Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ROLES OF H2S AND NO IN REGULATING THE ANTIOXIDANT SYSTEM OF VIBRIO ALGINOLYTICUS UNDER NORFLOXACIN STRESS
Review round: 1
Reviewer: 3

Output:
Basic reporting: All the comments are provided in comments to author section
Experimental design: All the comments are provided in comments to author section
Validity of the findings: All the comments are provided in comments to author section
Additional comments: I have now finished the review on the manuscript titled " Roles of H2S and NO in regulating the antioxidant system of Vibrio alginolyticus under norfloxacin stress”. The problem of antibiotics stress has worsened over the last decade particularly in the developing countries. Besides, the intensity to tolerate varying degrees of antibiotics stress is significantly different among different microbial and plant species. Therefore, there is a need to evaluate role of antibiotics and their tolerance potential among different organisms. The authors studied the interactive effect of H2S and NO in regulating antibiotics stress and it contains valuable information. However, the following comments may be incorporated to improve the quality of the publication.
There are few grammatical errors in sentence formation as well as spelling errors. I am not mentioning the line numbers here and suggest a through reading of the manuscript.
Materials and Methods part is very important and should be reproducible. I have several concerns regarding different protocols and execution of different work. My main points are as under;
How did authors dissolve antibiotics?
The authors did not provide the references of protocols they followed in a number of places in materials and methods.
The authors have several typographic errors throughout the manuscript. They are suggested to read the manuscript carefully for typographic errors and grammatical mistakes.
The results are presented nicely. However, the authors have big standard errors. Is there some problem with making graphs?
The discussion part should include logical reasoning of the results obtained rather than just comparing the results with the literature cited.
The conclusion part is weak. The authors are suggested to improve it and also include the contribution of the results obtained to the existing knowledge. The authors are also encouraged to include how researchers could get benefit and plan their future research based on this experiment.
This paper contains lot of data and information that could be useful for the readers. Therefore, I suggest a minor revision to improve the scientific quality of the manuscript.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ROLES OF H2S AND NO IN REGULATING THE ANTIOXIDANT SYSTEM OF VIBRIO ALGINOLYTICUS UNDER NORFLOXACIN STRESS
Review round: 2
Reviewer: 1

Output:
Basic reporting: OK
Experimental design: OK
Validity of the findings: OK
Additional comments: The authors have satisfactorily responded to all the questions and made the necessary changes to the manuscript. I have no further questions and suggest the acceptance of the revised manuscript.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: UNDERWATER LIGHT CLIMATE AND WAVELENGTH DEPENDENCE OF MICROALGAE PHOTOSYNTHETIC PARAMETERS IN A TEMPERATE SEA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript by Michel-Rodriguez presents a large scale study of the wavelength dependent photosynthetic properties of natural phytoplankton groups, by using wavelength-dependent chlorophyll fluorescence analysis. The manuscript presents a valuable large scale assessment of several natural locations supported by a rigorous statistical analysis. The manuscript is clearly written in professional manner. However, some clarifications, further details and rephrasing is necessary about the methodology of chlorophyll fluorescence analysis and the interpretation of photosynthetic processes and parameters.
Experimental design: The aims and the research questions of the work are clearly defined and the experimental design was carried out with high standards and with proper rationale. Some further improvements are suggested in particular to clarify the details and explanation of the methods, as detailed below.

line 89: multi-colour pulse amplitude modulation fluorometer could be called as ‘fluorometer’, not a sensor, to be more specific.

line 102 and other places – the naming of the colors is somewhat inconsistent, blue is called dark blue at some places, the 590 nm spectral region is amber or orange – please be more consistent with colour labelling

line 167-170: hypothesis is defined, but it is not clear in the context of Fig. 1 and the Figure 1 also needs further clarifications. In each panel, 2 lines are presented, but it is not explained what do these actually show. Why does light quantity and quality response decrease with wavelength? Photosynthetic organisms has a defined absorption spectrum as well as an action spectrum, which depends on the pigment composition, functional light absorption etc of the given species. These properties may of course change with the light climate, due to chromatic adaptation and adaptation to different irradiance levels and the authors may wanted to define the hypothesis accordingly, however this is not clear from Figure 1.

line 252: dark adaptation time should be explained and justified. Is 2.5 h dark adaptation is the right time period to obtain maximal Fv/Fm? In some algal species, very long dark adaptation can lead to the operation of chlororespiration, which might lead to over-reduction of electron transport chain, which eventually cause a decline in Fv/Fm even in darkness. Therefore the authors should give more details on the selected dark adaptation methodology to give a clear reference physiological state for the multi-wavelength Chl fluorescence analysis. Furthermore, it is not clear whether far-red light was used for the multicolour PAM measurements. Far-red light would serve the purpose of keeping the electron transport chain oxidized and thus it would help establishing reproducible control conditions for the measurements (Schreiber et al. 2012). This may or may not be beneficial for the presented work, however it should be clearly described whether such reference state conditions were applied.

line 278: for light curve measurements, the authors presumably applied 14 actinic light levels or steps, not pulses. Please clarify.

line 306: define Zumixl at its first mention.

line 367: the details of PCA should be described in Materials and Methods. Accordingly, in Figure 4 more explanation of the PCA analyis and the denotions and labelings on the X and Y axes should be explained in the figure legend as well.
Validity of the findings: The results presented in the work are properly discussed. All related data is provided with high details and the conclusions are well supported by the findings. Some further improvements are suggested particularly for the Discussion, as detailed below.

line 529: define briefly the fucoxanthin chlorophyll protein antenna complex.

line 538-542: in this study not a green microalga, but Nannochloropsis (Eustigmatophyceae) was studied. Nannochloropsis sp. lacks Chl b and it lacks strong absorption capacity in the 540 nm spectral region, so it shows a quite different Sigma(II) spectrum as compared to the Sigma(II) spectrum of planktonic diatoms. Therefore it is somewhat unclear what do the authors mean that the result 'does not agree with those of a green microalga'.  Other studies showed that the spectrum of the absorption cross-section did not change significantly as a result of chromatic adaptation or photoacclimation to various irradiance levels in Nannochloropsis (see e.g. Kandilian et al. Bioresource Technology 137 (2013) 63–73). Therefore the Discussion should be rephrased in this aspect when comparing different microalgae species with respect to their different inherent pigment composition.

line 545-550: the extended absorption of fucoxanthin into the 'green' spectral region is not only due to its specific location and high amount in the light-harvesting antenna, but also as a result of its specific molecular structure, as it contains a carbonyl moiety on a polyene backbone (Premvardhan et al. 2008 and other cited works). As the main phytoplankton group analyzed by the authors are diatoms and other Chl a/c containing organisms, authors should reflect on the basic structural and functional properties of fucoxanthin and FCPs.

line 554-557: ‘The theoretical maximum of Fv/Fm is 0.7 (in a dark-adapted state, as in this study), and its critical value is 0.3’ - for which species is this true? The absolute or 'healthy' level of Fv/Fm can be very different across species, for example in some cyanobacteria the optimal Fv/Fm level is 0.2-0.5, due to the contribution of fluorescence of phycobilisomes. Therefore, when defining a critical threshold of Fv/Fm for ‘healty’ values, it always has to be defined under which conditons and for which species this is valid for.

line 566-568: ‘Although r.ETRmax and r.alpha had a significant trend across wavelengths, they do not provide useful information about the spectral dependence of photosynthesis’. Some clarifications may be necessary here. The photosynthesis vs. irradiance curves, or 'ETR curves' should display wavelength specific features, because if the light absorption is higher at 440 vs 590 nm or 625 nm, alpha should be higher and consequently sub-saturating irradiance, Ek (and ETRmax) should be lower at 440 than at 625 nm (see e.g. Figs 4-5 in Schreiber et al. 2012). Therefore, the 'relative' ETR parameters have important wavelength specific features. When the incident PAR is multiplied with Sigma(II), the PAR(II), the quantum absorption rate can be determined, from which the absolute electron turnover rate of PSII could be determined (eqs. 7 and 8), which theoretically is independent of wavelength (provided that the Sigma(II) scaling is correct).

line 573: ‘These results were unexpected…’ could it be the reason that some of the photosynthetic parameters exhibited diurnal fluctuations? These might show different patterns in different species, therefore it is hard to reconcile its role in a phytoplankton community.

Figure 5: the deviation is high in nearly all parameters, therefore it is hard to see any wavelength dependent trend. Perhaps only Sigma(II) and NPQ shows some trend. Apparently ETR(II) is suitably equalized because of the Sigma(II)-scaled relETR.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: UNDERWATER LIGHT CLIMATE AND WAVELENGTH DEPENDENCE OF MICROALGAE PHOTOSYNTHETIC PARAMETERS IN A TEMPERATE SEA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper is written in a good English style, the figures and tables are okay and literature is well referenced. Sometimes the text might be shortened and results/conclusion should be accentuated, as they are lost in long methodological/statistical descriptive text.
Experimental design: The paper is in the scope of the journal and fill knowledge gap. Some information about light measurements is missing or is yet not clear. Also some of the fluorescence measurements still need additional information.

For further details, see review report
Validity of the findings: The paper shows new findings on community level sampled in the field. It supports knowledge already known on the species level, but extends it to natural communities. The statistical treatment I can’t judge, as I am not an expert on it, but it seems reasonable to me.
Additional comments: Although the results of your study are important for estimating primary production in different water bodies, the outcome is not amazing, as it follows what is already known about photosynthetic adaptations/acclimation. However, these results support the common knowledge on a species level now on the community levels in the field, which will be useful for oceanographic research. So a publication is recommended.
But from a photophysiological point of view, there evolve several questions for me, which need to  be answered before publication. There are also some interpretations which to my opinion deviated from the analysis, which look too much mechanistic for me. Also I miss some methodological informations.
To investigate communities consistent of such different algae groups is really a hard work.  There are multifactorial effects, including photoprotection, absorption cross section, sieve effects (population density), transmission spectrum, irradiance level, nutrient concentration, sun position, season etc.,  which need to be considered.  For that, interpretation is difficult and has to be done carefully, as outcome of this analysis.
If possible the text should be straightened, and results/conclusion could be more accentuated.

In details, I  will go sequentially  through the manuscript with questions  and comments:

Line 97: most photosynthetic studies are done with red light, also for in vivo fluorescence or gas exchange measurements. Blue LED are a recent technical development and are only recently  on the market.
Line 113ff: It is a misbelief that brown algae are more efficient in photosynthesis the red ones. Ecologically, red algae/cyanobacteria are still growing at water depth where irradiances are too low for brown ones, because the construction of their photosynthetic apparatus is more efficient in energy transfer, nearly without energy loss.  Moreover, the green enhanced spectrum  typically for coastal water is best absorbed by the phycobilines.
Line 123ff: Do not mix low and high light conditions in one sentence. It makes it difficult to follow. Larger antenna size is a result of low light, higher reaction center number with smaller antenna an acclimation to high light conditions.
Line 135: energy absorption of fucoxanthin does not change; however, energy conduction can change with different position in the antenna construction.
Line 169: It would be better to have a clearer indication of the axis in fig 1. The direction in which photosynthesis changes or where wavelengths are longer are not visible.
Line 201: Surface reference measurements are made above or below the water border? It is important because of reflection which depends on the sun position and sensor characteristic (refractive index). If you have considered only spectra close to the water surface, how did you determine the absorption characteristic of the respective water body? You certainly need a greater depth range, to see differences within the respective spectrum. From only PAR measurements this  will be not visible. Information is missing.
Line 213: Also here, atmospheric measurement or sensor covered by water?
Line 243: Did you control the phytoplankton diversity by a microscopic check?  The FluoroProbe check is not enough, as especially Haptophyceae pigments are similar to those of the Heterokonts. With the low number of detection channels of the instrument  you might see changes  within  a community, but really it is hard to discriminate between the different algae groups with a similar pigment content.
Line 250: 440 nm I would not call dark blue it is nice bright blue and 625nm is light red with a touch of orange. Real red would be seen at 650 nm.
Line 254: ?  Suggestion:  First, each sample was homogenized within an optical cuvette by  a magnetic stirrer and then the light sensor was placed into the center of a quartz cuvette to measure the photon flux density at each wavelength using a US-SQS/WB….
Line 262: Fluorescence level of the dark adapted samples?  It is not clear if dark or light adapted sample. You may have adjusted to a similar Fo by the gain or measuring light irradiance, to get a good signal-noise ratio.
Line 278: better: 14 pulses of increasing  actinic light intensity, each 20 s long,  as defined in the PAR-list file
Line 268: PSI and PSII absorb light equally?  This depends on the antenna size of each photosystem! You mean you need at least two photons to drive both photosystem (factor 0.5 for one photosystem). The light absorption/energy conduction of the respective photosystem depends also on state transitions.
Line 375: I miss methodological information how the spectra light ratios were determined  (see above) . It would be good to get information, in which extent this has changed at the different sample locations, as this is one important point of this study.
Line 393: from locations 10 to 22 ? (see fig 4B)

General comment to Fig.5 : Biophysically spoken, with constant photon number, red light effect should be twice as high as blue light, as half of the energy of blue photons is lost by heat dissipation (S2->S1 singlet state).  Thus, NPQ (heat dissipation) must be higher in blue than red light (Fig.5 K,L). So you might say, red light is more effective. However blue light also activates the sensor needed for photomorphological effects in algae, which use e.g. cryptochoms.  However, this is independent from the energy usage of the photosynthetic apparatus. Thus, the higher energy content of blue photons is wasted compared to those of red photons. This shows your analysis, if it is not standardize to the absorption cross section, as the antenna in brown algae absorb more blue than red light. Only in red algae, the higher Chl content of PS I increase the Chl absorption cross section of PS I compared to PS II in the blue and red waveband. Here, PS II absorbs more in the green/orange waveband, which therefore is more effective.
Eop and NPQ is in the orange waveband lowest, as in your brown community it is less absorbed, and less effective.

Line 418: (NPQ625 reached 0.2) at PAR 300.
Chapter 3.4. Sorry, I feel lost as I don’t know your analysis; can it be explained more clearly?

Chapter 4.1.2: Fv/Fm is the maximum quantum yield, a parameter only valid for dark adapted photosynthesis.  A decrease indicates chronic photoinhibition or photodamage, a severe harm to the photosynthetic apparatus. Do not confuse with the effective quantum light in light conditions, which shows photoacclimation or light stress effects. It is hard to depress Fv/Fm already by nutrients, as algae do not perform photosynthesis and growth will stop when storage compounds are consumed. Thus, low nutrients level do hardly affect it. Starving plants can have also a high Fv/Fm, but will show a low delta F/Fm’!

Line 509: Of course, a brown community absorbs more light in the blue/green waveband, as already visible in the absorption spectrum, what does not mean that this will be similar to  the photosyn-thetic action spectrum. And absorption cross section of a cell is different compared to  a community consistent of  different cell densities and cell size  (e.g influence of sieve effect).

Chapter 4.1.3  Blue light dominates under water, so this may induce rather photoprotection then red light, what your results show.  If there is no red light, no “red” photoinhibition can be caused. Photoinhibition is caused by high irradiances, which algae may not encounter in the lower euphotic zone.  Only at the water surface this might happen, after longer exposition time. For red light alpha should be higher, as this is similar to shade light conditions. However, the cell does not encounter monochromatic conditions under water, so you need to consider always polychromatic conditions with supporting effects (e.g. Emerson enhancement effect).

Line 635: ETR max you will get only at saturating light conditions, which might not be yet reached by only higher irradiances.

Chapter 4.3.: How do you define photoinhibition?  Dynamic photoinhibition is photoprotection and not a damage. Chronic photoinhibition is when reaction center function fails or is damaged.  Dynamic photoinhibition/photoprotection you see when the effective quantum yield decrease and chronic when Fv/Fm decreases.
Line 676: light absorption and photochemical quenching, where is this shown in your data?
Line 727: A photoreceptor is a molecule which is not involved in energy transport within the photosynthetic apparatus. Photoreceptors are e.g. phytochroms, cryptochrome etc, which serve for signal transfer/conversion.

Conclusions:
Of course, natural phytoplankton communities can photoacclimate to light quality dynamically under contrasting environmental conditions. Otherwise they would not grow there and would disappear.  Your data show that results found in lab experiments with single species are confirmed by field studies and that the blue waveband is the most important abiotic factor for photoacclimation.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: UNDERWATER LIGHT CLIMATE AND WAVELENGTH DEPENDENCE OF MICROALGAE PHOTOSYNTHETIC PARAMETERS IN A TEMPERATE SEA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript by Michel-Rodriguez et al. presents an interesting study on the application of multicolor PAM to study the wavelength dependency of natural phytoplankton communities in the English Channel. The manuscipt is quite lengthy but it is generally well-written for a data-intensive manuscript, having the detailed information included to demonstrate its rigour. I think the manuscript is consistent with the scope of PeerJ.

