Methods and Strategies to Uncover Coral-Associated Microbial Dark Matter

ABSTRACT The vast majority of environmental microbes have not yet been cultured, and most of the knowledge on coral-associated microbes (CAMs) has been generated from amplicon sequencing and metagenomes. However, exploring cultured CAMs is key for a detailed and comprehensive characterization of the roles of these microbes in shaping coral health and, ultimately, for their biotechnological use as, for example, coral probiotics and other natural products. Here, the strategies and technologies that have been used to access cultured CAMs are presented, while advantages and disadvantages associated with each of these strategies are discussed. We highlight the existing gaps and potential improvements in culture-dependent methodologies, indicating several possible alternatives (including culturomics and in situ diffusion devices) that could be applied to retrieve the CAM “dark matter” (i.e., the currently undescribed CAMs). This study provides the most comprehensive synthesis of the methodologies used to recover the cultured coral microbiome to date and draws suggestions for the development of the next generation of CAM culturomics.

E-mail: peerreview@asmusa.org  Reviewer comments: Reviewer #1 (Comments for the Author): Schultz et al. have reviewed various aspects about culturing microbes, specifically from corals. Overall, I liked the review and the strengths are that it is collecting and centralizing data on culturing coral-associated microbes (CAMs). The main weakness I see in this paper is that for some aspects it is very surface level and there's not enough depth to the some sections to really convey the complexity of culturing microbes. I am in no way saying that anything is wrong here, I'm mostly suggesting that some sections could have more depth and topics also be covered.
General comments: 1) Can the authors ensure they are using the already established terminology for certain things. I have more context in my specific comments.
2) There's no in depth/specific mention of enrichment of close culturing systems and the accumulation of toxic byproducts (apologizes if I missed this). There is some, but I'm referring to when the authors refer to simply adding sugars or chemicals like DMSP to media. There should be a caveat added of toxic byproducts accumulating from the metabolism of these additives. For example, it is known that glycerol present in growth media with certain Vibrio sp. will result in the accumulation of acidic byproducts that cause rapid culture crashes.
3) Maybe to add context, it would be good to add the relative year when these techniques were developed. For example, culture medias have not really changed in decades so it worth noting this is a major reason they should be updated.
Specific comments: Line 53-54: I would include the endosymbionts as well in this statement.
Line 139 (entire paragraph): Can there be more of a comparison of these techniques? Maybe pros and cons? I appreciate the overview, but the content/description is sparse here. I think this section can benefit from more elaboration.
Line 147: The authors are missing aspects about the toxic nature of lysed coral cells. What about the toxic montiporic acids released from Montipora spp.?
Line 183: The correct term is lysogeny broth. However, there are also modifications used instead, like LB + salt (LBS) that are actually being used for marine bacteria.
Line 184: This is more typically referred to as GASW Agar instead of GASWA. But the are different formulations and modification to this media as well (please note that).
Line 186: Is it actually NA or did the authors modifying? If is was modified, it can no longer be called NA.
Line 187: I'm not comfortable with this statement. First, there is a comparison of selective media vs general media. Second, this is from a literature review paper and the actual medias are not being directly compared. I this this section is a bit misleading.
Line 189 (paragraph): Can the authors please use the standard terms for media types? Basal/general media, enriched media, selective media, indicator media, transport media, and storage media.
Line 192-193: The wording is technically incorrect. This should be worded that you will increase the diversity of bacteria cultured. You increase the number of bacterial cells from growing them.
Line 197: "Promoters" This means something very different for genetics. Please use a different term. Or just say enrichment.
Line 201 (paragraph): The concept of nutrient shock should be mentioned. This is not a new phenomenon. This is was lead to the development of R2A.
Line 205: Again, I agree this was "new" for coral research, but these are known microbiology techniques and the original research should at least be acknowledged. Table: I like this table, but I think it would benefit from the relative year these technologies were developed. I'm always worried people will immediately disregard modification of culture medias and only focus on the fancy new toys available. But to give some context that culture medias are outdated and need improvement might make people realize that "simple" modifications can be combined with technology.
I would like to say that I enjoyed reading this manuscript and I wish you all the best in the revision process.

