Picoplankton Distribution and Activity in the Deep Waters of the Southern Adriatic Sea

Round 1

Reviewer 1 Report

The manuscript "Picoplankton distribution and activity in the deep waters of the southern Adriatic Sea" by Šantić and colleagues describe the comparative analysis of the biomass production, distribution and activity of picoplankten in the Eastern Mediterranean Sea. While the pre-winter and post-winter sampling points provide an interesting investigation about bacterial patterns in the deep sea, the manuscript should be improved to be considered for publication in Water. 

The objective of the study allows interesting views on this marine deep-sea habitat, however only a limited number of taxonomic groups were investigated. If the authors would have designed and analysed an amplicon study in addition, this would have allowed a comprehensive and comparative overview of the different patterns with interesting meta-data integration. Apart from the missing amplicon analysis, the experimental design of the study seems reasonable to me, but it is not thoroughly described. In addition, the introduction has an unclear structure (see more comments towards that below) and the Materials and Methods section is mixed with habitat description and motivation - two points that should rather be in the introduction paragraph. The statistics is only very shortly and not adequate described which makes a reproducibility of the statistical analysis almost impossible. The discussions and conclusions could be stated clearer to sell the message of the manuscript better to the reader. English language is adequate but should be improved at some points - a native speaker might be of help. And finally, the manuscript is divided in a high number of short paragraphs - this should be avoided and instead a clearer structure would improve the manuscript a lot. A golden thread through the manuscript, particularly introduction, discussion and conclusions is difficult to find. 

Specific comments: 

* abstract

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- page 1, line 12: the first sentence should be separated, first the background, then the objectives of the study should be described

- p1, l20: ...  (HNA bacteria) was found in deep waters ... -> were found

- p1, l25: ... abundance, and by water mass movements ... -> by is redundant

- p1, 27: ... web in deep-sea as well ... -> web in the deep sea ...

* introduction

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- p1, l32: ... became more impotant -> in relation to what more important? this should be clarified.

- p1, l35: ... Autothrophic members ... -> Introduction should rather start with this sentence

- p1, l43: what do the authors mean by ... absence of light, conditions that are necessary for the primary production? this is unclear. apart from that, the sentences should rather be separated. 

- p2, l45: the authors need to make clearer what they mean by that. It is well known that microbial communites are found in every depth of the ocean.

- p2, l46: remove year number

- p2, l47: why particularly Synechococcus? what is about other abundant and active bacteria that play a role in primary production? if the authors want to address specificially Synechococcus they should clearer explain why. 

- p2, l52: why now talk about photoheterotrophs? what is their role in the deep sea?

- p2, l61: reference missing

- p2, l62: this is a harsh break between the background and the objective of the studies. some rewriting is necessary to make the introduction clearer. 

- p2, l69 and following: The last two paragraphs of the introduction are very unclear. What is their purpose and why are they mentioned here? The introduction should rather end with line 68 (but then it would be really short). 

The introduction needs some work, right now it is very unclear. The authors jump between different aspects, such as deep- sea and photoheterotrophic bacteria - a clear structure (golden thread) is missing. The authors should try to avoid these short paragraphs entire the manuscript and extend or combine the existing ones. The last two paragraphs in the introduction are difficult to understand and to put them into the complete context. 

* material and methods

-------------------------------

- p3, l95: this belongs rather to the introduction than to materials and methods

- p3, l107: In fact, the primary goal of the ESAW cruise ... -> this is motivation, not methods, this should rather be at the end of the introduction

- p3, l114: CTD (conductivity-temperature-depth) -> switch text in and outside of brackets, the abbreviation belongs the first time it is mentioned in brackets, and the long version before the brackets, after that you can refer to the abbreviation

- p3, l121: ... international standard procedures ... -> to vague, this needs to be more precise. are there references, if not the protocol needs to be described more thoroughly

- p3, l126: - 20°C -> -20°C (remove space)

- p3, l142: 0.2-µm -> 0.2 µm (remove dash)

- p4, l158: flow cytometry -> why was no amplicon sequencing conducted? this should allow a broader overview

- p4, l166/167: abbreviation in brackets, see comment p3, l114

- p5, l183: ... slight adjustments: avoid repeitions to things that have already been described in [39], but clearly state what has been adjusted

- p5, l211: the statistics part is very short and not thoroughly described. this part should be clear enough so that the statistical analysis can be reproduced if necessary.

Also in the methods, there are too many paragraphs, that make it difficult to follow the methods clearly. Please combine where it is useful. 

* results

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- p11, l327: (heterotrophic bacteria, Synechococcus, Prochlorococcus, picoeukaryotes) -> this is a limited overview of the environment

- p11, l329 and following: maybe a table would make it easier to read and compare those numbers

- p12, l360: why is the data not shown? is this something that can be put in the supplements?

- p13, l375: members of picoplankton community. -> members of the picoplankton community.

Same conclusive comment as for the methods, please make less paragraphs to improve the readability.

* discussion
----------------

- p16, l425: reference missing

- p16, l427-429: Our results also revealed the presence (...) of picoautotrophic cells coming from the Eastern mediterranean and from the North Adriatic Sea. -> How was this shown in the analysis?

- p16, l431: The authors demonstrate in this paragraph that the findings agree with previous studies, but it should be more emphasized what the new findings in this study are.

- p16, l433-435: We suggest that picoautotrophs (...) could bee a significant carbon source (...) in the form of dead or live prey ... -> what is the basis for this assumption?

- p16, l463: We can conclude that the results of this study support the idea that deep ocean prokaryotes are active as those living in the epipelagic waters. -> This is difficult to judge if the amount of investigated microbial taxa is so low. 

- p17, l472: Heterotrophic bacteria contribute to a higher percentage of total picoplankton biomass ... -> higher than what?

- p17, l482: ... and that they can control the bacterial community in surface waters: what do the authors mean with control? in what sense?

- p17, l490: Therefore, AAPs might represent up to 10% of total prokaryotes in the open Adriatic Sea. -> where does this number come from? reference would be helpful

* conclusions

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the conclusions are a place in the manuscript to show the advances of this study to what has been done before to make the conclusions stronger. this could still be improved.

* tables, figures and  captions

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- table 2: this does not provide a lot of helpful information here, the table can be moved to the supplements

- figure 2: Panels d, e, f shows ... -> Panels (d), (e), and (f) show ...

- figure 3: see figure 2

- figure 4: caption does not describe clearly panels (a) - (f)

- figure 5: see figure 4

- figure 6: you should make clear what is Dec2015/Apr2016

- figure 9: why are the variance values on the axes missing?

- figure 10: regression lines have no formula and correlation coefficient, this should be corrected. Besides, the layout and quality of this image should be improved, not just copied from Excel.

Ten figures is a very high number, please consider if all of them are necessary for the main manuscript or if some of them can be moved to the supplements

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

Dear Authors,

I would like to thank you for the contribution you made in the sent manuscript. I have read it with great interest. I find the topic significant, especially that Authors included AAP and VLS in their studies. This is an indication of novelty in this paper.

Generally, whole manuscript is well planned and written. I have found only minor mistakes in grammar (see below). Nevertheless, I have several remarks and concerns that need to be cleared up before the publication.

Introduction:

This part is written very well. I don’t have much to add apart from the comments below

Page 2, line 46: A remnant of previous citation style. Please remove the crossed-out date.

Page 2, lines 69-84: In my pdf, most of the text has different font size like it was copied from somewhere else. Please unify the font size.

 

Materials and methods:

Generally there was a high workload to collect and evaluate the samples. I find the experiments well planned, although I am wondering why there is no analysis on ribosomal sequences?

Page 3: Please state the depth from which the sample was taken. There is an information in the results, but it should be stated in this section of the text. Please elaborate on how the samples were taken. Have you used a standardized device, or maybe you had something of your own design? This should be elaborated and clarified. Please add information how you made sure that your samples from depth water were not contaminated with material from shallow water, etc.

Page 4, lines 158-159: The sentence starting with “Samples of autotrophic...” is not grammatically correct. Please correct it.

Page 4, lines 166-167: I understand that the division into two groups is a common practice, but why didn’t you use more specific method, e.g. above mentioned ribosomal sequences?

Page 5: Why AAPs were sampled only in April. I think it’s one of the main flaws of this paper, because the comparison would greatly contribute in this case. Nevertheless, it’s very positive to see that you included AAPs in the studies!

Results:

The results are well organized. It’s quite clear how to get through the data.

Page 6, lines 225-226: It’s good that information is here, but I would rather see it in the materials section.

Pages 8-9: Why oxygen data was taken from station 7? On the Figure 2(f) station 8 is indicated. Please make it consistent. Why the data is missing? Please make the description of figure 3 consistent with fig. 2.

 

Page 10: Figure 4 and 5 look the same to me? It seems that the content planned for figure 5 is depicted also in figure 4. Please comment on this and correct it.

Pages 11-12: Why didn’t you use other method allowing to better characterize described microorganisms (e.g. FISH)? Even though they can be counted on a flow cytometry as a group it would be great to see which ones are abundant since different strains have different toxinogenic activity. Nevertheless, I understand that this was not the main focus in your project. Please share what you think in the topic.

Figure 7: Please add discrimination which graph is from which month. I can see Roman numerals in figures, but it's not obvious at a first glimpse.

Figure 9: please indicate a) and b) in the description.

Discussion and conclusion:

Discussion and conclusions are drawn in a good scientific way. I have not marked any places that crucially needed corrections in this part. Please let me just express that I am very happy to see results on AAPs (even though it’s a pity there is no results from winter sampling) and VLPs. From my perspective, I would elaborate a bit more on the activity of VLPs. The hypothesis of lysogenic development seems legit, although it is not the only scenario that can take place. Phages and bacteria live in certain balance in the environment. I would look more closely at correlation between biomass-associated factors and VLP in addition to sampling time.

References:

References list looks good. You did a good job in selecting many papers from impacted journals. The list also contains recent works which indicates that the results were compared up to date. You might increase the number of papers from the last five years, although it is not crucial in terms of this particular paper.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors have made extensive improvements for the manuscript. In general, I have no doubts for submission in Water, however a few minor things still need to be adressed:

Introduction p. 2, l. 83: SAP is not defined before use (The definition of this abbreviation is now at the end of the introduction, please change that) p. 2, l. 85: closing brackets missing p. 2, l. 87: repetition of the word "bring" in this sentence, rather use "... transporting southward colder and fresher waters" Methods p. 7, l. 277: Maybe I was not clear in the first review: I did not mean to "define" statistical standard methods (PCA does not need to be explained here), but rather describe why PCA was used (this is done now in l. 280 and following). I would remove the sentence "PCA analysis extracts the eigenvalues and eigenvectors from the covariance matrix of original variables, thus reducing the dimensionality of the data set. Starting from the first axis, the importance of principal coordinates decreases in the order of their decreasing eigenvalues." because this is just a definition of PCA, and this should be done. What is missing however, are more details in order to allow to reproduce analysis and results (for instance, if you were using R instead of CANOCO, you could share documented R scripts on Github). Discussion p. 20, l. 548: I would rather use "In this study" than "In this research" (but frankly, both formulations are fine) tables and figures Figure 6 is referenced in the text before Figure 5, please check all figures, tables and references that they are referenced in the text in the correct order The total amount of figures and tables is now thirteen. This is a very high number. I would like to ask the authors to either consider moving some of that into supplementary material or comment why all of them are necessary in the main manuscript. Figure 10, last review: the authors responded "the regression lines don't have formula, because they were taken from Gasol (1994)". Perhaps this should be mentioned in the caption.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf