Filtration effect of Cordyceps chanhua mycoderm on bacteria and its transport function on nitrogen

ABSTRACT Cordyceps chanhua is a kind of entomopathogenic fungus that has a long medicinal history in traditional Chinese medicine. In this study, we aimed to explore the role of C. chanhua mycoderm. Our results showed that C. chanhua mycoderm has a certain ability to transfer nitrogen, which can transfer nitrogen from the insect body to the external environment and transfer nitrogen from the environment to C. chanhua. By studying the filtration of bacterial mycoderm, it was found that the mycoderm of C. chanhua was able to filter out most of the bacteria in the environment. Using the high-throughput sequencing methods, we found that the abundance of bacterial community first increased and then decreased during the growth and development of C. chanhua. The bacterial richness and diversity of mycoderm of C. chanhua cultivated in sterile glass bottles were significantly lower than those cultivated in soil mulching. In addition, this study also found that the mycoderm of C. chanhua formed under both soil mulching cultivation and sterile glass bottle cultivation was hydrophobic. The mycoderm of C. chanhua, which occurred under sterile glass bottle cultivation, was closely and orderly arranged, while the mycoderm of C. chanhua cultivated in soil was quite the opposite. The density and thickness of the C. chanhua membrane in aseptic glass bottle culture were higher than those in soil-covered culture, but the dry/fresh weight was lower than those in soil-covered culture. IMPORTANCE During the natural growth of Cordyceps chanhua, it will form a mycoderm structure specialized from hyphae. We found that the bacterial membrane of C. chanhua not only filters environmental bacteria but also absorbs and transports nitrogen elements inside and outside the body of C. chanhua. These findings are of great significance for understanding the stable mechanism of the internal microbial community maintained by C. chanhua and how C. chanhua maintains its own nutritional balance. In addition, this study also enriched our understanding of the differences in bacterial community composition and related bacterial community functions of C. chanhua at different growth stages, which is of great value for understanding the environmental adaptation mechanism, the element distribution network, and the changing process of symbiotic microbial system after Cordyceps fungi infected the host. At the same time, it can also provide a theoretical basis for some important ecological imitation cultivation technology of Cordyceps fungi.


Specific comments and suggestions:
1. L68-71, it is confused for the researches on the morphological characteristics of the mycoderm in the sentence "most studies on the morphological characteristics of cordyceps fungi focus on the mycoderm, sclerotia, stroma and ascus structures of cordyceps (24,25,26).However, as a special structure of some entomopathogenic fungi, the study on the morphological characteristics of the mycoderm has not been reported."The authors should explain and revise.
2. L206, the subtitle for "Apparent Morphology of C. cicadae Mycoderm in Soil Atmosphere", it should be "Soil and Atmosphere".
3. L240-248, It is better to provide the morphology features of CFU (or at least several representative images for the morphology character of CFU).

4.
L497, "at the concentration of 500 mL", it is not concentration.

5.
L538-539, The details for equipment and parameter should be provided.

L863-866
What did the authors mean for the indicated arrow, the author should indicate.

7.
L881, the description should be used by English.

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Response to Reviewers
We sincerely thank the editor and all reviewers for their valuable feedback that we have used to improve the quality of our manuscript.The reviewer comments are laid out below in italicized font and specific concerns have been numbered.
--The manuscript is missing legends for figures 10-13.Please provide figure legends for all figures in the manuscript.
We apologize for the lack of legends in the manuscript, we have now placed the legends and table before the references in the manuscript and marked them in red font.
1.The authors should provide the responses in point-by-point.
2. The authors should provide the changes with highlighting in the revised manuscript, which can clarify the review's concerns.

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Response to Reviewers
We sincerely thank the editor and all reviewers for their valuable feedback that we have used to improve the quality of our manuscript.The reviewer comments are laid out below in italicized font and specific concerns have been numbered.Our response is given in normal font and changes/additions to the manuscript are given in the red text.
Reviewer #1 (Comments for the Author) It's not easy to understand the experimental and methodological methods.Maybe some figures or schematic diagrams should be added to make it clear, such as the filtration effect and N15 flow experiments.And also, the text is not set out well.