My main critism is that the Abstract is over simplified and does not really reflects the essense of the findings. The authors should provide example of photosynthetic paramaters with values to support the argument/theories especially those related to functional absorption cross section of PSII, which is mentioned in the title but absent in the Abstract. E.g. in "The natural phytoplankton communities ... " (Line 26-28) and in "Two wavelength ratios ..." (Line 30-32).

The authors need to tune down the usage of PETlambda and list out some of actual photosynthetic parameters from time to time throughout the manuscript. I suggest sticking to either PET or PETlambda as there are mixed usage of PET and PETlambda, which can be confusing.
Experimental design: LLine 180: RAMSES has several radiometers. Please provide model and the type of sensors e.g. irradiance, radiance. More information on the deployment of radiometer and data analysis is needed. E.g. How was it deployed? Were both upwelling or downwelling data used and were there binning of the data?

Line 190: You can calculate PAR from the radiometer measurements. Why do you need a separate sensor to measure PAR? Please provide the model and type (e.g. 2pi or 4pi) of the sensor.

Line 220-221: Were all samples subjected to similar dark-adaptation period and at what temperature?

Line 258-260: Can the authors clarify how long it took for each RLC measurement for each sample. With 14 pulses x 20 s x 5 wavelengths, I estimated that each sample will take about 23 minutes to complete. In this case, will the differences in dark-adaptation period affect the output of the photosynthetic parameters?

Line 267: Please provide references to support that PSII and PSI absorb light equally and justify the arbitary factor of 0.5.

Line 305-308: Were the data mean-centred and normalised prior to the PCA analysis?

Ek(II) is an important parameter in the manuscript, however, the information on how it is calculated is missing.
Validity of the findings: Phytoplankton samples were collected at 2 m (Line 157). Were all the sampling stations shallow and well-mixed? How representative are the surface samples for the study area? And how do you relate phytoplankton collected at 2 m with Zeu and Eavg?

Line 193: Surface waves can strongly affect surface PAR (I0) measurements Stramski et al. (2008) and many parameters presented in this manuscript are relying of surface PAR values. Was there any precaution taken or solution to tackle this issue?

Stramski, D., et al. (2008). Relationships between the surface concentration of particulate organic carbon and optical properties in the eastern South Pacific and eastern Atlantic Oceans. Biogeosciences, 5(1):171–201

Line 213: What were the precautions taken to make sure that phytoplankton composition were correctly estimated by the FluoroProbe sensor?
Additional comments: Line 40: "from day to year". The respond of phytoplankton to light can be as fast as minutes if not seconds.

Line 54-56: I think it is a bit inaccurate to cite Schreiber (2004) chapter for PE curves as the chapter is focusing on PAM fluorometry unless the authors were referring to rapid light curves by PAM-fluorometry. I suggest citing conventional PE curves articles e.g. Platt & Jassby (1976).

Line 135: Please provide country of manufacturer.

Line 173: Change "fiberglass" to glass fiber or microfiber

Line 220: What type of net?

Line 245-253: Please provide the equation to derive Sigma(II) and give a brief description of O-I1 especially when mention for the first time.

Line 251: "A" in QA should be subscript.

Section 2.4: As I commented earlier, this is lengthy manuscript. I am not sure if it is necessary to listed all the R packages, especially those used for plotting e.g. biplot (Line 308-309), ggplot2 and ggpur (Line 343-344).

Line 593: "where we collected communities" something is missing here

Figures:
Figure 1: It is not clear to me what is the different between to two lines in each plot.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: UNDERWATER LIGHT CLIMATE AND WAVELENGTH DEPENDENCE OF MICROALGAE PHOTOSYNTHETIC PARAMETERS IN A TEMPERATE SEA
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors revised the manuscript appropriately. However, the taxonomic names should be double-checked and corrected as necessary. 'brown-gold' algae is rather a general than a scientific name, therefore e.g. for Nannochloropsis the species name 'Nannochloropsis oculata' and its respective classifications (Eustigmatophyceae) should also be described.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: UNDERWATER LIGHT CLIMATE AND WAVELENGTH DEPENDENCE OF MICROALGAE PHOTOSYNTHETIC PARAMETERS IN A TEMPERATE SEA
Review round: 2
Reviewer: 2

Output:
Basic reporting: See below general comments
Experimental design: Re-evaluate formula units.
Validity of the findings: no comment
Additional comments: The manuscript is improved and purged from methodical vagueness. However, I still have some question about calculation of Sigma (4) and ETR II (8), see below, which should be cleared.
The authors should also consider, if they would speak from algae in general, this would concern also macroalgae, which as benthic organism would show a different response to light quality and quantity. So it is recommended to speak only  from phytoplankton or pelagic microalgae.

Minor comments and corrections are:
Abreviations in Tab.1 are missing and should be included: PE, Pmax, CCA, EC, Zeu,, Zumixl, Eavg, Kd(PAR), O and I1, E avg, DIN, TSS, CDOM. Some are common but even not explained in the text.

Line 84:  E is not the abbreviation for irradiance? The response would be named P, or?
Line 202 better: was not uncovered by the waves
Line 212: LI-193 4pi sensor is from LICOR not made by Biosperical Instruments

Line 257: without far red exposure (that would have locked the device for too long with respect to the many measurements required).
Tip: It is not necessary to give a long FR irradiation, to get a significant increase of Fm by release of qE or qT, about 15 s irradiation is generally sufficient. Next time you may check this for measurements. However, in this case with 2 h darkness photosynthesis is already in the dark acclimated state with max. Fm, if no chlororespiration (energetisation of the thylakoids) cause a decrease of Fv/Fm again.

Line 264-269: Here, a part of the old sentence is not deleted, so it is double.
Line 274: the same current fluorescence (F) level of 0.5,    here  better to write Ft
Line 285 ff: this is misunderstanding τ must be the time needed to reach the I1-level, otherwise the formula is meaningless, as you have always irradiated for 1ms. According to your text τ would be always 1ms.
Line 287: Avogadro's constant (6.022.1023 quanta.(mol photons)-1), are the units right? quanta or photons? I suppose you have to delete quanta. Shouldn´t be Sigma dimensionless or m-1? Please control  units.
Line 290f: there is some repetition to the sentences above.
Formula 8: It is curiously as you divide by L for determination of Sigma and multiply it again for PAR II
Why?

Line 564: increase in the “green” absorption capacity of fucoxanthin, Not better due to fucoxanthin,?  as the molecule itself will not change its  absorption characteristic.

Line 626ff: you should write brown microalgae or phytoplankton because in multicellular macroalgae you will  observe  different result. Generalization is here not appropriate. The same for the algae groups mentioned later, because benthic macro- or microalgae may respond differently.

Line 850. different ,  typing error.

Supplements:
Fig 4s : Labeling of axis is missing, figure not complete


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: UNDERWATER LIGHT CLIMATE AND WAVELENGTH DEPENDENCE OF MICROALGAE PHOTOSYNTHETIC PARAMETERS IN A TEMPERATE SEA
Review round: 2
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No comment


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: UNDERWATER LIGHT CLIMATE AND WAVELENGTH DEPENDENCE OF MICROALGAE PHOTOSYNTHETIC PARAMETERS IN A TEMPERATE SEA
Review round: 3
Reviewer: 1

Output:
Basic reporting: All misunderstandings an errors are resolved.
The publication is from my view ready for publication.
Experimental design: All misunderstandings resolved
Validity of the findings: See above
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: CDEV: A GROUND-TRUTH BASED MEASURE TO EVALUATE RNA-SEQ NORMALIZATION PERFORMANCE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is well-written and has a clear structure. Unfortunately, I could not find the GitHub repo and check that the data and code are available, although stated in the main text l. 153 (see "Validity of the findings" comments where I debate for the code’s availability and checking it up before publication). I apologize if it is my fault and the link is already present in the manuscript. I recommend making it less obscure for the reader then.

Although I find the main text figures essential and relevant for understanding the main text, I could not say so about Figures 8B, D. I find data representation in these figures redundant and its purpose unclear. These two figures demonstrate the matrices of pairwise cdev values between different matrices after normalization.
These matrices have ones on the main diagonal, as expected from the properties of cdev provided by the authors in l. 198 (cdev(X,X')=1 when X' is a scaled version of X; and on the main diagonal X'=X).
The matrix is symmetrical, as should be according to the symmetry property of cdev (l.192).
Thus the main diagonal and lower triangle can be excluded from the visualization without the loss of significant information to the reader.
Finally, the values of cdev between different normalizers are not discussed in the text. It is not clear what conclusions can be made from presenting this data. I suggest the authors retain the first row of matrices 8B and 8D in the main text (with the exclusion of cdev(raw, raw), and rather place the full versions to Supplementary materials. Alternatively, the full matrices’ results can be explained in the main text if they are indeed relevant.

