Apis cerana gut microbiota contribute to host health though stimulating host immune system and strengthening host resistance to Nosema ceranae

Gut microbial communities play vital roles in the modulation of many insects' immunity, including Apis mellifera. However, little is known about the interaction of Apis cerana gut bacteria and A. cerana immune system. Here in this study, we conducted a comparison between germ-free gut microbiota deficient (GD) workers and conventional gut community (CV) workers, to reveal the possible impact of gut microbiota on the expression of A. cerana antimicrobial peptides and immune regulate pathways. We also test whether A. cerana gut microbiota can strengthen host resistance to Nosema ceranae. We find that the expression of apidaecin, abaecin and hymenoptaecin were significantly upregulated with the presence of gut bacteria, and JNK pathway was activated; in the meanwhile, the existence of gut bacteria inhibited the proliferation of Nosema ceranae. These demonstrated the essential role of A. cerana gut microbiota to host health and provided critical insight into the honeybee host–microbiome interaction.


Comments to the Author(s)
Review on MS "Apis cerana gut microbiota contribute to host health though stimulating host immune system and strengthening host resistance to Nosema ceranae" by Wu, Yuqet al In this MS the authors quantified differences in immune gene regulation and Nosema resistance for the honey bee species A. cerana with respect to reduction of a natural microbiome. They found strong differences in a variety of gene expressions and Nosema tolerance. This is an interesting finding and mostly confirms what is known for the honey bee A. mellifera. The manuscript is well written, some parts could be adapted or expanded, see my suggestions below. I would like to see some more conservative term than "germ free", because there were still moderately dense loads of bacteria in the so called germ-free samples. Other than that I only have minor, textual comments and recommend publication.
Major: l 183: "cells per gut of CV bees and a total bacterial load of around 10^6 cells per gut of GF bees" I would not call the GF bees to be germ-free. This is a defined term. The authors should consider rephrasing this throughout the manuscript in as "underdeveloped microbiome" or similar. (also particularly in the discussion, e.g. "gut microbiota dysbiosis and germ-free are different situation" l 270) l. 186: "homogenized guts were successfully colonized with the characteristic microbiota." This means it has the same number of bacteria, but not necessarily the same identities/proportions of the community. As the authors didnt test for composition, this should be discussed.
Minor: whole MS: Sometimes it is confusing, when the authors use "honey bees", it is unclear (at points used arbitrarily) whether they refer to A. cerana, A. mellifera or the whole genus. This is particularly in the introduction and discussion, and clearing that out would make it easier to catch the novelities found here.
L. 43 Please consider this article and maybe rephrase : https://academic.oup.com/femsle/article/366/10/fnz117/5499024 l 115: "For GF and CV bees, six workers from each cage" Could the sampling design be explained a bit more, how many cages, same origin, same queen, how many bees in cage...? l 118: "immediately dissected in RNase-free water. Then sampled guts were immediately placed in chilled vials separately and used for DNA extraction" DNA/RNA typo? l. 159 "suspension containing 1 × 105 Nosema spores" it might be good to add a reference that this is a realistic load of Nosema observed also in the field.
Discussion: In some cases the study references and discusses microbiomes from very different insects, while other bees than honey bees are completely ignored. It might be good to set this also in context of other social bees (bumble bees, stingless) or even solitary bees.

Review form: Reviewer 2
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?
Major revision is needed (please make suggestions in comments) Comments to the Author(s) I think that this is an interesting study, especially given that relatively little microbiome work has been conducted with A. cerana, the bee species which N. ceranae is most closely associated. I think that the experiments seem well designed, though I have some concerns about replication and sample sizes, which are unclear in this manuscript.
1) The description of the same sizes is not adequate. How many colonies and how many cages were used? I suggest adding a table to show the sampling. In addition, it seems that the gut bacteria for all experiments obtained from just one colony? If so, this should be justified 2) The statistics are also not adequately reported. Only p-values are given without the detailed statistical test results (test statistic, degrees of freedom, etc.) These should be provided.
3) The language throughout should be thoroughly revised since there are multiple grammatical errors. I suggest that the authors consider using an online English editing service.