June 9, 2022
Dear editor, We are sending attached the revised version of the manuscript "Methods and strategies to uncover coral-associated microbial dark matter", and the associated point-by point answers to the reviewer, where we address all the suggestions and comments provided.
We would like to thank you and the reviewer for the detailed review and constructive suggestions provided.
Reviewer #1 (Comments for the Author): Schultz et al. have reviewed various aspects about culturing microbes, specifically from corals. Overall, I liked the review and the strengths are that it is collecting and centralizing data on culturing coral-associated microbes (CAMs). The main weakness I see in this paper is that for some aspects it is very surface level and there's not enough depth to the some sections to really convey the complexity of culturing microbes. I am in no way saying that anything is wrong here, I'm mostly suggesting that some sections could have more depth and topics also be covered.
A: Thank you very much for the positive feedback and suggestions to improve our manuscript.
General comments: 1) Can the authors ensure they are using the already established terminology for certain things. I have more context in my specific comments. A: Thank you. We have double checked the terminology used, as detailed below.
2) There's no in depth/specific mention of enrichment of close culturing systems and the accumulation of toxic byproducts (apologizes if I missed this). There is some, but I'm referring to when the authors refer to simply adding sugars or chemicals like DMSP to media. There should be a caveat added of toxic byproducts accumulating from the metabolism of these additives. For example, it is known that glycerol present in growth media with certain Vibrio sp. will result in the accumulation of acidic byproducts that cause rapid culture crashes.
A: Thank you for the note. We added more information regarding these points in the third paragraph of topic 2.2.1 "Culture media and growth conditions: regular and modified media", as follows: "Also, the selective DMSP-enriched culture medium seems to be a good option to increase the diversity of obtained CAMs [29], which is an example that, despite the potential accumulation of selective byproducts from the metabolism of culture media additives, such as sugars and or chemical compounds like DMSP [68], the selection of the media composition should be based on the scientific goals." We also mention other toxic compounds and conditions in lines 125-129; 161-165; 251-262, in Table 1 and in our conclusions.
3) Maybe to add context, it would be good to add the relative year when these techniques were developed. For example, culture medias have not really changed in decades so it worth noting this is a major reason they should be updated.
A: Thank you for the suggestion. We added this information in the revised manuscript, specifically in the first paragraph of topic 2.2.1. "Culture media and growth conditions: regular and modified media" and 2.2.3 "Alternative approaches", as follows: "Many resources are required for a microbe to be cultured, such as the correct physicochemical nutrients and energy sources. The most basic approach used to culture microbes from different sources is the use of nutrient-rich culture media (e.g., tryptic soy broth, lysogeny broth, marine broth, nutrient broth, to name a few). These culture media were developed decades ago (more than 50 years), and have been applied to grow and maintain microbial cultures from different environments and hosts, including the coral holobiont, using relatively few modifications that can increase the microbial diversity obtained. Here, we highlight that novel approaches developed for other hosts and samples should be adapted to increase CAM culturabilty."

Specific comments:
Line 53-54: I would include the endosymbionts as well in this statement. A: Good point. The endosymbionts are already included, as part of the microbiome, but it might be indeed good to highlight it. This sentence has been therefore edited, as follows: "Beneficial microbes are essential members of the coral holobiont (i.e., the coral host plus the associated microbiome, including their endosymbionts) [1, 2].
Line 139 (entire paragraph): Can there be more of a comparison of these techniques? Maybe pros and cons? I appreciate the overview, but the content/description is sparse here. I think this section can benefit from more elaboration.
A: We appreciated the suggestion and highlight that our goal here was indeed to be descriptive, as a comparison of these approaches would likely be somehow biased, due to the fact that these protocols were never directly compared, at least not all of them. We agree thought that some context/discussion on this direction could be provided, so we edited this paragraph as follows: "Microbes associated with mucus, for example, can be collected using sterile syringes to extract the liquid from the coral surfaces, or even from the whitish slurries that . Each of these protocols present associated challenges and limitations, including the amount of biomass that is obtained, potential microbial contamination from the surrounding seawater and/or cross-contamination among coral compartments. The selected technique will therefore likely rely on the available expertise, logistics and research goals." Line 147: The authors are missing aspects about the toxic nature of lysed coral cells. What about the toxic montiporic acids released from Montipora spp.? A: Very interesting point we were not aware of, thank you for including it. This information is now available in the third paragraph of the topic 2.1 "Sample processing interferes with the culturability of the coral microbiome". Check the new sentences below: "It is important to mention the potential effect of coral holobiont compounds on the culturability of specific CAMs. The production of mantiporic acids by the stony coral Montipora spp., [63, 64], can, for example, affect cultured cells (e.g., zooxanthellae), due to its antimicrobial activity and cytotoxicity [64, 65]." Line 183: The correct term is lysogeny broth. However, there are also modifications used instead, like LB + salt (LBS) that are actually being used for marine bacteria. A: Thank you for the note. It was a typo and we corrected it in the revised manuscript. The term "lysogen broth" was corrected to "lysogeny broth"