Widespread infection of Areospora rohanae in southern king crab (Lithodes santolla) populations across south Chilean Patagonia

Cottage cheese disease is caused by microsporidian parasites that infect a wide range of animal populations. Despite its potential to affect economically important activities, the spatial patterns of prevalence of this disease are still not well understood. Here, we analyse the occurrence of the microsporidian Areospora rohanae in populations of the king crab Lithodes santolla over ca 800 km of the southeastern Pacific shore. In winter 2011, conical pots were deployed between 50 and 200 m depth to capture crabs of a wide range of sizes. The infection was widely distributed along the region, with a mean prevalence of 16%, and no significant association between prevalence and geographical location was detected. Males, females and ovigerous females showed similar prevalence values of 16.5 (13–18.9), 15 (9.2–15) and 16.7% (10–19%), respectively. These patterns of prevalence were consistent across crab body sizes, despite the ontogenetic and sex-dependent variations in feeding behaviour and bathymetric migrations previously reported for king crabs. This study provided the first report of the geographical distribution of A. rohanae infecting southern king crabs.


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? No

Recommendation? Accept with minor revision (please list in comments)
Comments to the Author(s) I think that it would have been necessary to examine sub samples of L. santolla by histology, including those do not display clinical signs of infection. Clinical signs as whitish nodules arising from the sub-cuticular tissues are macroscopic signs of probably late-stage/patent infections (e.g. Field et al. 1992, Shields & Behringer 2004, Stentiford et al. 2010, 2014, and use of gross visual assessment methods may significantly underestimate the actual prevalence of A. rohanae. May be to save this point (since the sampling was already done), you could little explain in discussion section. Prevalence associated with geographic locations was examined. Did you study it with respect to depth of samples? No association between prevalence and 36 geographic location was detected, may be it could be explained by the different sampling sizes at each location (e.g. in Grupo Solari only 7 king crabs were collected whilst in Isla Toro 34 king crabs were collected). It might be discussed in the corresponding section. A table of these results presenting sampling size, sex proportion and their prevalence (besides the map) is suggested to be provided.
A co-habitation experiment with infected crabs and uninfected ones to explore potential for crabcrab transmission could be addressed. Page7-Line111: Replace "oocytes" by "eggs" Line 112: Idem Page 10-Line 165: Replace "larger" by "higher" What is the commercial size of L. santolla? It is suggested to provide this information.

31-Jul-2019
Dear Dr Rodríguez, The editors assigned to your paper ("Widespread infection of Areospora rohanae in southern king crab (Lithodes santolla) populations across south Chilean Patagonia") have now received comments from reviewers. We would like you to revise your paper in accordance with the referee and Associate Editor suggestions which can be found below (not including confidential reports to the Editor). Please note this decision does not guarantee eventual acceptance.
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If you wish to submit your supporting data or code to Dryad (http://datadryad.org/), or modify your current submission to dryad, please use the following link: http://datadryad.org/submit?journalID=RSOS&manu=RSOS-190682 • Competing interests Please declare any financial or non-financial competing interests, or state that you have no competing interests.
• Authors' contributions All submissions, other than those with a single author, must include an Authors' Contributions section which individually lists the specific contribution of each author. The list of Authors should meet all of the following criteria; 1) substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; 2) drafting the article or revising it critically for important intellectual content; and 3) final approval of the version to be published.
All contributors who do not meet all of these criteria should be included in the acknowledgements.
We suggest the following format: AB carried out the molecular lab work, participated in data analysis, carried out sequence alignments, participated in the design of the study and drafted the manuscript; CD carried out the statistical analyses; EF collected field data; GH conceived of the study, designed the study, coordinated the study and helped draft the manuscript. All authors gave final approval for publication.
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. Given the variety of comments it seems likely that the authors can address the concerns in a reasonable timeframe, but if more is needed, please let us know. Thanks for submitting.
Reviewers' Comments to Author: Reviewer: 1 Major: This is a simple study that would be better presented as a shorter paper/note in a specialist journal. 57 cited references are overkill for a study that only assessed prevalence over a single sample of crabs from one time point (winter 2011). So little is known of this parasite (described in 2014), particularly its mechanisms of transmission, dispersal, seasonality etc., so most text hypothesizing mechanisms driving prevalence/distribution are supposition at best. I suggest the authors restructure the paper as a short report-the prevalence/distribution data is valuable, but the context needs to be changed.
It is not clear to this reviewer that the pathognomonic sign for infection of A. rohanae has been well established and tested, yet the gross presentation has been used as a diagnostic tool in the current study (lines 113-118). I have read Stentiford et al. (2014) study and nowhere in that paper are the number of crabs that were used to describe the parasite presented. PCR data was only generated from two independent samples. Stentiford et al do not indicate that no other infections can be associated to these symptoms in king crabs (incorrectly stated by the authors in lines [117][118]). Further, cottage cheese disease (pathogen not described to species level) has previously been reported from king crabs in the northern hemisphere. Clearly further work needs to be carried out to establish, beyond doubt, that the presented symptoms are unique to A. rohanae infection.