Few unclearly defined terms make the reader stumble and look for additional literature or other clues in the text:
- n and m in l. 155 presumably represent the number of genes and the number of samples, correspondingly. This is not stated in the text, though.
- "Oracle" (a title used for ground-truth normalization in the main figures) is not defined in the main text.
- For the simulations’ description, the authors use the terms "simplistic" and "realistic.” This corresponds to "single" versus "continuous" fold change in Table 1, correspondingly. However, to my understanding of Figure 2, these two regimes are also named "homogeneous" versus "heterogeneous" fold change. Intuitively, this seems natural renaming of these regimes. However, this confuses the reader as it also sounds like the introduction of the new entity in simulations. I suggest renaming the titles in Figure 2 and Table 1 to be the same.

There are few typos that can be easily checked for and corrected. I provide what I noticed:
- Figure 2 title "realistic" instead of "realistic"
- l. 381 "that will requires"
- l. 120-122 "we elaborate", but "We demonstrated" in the next sentence.
Experimental design: The paper's experimental setup allows for comprehensible and unbiased testing of both cdev performances as the measure of normalization quality and performance of different non-ground truth normalizers. However, because both of these aims are strived for, the authors' debate is not completely persuasive.

For example, in Figures 4, 5 the authors demonstrate the number of tests on simulated and real data that demonstrate that cdev sometimes produces results similar or better to the traditional FC MSE approach on ground-truth normalizations. Next, for testing different normalizers, the authors use only cdev as they assume its benefits proved.
Indeed, the authors demonstrate that there are cases when FC MSE does not detect the bad normalization (Figure 6), and cdev has great discriminative power. However, the authors do not describe the opposite cases clear from Figure 6A and Figure 5 "Heart-Lung" comparison, when cdev is closer to 1. At the same time, FC MSE is large and clearly demonstrates the low quality of normalization.
To solve this conundrum, I would suggest one of two (or both) improvements:
1. reporting a close-up examination of one opposite case in Supplementary materials (e.g., demonstration of normalization and expression heatmap before and after normalization for the case with FC MSE~1 and cdev ~4 in "Heart-Kidney" dataset);
2. providing the FC MSE alongside Figures 7 and 8 in Supplementary materials so that the reader can assess all normalizers' performance in an unbiased manner.

As a researcher who will potentially use cdev to assess normalization purposes in my experiments, I find it hard to judge when the normalization is good or not. In Figure 7A, one can observe that number of DE errors increases with larger cdev, but is there a threshold above which I should consider my normalization as "bad"?
I believe that the authors have all the power to take one step further toward this naturally arising "real-life" problem. I suggest the authors can propose the threshold value of cdev given the number of total and DE genes that will notify the researcher whether the normalization was "good" or "not".

Finally, if I do not have extensive computation powers to calculate cdev (as cautioned by the authors in l. 377-378), and I have access only to simplistic FC MSE, how can I rely on the results of this study to decide on the success of normalization? If not introducing the same threshold definition for MC MSE, I would suggest providing Figure 2 with FC MSE instead of cdev in Supplementary materials.

The author's selection of normalizers is made clear in l. 207: "These methods are chosen due to the simplicity of computation and/or availability of R implementation, which allows quick integration to the current analysis." However, the selection does not include the best existing normalizer called graph-based normalization (proposed by the authors earlier, Tran et al. 2020), probably due to its computational complexity. Nevertheless, I find it important (although optional) to add this normalizer and test cdev not only on mediocre normalizers but also for a range of real datasets with the best normalizer known to the literature (although only part of datasets can probably be processed with gbnorm).

The last unclear point on experimental design, can the authors discuss why the uniform distribution of fold change values was selected for simulations? Is it close to the distribution observed in real datasets?
Validity of the findings: The authors test a cdev method to assess the quality of normalization, which was proposed by the same team earlier (Tran et al. 2020). The usage of the already-published method debates for its technical validity, although I noticed two potential weak points.

First, when cdev is computed, it uses a conditional number of Moore-Penrose pseudoinverse multiplied with the expression matrix, or kappa. In R, there are several parameters of the kappa function that control the properties of calculation [1], including whether the exact or approximate result is returned. If the approximate calculation was used, can the authors guarantee its adequacy and numerical stability?
To my experience, the calculation of the smallest singular values may be poorly implemented in some programming languages and result in inadequate results. If the cdev code was made available, I would try to validate the kappa method on a number of datasets and check its numerical stability.

1. https://stat.ethz.ch/R-manual/R-devel/library/base/html/kappa.html

Second, cdev relies on the assumption that the normalization is agnostic to the scaling (l. 177-179). However, to my understanding, some DE calling algorithms rely on the absolute count of the gene expression to derive the statistical significance of expression changes (e.g., DESeq Anders & Huber 2010).
As I am not a specialist in limma toolkit used by the authors, can they comment whether it is not the same for calling DE genes with limma?
Also, I suggest discussing this problem when explaining the principles of cdev, since a perfectly normalized dataset by cdev criterion might require additional scaling to produce valid results with such a popular tool as DEseq.
Additional comments: I find the research an important contribution to the field, an extensive benchmark of two normalization criteria (cdev and FC MSE) for several normalizers and various simulated and real datasets. Although both testings against ground-truth with ERCC spike-ins (Tran et al. 2020), cdev, and FC MSE were introduced before, there is a clear need for a study like this one will describe the properties and behavior of these entities on a set of simulated and real-life datasets. Although not new, this paper's theoretical contribution is clear, as it provides the strict formulation and practical demonstration of cdev.
In my opinion, the paper lacks few improvements and additional validations before the publication.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: CDEV: A GROUND-TRUTH BASED MEASURE TO EVALUATE RNA-SEQ NORMALIZATION PERFORMANCE
Review round: 1
Reviewer: 2

Output:
Basic reporting: 0) l171-202: description of sdev is no clear. Authors introduce expression matrix as n*m matrix where n is number of genes and m is number of samples (l155). Then they define matrix B as XB=A where X and A are expression matrices of the same size. But on line 192 matrix B is n*n matrix while to make XB=A correct B should be m*m. Maybe authors use same variable in different places for different porpoises, but in this case it is still unclear whether number of rows (columns) in B is equal to number of genes or samples. From the text it look like latter is correct, if so B should be not a solution of XB=A but approximation, but it is not discussed. Authors suppose that B is diagonal, but in reality it obviously never will be diagonal at least due to random noise.
1) Authors should provide access to code: functions to compute sdev and examples of it application. Authors say that all code is available on GitHub but provide no links. It is very important taking into account uncertainties in description of the method.
2) l213: "The resulted log-cpm matrix was then passed to limma functions, along with the per-sample scaling factors computed according to each method." - sclaing factors usually used under cpm calulation (and cpms usually already scaled for them). So. mostl likely scaling factors were not supplied separately from cpms, please clarify
3) It is commonly assumed that count-based methods such as edgeR or DESeq are prefered under methods based on normal distributions such as limma. While I do not think that it affect result too much I would suggest to use more convenient methods or at least explain why limma was used.
4) S2: x-axis show samples and different spike-ins are shown by lines, right? I suggest to add this information into legend. Figure title is misleading because it shows read counts rather than correlation, one can show distribution of all correlation coeeficients as well. I would suggest to substract mean spike-in expression at least from dmelAC dataset to make agreement more visible
5) It is completely unclear what is shown on fig 2. Authors should explicitly describe the procedure. What "fraction of differential genes in scaling" stands for?
Experimental design: authors use proper experimental design
Validity of the findings: taking into account incomplete description of the methods it is hard to conclude on validity
Additional comments: Authors introduce new method sdev to compare two expression matrices for evaluation of different normalization procedures. I cannot see applications for the method in majority of RNA-Seq experiments because most of them do not use spike-ins that can be used to check normalization quality. But maybe sdev can be used to develop new normalization procedures while I'm not sure that they are needed. In any case currently it is hard to conclude on manuscript quality because source code is not available and methods description is not clear


