Distribution of Toxigenic Halomicronema spp. in Adjacent Environments on the Island of Ischia: Comparison of Strains from Thermal Waters and Free Living in Posidonia Oceanica Meadows
, Mirko Mutalipassi 1, Nadia Ruocco 1, Francesca Glaviano 1, Antonino Pollio 2, Antonio Luca Langellotti 3, Giovanna Romano 1 and Maria Costantini 1
Round 1
Reviewer 1 Report
An interesting study on toxicity of Halomicronema strains. It is well-written, English is good. I have some issues to be addressed during revision:
As long as I find the background on taxonomical issues interesting, I don't think the description of them benefits this particular manuscript in any way
In other words, the background of this paper requires revision/reformulation, it should focus more on Halomicronema instead of introducing various general issues such as cyanobacteria in general etc. I believe it would be good to start Introduction by discussing toxicity of various species, giving examples of toxins produced by cyanobacteria, and giving a background on their postulated ecological role (e.g. allelopathy, defense against grazing etc.).
There is no need to spell "Extracellular Polymeric Substances" from capital letter in the middle of sentence.
Some cyanobacteria has been experimentally reported to use toxins as response to nutrient-depletion and related allelopathic interactions, and these can contribute to their ongoing expansion even when nutrient loading is limited (eutrophication is one of main contributing factors to cyanobacterial growth): https://doi.org/10.1016/j.hal.2014.03.002
For clarity, I suggest to provide a separate pararaph that describes aims of this study, at the end of Introduction.
Report a final pH of BG-11 used in cultures
Make sure that all abbreviations are explained at first mention (e.g. TEM)
L.398-399: ?? I don't quite understand how apoptosis was identified. Specify.
I think a great limitation of your study is lack of analytical work on toxin identification of studied strain. Elaborate more on this in discussion.
There is no proper conclusions at the end of the paper - please formulate as a separate section.
Author Response
Answer to the reviewer 1
An interesting study on toxicity of Halomicronema strains. It is well-written, English is good. I have some issues to be addressed during revision
We thank the reviewer for his kind evaluation
As long as I find the background on taxonomical issues interesting, I don't think the description of them benefits this particular manuscript in any way. In other words, the background of this paper requires revision/reformulation, it should focus more on Halomicronema instead of introducing various general issues such as cyanobacteria in general etc. I believe it would be good to start Introduction by discussing toxicity of various species, giving examples of toxins produced by cyanobacteria, and giving a background on their postulated ecological role (e.g. allelopathy, defense against grazing etc.).
All this has been changed accordingly. In particular, given also the comments of other reviewers, we left in the introduction only part of the taxonomical descriptions, revising them all. Of course we have also added information on the required topics, about toxicity of various species, categories of toxins, etc.
There is no need to spell "Extracellular Polymeric Substances" from capital letter in the middle of sentence.
Revised
Some cyanobacteria has been experimentally reported to use toxins as response to nutrient-depletion and related allelopathic interactions, and these can contribute to their ongoing expansion even when nutrient loading is limited (eutrophication is one of main contributing factors to cyanobacterial growth): https://doi.org/10.1016/j.hal.2014.03.002
Done, as required in the introduction and cited it properly.
For clarity, I suggest to provide a separate pararaph that describes aims of this study, at the end of Introduction.
Done
Report a final pH of BG-11 used in cultures
Done at line 358
Make sure that all abbreviations are explained at first mention (e.g. TEM)
We did it along the text and hopefully revised at all
L.398-399: ?? I don't quite understand how apoptosis was identified. Specify.
Done at lines 427-428
I think a great limitation of your study is lack of analytical work on toxin identification of studied strain. Elaborate more on this in discussion.
Biochemical analytical investigations (still in progress) are a different topic, not evaluated in the present study. As suggested, the topic was elaborated further, within the discussion.
There is no proper conclusions at the end of the paper - please formulate as a separate section.
Done at line 334 and revised further sections
Reviewer 2 Report
The manuscript entitled "Distribution of toxigenic Halomicronema spp. in adjacent environments in the Island of Ischia: comparison of strains from thermal waters and free living in Posidonia oceanica meadows" refers to an important aspect of different pattern concerning toxicity of two congeneric cyanobacteria species living in different environments, i.e. marine waters and thermal waters, but in the same area.
However, a minor revision is necessary concerning primarily some mistakes or confusion and correction of English style and grammar throughout the whole text.
The full taxonomic names should be used concerning e.g. Posidonia oceanica - Posidonia oceanica (Linnaeus) Delile and Halomicronema metazoicum - Halomicronema metazoicum C.Caroppo, P.Pagliara & P.Albertano when you mentioned for the first time. The same matter concerns other species if, of course, it is possible to give full taxonomic names.
Lines 31-32: „Filamentous cyanobacteria that do not produce heterocysts are traditionally included in the Oscillatoriales, one of the five subsection in which cyanobacteria were grouped by Ripka et al. [1].”
Recently, the cyanobacteria taxonomy has changed. Please verify according to the latest classification e.g. according to classification included in AlgaeBase (http://www.algaebase.org).
Figures 1 and 2 – please correct the title, primarily in case of: “Photomicrophraphs” or “Photomicrographs”, using small/big letters, unnecessary dots and so on.
In taxonomic names sp. should be written without italics.
Figures 5, 6, 7, 8 – please add the explanation to the abbreviation Ctrl(-).
Please unify the following phrases:
line 320: and 12/12 dark/light cycle
line 332: and a dark:light cycle of 12:12 hours
Conclusions are missing. Thus, I suggest adding the conclusions which will summarize this study.
Author Response
Answer to Reviewer 2
The manuscript entitled "Distribution of toxigenic Halomicronema spp. in adjacent environments in the Island of Ischia: comparison of strains from thermal waters and free living in Posidonia oceanica meadows" refers to an important aspect of different pattern concerning toxicity of two congeneric cyanobacteria species living in different environments, i.e. marine waters and thermal waters, but in the same area.
We thank the reviewer for the positive evaluation
However, a minor revision is necessary concerning primarily some mistakes or confusion and correction of English style and grammar throughout the whole text.
English text was revised by a mother-language person, now acknowledged
The full taxonomic names should be used concerning e.g. Posidonia oceanica - Posidonia oceanica (Linnaeus) Delile and Halomicronema metazoicum - Halomicronema metazoicum C.Caroppo, P.Pagliara & P.Albertano when you mentioned for the first time. The same matter concerns other species if, of course, it is possible to give full taxonomic names.
Done along the text and especially at line 109, and 107
Lines 31-32: „Filamentous cyanobacteria that do not produce heterocysts are traditionally included in the Oscillatoriales, one of the five subsection in which cyanobacteria were grouped by Ripka et al. [1].”
Revised
Recently, the cyanobacteria taxonomy has changed. Please verify according to the latest classification e.g. according to classification included in AlgaeBase (http://www.algaebase.org).
Done
Figures 1 and 2 – please correct the title, primarily in case of: “Photomicrophraphs” or “Photomicrographs”, using small/big letters, unnecessary dots and so on.
Done
In taxonomic names sp. should be written without italics.
Done
Figures 5, 6, 7, 8 – please add the explanation to the abbreviation Ctrl(-).
Done
Please unify the following phrases:
line 320: and 12/12 dark/light cycle
line 332: and a dark:light cycle of 12:12 hours
Done
Conclusions are missing. Thus, I suggest adding the conclusions which will summarize this study.
Done at line 340
Reviewer 3 Report
I find the subject of your study very interesting and definitely should be shared with the scientific community, but there are some issues that I think have to be addressed.
Although I think your premise is quite probable, I think that general conclusions should not be made using just a pair of cyanobacteria strains. Is there any other publication supporting your premise?
Another issue I have is the limited scope of your toxicity assays, all based on a single organism, when there are some very affordable and simple tests with other organisms (as an example Artemina salina or Daphnia), which is imperative when you are inferring to the general toxicity of the studied strains in your conclusions.
Besides these two main issues, here is a list of smaller problems that I think should be addressed:
-line 53: there is a mistake in "to the highest (Antarctic; [10]) or lowest (thermal environments; [11]) temperatures" - exchange the environment to the correct temperature range.
-Figure 1c: is it possible to use a better photo? The necridic cells are not very clear (alternatively, include an arrow or similar graphic to point them out.
-line124: typo: Should be "mat", not "matte".
-line 130: some more information on the environment would be usefull: temperature and pH?
-line 149: typo: Should be "phylogenetically", not "phylogenitically".
-Figure 3: Can you increase the number of reference strains in the study? Also, the tree suggests high similarity between your strain Cyano_Pos and H.sp. Goniastrea-1. You did not comment on this or indicated there similarity. Should this two strains also be classified as H. metazoicum?
Figure 4: It is not clear if tests were carried out for H. metazoicum for higher concentration. Is so the graphic should be clearer pointing out the result (even if 0%).
Figure 8: The behavior of the assay for H. metazoicum is not dose dependent. It should be commented in the discussion.
Figure 10: same comment as in figure 4.
line 255-258: Your comments have a general scope, but the toxicity assays carried out are limited the a single organism that does not even coexist in the same environment - my issue here is connected to one of the main questions I indicated at the start of my comment.
line 261-262: Your comments have a general scope, but the toxicity assays carried out are limited the a single organism.
line 263-264: The toxicity of the H. metazoicum homogenate of is only displayed at high concentrations. Again your comments suggest general toxicity (that might be present) but it was only tested on one organism.
line 383: possible typo: "shaken"
line 406: you should point out the resulting acceleration or provide us with the centrifuge arm length.
line 406 and 408: I need some clarification; you are collecting the liquid over the solid pellet (supernatant) or the liquid under floating solids (surnatant).
I have a final question regarding the SEM chemical analysis: does it collect information regarding the chemical composition of the surface of your sample or of the whole of the sample? If the answer id the second case, I find it very strange not to find any Phosphorous in one of the strains, as it is one of the essential elements for life (in DNA/RNA as an example).
I hope you find this comments helpful.
Best regards
Author Response
Answer to Reviewer 3
I find the subject of your study very interesting and definitely should be shared with the scientific community, but there are some issues that I think have to be addressed.
We thank the reviewer for the positive evaluation
Although I think your premise is quite probable, I think that general conclusions should not be made using just a pair of cyanobacteria strains. Is there any other publication supporting your premise?
We reported such cases in the revised introduction (e.g., among others, Rzymski et al, 2014)
Another issue I have is the limited scope of your toxicity assays, all based on a single organism, when there are some very affordable and simple tests with other organisms (as an example Artemina salina or Daphnia), which is imperative when you are inferring to the general toxicity of the studied strains in your conclusions.
As now explained in the methods (according to Romano et al 2011), P. lividus embryos are formally considered to provide a standard toxicity test to demonstrate both anti-mithotic and theratogenic power. For this reason this standard test has been adopted here as a mean to immediately detect the toxigenic properties of exudates. Further studies will take into account different models, to explain various activities also in view of the structure elucidation of specific compounds. However, in this study such details could be superfluous if not confounding and for this reason they were not taken into account.
Besides these two main issues, here is a list of smaller problems that I think should be addressed:
-line 53: there is a mistake in "to the highest (Antarctic; [10]) or lowest (thermal environments; [11]) temperatures" - exchange the environment to the correct temperature range.
Corrected
-Figure 1c: is it possible to use a better photo? The necridic cells are not very clear (alternatively, include an arrow or similar graphic to point them out.
-line124: typo: Should be "mat", not "matte".
Done
-line 130: some more information on the environment would be usefull: temperature and pH?
These details were given in the methods, lines 360 and further
-line 149: typo: Should be "phylogenetically", not "phylogenitically".
DONE
-Figure 3: Can you increase the number of reference strains in the study? Also, the tree suggests high similarity between your strain Cyano_Pos and H.sp. Goniastrea-1. You did not comment on this or indicated there similarity. Should this two strains also be classified as H. metazoicum?
The reference strains used for the tree are those usually used for the phylogenetic analysis of Cyanobacteria. In particular, according our previous work (see Figure 3 in Ruocco N., Mutalipassi M, Pollio A., Costantini S., Zupo V., Costantini M. (2018c) we have chosen the strains belonging to the subgroup C, phylogenetically related to Cyano_Pos and H. metazoicum. Cyano_Pos and H.sp. Goniastrea-1 have very high similarity (99%), as now reported in the revised version of the manuscript (see also Table_S2). These two strains can be classified as H. metazoicum.
Figure 4: It is not clear if tests were carried out for H. metazoicum for higher concentration. Is so the graphic should be clearer pointing out the result (even if 0%).
Bars pointing at 0 indicate 0. It is uneasy to indicate it differently using the capabilities of our standard graphing software. However, we added now a clear description in the captions and we are keen to accept any further editorial suggestion about this, if it will clarify the issue
Figure 8: The behavior of the assay for H. metazoicum is not dose dependent. It should be commented in the discussion.
As shown in figure 7, H. metazoicum produced 100% of apoptotic embryos up to 103. The next results in figure 8 are a percentage of the survivors and they are dose dependent, thus. It has been highlighted in results and discussion
Figure 10: same comment as in figure 4.
Same caption revision as in figure 4
line 255-258: Your comments have a general scope, but the toxicity assays carried out are limited the a single organism that does not even coexist in the same environment - my issue here is connected to one of the main questions I indicated at the start of my comment.
The discussion has been improved now and enriched with some details. However, as above specified, the choice of the model organism was due to its general and well recognized value for the investigated factors. Other organisms could add confusion to the simple and straight description of toxigenic activity.
line 261-262: Your comments have a general scope, but the toxicity assays carried out are limited the a single organism.
As above, and as now highlighted in the introduction, the main scope was not to detect general patterns of toxicity to be applied to their original environments. The toxicity tests served to characterize their toxigenic power using a generally adopted model organism, to standardize the results and make them comparable also in future, with other species.
line 263-264: The toxicity of the H. metazoicum homogenate of is only displayed at high concentrations. Again your comments suggest general toxicity (that might be present) but it was only tested on one organism.
As above
line 383: possible typo: "shaken"
Corrected
line 406: you should point out the resulting acceleration or provide us with the centrifuge arm length.
Revised
line 406 and 408: I need some clarification; you are collecting the liquid over the solid pellet (supernatant) or the liquid under floating solids (surnatant).
We collected over. Revised now as suggested
I have a final question regarding the SEM chemical analysis: does it collect information regarding the chemical composition of the surface of your sample or of the whole of the sample? If the answer id the second case, I find it very strange not to find any Phosphorous in one of the strains, as it is one of the essential elements for life (in DNA/RNA as an example).
Being an analysis at SEM it collects information at the surface of the sample and it is of course an indicative measure, especially for very abundant elements, over the sample’s surface
