All reviews of published articles are made public. This includes manuscript files, peer review comments, author rebuttals and revised materials. Note: This was optional for articles submitted before 13 February 2023.
Peer reviewers are encouraged (but not required) to provide their names to the authors when submitting their peer review. If they agree to provide their name, then their personal profile page will reflect a public acknowledgment that they performed a review (even if the article is rejected). If the article is accepted, then reviewers who provided their name will be associated with the article itself.
Dear Author’s,
Thank you for submitting your manuscript to PeerJ. Following thorough peer review and consideration of the final revisions, I am delighted to inform you that your manuscript has been accepted for publication.
The reviewers and I found your study to be an original and well-executed contribution to the understanding of microbial associations in elasmobranch sensory systems. Your findings provide valuable insight into the holobiont concept as applied to sharks and will be of broad interest to marine microbiologists and evolutionary biologists alike.
Congratulations to you and your co-authors on this achievement. We look forward to seeing your article published soon in PeerJ.
Warm regards,
Armando Sunny
[# PeerJ Staff Note - this decision was reviewed and approved by Patricia Gandini, a PeerJ Section Editor covering this Section #]
I found the writing to be clear and unambiguous throughout. Sufficient background and context were provided, and appropriate references were cited. The figures and structure of the article were professional. I think an additional figure (or table) showing the whole genome results (and the highlighted genes/functions) would be helpful instead of listing them in the results as they are currently, but the other figures were straightforward. The results, although somewhat preliminary, supported their questions and they clearly outlined the limitations of their approach, primarily with respect to using a selective media.
The research falls within the Aims and Scopes of PeerJ and the question was clearly defined and placed within the context of the field. The results of this study, while preliminary, do lay a foundation for future research into the microbial communities and their role within the AoL. The methods have some limitations, but the authors state this in their discussion. Otherwise, the methods were clearly described with sufficient detail.
The results were novel in that this is the first description of the microbial community associated with the AoL. Unfortunately, the methods used were limited in the ability to fully characterize the community, but the authors are aware of this and plan to expand upon this preliminary study in the future. The authors do a nice job of discussing the results with potential implications of the microbial community within the AoL, given the limitations of their data.
This revised version of the manuscript is much improved. The authors did a nice job addressing previous concerns.
Thank you for submitting your manuscript.
We appreciate your contribution and the effort you have put into this research.
Following a thorough review, we have received comments from both reviewers, who acknowledge the potential significance of your study. However, they have also identified key areas that require substantial revision before the manuscript can be considered for publication. Specifically, they suggest that the Materials and Methods section needs a more detailed and precise explanation to enhance clarity and reproducibility. Additionally, they have raised concerns regarding the validity of the findings, recommending further clarification and stronger support for the conclusions.
We encourage you to carefully address these points and submit a revised version of your manuscript. Strengthening the methodological descriptions and providing additional validation of the results will be crucial to ensuring the manuscript meets the journal’s standards.
Best regards,
Armando Sunny
While some of the previous comments were addressed, I still find some of the authors conclusions and assumptions to be misleading, and at times false, particularly with respect to their claims of bioluminescent bacteria and the potential mutualism (which they compare to the squid-vibrio symbiosis throughout). First, the genomes presented are non-luminous bacteria as they do not contain the light-producing lux operon in them. This is further supported by their luminosity measurements, for for which the bacteria produced RLU values of other non-luminous bacteria. They also fail to discuss the ecologies of the bacteria identified, which are common marine microbes that are opportunistically pathogenic (this does no mean they are always acting as pathogens). While there are some interesting results in the manuscript, I am still struggling with the overall messaging and packaging of the study as a potential biolumienscent association with bacteria.
Much of the microbiological assays were lacking sufficient details and done in somewhat non-conventional ways. For example, when looking for bioluminescent bacteria, isolates are usually identified in the dark to confirm their ability to produce light. There was no mention of this being done. Furthermore, details such as pelleting the liquid culture before DNA extraction were lacking. I also found some of the details needed to interpret the results presented in Figs 5&6 to be missing (see details below).
Some of the results presented are interesting and lay a foundation for future research, however the study itself does not support all of the claims the authors are making.
More detailed comments below:
Line 106: “This suggests…”
Re-word this transition. The previous sentence refers to counterillumination, which is different from communication, attraction, and hunting.
Line 109-112: This sentence is a bit confusing. The function of bioluminescence is different than bioluminescent symbiosis in which the host provides nutrients, etc. The bacterial cells can produce light outside of the host environment and are typically facultative symbionts. Re-word for clarity.
Line 123: italicize lux
Lines 165-166: what is meant by “placed”? Did you streak out the sample? Is the extracted jelly the same as the sample that was placed on the petri dish? Please clarify.
Lines 241-242: which program did you use to create the ML tree? And which nucleotide substitution model was implemented?
Lines 246-247: how did you select these eight particular isolates? Did you check for luminescence when you selected them? Based on your whole genome sequences, I am highly skeptical that these strains are bioluminescent, as they do not contain the lux operon responsible for light production. They are likely non-luminous members of the described genera.
Line 248: Did you spin a pellet first?
Line 271: It is unclear which “Vibrio isolates” you used to measure luminosity. Were these any of the isolates that were sequenced? How did you select these isolates? Did you check for luminosity on the plates before growing them in liquid media? In the figure you include
Lines 276-277: RLUs aren’t a measure of fluorescence, but luminescence
Lines 307-309: Did you homogenize the biofilm in the water, or did it remain a film? Please elaborate here.
Line 380: This low level indicates you have isolated non-luminous bacteria. This is also the incorrect figure reference – it should be Fig 4.
Lines 381-382: Are these the genomes of the same bacteria for which luminescence was measured. The current text make it seem that way, but the methods are very vague. These lux genes are involved in quorum sensing and the regulation of light production, but none of them directly produce light. Without lux AB (which encode for luciferase) and luxCDE, the bacteria cannot make light. The sentence is very misleading. You have likely isolated and sequenced non-luminous Vibrios from your sample.
Lines 324-326: The way in which these results are stated are slightly misleading. Because your approach was not quantitative, you need to state this more cautiously. Most of the bacteria that you cultured and sequenced from the various samples belonged to these groups.
Line 341: “both”
Line 352-3: check grammar
Line 380: this measurement of RLUs is consistent with a non-luminous bacterium
Line 424: italicize genus
Line 436-437: These two processes are only similar in that they are environmentally acquired. The bobtail squid – V. fischeri symbiosis is much more specific and intricate, with a series of highly controlled steps that occur during the colonization process ensuring that only V. fischeri end up as the sole occupants of the specialized light organ. Comparing them here is misleading. Your system is more similar to any environmentally acquired marine bacterium in a less-specific community association.
Line 459: Yes – very low and consistent with non-luminous bacterial species.
Line 461-7. I suspect it is more likely the case that you did not test the same bacteria in these two assays. There very well could have been luminous bacteria in your AoL samples, but you did not isolate, culture, and sequence such isolates. When working with bioluminescent bacteria you should verify in the plating step that the colonies selected do indeed produce light by observing the plates in the dark. There was no mention of this being done. Additionally, your genomes all lacked the lux genes involved in light production, thus they are non-luminous strains, consistent with your RLU readings. These results need to be updated to more accurately reflect what your results show.
Lines 598-600: Based on the genomes of the bacteria that you isolated and sequenced, you do not provide direct evidence of bioluminescent bacteria in the AoL. Your image indicates that the AoL are bioluminescent, however the bacterial sequences presented here are all non-luminous, and you provide no evidence that they produce light based on your luminescence assay of the bacterial cultures. You should carefully re-word this discussion to reflect what your actual results show and not over-state or make assumptions about this potential relationship. Have you also considered the possibility that the shark itself could be producing the light observed as is the case for many autogenously luminous species?
Lines 612-613: Do you have reason to believe that the AoL is a low oxygen environment? The luciferase reaction requires O2, thus bioluminescence typically does not occur in such low-O2 environments.
Line 624-626: While this may be the case, you still avoid any discussion of the fact that both Vibrio species that you sequenced are opportunistic pathogens and generally not considered to be mutualists. You go into great depths on the potential metabolisms of the bacteria (based on gene content alone), but fail to describe their ecologies or natural histories. I think this is an important part of the story. These bacteria are fairly common on marine organisms, and in times of stress they are there and ready to cause disease (vibriosis).
Line 651: You still haven’t shown that the light produced was from the bacteria and not the shark itself.
Table 1 caption: check spelling and grammar
Figure 2 caption: which model of substitution did you use?
Figure 5: You cite two shark species but only show one profile. Did you pool the samples? Your methods made it sound as if you analyzed many samples – how are they represented on this figure? Make sure your methods/results/figures align with enough detail to understand how the data were produced.
Figure 6. Similar to Fig 5. Is this a composite result of all of your samples or were they pooled? Please provide more details.
Dear Authors,
Thank you for submitting your manuscript to PeerJ. Both reviewers have noted that the current methods section is quite limited. As a result, they recommend significant revisions to enhance the descriptions of the materials and methods. Additionally, they suggest performing further analysis where possible.
We appreciate your attention to these recommendations and look forward to your revised manuscript.
Best regards,
Armando Sunny
The language was clear and unambiguous throughout. There were appropriate citations, however the discussion could use more mention of other studies relating to many of the vibrio spp. sequenced as well as other bioluminescent symbioses to provide more context for this study. The structure of the manuscript was good. The results were presented in a clear way, although I am not sure they support all of the claims made.
The experimental design was mostly clear, although there were a few places where more details were needed (see details below). Additionally, some justification for the methods would be helpful. For example, why did the authors choose to only sequence 16s from select isolates and not use whole community 16s metabarcoding of the AoL? That approach would had provided more conclusive results and the different shark samples could be compared better. Selecting isolates at random introduces biases from culturing methods. In this regard, I think the paper is limited in it's ability to make certain conclusions and is primarily descriptive in its nature, lacking high technical standard.
The results presented are clear and their data are robust, but they lack any ability to provide conclusive evidence of some of their hypotheses (bioluminescent symbiosis) as the results are merely a description of some of the species that were present in the AoL that they selected for culturing. The authors also fail to provide any details regarding the known biology of the isolates for which they sequenced the whole genomes, many of which are known marine pathogens.
Overall, the paper is clear and well-written, but could benefit from some more integration of their results with the existing literature. Some of the methods also need more clarification and details. For example, it was not clear why/how they chose to do whole genome sequencing of 8 isolates - why those 8? Also, if the authors wanted to profile the bacteria in the AoL jelly, why not do whole community metabarcoding of 16s? Why only sequence select isolates? More details in the methods are needed to better understand the choices made by the authors as well as the experimental details of certain assays used, particularly for measuring luminosity. How did you do your dilutions to compare the jelly to individual isolates? Were they in the same broth? If so, how do your conclusions that the jelly provided more nutrients hold up?
The discussion could benefit from more integration of species’ identities with known function in the literature. For example, many of the Vibrios sequenced here are known marine pathogens. What is known about the species that you have identified in these other systems? They are still bioluminescent, but not in a mutualistic way. The discussion of the potential use of bioluminescence is not well developed and overlooks many of the other existing fish systems, which are typically a one host-one bacterium association isolated from the rest of the microbial community over which the host has control over light emittance via a shutter/flap over the light organ. These points should be discussed in more depth and compared to the very different results presented here. Why would this system be so different?
More specific comments:
Introduction
Line 46: Remove “Add your”
Line 95: attraction to what? Each other? Prey? Or both? Please elaborate.
LInes 97-98: Also as a way for the cells to prevent oxygen toxicity (Timmins et al. 2001, J. Mol. Evol.)
Methods:
Line 122: Remove “Add your materials and methods”
Lines 127-130: Be consistent with how you write number of individuals
Line 129: “y” --> “and”
Line 131: Did you do anything to sterilize the surface of the sharks before extracting the jelly?
Line 133: What does this mean? What does “carefully isolated” mean? Were they dissected from the shark?
Line 137: Did you plate different dilutions of the jelly? How much volume of jelly was plated?
Lines 143-144: The current wording sounds as if you made 2 separate frozen cultures. One in just 80% glycerol and another with a 1:1 of media and glycerol (what percentage?). Please re-word for clarity.
Line 146: Is this from all of the sharks combined?
Line 174: How did you select which isolates to sequence? Were these all bioluminescent?
Line 184: “by with” - choose one
Line 188: “search manual” --> “manual search”
Line 188: “Lux” --> “lux”
Line 196: Do you mean “Luminescence” (not “Fluorescence”)?
Line 199: How long were they grown up for? What was the initial vol. of the inoculum vs the LB broth?
Line 200: Did you dilute in LB broth again? Please elaborate.
Line 220: Why the difference in drying methods? Was this intentional? How/why did you select some to be air dried vs. 100C?
Line 224: Why was this species/specimen treated differently than the others?
Line 228: Where did the V. harveyi cells come from? Were these from a stock culture? What was the biofilm gown on? Please elaborate.
Results
General comment: Wondering why the authors chose to use 16s RNA sequencing from isolates only and not of the whole jelly metacommunity in combination with culturing. This method is biasing which microbes were screened/identified and ignoring any unculturable spp.
Lines 255-257: This kind of conclusion is difficult to make with your sampling approach because it is not quantitative. You selected which isolates to sequence from each sample and did not capture the whole microbial community in the AoL.
Line 276: Italicize “alginolyticus"
Line 282: Check sentence structure
Lines 267-285: This information is easier to read/interpret in your table format and simply summarize/highlight some of them in the text here.
Lines 286-296: Same comment.
Line 297: What does “and zoom” mean?
Line 303: Should this be “RLU”?
Line 310: What about luxCDABE(F)G? The lux genes reported are part of the signaling/quorum sensing pathway, but not the luciferase reaction itself.
Line 311-312: Check grammar/structure
Consider combining Figs 6/7 --> 6a/b or moving Fig 6 to Supplementary Materials since it is harder to interpret
Discussion
Line 361: Could this be associated with the amount of time the specimens were in the market before sampling? Staphylococcus is typically an opportunistic pathogen in marine systems and could have increased in abundance after the shark was captured.
Line 364: This is likely also true for the other two locations, if you had water samples from there too. I suspect that the differences in dominant bacteria is more a result of time between capture and sampling (ie: amount of time in the market).
Line 366: These are not cuttlefish – they are bobtail squid
Line 367: They only acquire a single species of bacteria, not the entire community --> it is a highly selective process
Line 368: squid
Line 372: “the effect of the ampullae jelly in such microbes” - I’m not sure what you mean here. Please re-word/explain.
Lines 379-380: But you diluted the jelly sample in LB broth for these assays, correct? How much of the jelly substrate was in the sample for this assay? Could this not be a result of a mixed bacterial community vs. single isolate?
Lines 384-386: This could be true, but your methods currently lack enough detail to draw this conclusion. You need to more clearly explain your dilutions and the methods by which you cultured your samples for this assay to compare.
Lines 388-390: Fish that use bioluminescence for signaling typically have some control over how much light they emit (such as a shutter or flap over the light organ that can open or close). How would they use the light in the pores here to signal?
Line 391-392: This result seems out of place here. Can you elaborate more on the significance of this result? How does it connect to the use of bacterial luminescence?
Line 410: Mediated how? Are you suggesting that bacterial biofilms increase the conductivity of the gel? You would need to compare sterile jelly to that with the bacteria to make this conclusion. Please elaborate.
Line 417-418: I’m still unclear on what you mean by “mediate” here. How are they mediating sensory capabilities?
Lines 426-428: Many Vibrios are opportunistic pathogens and this is typical of their genomes. It’s difficult to tease apart a beneficial symbiont from a pathogen in this regard.
Lines 441-443: It is also important to mention that the bobtail squid houses only a single species of bacteria in its light organ, which it depends on for its survival, whereas this is a mixed bacteria community in the AoL, and is thus, unlikely a similarly evolved system.
Line 462: Many Vibrios (including species that you identified in your samples such as V. owensii, V. rotiferianus , V. harveyi) are known marine pathogens, so this hypothesis does not hold true. They also produce light, but do not provide this function to their hosts in a beneficial manner.
Lines 476-478: This point has already been made and is repetitive here. Moreover, there is little evidence that this is the case when compared to other bioluminescent systems that have evolved for these various purposes (they have distinct organs designed to only house a particular sp. of bacteria and a mechanism for controlling the amount of light emitted). Many Vibrio are bioluminescent but they do not provide this as a beneficial function to their host. I suggest you discuss this alternative as well, including more details on what is known of the Vibrio spp. That you did whole genome sequencing on, none of which have been seen as beneficial mutualists in other marine squid or fish hosts.
What about the Exiguobacterium sp. That you sequenced. There is no mention of it in the discussion. How do your findings on that species incorporate into the rest of your study?
Line 488-489: I think this conclusion is unlikely without any real support in this study for this phenomenon.
Figure 2 caption: “iin situ“ --> “in situ”. This is a dead specimen, correct? Do you know how long the specimen has been dead for and at what temperature it was maintained? This caption should have more detail to make clear whether this is observed in a living shark or not. There’s also a lot of black space with nothing visible in it and could be made much smaller. Please include scale bars for reference.
Figure 3. Was this re-created based on any actual observations in the wild, or is this simply speculation based on where the AoL are located? Please elaborate for clarity.
Figure 5. The different heights of the boxes around compounds are a little confusing here. It also appears like some of the boxes highlighted don’t have peaks in those areas (ie: Amide A, Amide I?), which you state was in the AoL in your results.
This paper takes unique samples of the AoL and samples their bacteria using 16S rRNA, whole genome sequencing, and chemical and refraction measurements. While the authors seem well-versed in shark physiology (which is outside my area of expertise), the paper is lacking in their understanding and their sophistication in discussing host-associated bacteria, especially bioluminescent bacteria. The methods used make me highly suspect of the results presented.
Various errors that make me doubt the conclusions drawn given the high likelihood of contamination that would lead to spurious conclusions. These include:
1) The animals were not surface sterilized before the samples were taken
2) The samples though stored in glycerol were not noted to be flash-frozen or kept at -80C. This would bias the populations that would culture.
3) Culture methods are not a reliable method for evaluating the relative proportion of bacteria, so in theory there could be one contaminant on the skin (which is common for fish to harbor these sorts of bacteria) to lead to the conclusion that this bacteria is found within the AoL
4) It is unclear if all samples were taken from dead animals collected from fish markets, which will also bias the materials gathered.
5) The sterile method to culture isolates is not described and thus I am assuming it was not performed
6) Was the PCR product not cleaned? This can lead to some to some issues as well with the sequencing results.
7) We don’t usually use neighbor-joining trees for 16S analysis as it can lead to dubious results, maximum likelihood is a better method
8) The data is not in the NCBI for the 16S sequences (though can confirm for the whole-genome work)
9) The link for their whole-genome gene searches is depreciated and unclear how they performed this using https://www.healthtech.dtu.dk/
The result section was lacking and the methods sections were so dubious that I did not fully review the results.
Line 30: Be clear in the abstract that you cultured these bacteria. You also do not establish from your introduction why looking for bacteria in AoL is important, the two topics are handled too separately for this paper.
Line 35: remove all instances of “host-guest” for “host-associated” (because just not a common phrase used in symbiosis literature)
Line 36: overall the sentence in the abstract where this occurs is confusing. Be clear that you did whole genome sequencing to find host-association genes
Line 37: The sentence after that is too long and is confusing, either turn into a list with “;” or break into separate sentences
Line 40: Remove the phrase “We believe we documented here for the for the first time” to “This study is the first documented occurrence of”
Line 46: remove "Add your”
Line 70: There is very little research in this area and this is too narrow a discussion of it. It is also unfocused, limit your recap to like organs and shark studies and look at the evenness as well as the diversity of these bacteria.
Line 84: Remove “On the other hand”. Also overall I found this summary of the importance of bioluminescence to be confused and incomplete. There are species that are harmful and there are species that are mutualistic. These are seldom the same species. As written this is confusing and may mislead readers to a misunderstanding of the complexities of these interactions.
Line 87: It is unclear why the authors are limiting the discussion to these members, there are many more species found in marine fishes
Line 96: “while the relevance of bioluminescence for marine bacteria is still not understood” is this positing that we don’t know why bacteria evolved to bioluminese—because we do have various hypotheses. If you’re positing that we don’t know why the host would select for bioluminescent bacteria, we have several hypotheses for that as well.
Line 100: You need to clearly differentiate between the shark luminescence and possible bacterial sources. Also you need to establish why you’d think their AoL would be luminescing by bacteria. Also, it is unclear if you’re looking at deep sea shark species—how is this related to the species you’re investigating in Mexico?
Line 122: Remove “Add your materials and methods”
Line 133: unclear how the bacterial loop was sterilized
Line 135: unclear why would place in glycerol if they’re goint to “culture fresh” and unclear why this would lead to axenic cultures. The usual method is the three-streak method to ensure there are single isolates. This is not noted in the paper.
Line 137: Spell out what TCBS is
Line 150: spell out minute and underscore for magnesium chloride in this paragraph
Line 194: did you mean Exiguobacterium, also telling that this bacteria is found in the gastrointestinal tract of marine fish, which suggests that may be a skin contaminant
Line 198: Vibrio is capitalized and italicized
Line 199: unclear what you mean by “Both jelly and bacterial isolates”
Line 223: I am very confused what this is describing. Is this supposed to be in this section?
Line 243: The result section does not describe all of the methods outlined.
All text and materials provided via this peer-review history page are made available under a Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.