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: CDEV: A GROUND-TRUTH BASED MEASURE TO EVALUATE RNA-SEQ NORMALIZATION PERFORMANCE
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors use clear, unambiguous and professional English in their paper. The background of the problem is well explained and supported with relevant literature references.
The structure of the article is clear and comprehensive. Provided tables and figures sufficiently illustrate statements authors make in manuscript.
Authors did not provide any links to code or generated counts data they used for the analysis.
Experimental design: The focus of the research is evaluating different normalization methods for RNA-seq based data, which is an important question in the field of Differential Gene Expression. It fits well the scope of the journal. Authors suggest a method that they claim outperforms existing popular normalization approaches.
Authors have used acceptable number of datasets differing in groups heterogeneity and effect size. Sufficient number of state-of-art normalization techniques were employed for the comparison.
Description of applied methods lacks detail and does not give sufficient information for one to reproduce the analysis. The main theoretical concept authors propose, in turn, is described thoroughly and in detail.
Validity of the findings: Authors compiled several RNA-seq experiments with ERCC spike-ins from several organisms, of different size, structure and with addition if different spike-in mixtures. On real data sets, it was shown that cdev shows results following the theoretical predictions. Also, based on the comparison of cdev results with log-fold MSE, authors claim that it is a more robust alternative to the latter one.
Conclusions of the paper are well-supported by the provided analysis results.
Additional comments: I suggest several things that can improve the work.
Counts data and code used for the analysis should become accessible, and more detail of the analysis to the methods section should be added.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: CDEV: A GROUND-TRUTH BASED MEASURE TO EVALUATE RNA-SEQ NORMALIZATION PERFORMANCE
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors resolved all the comments.
Like an overlay of page number and the figure caption (p.5), some stylistic problems should be double-checked. Also, the GitHub links still cannot be found in the text. Clearly, it's a problem with either compilation of the file or the submission system, as the authors have readily provided the links with the rebuttal letter. This can be double-checked with the editor or support staff.
Experimental design: The authors resolved all the comments.
Validity of the findings: The authors resolved all the comments.
Additional comments: All the concerns were thoroughly investigated and clarified. I believe that the manuscript is ready for publication with PeerJ.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: CDEV: A GROUND-TRUTH BASED MEASURE TO EVALUATE RNA-SEQ NORMALIZATION PERFORMANCE
Review round: 2
Reviewer: 2

Output:
Basic reporting: while in general the paper is clearly written and easy to follow it seems to over-complicates quite simple idea (see below).  Under used assumptions proposed method can be described in a much simpler way (ratio of maximal and minimal normalization coefficients) and implemented much efficiently (see attached R markdown).
Experimental design: study design is appropriate
Validity of the findings: findings are valid
Additional comments: Thanks to provided code it is now clear how cdev works. In general authors consider family of read count matrix normalization procedures that can be expressed as XB=A where X and A are read count matrices and B is m*m  normalization matrix where m is number of samples. So, this family of normalization methods include all possible linear transformation but do not include more advanced approaches that uses different normalization coefficients for different genes (as SCT from  Seurat for instance). As far as I know  out of the whole considered family of normalization methods only simple per-sample scaling (that corresponds to the diagonal B) is used in practice, and it is the only case considered by authors, most important properties of cdev take place only under this assumption. So, only diagonal B are reasonable and only them are considered in the paper. Diagonal matrix is easy to consider as a vector of normalization coefficients. Since singular values of diagonal matrix are equal to its diagonal elements, cdev is simply a ratio of maximal and minimal diagonal element (normalization coefficient). In attached R markdown I show that one-row function can calculate cdev 500-time faster, than method proposed by authors.
While cdev is rather trivial method, it still might be useful, the paper clearly show that it correlates with other measurements of normalization quality. But current description of the method is foolishly over-complicated and implementation is obviously inefficient.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: CDEV: A GROUND-TRUTH BASED MEASURE TO EVALUATE RNA-SEQ NORMALIZATION PERFORMANCE
Review round: 3
Reviewer: 1

Output:
Basic reporting: all my comments are addressed in the revision
Experimental design: design is correct
Validity of the findings: findings are valid
Additional comments: I still think that general case of sdev is emphasized too much in the paper taking into account that only special case of diagonal B is considered. But since it doesn't affect validity of the paper i think that whether this part should be shortened or not must be decided by editor or/and authors.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: THE CORRELATION OF WDR76 EXPRESSION WITH SURVIVAL OUTCOMES AND IMMUNE INFILTRATES IN LUNG ADENOCARCINOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: In the submitted manuscript, Fang et al. analyzed the expression of WD repeat domain 76 (WDR76) with tissues from Lung Adenocarcinoma patients to understand the correlation of WDR76 with cancer progression. At the same time, the authors analyzed the WDR76 levels to various databases to understand its correlation with various protein expressions related to cancer progression and immune infiltrates to suggest the potential functions of WDR76 in the regulation of immune infiltrates and cancer progression.
Overall, the manuscript has been with explicit language, providing a clear introduction of the significance of the study.
Experimental design: The experiments designed are well defined, addressing relevant questions. In addition, the authors provided detailed descriptions of the methodologies performed for the rigorous investigation.
Validity of the findings: The findings are interesting and highlight a current gap of knowledge of how WDR76 expression influences cancer progression.
A minor comment on the conclusion of the TIMER analysis is that while stating that WDR76 expression induces infiltration of CD8 T cells is counter-logical. Understandably the data show a positive correlation between WDR76 expression and CD8 T cells’ presence in the tissue. Infiltration of CD8 T cells indicates a positive immune response, which the data do not show. A suggestion for the authors will be to conclude the data as a positive correlation between WDR76 expression and CD8 T cells’ presence rather than using a strong word as ‘infiltration’.
Additional comments: However, certain points warrant further attention from the authors for a better reading of the manuscript.
Figure 1 is broken into two parts and discussed in two sections in the results, creating a discontinuity. Consider changing the text or figure sequencing for a better and linear flow of the paper for the readers.
Several times, the authors have used abbreviations without stating the full form. For terms like OS, DSS, and PFS, the authors used the full forms in the methods section, but please use the full forms first in the result section for better readability.
For other jargons, such as P, T, M stages, define the term and provide the context for addressing those stages in the analysis.
In figure 4, Uni or multivariate Cox analysis, further detailed analysis of the result is warranted. There are various parameters shown in the figure that was never discussed during the analysis.
In Figure 5, please consider listing the proteins based on the scores rather than alphabetic order for a better sense of the figure.
In the TIMER database analysis, the authors analyzed the WDR76 expression with the levels of various immune infiltrates. Simultaneous analysis of the cancer progression with the immune infiltrates is strongly advised to differentiate between the effects of tumor progression versus WDR76 expression. For example, it is not clear from the analysis if the CD8 T cell infiltration is the effect of tumor progression or WDR76 expression. This will also help understanding if the WDR76 expression of the CD8 T cell infiltrates or only the exhaustion of the CD8 T cells, as shown with GEPIA analysis.
Similarly, for CD4 T cell infiltration in TIMER analysis, consider analyzing Th1 cells and Treg cells separately. That would provide a better opportunity for parallel analysis of gene expression as done with GEPIA database.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: THE CORRELATION OF WDR76 EXPRESSION WITH SURVIVAL OUTCOMES AND IMMUNE INFILTRATES IN LUNG ADENOCARCINOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this manuscript, Fang et. al, investigate the role of WDR76 as a potential therapeutic target for LUAD. Overall, the article is well written, clear and unambiguous in most parts, though I would recommend proofreading by prefessional copywriter for occasional grammatical mistakes and inaccurate sentence construction. Proper background, literature references and context have been provided where relevant. I have some comments which I think the authors should work on before the manuscript is ready for publication.
Experimental design: All the bioinformatics and statistical analyses have been performed logically and accurately. The question is well defined and the analyses they perform accurately test their hypotheses. The method of analyses have been described with sufficent details in the method section.
Validity of the findings: I have major concern about how the authors interpreted their analyses of the TIMER and GEPIA database (Figure 6). They conclude that WDR76 expression showed positive correlation with some infiltrating immune cells (CD8+ T cells, macrophages, neutrophils), but negative with some (B cells and CD4+ T cells) but offer no explanation regarding why this is happening. Also the authors claim that WDR76 expression can be helpful in identifying patient response to immunotherapy. While this might be true, the contradictory role of WDR76 in increasing CD8+ T cell infiltration and at the same time increasing the markers of T cell exhaustion is confusing and not backing up the authors' claim.
This issue needs to be addressed more clearly in the discussion section (Lines 216-226), More emphasis should given on observations that back their claim (T reg cells, exhaustion markers, etc) and mention the contradictory effect of CD8 infiltration at the end.

Some minor comments:
1. Line 146 - Please write the full form of OS, HR, DSS, PFS and CI here.

2. Figure 1 and figure 2, better to show the individual data points for the graphs.

3. Do the authors have any insight on why in fig 1E, the only significant difference is observed between T1 and T2 stage and not between T1 and the subsequent advanced stages? This should be addressed either in the results or in the discussion.

4. In figure 4, it will be helpful if the authors have a subheading above the tables indicating that A and B show analyses for OS, C and D for PFS and E and F for DSS.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: THE CORRELATION OF WDR76 EXPRESSION WITH SURVIVAL OUTCOMES AND IMMUNE INFILTRATES IN LUNG ADENOCARCINOMA
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Fang et al investigated the role of WDR76 regulation in LUAD and its microenvironment and suggested that the expression of WDR76 was upregulated in lung tumors may have predictive value for LUAD prognosis. The manuscript requires major revision. One of the major concerns is that the authors made bold conclusions, such as “WDR76 was involved in the regulation of immune infiltrates and a potential therapeutic target”, based on preliminary correlation analyses without further functional validation.

1.	The authors compared the expression of WDR76 between tumor (n=535) and normal samples (n=59) from the TCGA cohort. The sample size of matched normal samples is much smaller than the tumors, and the sample size of the two validation data sets (GSE10799 and GSE27262) was not mentioned in the text. It is important to exclude tumors without matched normals and perform a paired tumor-normal comparison based on a combined cohort (TCGA, GSE10799 and GSE27262) to confirm the increased expression of WDR76 in the tumors.
2.	It is unclear how the WDR76 expression high and the expression low group was defined. It is particularly important for the survival analyses, as the two groups didn’t separate the overall survival very well, even though the p value was significant.
3.	Multiple testing correction is needed when testing WDR76 and immune cell markers in GEPIA.
4.	WDR76 may have predictive value for prognosis, but the authors didn’t test WDR76 inhibition in cancer cells, and therefore it is hard to conclude that WDR76 can be a potential therapeutic target as stated in the abstract.
5.	The authors need to be careful drawing bold conclusions without sufficient experimental evidence. As an example, data supporting the conclusion “WDR76 was also confirmed to be involved in the regulation of immune infiltrates in LUAD” is based on some correlation analyses without experiments testing the causality. WDR76 might be a biomarker but its association with immune infiltration does not necessarily mean that WDR76 directly regulates tumor immunogenicity.
6.	The writing of the manuscript needs improvement. P values and effect sizes should be cleared stated following every association test.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: THE CORRELATION OF WDR76 EXPRESSION WITH SURVIVAL OUTCOMES AND IMMUNE INFILTRATES IN LUNG ADENOCARCINOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have responded sufficiently to the issues raised in the review.
Experimental design: Experimental designs were rigorous and well defined.
Validity of the findings: The authors' response to the review and corresponding changes in the manuscript has strengthened the paper.
Additional comments: With the additional revisions, the manuscript is ready to be accepted for publication.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: THE CORRELATION OF WDR76 EXPRESSION WITH SURVIVAL OUTCOMES AND IMMUNE INFILTRATES IN LUNG ADENOCARCINOMA
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors have addressed all my concerns and the manuscript is ready for publication.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EFFECTS OF A NATURAL PRECIPITATION GRADIENT ON FISH AND MACROINVERTEBRATE ASSEMBLAGES IN COASTAL STREAMS
Review round: 1
Reviewer: 1

Output:
Basic reporting: As far as I can judge, professional English is used throughout the manuscript. However from place to place, authors have made either typographical errors (and miss words sometimes) or do no use the right tense.
li 24. why "also"?
li 43. "changes in water temperature "
li 93. "for" evaluating.
li 95. replace "to climate" by "the climate"
li 125. "was"
li 138. "were"
li 150. "field rinsed"???
li 195. "we need to know if this was "all" environmental variables or "each" environmental variables (it seems it is all.
li 219. "positively related with precipitation"
li 240. "included"
li 286. "Pisidium" in italics
li 290. Oksanen (2013) is not in the references. I guess you mean Oksanen et al. (2019)
In some place the paper is rather heavy on-going. For example the long description at li 223-230 or at li 239-250 are not very useful as they are and could be simplified. In fact li 251-260 are a direct product of what is described in li 239-250 and the two parts could be mixed and lighten.
Li 378-384. What a very, very long sentence.

Li 404-406. I hardly see the strength of this sentence. Do we need it? Either too mich or not enough. Why do not you use traits in this paper? The study would be probably benefit from this.

Li 409. Acknowledgements look strange?

Li 442. Strange references. Is it complete?

In the version I have label of Figure 1 is for Figure 2 and vice-versa? Generally speaking I find the references very "US"oriented. You may take advantage of literature abroad. May I suggest to have a look at DOI: 10.1111/j.1365-2486.2007.01375.x (sorry if I  indicate a paper I contribute to. It is just an example, there are probably many others ) and also DOI: 10.1007/s10750-012-1244-4.

Raw data Are shared including the statistics, which is very nice from authors. Almost nothing is hidden and the objectives of the paper are clear but probably too ambitious with the present data set. On li 47-49 "clarifying mechanistic links between climate drivers and instream biological communities will improve our ability to predict to the effects of anthropogenic climate change on lotic ecosystems." In the present version, the approach is rather correlative than causative. This is already OK to show correlations and it is necessary to have large scale observational studies but it cannot be further argued that causal relationships are detected there.

As a result, I totally disagree with the statement on li 314-317. Correlation is not causality. By the way the observed correlation is notre straightforward. Why should precipitation influence conductivity and nutrients directly? Is it because precipitation wash of ions? In addition the next sentence is ackward as you tell us here that one of your site is biased.
I am also not convinced by you sentence on li 324-327 for the same reason. In addition could we have similar compositional shift in other environmental situations?
Experimental design: Li 158. In the appendix it is strange to see the same numbers for different taxa and different sites. Why is this?

This study is original though it would gain to add some literature that has addressed similar topic with different angles.The research question are well-defined and consist mainly in identifying patterns of diversity and composition of fish and invertebrate communities associated to changes in precipitation and identify environmental drivers that mediate the effets of climate on community processes. From the the first objective I do not see why precipitation should have a direct effet on diversity and composition. It seems that the answer lies in the second objective but it is a bit tricky and the present version is not that convincing. We would probably need some model of the cascading effects from precipitation toward aquatic communities to better catch the ideas of authors. By the way, I do not see which community processes are addressed in the present version (dispersion? reproduction)

My main concern goes to the number of sites used to address this ambitious questions. I do not think at this scale with 10 sites one can catch all the natural variability of aquatic biota even if other factors are controlled (geology, elevation, temperature).
Validity of the findings: I am quite skeptical about the validity of findings because the statistical analyses lacks  strength. As I said above, authors sampled 10 sites all possible regression between pairs of environmental variables and between environmental variables and biological metrics. In the appendix-regression 125 linear regression were performed. We have another set of 52 p-values in the appendix-ordination. The chance that one p-value become <0.05 just by chance increase with the number of tests. Authors should thus use a correction to the p-values. I corrected the 125 p-values of their appendix-regression and found that from the 29 significant results, false discovery rate adjustment put to the fore only 6 pairs. I think the NMDS approach is much more useful and consistent than the single regression approach. By the way why not a multiple regression approach which would help to  answer the second objective. Of course with only 10 sites, no more than 3 uncorrelated environmental drivers should be used in the model.
Additional comments: There is the general interest in using space-for-time studies to address potential environmental shifts in aquatic biota. However, in this paper,  I think we need a better integration of the statistical testing and underlying hypotheses. Maybe an additional tables showing the mechanistic relationship among environmental variables could help to better discuss results and how these relationships scale up to aquatic biota would be helpful. The question remains whether 10 sites (representing a substantial amount of work) are sufficient whereas  large data surveys are currently made in the US and could provide much more sites along the precipitation gradient. Maybe the NMDS thoroughly commented would be enough. Finally, the introduction addresses the environmental filers framework. It is strange that natural disturbance is not mentioned. Precipitations will change water quantity but also the extreme events will be accentuated by climate change. Natural disturbance is a very highly structuring factors of aquatic communities and dozen of papers have addressed this. This driver could be better addressed in the present paper.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EFFECTS OF A NATURAL PRECIPITATION GRADIENT ON FISH AND MACROINVERTEBRATE ASSEMBLAGES IN COASTAL STREAMS
Review round: 1
Reviewer: 2

Output:
Basic reporting: I thank the opportunity to review the manuscript by Kinard et al. In this study the authors conducted field samplings for fish and invertebrates in 10 wadeable streams along a gradient of natural precipitation. They considered it as an interesting natural experiment since other environmental variables are varying little in the region, not confounding the causal linkages between precipitation and biological changes. They found a strong compositional shift in both biological groups but only fish diversity responded positively to rainfall increase.

Overall, this is a robust study in terms of field methods, biological sampling, and processing, as well as taxonomic identification and counting. The experimental design looks indeed interesting and the results are promising. I commend the authors for the detailed biological consideration of each species found, as well as for the detailed description of biological processing. However, I have some concerns regarding the chosen analyses and mostly with the discussion. The analyses cannot fully show what the authors claim during discussion whereas the discussion itself is too focused on subjective links between species and habitat characteristics. It could be better to focus on the main point of the introduction, talking about communities in the general terms, as well as to making inferences about the future impacts of climate change.

Answering to the specific points by PeerJ:

English: English is good, but some subjective adjectives used could be avoided.

Literature: Literature is updated, but I miss some key references when discussing intermittent streams (e.g. T. Datry, N. Bonada).

Structure: Some parts of the introduction could be shorter and only included in methods. The figure must be improved and better organized in rows and columns.

Results and hypotheses: Results seem robust and well sampled, but the chosen analyses made understanding difficult.

My detailed comments can be found in the attached document that I hope can be helpful.

Regards,
Victor Saito
Experimental design: This is one of the strengths of the study. The experimental design is within a natural experiment and the samples were based on well-known protocols with large evidence of suitability to the questions made. Only small detailing is needed.
Validity of the findings: The data seem robust, but could be better presented. The NMDS is not informative on the relative abundance and fidelity of taxa to a given type of stream. I include some ways to improve it in the attached document.
Additional comments: This is an interesting study and can be a good contribution to stream ecologists. I hope you can modify it in a way that strengthens your findings and make the conclusion clearer.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EFFECTS OF A NATURAL PRECIPITATION GRADIENT ON FISH AND MACROINVERTEBRATE ASSEMBLAGES IN COASTAL STREAMS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript has an interesting focus. It is well written, but the introduction lacks context. A more robust hypothesis would make the authors' objective clearer.
More details in the PDF file.
Experimental design: There are some problems with the methods. lack of references. and the analyzes used are not the most suitable for the data. See comments and suggestions for analysis in the annexes.
Validity of the findings: At some points the conclusions are vague. they need to be more connected with the results obtained and the objectives of the study.
Additional comments: The theme of the manuscript is current and the way in which the authors justified the need to assess climate gradients in the face of climate change is very interesting. However, the data still need to be better organized to show the existing patterns. I strongly suggest rethinking some of the analyzes used. It is a robust database and contains important information about biological community changes in streams.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EFFECTS OF A NATURAL PRECIPITATION GRADIENT ON FISH AND MACROINVERTEBRATE ASSEMBLAGES IN COASTAL STREAMS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The study largely improved from the previous version. The authors did a good job in addressing the major points from my review. There are however some points that still need consideration (see other boxes).

1- English and writing are clear and professional.
2- Literature is appropriate.
3- The structure and figures are much better than the previous ones.
4- Results are relevant and can be used to tackle the hypotheses. Some parts are too speculative, but I detail them below.
Experimental design: The study design is much clearer now and the advantages of the study area for the hypotheses tested very well written.

1- The study is an original primary research.
2- The research question is well defined.
3- The investigation methods and analytical techniques are appropriate.
4- Methods are well described, but I miss a reference or more information for the constrained RDA.
Validity of the findings: The findings are sound in general, but I have one point of concern. The discussion around invertebrate community changes is too speculative and not based on proper results. The idea of an abrupt change is also not well based or explained. Moreover, the correlation between precipitation and diversity seen to be impacted by an outlier that could be tackled by robust regression.

1- Replication within a stream is appropriate. Replication among streams is small but the discussion around the results are appropriate and concern mostly the studied region.
2 - Necessary data was clear for this assessment.
3 - Only the discussion of invertebrates' abrupt changes are too speculative.
4 - Speculative about invertebrates are taken too far supporting the conclusion.
Additional comments: Abstract:
-change USGS gauged to ‘monitored streams’
-remove ‘significantly’
-‘a variety of omnivores and piscivores fish’
-why consider the changes abrupt?

L47: ‘in general’
L 68: Perhaps complement with 'as long as species can disperse to suitable habitats'
L 114: ‘characterization of fish and invertebrate communities and quantification of environmental variables.’
L 121: ‘The link between stable conditions and habitat heterogeneity is not very clear.’
L 147: Ok, so the sampling effort was proportional to stream width, but not equal among sites. Am I correct? How do you compare abundances among sites? Using individuals per square meter?
L 169: You need to describe this type of information for fish as well.
L 186: gathered?
L 216: Because of this property, diversity indices are commonly criticized because the changes you are seeing cannot be interpreted as pure changes in richness or equitability (e.g. Magurran and McGill's book - Measuring Biological Diversity). I would recommend you to have also a pure metric of evenness, even if only to show on the Sup. Material. This would allow us to understand the changes in Shannon Diversity.
L 233: And which function for the constrained RDA? Also, I think the specific method for constraining an RDA is new (I did not find the information on the two references provided), so a reference would be nice.
L 250: How correlated they are? Some of the Shannon diversity changes could be due to changes in evenness and this would be nice to show.
L 261: This is because it is constrained before the RDA, right?
L 277: One site with very low precipitation looks like an outlier here. You could try a robust regression that accounts for extreme values because overall the scatterplot shows a decreasing trend in diversity with increasing precipitation.
Paragraph 401: This paragraph is mostly based on non-gathered results. You do not have data on basal resources, competitive specialization, competition among species, and predation pressures. Altogether, this logical construction is too fragile. Moreover, it is not well explained why these changes could be considered as abrupt ecosystem shifts. Abrupt shifts have breaking points and your RDA shows a continuous change in composition.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EFFECTS OF A NATURAL PRECIPITATION GRADIENT ON FISH AND MACROINVERTEBRATE ASSEMBLAGES IN COASTAL STREAMS
Review round: 2
Reviewer: 2

Output:
Basic reporting: "no comment"
Experimental design: Materials and methods:
Line 158:  sweep (15 s duration) sampling from a representative distribution of best available
R: Isn't it a short time? is there any reference that can help saying that this time is enough for the collection?
Line 162-164: In the lab, samples were spread across a gridded sampling tray and randomly selected grid cells were picked to completion until the total count was > 300 individuals (USEPA 2015). Samples containing less than 300 individuals were

R: see this sentence with a different font.

R: In the end of the “Analyses” section authors can add that all scripts were available in the supplementary material.

R: In figure 3, the graphs could be enlarged. They were very good, but small.
Validity of the findings: Line 347-349: (Williams 1999; Dehedin et al. 2013). We also noted but did not quantify higher concentrations of silt in the semi-arid streams with prohibitive implications for nesting species (Jones et al. 2015).
R: I think this is speculation, maybe I would remove that sentence or identify.

R: The result of the regression between canopy cover and shannonfish remained to be discussed further. I suggest that it is not an expected result (Dala-Corte et al. 2020, Journal of Applied Ecology), but the authors need to discuss more about this because this result brings new information that is the influence of the precipitation gradient associated with canopy cover. Are precipitation and canopy cover correlated variables? It would be relevant information. Until maybe authors use the factor as a covariable. So authors can see if the canopy cover can influence the fish divesity however it depends on precipitation gradient. Also, this result “fish shannon negatively with canopy” is in the abstract ... I think that the authors can remove from abstract and discuss more about it in the discussion section.

Line 371-372: Red shiners (Cyprinella lutrensis) were curiously absent from semi-arid sites and were only present in four mesic and sub-humid sites.
R: I suggest modifying the word "curiously". Example: “Contrary to expectations Red shiners are absent….”  I think the language gets more scientific.

Line 374:  from the rainfall-gradient effects and coextended with Rosgen (stream morphology) and
R: I suggest removing "Rosgen" leaving only "stream morphology", since the authors have already explained that it is a proxy in the material and methods.

Line 445 -450:  hey warn that regions expected to become more arid, like Central and Western Texas (Jiang and Yang 2012), could expect a loss of competitive taxa with low environmental tolerances as observed here with centrarchids, ephemeropterans, and trichopterans. And that in their absence, rugged and euryhaline taxa (like livebearers, burrowing gastropods and predatory invertebrates) flourish.
R: This part need to be summarized on the abstract it is a very important conclusion of the research.
Additional comments: Abstract:
Line 36-37: These results indicate that small future changes in precipitation regime in this region may result in abrupt transitions into new community states.
But what would these new communities look like? Modify something? I think here authors have to make it clear that the modification would not be very good as it would increase/decrease diversity? Or change competition? Function on community?

