PONE-D-22-12849Dissecting plankton patchiness through autonomous platforms and in-situ optical sensors.PLOS ONE

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"Partial data, including one of the AUV missions and tracking of subsurface chlorophyll maxima has been published in Fossum et al 2019 Science Robotics. Fossum et al is a technical paper focused on the adaptive sampling of subsurface chlorophyll maxima. Our article use parts of his data, such as the 3D plots of chlorophyll distributions (Fig 3b,d) and parts of the Silcam image (Fig 6, 30m MLD only - although we pooled the data at different depths, so it is not an identical figure) in region B. In our manuscripts, we add unpublished data (data from region A) to compare the contrasting patterns of patchiness between the two regions. Besides, we add unpublished photo-physiological data from the FRRf (Fig 7), nutrient data (Fig 4) and phytoplankton and meso-zooplankton counts (Fig 5). While Fossum et al is a technical and methodological paper for engineers, our paper is more interdisciplinary and the discussion is focused on the bio-physical interactions that shape plankton patchiness."

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Reviewers' comments:

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Comments to the Author

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: N/A

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: No

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5. Review Comments to the Author

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Reviewer #1: This manuscript reports the results of detailed surveys of the Norwegian coastal sea. It is an exemplary combination of conventional oceanographic sampling, AUV profiling and “in situ” methods. The results from the two contrasting study areas highlight the importance of episodic mixing events in the productivity of shelf seas and outline key adaptations, including photo-physiology of the dominant species involved. While the study highlights the importance of subsurface chlorophyll maxima (SCM), it also draws attention to the significance of episodic mixing events in recharging nutrients. As such the study is a really useful addition to the literature on the biogeochemistry of coastal seas.

On the whole, the manuscript is put together well, however there is some unnecessary confusion regarding the labelling of the different study areas and the methods of “pooling” of data. More detailed recommendations regarding this are given below, together with a list of minor points of phrasing or ambiguity that require correction.

“Pooled stations” and location of Fig 3 data – these issues are confusing.

This requires some revision of Figures 1 and 3 and captions and in the text.

To avoid confusion, the survey areas should be designated A and B at the outset and should be indicated as such on Figure 1.

The “pooling” concept is currently mentioned in the manuscript in the following order:

In the “Materials and Methods” section under the “Sampling” heading, it is stated that vertical profiles were taken at five stations, presumably the stations numbered 1 through 5 on Figure 1.

“Pooling” is first mentioned in the caption to Figure 1:

“c, Detailed map showing the area where the AUV mission was conducted and stations pooled based on similar characteristics (for more details, see results section)”

In the key to Fig. 1 “pooled stations” has black line, whereas the stations appear to be enclosed with lines of different colour (grey and red). In the absence of further clarification, this is confusing. There is a small black line to the SE of station 5, but I assume this is not what is meant. It would be better to indicate “pooled stations” in the key with a closed loop.

In fact, the “more details” are given, not in the results section (as stated in the caption to Fig. 1), but in the “Materials and Methods” section under the “Data Analyses” heading, where it is stated that the station profiles:

“were pooled (averaged by depth) as stations 1 and 2, and stations 3, 4 and 5. Pooling of these stations were (sic) based on similar hydrographical structure ….”

It would be clearer if the “pooling” or averaging was clarified earlier.

The case for “pooling” would be stronger if the salinity/ temperature/ chlorophyll – depth profiles of the five individual stations were shown in the supporting information.

This confusion is further compounded by the lack of adequate detail in Figure 3 and its caption. Figure 3a and 3b are the AUV profiles and are labelled A and B in Fig. 3, but are not labelled as such on Fig. 1. In the caption to Fig. 3, the two AUV survey areas are designated “A-left and B-right” but there is no A or B label on Fig 1. Do A and B refer to the NE and SW AUV surveys respectively? This needs to be clarified in the figures and captions.

Do Fig 3c and 3d represent the multiple AUV profiles from the two AUV profile areas? It would help to clarify this – also the caption incorrectly has these as Fig 3 b,c.

Finally, the single panel in Fig 3e contains what I assume to be the shipboard profiles

from both pooled stations 1 and 2 and from pooled stations 3, 4 and 5. It would be clearer have these as two separate panels beneath the corresponding AUV survey panels above, depicting results from areas A and B.

Also, it looks as if the plots in Fig 3e contain multiple superimposed profiles and not and averaged profile for the two pooled areas (as suggested in the text). This should be clarified.

Minor points:

Line 19: replace “at” with “off”

Line 24: should this not be “observed above the base of the mixed layer”?

Line 27: does “twice lower” mean “half”? – if so best to change

Line 37: insert “a” after suggesting

Line 38: should read “Our results emphasize”

Line 46: delete “by”

Line 54: delete “water layer, namely”

Line 60: add “the development of” before SCMs

Line 62 replace “is” with “are”

Line 71: replace “vastly” with “extensively”

Line 80: insert “the dinoflagellate” before Tripos – to be consistent with diatom mention

Line 82: insert “the” before “main”

Line 122: “close in time” – vague

Line 153: “mode strategies” – meaning unclear

Line 157: unclear to what this velocity refers

Line 162: double bracket

Line 165: replace “information” with “determination”

Line 226 delete “This means that”

Lines 238-239; need to mention areas A and B – se detailed comments above.

Line 330: “(and vice versa)” – unclear to what is meant here

Line 337: insert “for” before “which”

Line 409: insert “an” before “episodic”

Line 410: insert “an” before “event”

Line 413: should be “layers”

Line 414: should be “dinoflagellates”

Line 422: replace “on” with “within”

Line 423: insert “a” after “Such”

Line 437: replace “has” with “have”

Line 443: should be “suggests”

Lines 452-454: confused sentence – need re-write

Line 456: replace “mixing” with “mixed”

Line 460: should be “conditions”

Line 479: should be “ciliate”

Line 487: delete “even”

Line 497: replace “was noticed” with “occurred”

Line 499: delete “were”

Line 506: should be “A shallow…”

Reviewer #2: Review of the manuscript entitled « Dissecting plankton patchiness through autonomous platforms and in-situ optical sensors” presented for consideration in Plos One by Fragoso et al.

General remarks.

In this manuscript, Fragoso and co-authors reports the use of various instruments during a sampling effort in northern Norway, following a sampling from inshore to offshore waters. During this sampling, AUV have been deployed to measure classical T,S, chla measurements while other instruments were deployed from the boat itself, including an imaging instrument (silcam) and an optical one (FRRf). The course of the sampling was perturbated by a strong mixing event (without clear definition of its timing compared to the cruise. Was it before or during? If during, I would say between station 2 and 3 but this remains a guess), which adds to the duality offshore/inshore a second level of duality (mixed vs non-mixed) which do not ease the interpretation.

The aim of the dataset gathered and the purpose of the sampling cruise are not clearly defined and may look as the result of a test deployment (or maybe it was not the case and this was omitted or got aborded as a result of the “strong mixing” happening between the two group of stations). Consequently, the observations do not clearly target any precise process and are hardly demonstrating clear interactions between the different measurements conducted (see below on the mismatch between some measurements cross-interpreted). Moreover, there is a clear decoupling between the title (patchiness is nearly not studied nor dissected here, only the presence/absence of a sub-surface chlorophyl maxima -SCM -is reported), the introduction (with a clear demonstration of the importance of different processes to control the SCM) and the latter lack of discussion or exploitation of those processes in regards to the results. Even the AUV results (done to “track the tri dimensional chl a patchiness”, line 75) are only used to confirm the mixed vs non mixed nature of both environments (fig 3) but is never used to explore the said patchiness.

This said, at sea everything happens and the best should be tried to use the collected data. However, the manuscript also suffers from three large weaknesses:

1) First of all, the quality of all figures is just unacceptable as it. They arrived really pixelated and at least Fig 6 is purely not usable: plankton images are not visible: is it really plankton that we are supposed to see? Fonts are too small to not be blurred pixels- on a full-page figure-. Some axes do not make sense (see vertical axe on figure 5e) or are missing critical information (where are A and B zones on figure 1; how are located currents mentioned in the text?). All this also cast doubts also on the quality of image collected and how they are identified or used, and is not helped by the fact that it seems that the images were only classified by an algorithm (line 167-171) without human expertise and without estimation of the efficiency of classification. This is clearly not acceptable without an estimation of the error made or at least a verification of a subset of the image collected.

2) Most of the results and conclusions relies on the discrepancies between counts of large phytoplankton species (Tripos from both Silcam and microscope counts; Proboscia and ciliates from microscopy) and other measurements relying on in-situ Chla observed by optical means of through filtration on GF/F filters (0.7µm) and chla measurements. However, this left most of the phytoplankton, and their associated chlorophyll a or optical photosynthetic parameters, from 0.7µm to several tens of microns unmeasured by counts. Therefore without at least realizing this discrepancy and recognizing this bias, any conclusion based on such uncoupling between counts and chlorophyll / optical measurements are purely speculative if not purely wrong…. since they could not be coupled together. At least using flowcytometry to fill this size gap should have been conducted, but other methods also exist.

