better understanding of this reproductive tactic by the readers.

MATERIAL AND METHODS

The dataset and analysis presented by the authors strongly support the goals of the study, reflecting huge experience of the authors within these techniques. The methodology is well detailed and the replication of this study is possible. On line supporting information contains the protocols and algorithms used in this study, allowing their application in future research.

Line 121: Please, include the meaning of SNPs in the first time that this abbreviation appears in the text.

DISCUSSION

This section is short, however interesting and well written. However, I suggest the inclusion of some recent publications on uterine muscular twisting (UMT) and infundibular sperm storage in Viperidae snakes to improve this section.

Line 217-219: This paragraph may be improved with the inclusion of these recent publications (Silva et al 2019; Silva et al., 2020, Barros et al., 2020; Souza and Almeida-Santos, 2020) on sperm storage in Bothrops and Lachesis (closely related taxa). Recent data show that the UMT does not prevent the passage of spermatozoa to the infundibulum after mating as it previously hypothesized for some Viperidae snakes. The UMT has been described in vitellogenic, non-vitellogenic and pregnant females, with and without the presence of sperm, raising many doubts about the function of this structure. Recent findings showed that sperm storage in Bothrops and Lachesis occur in the furrows of the útero-vaginal junction or in the UMT, and in infundibular sperm receptacles. It suggests that Crotalus may also have infundibular sperm receptacles. Please, find the references for the publications below:

SILVA, KARINA M. P.; BARROS, VERÔNICA A.; ROJAS, CLAUDIO A.; ALMEIDA'SANTOS, SELMA M. .Infundibular sperm storage and uterine muscular twisting in the Amazonian lancehead,. Anatomical Record-Advances in Integrative Anatomy and Evolutionary Biology, v. 2019, p. ar.24309-1-10, 2019

BARROS, VERÔNICA A.; ROJAS, CLAUDIO A; ALMEIDA'SANTOS, SELMA M Biologia Reprodutiva das Serpentes Jararacas: Ciclos e Comportamentos, Dimorfismo e Maturidade sexual. 1. ed. Ilhas Mauricio: Novas Edições Acadêmicas, 2020. v. 1. 173p

SILVA, KARINA M.P.; BRAZ, HENRIQUE B.; KASPEROVICZUS, KARINA N.; MARQUES, OTAVIO A.V. ; ALMEIDA-SANTOS, SELMA M. Reproduction in the pitviper Bothrops jararacussu: large females increase their reproductive output while small males increase their potential to mate. Zoology, v. xx, p. 125816, 2020

SOUZA, ELETRA; ALMEIDA'SANTOS, SELMA MARIA. Reproduction in the bushmaster ( Lachesis muta ): Uterine muscular coiling and female sperm storage. Acta Zoologica ONLINE, v. 2020, p. 1-12, 2020

REFERENCES

This reference was not cited in the manusccript

52 -Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MAR, Bender D, Maller J, Sklar P, de Bakker PIW, Daly MJ, Sham PC. 2007 PLINK: a toolset for whole-genome association and population-based linkage analysis. A. J. Hum. Genet. 81, 559–575.

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Exceptional long-term sperm storage by a female vertebrate

PONE-D-21-10790R1

Dear Dr. Booth,

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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Stefan Schlatt

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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

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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: (No Response)

Reviewer #2: Yes

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

Reviewer #1: (No Response)

Reviewer #2: 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: (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: (No Response)

Reviewer #2: Yes

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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: The authors of the present study “Exceptional long-term sperm storage by a female vertebrate” followed all the sugestions indicated in the review and especially regarding the inclusion of more recent hypotheses about the long-term sperm storage (LTSS) in crotaline snakes. The insertion of this information aimed to improve the quality of the manuscript from the morphophysiological point of view. The study is of high quality and will stimulate new research on the storage of sperm in vertebrates. Minor sugestions are included below:

References

Line 144: change Souz by “Souza” and Almeida Santo by “Almeida-Santos”

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Reviewer #1: No

Reviewer #2: No