06-Apr-2020
Dear Dr Wu, The editors assigned to your paper ("Apis cerana gut microbiota contribute to host health though stimulating host immune system and strengthening host resistance to Nosema ceranae") 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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For information about language editing services endorsed by the Royal Society, please follow the link below: https://royalsociety.org/journals/authors/language-polishing/ Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) Review on MS "Apis cerana gut microbiota contribute to host health though stimulating host immune system and strengthening host resistance to Nosema ceranae" by Wu, Yuqet al In this MS the authors quantified differences in immune gene regulation and Nosema resistance for the honey bee species A. cerana with respect to reduction of a natural microbiome. They found strong differences in a variety of gene expressions and Nosema tolerance. This is an interesting finding and mostly confirms what is known for the honey bee A. mellifera. The manuscript is well written, some parts could be adapted or expanded, see my suggestions below. I would like to see some more conservative term than "germ free", because there were still moderately dense loads of bacteria in the so called germ-free samples. Other than that I only have minor, textual comments and recommend publication.
Major: l 183: "cells per gut of CV bees and a total bacterial load of around 10^6 cells per gut of GF bees" I would not call the GF bees to be germ-free. This is a defined term. The authors should consider rephrasing this throughout the manuscript in as "underdeveloped microbiome" or similar. (also particularly in the discussion, e.g. "gut microbiota dysbiosis and germ-free are different situation" l 270) l. 186: "homogenized guts were successfully colonized with the characteristic microbiota." This means it has the same number of bacteria, but not necessarily the same identities/proportions of the community. As the authors didnt test for composition, this should be discussed.

Minor:
whole MS: Sometimes it is confusing, when the authors use "honey bees", it is unclear (at points used arbitrarily) whether they refer to A. cerana, A. mellifera or the whole genus. This is particularly in the introduction and discussion, and clearing that out would make it easier to catch the novelities found here.
L. 43 Please consider this article and maybe rephrase : https://academic.oup.com/femsle/article/366/10/fnz117/5499024 l 115: "For GF and CV bees, six workers from each cage" Could the sampling design be explained a bit more, how many cages, same origin, same queen, how many bees in cage...? l 118: "immediately dissected in RNase-free water. Then sampled guts were immediately placed in chilled vials separately and used for DNA extraction" DNA/RNA typo? l. 159 "suspension containing 1 × 105 Nosema spores" it might be good to add a reference that this is a realistic load of Nosema observed also in the field.
Discussion: In some cases the study references and discusses microbiomes from very different insects, while other bees than honey bees are completely ignored. It might be good to set this also in context of other social bees (bumble bees, stingless) or even solitary bees.
Reviewer: 2 Comments to the Author(s) I think that this is an interesting study, especially given that relatively little microbiome work has been conducted with A. cerana, the bee species which N. ceranae is most closely associated. I think that the experiments seem well designed, though I have some concerns about replication and sample sizes, which are unclear in this manuscript.
1) The description of the same sizes is not adequate. How many colonies and how many cages were used? I suggest adding a table to show the sampling. In addition, it seems that the gut bacteria for all experiments obtained from just one colony? If so, this should be justified 2) The statistics are also not adequately reported. Only p-values are given without the detailed statistical test results (test statistic, degrees of freedom, etc.) These should be provided.
3) The language throughout should be thoroughly revised since there are multiple grammatical errors. I suggest that the authors consider using an online English editing service.
Author's Response to Decision Letter for (RSOS-192100.R0) See Appendix A.

Decision letter (RSOS-192100.R1)
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Dear Dr Wu, It is a pleasure to accept your manuscript entitled "Apis cerana gut microbiota contribute to host health though stimulating host immune system and strengthening host resistance to Nosema ceranae" 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. As you have been requested to edit the written English, you must provide proof that you have done so: acceptable proof includes a certificate of language-editing from a language editing service or a signed letter from a native speaker of English. If you do not provide this proof, your manuscript may be returned to you. For information about language editing services endorsed by the Royal Society, please follow the link below: https://royalsociety.org/journals/authors/language-polishing/ Reply: We have had our manuscript edited for correct English language usage, grammar, punctuation and spelling by qualified native English speaking editors at Charlesworth Author Services.
Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) Review on MS "Apis cerana gut microbiota contribute to host health though stimulating host immune system and strengthening host resistance to Nosema ceranae" by Wu, Yuqet al In this MS the authors quantified differences in immune gene regulation and Nosema resistance for the honey bee species A. cerana with respect to reduction of a natural microbiome. They found strong differences in a variety of gene expressions and Nosema tolerance. This is an interesting finding and mostly confirms what is known for the honey bee A. mellifera. The manuscript is well written, some parts could be adapted or expanded, see my suggestions below. I would like to see some more conservative term than "germ free", because there were still moderately dense loads of bacteria in the so called germ-free samples. Other than that I only have minor, textual comments and recommend publication. Reply: We thank the reviewer for the compliments on our study. We have revised and improved our manuscript according to the reviewer's comments.
Major: l 183: "cells per gut of CV bees and a total bacterial load of around 10^6 cells per gut of GF bees" I would not call the GF bees to be germ-free. This is a defined term. The authors should consider rephrasing this throughout the manuscript in as "underdeveloped microbiome" or similar. (also particularly in the discussion, e.g. "gut microbiota dysbiosis and germ-free are different situation" l 270) Reply: We appreciate the suggestion from reviewer and have rephased germ-free worker as Gut microbiota deficient (GD). l. 186: "homogenized guts were successfully colonized with the characteristic microbiota." This means it has the same number of bacteria, but not necessarily the same identities/proportions of the community. As the authors didnt test for composition, this should be discussed.