Introduction:
Line 112-119:
This study conducts the first bioassessments of stream biota in this region. So, we established a framework for discussing abiotic and biotic filtering processes in community assembly, followed up by several expectations based on existing literature and precipitation-driven patterns that can be observed at a glance within the region.
R: I understand that this is a pioneering work. But it does not justify the lack of some information. Throughout the introduction, the authors talk about changes in the biota of fish and macroinvertebrates. I think the authors can be more specific on this issue. for example: diversity increases with rainy and decreases in the dry region ... Or decreases the richness of fish species in a given place. I want to make it clear that the authors have already put references on this, I just think that adding specific information, like the one I mentioned above, would help to understand the objectives and predictions of the manuscript. In my opinion, using results from work done in drought and wetter regions would only improve the introduction of the manuscript.

References:
R: The manuscript needs a revision of the references because they are not standardized with the journal's norms.
R: For example: names are different abbreviated in the first and write out in the second.
Line 510 - Fetscher, A. Elizabeth, Meredith D. A. Howard, Rosalina Stancheva, Raphael M. Kudela, Eric D. Stein, Martha A. Sutula, Lilian B. Busse, and Robert G. Sheath. 2015. “Wadeable Streams as Widespread Sources of Benthic Cyanotoxins in California, USA.” Harmful Algae 49 (November): 105–16. https://doi.org/10.1016/j.hal.2015.09.002.

Line 514 Fréjaville, Thibaut, Albert Vilà‐Cabrera, Thomas Curt, and Christopher Carcaillet. 2018. “Aridity and Competition Drive Fire Resistance Trait Covariation in Mountain Trees.” Ecosphere 9 (12): e02493. https://doi.org/10.1002/ecs2.2493.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: CROSS-REACTIVITY AND INCLUSIVITY ANALYSIS OF CRISPR-BASED DIAGNOSTIC ASSAYS OF CORONAVIRUS SARS-COV-2
Review round: 1
Reviewer: 1

Output:
Basic reporting: •	The introduction and background nicely presented and relevant literature cited. The sentence describing the study objective is too long. Plz simplify it and if possible divide it into two sentences. (line # 91-93)
Experimental design: •	The authors have listed various freely available softwares for in silico analysis for detection of mutations in the existing CRISPR-Cas-based diagnostic assays. However, considering the constant addition of SARS-CoV-2 sequences from around the globe, I highly recommend the authors to make a web-based tool. The user interface of this web tool incorporates all the free softwares enlisted/required in the assessment of mutations. This will allow clinicians and researchers to quickly assess the presence or absence of mutations in target regions of any future diagnostic assay.

•	Alternatively, to automate the pipeline for basic scientists and clinical researchers the authors can make their methods pipeline in the form of a script to be run using command line (cmd for windows/Terminal for mac).

•	The analysis part seems quite laborious and require manual inspection of thousands of sequences.

o	The sequence alignment can be easily replaced by ‘R’-based script which performs the sequence alignment (‘msa’ package). The script can also include code for inspection of sequences and determine the variability. Furthermore, PNNS can also be incorporated in the script. For more information please read the following links.
https://www.bioconductor.org/packages/release/bioc/vignettes/DECIPHER/inst/doc/ArtOfAlignmentInR.pdf

https://bioconductor.org/packages/release/bioc/vignettes/msa/inst/doc/msa.pdf

https://cran.r-project.org/web/packages/ggmsa/vignettes/ggmsa.html
Validity of the findings: Not applicable at this time.
Additional comments: In my professional opinion the authors should automate their methods either by making a web-based portal or making an 'R'-based script. However, if they feel that at this time they do not have the expertise or time to make an automated solution, the current methodology is totally acceptable for the purpose of publication.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: CROSS-REACTIVITY AND INCLUSIVITY ANALYSIS OF CRISPR-BASED DIAGNOSTIC ASSAYS OF CORONAVIRUS SARS-COV-2
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript proposes a bioinformatic pipeline for analyzing the specificity of CRISPR gRNAs used in CRISPR-based SARS-CoV-2 diagnostics. However, no actual sequence analysis is performed and no results are obtained and no original data presented. This manuscript would fall under the category of "Hypothesis Papers".
Experimental design: Not applicable - the manuscript does not present original results.
Validity of the findings: Not applicable - the manuscript does not present original results.
Additional comments: Some suggestions:

1. This manuscript proposes to assess the on-target specificity of CRISPR gRNAs used for SARS-CoV-2 diagnostics. This bioinformatic analysis could be improved by incorporating a counter selection against sgRNAs to exclude those that cross-react with other viral, human or bacterial genes.

2. Line 80-83: The author states, “One of the focus of CRISPR-diagnostics remained to improve assay specificity as tolerance of mismatches by Cas9 can result in a false-positive diagnosis. This risk has been minimized with the newer Cas proteins, Cas12 and Cas13, that have a lower tolerance for mismatches compared to Cas9 especially in the “seed” region.” .

This is not accurate — Cas9 is not used in CRISPR diagnostics because, unlike Cas12 and Cas13, Cas9 does not exhibit non-specific trans-activity upon target sequence binding. It is the trans-activity of Cas12 and Cas13 that enable these proteins to trigger a detectable signal upon target sequence binding/identification.

3. Line 58: the author gets the nomenclature between Cas12a and Cas13a incorrect. Cas12a = Cpf1, Cas13a = C2c2.

4. Line 15: what is a stage 1 protocol?


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: CROSS-REACTIVITY AND INCLUSIVITY ANALYSIS OF CRISPR-BASED DIAGNOSTIC ASSAYS OF CORONAVIRUS SARS-COV-2
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The author is going to use the protocol he used already for qPCR test systems. He made corrections accordingly to suit the new test target and offered the new design to incorporate a broader virus sequence set. Overall, the intro is sound. There was one technical typo: in line 58, Cas12a, and Cas13a former names are mixed up. Author might want to add the reference to  another Cas13a-based assay CARMEN by Ackerman et al., Nature, 2020 (nature.com/articles/s41586-020-2279-8).

Although comparing the protospacer to the alignment is more straightforward than comparing three-component qPCR oligo sets, several concerns should be addressed:
1	PAM may have degenerate bases by its nature;
2	PAM length may vary from one-two bases to six-seven for different Cas proteins and mutant derivates thereof (https://www.addgene.org/guides/crispr/#rna-targeting);
3	PAM orientation is relevant to protospacer;
4	Seed regions, as pointed by author, differ for Cas proteins, so the influence of mismatch position and mismatch count varies;
5	Different Cas proteins tolerate mismatches differently;
6	SNP frequency should be assessed correctly after batch alignments concatenation.
As it is  only planned to study around a dozen of protospacers, all these questions can be easily addressed.
“More than one mismatch” threshold offered by Bioinformatics Institute for CoVsurver author is planning to use in this study might be to broad. If 60000 isolates would be used, two sequences with the same mismatch will represent only 0.003% of the set. For CoVsurver, authors use a threshold of more than one functional mutation, i.e., leading to an amino acid substitution. Variation type does not play a role for Cas-based test purposes. This strict threshold may lead to wrong conclusions.
From the Cas-based SARS-CoV-2 detection assays designer standpoint, it should be mentioned, that they had small maneuver space, as they should find protospacer common to all SARS-CoV-2 strains, yet unique to SARS-CoV-2 coronavirus, and Cas-specific PAM should be adjacent to the target region. Compared to a qPCR test, for Cas-based tests, specificity depends only on one oligo strain of approximately 20 bases, but on the other hand, it makes Cas-based tests less prone to mismatches.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: STRESS DISTRIBUTION IN THE BONOBO (PAN PANISCUS) TRAPEZIOMETACARPAL JOINT DURING GRASPING
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Overall, this is an original study that provides needed information on the form-function relationship between the TMC joint morphology and hand grip use in bonobos; a primate species that is very under-represented in anatomical and biomechanical studies on great ape hand use. The manuscript is written in a clear and accessible style, enabling relatively easy appreciation of what can sometimes be an overly-technical topic. The small sample size is quite understandable, given the difficulties in obtaining cadaveric specimens of captive bonobos. Despite this limitation, the methods of data collection and analysis are appropriate, increasing the power of this study. The figures illustrate the text well and present the data clearly. My only substantive comments are quite minor.