3) Finally, most of the discussion seems to be either very speculative or to ignore potential other hypothesis. Indeed, most of the results are interpreted as if the two zones were relatively similar in their structure prior to the wind mixing. Therefore, all explanations to their potential differences are interpreted to be the result of the mixing event (that have happened during or before the cruise? This is not specified…) and to their protected/exposed nature. However, without measurements showing that they were similar in their nature before such interpretation is just purely speculative. There is an alternative explanation: that they were already different prior to the mixing. Indeed, most of the observed uncoupled associations between nutrients/phytoplankton/zooplankton seems to be coherent with a top-down control (from zooplankton on large phytoplankton? From larger predators on zooplankton?). Without exploring other hypothesis and processes which may have caused the observed differences between the two sites, the discussion is not only speculative but also very partial and oriented in the presented conclusions.

Because of the above-mentioned weaknesses, I could not recommend publication, at least with a clearly major change in results uses and discussion.

Detailed remarks

Title: I am not sure after having read the full manuscript that any results were really exploiting the “patchiness” aspects of the data (except maybe the fact that there is a deep chlorophyl maxima), not to talk about “dissecting” this patchiness. On the same aspect, I was really enthusiast about the title which let me think that in-situ optical sensors were used onboard autonomous platforms (gliders)…. While in fact it is not at all the case. The abstract did not clearly correct this and this is only in the material and methods that I finally understood that it was not the case. Because of those two points, the title is misleading and should be changed or corrected.

Line 19: adaptative sampling should be understandable by someone that are not familiar with gliders deployments: please explain here.

Line 26: “exposed” exposed to what? (maybe just used inshore/offshore)

27: if sub-surface chlorophyll maxima is correlated with a decrease of abundance of those two large phytoplankton cells, maybe this should indicate that other phytoplankton are responsible for this:

Line 91 (section study area): most of this section present why the study area is a great study site in term of scientific background (reduction of seabird etc…). Therefore I suggest moving most of this section to introduction.

Line 95-96: Could you please indicate those two currents on the Fig 1 to guide the reader?

Line 118: Fig 1 should be called since there.

Figure 1: A and B boxes (used latter) should be indicated here. The line style and line width of those boxes should be darker and larger to be easily spotted (this is not the case here). Consider as well that the color choice for depth is potentially bad: impossible to disentangle depth 225 from depth 600. Additionally, I guess those depth should be rather explained with range of depth (e.g. 200-300; 300-900m), except if the Norwegian seafloor is peculiar in its bathymetry.

In what extend data collected with the boat and with the glider could be compared (notably true since the glider in box A operated in deeper waters (225? 600?) than the close-by sampling stations (125m).

Line 143: AUV date deployment? Was it the same AUV deployed two times over short period or two deployed over a longer period?

Line 167-171: automatic recognition is often not enough (but see https://doi.org/10.1146/annurev-marine-041921-013023 ). Were the images manually inspected ? Was the efficiency of the image recognition evaluated by any means? The images provided as examples in figure 6 do not allow any understanding on the quality of the process. How much images were acquired? How much classified/ verified by an human expert?

Line 180: ST is an acquisition protocol not a measurement (line 176), please rephrase.

Line 186: nice explanation, but why not having repeated the same kind of explanations for other parameters bellow. (And no, table 1 does not further demystify those)

Line 210: this protocol leads to measure chl a in >0.7µm organisms. Which could potentially explain much difference in between some given parameters (see bad coupling with large phytoplankton reported in abstract). Having this compared with Lugol staining and microscopic counts without explaining what count was operated do not help the reader. What minimal size was considered? Or did only the two target phytoplankton species were counted (which have size >20µm at least)?

Line 216-219: Ethanol fixation can cause shrinkage of organisms and potentially bias the samples. How this may bias the results or did only a count was done?

Line 243: why did you pooled those samples? Was the station 5 not acquired? If yes, this should be mentioned since the material and methods. From the fig 1, it seems more logical to pool station 4&5 to compare with the glider, station 3 been right in between the two glider areas. I do understand that this grouping was made on hydrographic structure … which is never shown on a per profile basis.

Results:

Line 266-267:there is no way to say that the vertical structure of AUV and CTD show similar patterns here, and it is impossible to judge this without plotting those side by side with similar measurements: here temperature, chlorophyl and salinity are shown from AUV while density is displayed only from boat measurements (no temperature/salinity, chl a?).

Is the difference of MLD due to the inshore/offshore nature of the stations or did the wind mixing occurred somehow between station 2 and 3?

Line 290: add space

Line 302 and onward (not a comment but important for latter comments): here is related that deeper-mixed waters have less nitrates/ higher (large) phytoplankton (but no SCM) and lower zooplankton. On the contrary shallow mixed waters have higher nutrients, low (large) phytoplankton (but high Chla) and high zooplankton. All this looks like the usual symptoms of an ecosystem under top-down control.

Discussion

Line 410-411: very speculative, from the results obtained, this could also be due to a trophic cascade (top down effect) rather than the bottom up effect proposed. But without temporal observations, this is hard to say. Did the AUV were at sea a long time and could provide such temporal vision?

Line 431: reference 41 format in different.

Line 442-458: this part is really speculative since those observations could be the results of several inhomogeneous measurements: Chla both seen by sensors and measured on filters is usually from small size fraction (filtered onto 0.2µm filter). The fact to have measured low abundance of two large phytoplankton is not an indication that all phytoplankton were low (and higher Kd seem to indicate larger particles load….. usually correlated with small phytoplankton or sediments). All this part relies on the fact that all phytoplankton are supposed to be represented by the two target species identifies and counted on microscope. Since this is very likely not the case, the whole part needs to be modified to take this into account.

Another important thing is that those high chla/ low phytoplankton have large amounts of zooplankton (which prefers larger preys) and may help in amplifying this discrepancy.

Line 472-484: all this explanation (on differences between zooplankton and phytoplankton) relies on the assumption that all zooplankton reacts like ciliates (with rapid respond to phytoplankton bloom). However, half of zooplankton measured are instead copepods, which does have a lifetime of the level of months and then could not respond within the (ungiven) timeframe between mixing and sampling. Therefore, this part sound really speculative. Moreover, it relies on the untold assumption that conditions were about similar before the mixing, which is again unlikely to be the case. One other explanation is that deeply mixed waters stations are naturally low in abundance of zooplankton…. And this could be due to higher predation onto zooplankton (fishes?). Since the described process sounds really in line with top-down control of those ecosystems, I suggest the authors to rethink their results in this respect and not necessarily try to explain those by bottom up control processes (note that both could happens together).

Figure 5: the bottom panel (e) have a depth axis together with taxonomy

Figure 6: this figure has really a bad quality and do not allow to neither read correctly the axis not see the images that are supposed to represent plankton species … is the present state, this is not possible at all and could not be used at all to understand results. Such low quality figure (not that all the other ones were badly pixeled too) is unacceptable and could not be reviewed.

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Reviewer #2: No

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PONE-D-22-12849R1Contrasting phytoplankton-zooplankton distributions observed through autonomous platforms, in-situ optical sensors and discrete sampling.PLOS ONE

Dear Dr. Fragoso,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Sep 09 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

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Emmanuel S. Boss

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

Dear authors,

I have read the reviewers comments and your answers as well as the revised manuscript and found your answer and the manuscript adequate.

One minor point you may want to revise is the interpretation of the data as suggesting either top-down control or bottom up control in each of the two cases.

I think that looking at the ocean as usually being close to steady state (loss=growth) with perturbation in the system allowing for improved growth (increased biomass) or with stagnation resulting in reduced growth (and hence decreasing biomass) with mixotrophic organisms contributing to tip further the balance may be a useful framework.

Thus when NPP is strong grazing maybe equally strong resulting in no net phytoplankton biomass accumulation but high concentration of grazing indicators. Similarly, when NPP is weak, there can be no net phytoplankton biomass accumulation, but low concentration of grazing indicators.

It is very rare (e.g. harmful algal blooms) where only one side is winning (hence observed accumulation of phytoplankton in the field are typically an order of magnitude smaller than their specific growth rates).

Feel free to not revise based on this advice.

Best, Emmanuel

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Contrasting phytoplankton-zooplankton distributions observed through autonomous platforms, in-situ optical sensors and discrete sampling.

PONE-D-22-12849R2

Dear Dr. Fragoso,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Emmanuel S. Boss

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Dear authors,

I am happy to accept your important contribution to PLOS One. 

Also, thank you for taking my non-binding comments into heart.

I was NOT fishing for citations, but if you found the BB2018 paper of relevance that is great.

One more comment: if the growth rate of phytoplankton gets a boost (due to nutrient input), it will likely, in a first stage cause growth processes > loss processes (resulting in accumulation, or dP/dt>0).

This is likely to be followed by increase in losses (due to increased contact rate) bringing things back to quasi steady-state (growth=loss).

Hence to keep accumulating over time (long term 'blooming') growth needs to continuously improve relative to loss. This does not mean that loss is not decreased during mixing (it might very well do so) but simply that what we observed is the imbalance between growth and loss processes, never one in the absence of the other (except, maybe during HABs).

All the best, Emmanuel

Reviewers' comments: