Haze Exposure Changes the Skin Fungal Community and Promotes the Growth of Talaromyces Strains

ABSTRACT Haze pollution has been a public health issue. The skin microbiota, as a component of the first line of defense, is disturbed by environmental pollutants, which may have an impact on human health. A total of 74 skin samples from healthy students were collected during haze and nonhaze days in spring and winter. Significant differences of skin fungal community composition between haze and nonhaze days were observed in female and male samples in spring and male samples in winter based on unweighted UniFrac distance analysis. Phylogenetic diversity whole-tree indices and observed features were significantly increased during haze days in male samples in winter compared to nonhaze days, but no significant difference was observed in other groups. Dothideomycetes, Capnodiales, Mycosphaerellaceae, etc. were significantly enriched during nonhaze days, whereas Trichocomaceae, Talaromyces, and Pezizaceae were significantly enriched during haze days. Thus, five Talaromyces strains were isolated, and an in vitro culture experiment revealed that the growth of representative Talaromyces strains was increased at high concentrations of particulate matter, confirming the sequencing results. Furthermore, during haze days, the fungal community assembly was better fitted to a niche-based assembly model than during nonhaze days. Talaromyces enriched during haze days deviated from the neutral assembly process. Our findings provided a comprehensive characterization of the skin fungal community during haze and nonhaze days and elucidated novel insights into how haze exposure influences the skin fungal community. IMPORTANCE Skin fungi play an important role in human health. Particulate matter (PM), the main haze pollutant, has been a public environmental threat. However, few studies have assessed the effects of air pollutants on skin fungi. Here, haze exposure influenced the diversity and composition of the skin fungal community. In an in vitro experiment, a high concentration of PM promoted the growth of Talaromyces strains. The fungal community assembly is better fitted to a niche-based assembly model during haze days. We anticipate that this study may provide new insights on the role of haze exposure disturbing the skin fungal community. It lays the groundwork for further clarifying the association between the changes of the skin fungal community and adverse health outcomes. Our study is the first to report the changes in the skin fungal community during haze and nonhaze days, which expands the understanding of the relationship between haze and skin fungi.

2. Reorder the results so that it goes diversity, differential abundance, networks, then culture. By starting with networks, I have little frame of reference for what differences are being analyzed. similarly, I would recommend starting with the paragraph describing the taxa identified (starting on line 153) as this gives context for everything else. 3. It is not clear how many samples are in each group, in particular when building the networks. It looks like there are about 18 samples used for each network, which is not enough to draw strong conclusions about network topography. However, network construction is missing from the methods section and this could be addressed in that section. 4. The discussion section is missing some context or explanations beyond citations, particularly around cleaning the retroauricular crease (lines 260-261), airborne fungi (lines 269-277),and the neutral model (lines 314-319).
Smaller edits include: 1. Report all p-values as values rather than > or < 0.05. 2. Co-culture usually implies multiple microbes but it appears to mean one fungal strain with particulate matter in this case. For this situation, you can either use "culture" or define co-culture when it is first introduced. 3. In the culture section the abbreviation HC is used but not defined. HC, MC, and LC are all used in Fig 5 without definitions.
Reviewer #2 (Comments for the Author): The authors describe here an interesting study where they assessed the influence of haze pollutants on the skin fungal communities depending on the host sex, as well as the seasons (spring vs. winter). The genus Talaromyces was found to be enriched during haze days. Moreover, the authors were able to isolate five Talaromyces strains and further showed that their growth was promoted by a high concentration of particulate matter.
There are a lot of parts in the manuscript that I could not properly understand. Comments and suggestions can be found in the review attachment file.

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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. L1 : remove « the » in the title. L24, 26, 27 & 30: remove "the". L31: what does "PD" stand for? Explain the acronym at the first instance. L36: write "5" in full letters. L39: replace "furtherly" by "furthermore". L40-41: "The genera significantly enriched…", please rephrase. L46: replace "on" by "in". L48-49: please rephrase. L50: please define the acronym "PM" in line 46, e.g. Particular matter (PM). L52: replace "thoughts" by "insights on the role". L52-54: split the sentence in two. L61: add "the" before "host" and "skin". L62-64: please rephrase. L65: I would suggest to replace "stay in temporal stability" by "is stable overtime". L66-68: please rephrase in order to avoid the repetition of "factors". L82-83: please rephrase the sentence "PM deposits …". L91: please replace "to our best" by "to the best of our". L95: please rephrase "genera in strain-level". L100: please rephrase "… passed quality filtering, denoised, merged, chimeras filtering, …". L103-107: please rephrase. L113: what do you mean exactly by "classified". Please clarify. L140: please define "PD whole tree index". L165: remove "s" to "examples". L173: please define what a LEfSe analysis is. L189: please rephrase "which was cluster to four groups". L193-194: please rephrase. L195-196: please define the acronyms "LC", "MC", and "HC" at their first instances. L204: please define "AIC". L220-339: please review thoroughly all the discussion and conclusion so that the reading is smoother. L358-360: please write the date in the following format "on March 14" or "on the 14 th of March". L373: please describe the PCR conditions that were used. L375-377: How was the library preparation for the Illumina sequencing done? L379: write "UNITE" in capital letters. L394: "The swab sample was diluted 6 times…" in what was it diluted? L395: what is the brand of the SDA? Is it also OXOID? If yes, please specify again. L396: change from "To inhibit the bacteria growth" to "To inhibit bacterial growth". L401: again here, please indicate the PCR conditions and which sequencing technology was used.

strains" (Manuscript ID: Spectrum01188-22)
Reviewer #1 (Comments for the Author): The authors have characterized the relationship between the human skin mycobiome and haze exposure, or particulate matter. They found that haze exposure does alter the composition and network properties of the mycobiome, and specifically increases abundance of Talaromyces. Increase in Talaryomyces was confirmed by culturing isolates with PM.
Thanks for helping us to improve our manuscript. According to your comments, we revised the manuscript and uploaded our revisions and responses. The answers to the comments are as follows.
While the science seems mostly fine, the manuscript was difficult to read. The following suggestions would help improve readability: 1. Review for grammar -there are a large number of errors, particularly with the use of "respectively" and "furtherly".
Thank you for your suggestions. We checked the manuscript carefully and found an experienced professor to revise our manuscript for correcting the English.
The incorrect uses of "respectively" were deleted in Line 117 and 192. The words "furtherly" were replaced with "furthermore" in Line 38, 160, and 320.
2. Reorder the results so that it goes diversity, differential abundance, networks, then culture. By starting with networks, I have little frame of reference for what differences are being analyzed. similarly, I would recommend starting with the paragraph describing the taxa identified (starting on line 153) as this gives context for everything else.
OK, we reorganized the results according to your suggestions. The contents of results go composition, diversity, differential abundance, networks, then culture.
3. It is not clear how many samples are in each group, in particular when building the networks. It looks like there are about 18 samples used for each network, which is not enough to draw strong conclusions about network topography. However, network construction is missing from the methods section and this could be addressed in that section.

Thank you for your suggestions, which remind us to improve our research. The samples numbers were listed in Line 97-100. And we revised the method of network to '19 samples (haze days in spring group), 19 samples (non-haze days in spring group), 18 samples (haze days in winter group), and 18 samples
(non-haze days in winter group) were used to construct four networks (Fig 2A).

The networks were constructed using the Cytoscape (version 3.8.2) based on
Spearman correlation index (cutoff value > 0.7) (80,81). Using R software, the network structure was described with natural connectivity in response to node removal and degree distribution.' in Line 384-390. (1) 'cleaning the retroauricular crease (lines 260-261)': We deleted the sentence 'Secondly, the males may have low frequency of cleaning the retroauricular crease, which lead to the longer times to haze exposure.', because no reference was found to support our speculation. 2. Co-culture usually implies multiple microbes but it appears to mean one fungal strain with particulate matter in this case. For this situation, you can either use "culture" or define co-culture when it is first introduced.
Thank you for your suggestions, we revised 'co-culture' to 'culture' in Line 36, 320, 330, 420, and 426. The authors describe here an interesting study where they assessed the influence of haze pollutants on the skin fungal communities depending on the host sex, as well as the seasons (spring vs. winter). The genus Talaromyces was found to be enriched during haze days. Moreover, the authors were able to isolate five Talaromyces strains and further showed that their growth was promoted by a high concentration of particulate matter.
Thanks for your suggestions. According to your and other reviewer's comments, we have revised our paper carefully. Thank you for helping us to improve the manuscript.
There are a lot of parts in the manuscript that I could not properly understand.
Comments and suggestions can be found in the review attachment file. Thank you for addressing our concerns in this revision, the manuscript is much improved. I appreciate the repeated reminders of the sample size. However, given the small sample size and large number of ASVs, I feel that the network analysis is inappropriate and should be removed. Spearman correlation is not appropriate for abundance data without a transformation (many references exist about this in the bacteria literature) and the small sample size limits any conclusions that can be drawn about a network. That said, the paper is strong without this analysis.
Thanks for your suggestions. According to your comments, we have revised our paper carefully.
We removed all the sentences related to the network analysis in Abstract (Line  Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. You will be notified when your proofs are ready to be viewed.
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