Response:
We feel great thanks for your professional review work on our article.
As you are concerned, there are several problems that need to be addressed.
According to your nice suggestions, we have made corrections to our previous draft, the detailed corrections are listed below.
(1) We have corrected some textual errors in the article, and the changes to the manuscript have been shown in red text.
(2) In order to better understand the experiment and methodological methods, we drew some schematic diagrams in the manuscript.We have put schematic diagrams in the corresponding position in the manuscript.

The Microbial Diversity is the diversity of mycoderm? Please specified
throughout the text.
We apologize for not specifying the meaning of microbial diversity in the text， and the microbial diversity in this study refers to the bacterial community diversity of internal sclerotium and mycoderm of Cordyceps Chanhua at different growth stages and under different cultivation environments, we have specified in the manuscript.

Why there is no results of fungal diversity.
In manuscript L71~73, previous studies have found that the relative abundance of other fungi in the sclerotia of C. chanhua was relatively low, while the bacterial community was relatively rich, therefore, we didn't study fungal diversity.

C. cicadae can develop normally? It should be discussed.
Thank you very much for your valuable advice on our manuscript，to better answer this question, we designed an experiment on the effect of complete disinfection of the pupae of Antheraea pernyi on the growth and development of C. chanhua.We designed a total of 4 groups of experiments, namely (1) disinfection was carried out according to the disinfection method of the pupae of Antheraea pernyi in the original manuscript (d 1 ): wash the surface of A. pernyi with tap water and dry it naturally.Soak in 75% alcohol in the sterile super clean table and scrub the insect surface with cotton for about ten seconds, then rinse it with sterile water, clip it into a white porcelain plate covered with sterile paper towels, and dry the surface water; (2) the sodium hypochlorite solution was added on the basis of the experimental group d 1 (d 2 ): First rinse the surface of the surface of A. pernyi with tap water, dry naturally; the insect body surface was soaked with 75% alcohol and scrubbed with cotton for about 10 s in a sterile ultra-clean table, and then washed with 5% sodium hypochlorite solution for 10 s.The most sterile water was used for washing, and then placed on a white porcelain plate covered with sterile paper towels to absorb the surface moisture; (3) a slight improvement was made on the basis of experimental group d 2 (d 3 ): First rinse the surface of the surface of A. pernyi with tap water, dry naturally; the insect body surface was soaked with 75% alcohol and scrubbed with cotton for about 20 s in a sterile ultra-clean table, and then washed with 5% sodium hypochlorite solution for 20 s.The most sterile water was used for washing, and then placed on a white porcelain plate covered with sterile paper towels to absorb the surface moisture; (4) the surface of A. pernyi was completely sterilized at 121 ℃ for 30 min (d 4 ).The growth and development of C. Chanhua 25 days after inoculation were shown in Table 1.

Specific comments and suggestions:
1. L68-71, it is confused for the researches on the morphological characteristics of the mycoderm in the sentence "most studies on the morphological characteristics of cordyceps fungi focus on the mycoderm, sclerotia, stroma and ascus structures of cordyceps (24, 25, 26).However, as a special structure of some entomopathogenic fungi, the study on the morphological characteristics of the mycoderm has not been reported."The authors should explain and revise.
Response: Thanks for your careful checks.We are sorry for our carelessness.
Based on your comments, we have made the corrections to make the word harmonized within the whole manuscript.It is not appropriate to have quite a few (small) Tables, Table 8 should go to supplementary.What are the relationships regarding Table 6 vs. Fig.7, and Table 7 vs.Fig.9, the same things?If being similar, please move Tables 6 and 7 to supplementary as well.
Thank you for submitting your manuscript to Microbiology Spectrum.When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues raised by the reviewers as file type "Response to Reviewers," not in your cover letter, and (2) a PDF file that indicates the changes from the original submission (by highlighting or underlining the changes) as file type "Marked Up Manuscript -For Review Only".Please use this link to submit your revised manuscript -we strongly recommend that you submit your paper within the next 60 days or reach out to me.Detailed instructions on submitting your revised paper are below.

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• Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file.
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Response to the reviewer on October 10
1.The Microbial Diversity is the diversity of mycoderm?Please specified throughout the text.
We apologize for not specifying the meaning of microbial diversity in the text， and the microbial diversity in this study refers to the bacterial community diversity of internal sclerotium and mycoderm of Cordyceps Chanhua at different growth stages and under different cultivation environments, we have specified in the manuscript.
2. Why there is no results of fungal diversity.
In manuscript L71~73, previous studies have found that the relative abundance of other fungi in the sclerotia of C. chanhua was relatively low, while the bacterial community was relatively rich, therefore, we didn't study fungal diversity.

Specific comments and suggestions:
1. L68-71, it is confused for the researches on the morphological characteristics of the mycoderm in the sentence "most studies on the morphological characteristics of cordyceps fungi focus on the mycoderm, sclerotia, stroma and ascus structures of cordyceps (24, 25, 26).However, as a special structure of some entomopathogenic fungi, the study on the morphological characteristics of the mycoderm has not been reported."The authors should explain and revise.
Response: Thanks for your careful checks.We are sorry for our carelessness.
Based on your comments, we have made the corrections to make the word harmonized within the whole manuscript.
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex.Go to Author Tasks and click the appropriate manuscript title to begin the revision process.The information that you entered when you first submitted the paper will be displayed.Please update the information as necessary.Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER.
• Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file.
• Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file.For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process.Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript." Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me.If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail.Arrangements for payment must be made before your article is published.For a complete list of Publication Fees, including supplemental material costs, please visit our website.
Corresponding authors may join or renew ASM membership to obtain discounts on publication fees.Need to upgrade your membership level?Please contact Customer Service at Service@asmusa.org.
Thank you for submitting your paper to Microbiology Spectrum.

Response to Reviewers
We sincerely thank the editor and all reviewers for their valuable feedback that we have used to improve the quality of our manuscript.The reviewer comments are laid out below in italicized font and specific concerns have been numbered.Our response is given in normal font and changes/additions to the manuscript are given in the red text.
Response to the reviewer on October 14    4.It is not appropriate to have quite a few (small) Tables, Table 8 should go to supplementary.What are the relationships regarding Table 6 vs. Fig.7, and Table 7 vs.Fig.9, the same things?If being similar, please move Tables 6 and 7 to supplementary as well.
Response: Thanks for your careful checks.We have moved Table 8 into the supplementary, and the corresponding number in the supplementary material is Table S3.In addition, Table 6 and Table 7 show the 15 N and total nitrogen content of different samples in experiment D and experiment E, respectively.Fig. 8 and Fig. 9 (Now merged into Fig.8) respectively show the enrichment degree of each sample of 15 N in experiment D and experiment E. We have now moved Table 6 and Table 7 into the supplementary, and the corresponding number in the supplementary material is Table S1 and Table S2.
Reviewer #1 (Comments for the Author) It's not easy to understand the experimental and methodological methods.Maybe some figures or schematic diagrams should be added to make it clear, such as the filtration effect and N15 flow experiments.And also, the text is not set out well.

Response:
We feel great thanks for your professional review work on our article.
As you are concerned, there are several problems that need to be addressed.
According to your nice suggestions, we have made corrections to our previous draft, the detailed corrections are listed below.
(1) We have corrected some textual errors in the article, and the changes to the manuscript have been shown in red text.
(2) In order to better understand the experiment and methodological methods, we drew some schematic diagrams in the manuscript.We have put schematic diagrams in the corresponding position in the manuscript.
Response to the reviewer on October 10 1.The Microbial Diversity is the diversity of mycoderm?Please specified throughout the text.
We apologize for not specifying the meaning of microbial diversity in the text， and the microbial diversity in this study refers to the bacterial community diversity of internal sclerotium and mycoderm of Cordyceps Chanhua at different growth stages and under different cultivation environments, we have specified in the manuscript.
2. Why there is no results of fungal diversity.
In manuscript L71~73, previous studies have found that the relative abundance of other fungi in the sclerotia of C. chanhua was relatively low, while the bacterial community was relatively rich, therefore, we didn't study fungal diversity.

Specific comments and suggestions:
1. L68-71, it is confused for the researches on the morphological characteristics of the mycoderm in the sentence "most studies on the morphological characteristics of cordyceps fungi focus on the mycoderm, sclerotia, stroma and ascus structures of cordyceps (24, 25, 26).However, as a special structure of some entomopathogenic fungi, the study on the morphological characteristics of the mycoderm has not been reported."The authors should explain and revise.
Response: Thanks for your careful checks.We are sorry for our carelessness.
Based on your comments, we have made the corrections to make the word harmonized within the whole manuscript.

Reviewer # 2 (
Comments for the Author): In this manuscript, the authors analyzed the bacterial community in different parts of C. cicadae in different production stages and different environments by high-throughput sequencing.They also demonstrated that the filtration effect of C. cicadae membrane on bacterial communities.Furthermore, they also found the nitrogen transport function of the membrane of C.cicadae.The obtained information provide a basis for understanding the mechanism of the internal microbial community maintained by C. cicadae and nutritional balance by self-regulation in C. cicadae.
We have merged Fig. 8 and Fig. 9 into a figure with two panels, and the new number is Fig. 8 in the manuscript.The corresponding figures and legends have been placed after the references in the manuscript.The final result is shown below.

Fig. 8
Fig. 8 δ 15 N values in different samples in experiment D and experiment E. A: δ 15 N flows from Cordyceps chanhua to soil samples.B: δ 15 N flows from soil to Cordyceps chanhua samples.
Thanks for your advice and we have moved the Figures 10-13mentioned in Methods to Supplementary and re-order them.And Figures 10-13Corresponding numbers in supplementary are shown in Fig. S6-S9.

4.
Cordyceps cicadae should be Cordyceps chanhua.As you are concerned, Cordyceps cicadae should be Cordyceps chanhua.The reason is that Chinese scholar Li Zengzhi found the sexual Cordyceps chanhua, classified it as a new species of the genus Cordyceps, and named it Cordyceps chanhua with the ancient name of Cordyceps cicadae (1).REFERENCE 1. Li ZZ, Luan FG, Nigel HJ, Zhang SL, Chen MJ, Huang B, Shun CS, Chen ZA, Li CR, Tan YJ, Dong JF.Biodiversity of cordycipitoid fungi associated with Isaria cicadae Miquel Ⅱ: Teleomorph discovery and nomenclature of chanhua, animportant medicinal fungus in China.Mycosystema, 2021, 40(2): 1-12.https://doi.org/10.13346/j.mycosystema.200119.Thank you again for your careful checks.Based on your comments, we have corrected some other minor errors in the manuscript.Reviewer #2 (Comments for the Author): In this manuscript, the authors analyzed the bacterial community in different parts of C. cicadae in different production stages and different environments by high-throughput sequencing.They also demonstrated that the filtration effect of C. cicadae membrane on bacterial communities.Furthermore, they also found the nitrogen transport function of the membrane of C.cicadae.The obtained information provide a basis for understanding the mechanism of the internal microbial community maintained by C. cicadae and nutritional balance by self-regulation in C. cicadae.
• Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred

Table 1 Growth information of Cordyceps Chanhua under different disinfection methods of the pupae of Antheraea pernyi
As can be seen from Table1, 25 days after inoculation, d 2 disinfection treatment had the highest rate of C. Chanhua rigidor and the highest number of fruite bodies, however, d 4 had the lowest ossification rate and number of fruites.Therefore, the pupae of A. pernyi sterilization is not suitable for C. Chanhua cultivation.4. Cordyceps cicadae should be Cordyceps chanhua.As you are concerned, Cordyceps cicadae should be Cordyceps chanhua.The reason is that Chinese scholar Li Zengzhi found the sexual Cordyceps chanhua, classified it as a new species of the genus Cordyceps, and named it Cordyceps chanhua with the ancient name of Cordyceps cicadae (1).REFERENCE 1. Li ZZ, Luan FG, Nigel HJ, Zhang SL, Chen MJ, Huang B, Shun CS, Chen ZA, Li CR, Tan YJ, Dong JF.Biodiversity of cordycipitoid fungi associated with Isaria cicadae Miquel Ⅱ: Teleomorph discovery and nomenclature of chanhua, animportant medicinal fungus in China.Mycosystema, 2021, 40(2): 1-12.https://doi.org/10.13346/j.mycosystema.200119.high-throughput sequencing.They also demonstrated that the filtration effect of C. cicadae membrane on bacterial communities.Furthermore, they also found the nitrogen transport function of the membrane of C.cicadae.The obtained information provide a basis for understanding the mechanism of the internal microbial community maintained by C. cicadae and nutritional balance by self-regulation in C. cicadae.
Thanks for this revsion.Many improvements are still required before further suggestion, especially regarding the Table and Figure numbers: 1. Cordyceps cicadae is still used in Supplementary. 2. Merge Fig. 8 and Fig. 9 into a figure with two panels.3. Move the Figures 10-13 mentioned in Methods to Supplementary and re-order them.
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of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. "
• Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process.SubmissionsPlease return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me.If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.

Antheraea pernyi is sterilized thoroughly before being infection, C. cicadae can develop normally? It should be discussed.
First rinse the surface of the surface of A. pernyi with tap water, dry naturally; the insect body surface was soaked with 75% alcohol and scrubbed with cotton for about 10 s in a sterile ultra-clean table, and then washed with 5% sodium hypochlorite surface with cotton for about ten seconds, then rinse it with sterile water, clip it into a white porcelain plate covered with sterile paper towels, and dry the surface water; (2) the sodium hypochlorite solution was added on the basis of the experimental group d 1 (d 2 ): the surface of A. pernyi was completely sterilized at 121 ℃ for 30 min (d 4 ).The growth and development of C. Chanhua 25 days after inoculation were shown in Table1.Table 1

Growth information of Cordyceps Chanhua under different disinfection methods of the pupae of Antheraea pernyi
As can be seen from Table 1, 25 days after inoculation, d 2 disinfection treatment had the highest rate of C. Chanhua rigidor and the highest number of fruite bodies, however, d 4 had the lowest ossification rate and number of fruites.Therefore, the pupae of A. pernyi sterilization is not suitable for C. Chanhua cultivation.classified it as a new species of the genus Cordyceps, and named it Cordyceps chanhua with the ancient name of Cordyceps cicadae (1).
4. Cordyceps cicadae should be Cordyceps chanhua.As you are concerned, Cordyceps cicadae should be Cordyceps chanhua.The reason is that Chinese scholar Li Zengzhi found the sexual Cordyceps chanhua, Thank you again for your careful checks.Based on your comments, we have corrected some other minor errors in the manuscript.
• Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred

1. Cordyceps cicadae is still used in Supplementary.
Thank you for your careful checks.And we've already changed Cordyceps cicadae to Cordyceps chanhua in the Supplementary. Response:

Antheraea pernyi is sterilized thoroughly before being infection, C. cicadae can develop normally? It should be discussed.
Thank you very much for your valuable advice on our manuscript，to better answer this question, we designed an experiment on the effect of complete disinfection of the pupae of Antheraea pernyi on the growth and development of C. chanhua.We designed a total of 4 groups of experiments, namely (1) disinfection was carried out according to the disinfection method of the pupae of Antheraea pernyi in the original manuscript (d 1 ): wash the surface of A. pernyi with tap water and dry it naturally.Soak in 75% alcohol in the sterile super clean table and scrub the insect surface with cotton for about ten seconds, then rinse it with sterile water, clip it into a white porcelain plate covered with sterile paper towels, and dry the surface water; (2) the sodium hypochlorite solution was added on the basis of the experimental group d 1 (d 2 ): First rinse the surface of the surface of A. pernyi with tap water, dry naturally; the insect body surface was soaked with 75% alcohol and scrubbed with cotton for about 10 s in a sterile ultra-clean table, and then washed with 5% sodium hypochlorite solution for 10 s.The most sterile water was used for washing, and then placed on a white porcelain plate covered with sterile paper towels to absorb the surface moisture;(3) a slight improvement was made on the basis of experimental group d 2 (d 3 ): First rinse the surface of the surface of A. pernyi with tap water, dry naturally; the insect body surface was soaked with 75% alcohol and scrubbed with cotton for about 20 s in a sterile ultra-clean table, and then washed with 5% sodium hypochlorite solution for 20 s.The most sterile water was used for washing, and then placed on a white porcelain plate covered with sterile paper towels to absorb the surface moisture; (4) the surface of A. pernyi was completely sterilized at 121 ℃ for 30 min (d 4 ).The growth and development of C. Chanhua 25 days after inoculation were shown in

1 Growth information of Cordyceps Chanhua under different disinfection methods of the pupae of Antheraea pernyi
As can be seen from Table1, 25 days after inoculation, d 2 disinfection treatment had the highest rate of C. Chanhua rigidor and the highest number of fruite bodies, however, d 4 had the lowest ossification rate and number of fruites.Therefore, the pupae of A. pernyi sterilization is not suitable for C. Chanhua cultivation.