Minor:
Line 35-36, expand on the stated significant association between prevalence and geographical location as there is no mention of this in the results section, page 8??
Lines 67-68, FYI conversely, many crustaceans show a higher prevalence of pathogens in earlier life stages.
Line 71, rewrite to read "In the case" Line 81-82, better describe "depolymerization of the contractile apparatus" as I don't understand what the authors mean. How do spores do this?
Line 86, do Lithodid crabs harbor diverse microsporidian parasites? I have only seen very few microsporidian species mentioned in the associated king crab literature? Can the authors elaborate?
Line 105, what is a conical plot? Do the authors not mean "pots"? Provide mesh width of traps/pots. from the sub-cuticular tissues are macroscopic signs of probably late-stage/patent infections (e.g. Field et al. 1992, Shields & Behringer 2004, Stentiford et al. 2010, 2014, and use of gross visual assessment methods may significantly underestimate the actual prevalence of A. rohanae. May be to save this point (since the sampling was already done), you could little explain in discussion section. Prevalence associated with geographic locations was examined. Did you study it with respect to depth of samples? No association between prevalence and 36 geographic location was detected, may be it could be explained by the different sampling sizes at each location (e.g. in Grupo Solari only 7 king crabs were collected whilst in Isla Toro 34 king crabs were collected). It might be discussed in the corresponding section. A table of these results presenting sampling size, sex proportion and their prevalence (besides the map) is suggested to be provided.
A co-habitation experiment with infected crabs and uninfected ones to explore potential for crabcrab transmission could be addressed.
Page7-Line111: Replace "oocytes" by "eggs" Line 112: Idem Page 10-Line 165: Replace "larger" by "higher" What is the commercial size of L. santolla? It is suggested to provide this information.

Author's Response to Decision Letter for (RSOS-190682.R0)
See Appendix A.

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

Recommendation?
Accept as is Royal Society Open Science operates under a continuous publication model (http://bit.ly/cpFAQ). Your article will be published straight into the next open issue and this will be the final version of the paper. As such, it can be cited immediately by other researchers. As the issue version of your paper will be the only version to be published I would advise you to check your proofs thoroughly as changes cannot be made once the paper is published. Reviewers' Comments to Author: Reviewer: 1 "Major: This is a simple study that would be better presented as a shorter paper/note in a specialist journal. 57 cited references are overkill for a study that only assessed prevalence over a single sample of crabs from one time point (winter 2011). So little is known of this parasite (described in 2014), particularly its mechanisms of transmission, dispersal, seasonality etc., so most text hypothesizing mechanisms driving prevalence/distribution are supposition at best. I suggest the authors restructure the paper as a short report-the prevalence/distribution data is valuable, but the context needs to be changed." R: Thank you for this comment. We agree with Reviewer #1 on the fact that our study is based upon a single sample and one point of time. However, we would like to stress that this contribution is the first report of the spatial distribution of the infection, covering circa 800 km of the southeastern pacific shore. Moreover, the sample encompassed 3000 host individuals. Also, and perhaps more importantly, Lithodes santolla is an economically important natural resource of local communities in Magallanes and elsewhere. Thus, we see this contribution and a relevant benchmark to further develop a research agenda on the parasite-host dynamics of Aerospora rohanae and L. santolla. For those reasons, we have shortened the manuscript, reduced too speculative statements and sloppy citations, and also removed redundant literature (43 references), but with an eye in keeping the paper rather than the short note format. With this, we hope Reviewer #1 understand our concerns related to moving our paper to a note.