Seasonal Changes in the Size Distribution of Copepods Is Affected by Coastal Upwelling

Round 1

Reviewer 1 Report

While the authors concede that refinement is needed, the method used is innovative and promising. I wish something like it was available in my graduate school days when I spent endless hours counting copepods and other meiofauna.

I think it would help those of us not familiar with FlowCAM to visualize this method if some microscope images of copepods and their conversion into ellipses were included.  

I've indicated in the marked manuscript some suggestions for English and clarity.

 

Comments for author File: Comments.pdf

Author Response

Dear reviewer, thank you for your valuable suggestions, which have contributed greatly to the improvement of our study. So, below are the indications and changes according to your suggestions. The comments of the reviewer indicated in the text lines, and my revisions in Answer.

 

Line 29 - planktoni has been changed to plankton.

Line 64 - Delete OK

Line 72 - Yes it makes sense and we change it to: Despite copepods being highly diverse and abundant, ...

Line 119 – Thisstudy was changed to This study

Linha 146-147- (Linhas atuais 148-149) É necessário manter esta sentença porque as informações de espécies e classes de tamanho foram realmente apresentadas na Tabela 1. Com o microscópio, todos os copépodes foram identificados até o nível taxonômico mais baixo e medidos com a ajuda do Zeiss© Zen ImageLine 281-283 Foi retirado do texto.

Linha 301 (Linhas Atuais 298) - picos foi alterado para pico

Linha 307-313 - Foi retirado do manuscrito a pedido de outro revisor.

Reviewer 2 Report

Based on abundant environmental and planktonic data from 2010 to 2014, this paper investigated the effectiveness of FC as a tool for classifying copepod size and abundance, and the effect of sea temperature cycles on copepod assemblage. The experiments have been rigorously designed and conducted. However, there seems to be quite a few misleading citations of figures and tables in the result part, and it will be better if there could be more about the findings about the effects of environmental changes on the copepods in the abstract (which seems to be the main object of the paper inferred by the title). Comments have been marked in the attached PDF.

Comments for author File: Comments.pdf

Author Response

Dear reviewer, thank you for your valuable suggestions, which have contributed greatly to the improvement of our study. So, below are the indications and changes according to your suggestions. The comments of the reviewer indicated in the text lines, and my revisions in Answer.

 

Line 37- From CF to FC (Current line 39).

Line 119- Thisstudy to This study.

Line 156- (Current line 157-158) Because it was the size of organism that the FlowCam was able to analyze without obstructing the flow.

Line 257 (Current line 260) The figure was updated.

278 - It seems that the abundacea data in figure 6 are from eit her optical microscopy or FC. No correlat is show in Fig 6.

Answer: (Current line 274) The information passed on line 278 is not really from figure 6. Therefore, I placed the text at the top, which corresponds to figure 4.

Line 281- It says tabela 1 shows microcopy measurements, but why is it cited here to demonstrate the FC results?

Answer: (Current line 271) Table 1 is actually showing the results of the conventional optical stereomicroscope. The call to Table 1 has been removed from this part of the text and placed in the appropriate part.

Line 282- Are these two R2 values from linear correlation analisis of abundance data from microcory and FC? Then, it should indicat e the FC abundance data are reliable when r2 is close to 1. How ever the two r2 values seems to be too low.
Answer:  Indeed the R2 values were low and the idea was to show that the smaller copepods were easier to process than the large ones. However, there is no reason to use R2 for the organisms since it was presented below to show the efficiency of the method for the copepod assembly as a whole.

Line 286 - Also it is very confusing that fig 6 was cited herein.

Answer: Yes, it really was in the wrong place.

Line 335 - Maybe it will be better if the authors can explain a bit about how to read this figure

Answer: (Current line 323) Correlations between Chl-a and temperature with different size classes of copepods. Above and next to the scatter plots are histogram plots with the size classes of the copepods and the linear correlations are showing the positive or negative relationship between the size classes.

Line 317-319 – Little or no difference in percentage between copepods with prosome lengths ranging from 200 µm and 500 µm occurred during the first semester, but during winter and spring, more than 55% of copepods were very small with a prosome 319 length between 100 and 300 µm.

Answer: Actually, the sentence was confusing, but it was rewritten.

We changed to: (Current line 307-308) During winter and spring, more than 55% of copepods were very small with a prosome length between 100 and 300 µm.

Lina 453 –Paracalanus quasimodo: It was put in italics (Current line 450).

Reviewer 3 Report

Thank you for the study on so relevant topic - getting the very much needed numbers out of the pelagic system to comprehend the functioning of the food-webs in the situation of upwellings. Use of automated counting possibilities is a way to future, although a lot of small and hugely important developments are needed here. Your manuscript is definitely a contribution in this sector. Also - the automated counting based on size definitely increases the number of counted samples but can’t be used as the exclusive method of sample analysis. Species composition should be checked regularly as it is not only the body size but also ecology of species impacting the functioning of food-webs.

Still, there are some issues to be improved or corrected within your manuscript for better consistency and accuracy. My suggestions and questions to consider are following the order of manuscript chapters.

Abstract

28 -29  Isn’t a noun missing after “ planktonic”?

37 - Abbreviation CF is not explained anywhere in the abstract

The last sentence of the abstract is not supported by conclusions.

 

Introduction

79 - 80 Please rephrase:  It is true that smaller size copepods inhabit the upper layers but statement “large copepods can perform diel vertical migration efficiently” doesn’t explain that the larger copepods are dominating in deeper layers. Besides, also small copepods are performing dial migrations. 

83-92  If the seasonal variation of body size is known then it is possible to apply the seasonal values of individual body mass for total biomass calculations, without regular size measurements. This approach is used in the Baltic Sea already for many years. https://helcom.fi/wp-content/uploads/2019/08/Guidelines-for-monitoring-of-mesozooplankton.pdf

 

Materials and Methods

Starting at 126 - please rephrase, the sentence is difficult to capture - "They both were considered proxies for shifts in the ecosystem status, being upwelling (<20°C) [37] and bloom of phytoplankton (>2.0 mg.m-3)." 

In 129  - “while zooplankton samples were analyzed only on full moon days, in the morning.”  I suppose, it is “sampled” instead of “analyzed”? And if so, why only at full moon days?

What was the speed of horizontal hauling and what was the type of the net? Was a flowmeter used when hauling? Were the samples fixed (preserved)?

What is the total depth of the sampling station?

 

Results

The question arises whether the estimation of the mean values is so important if you were looking for seasonal differences. Could you reflect on that?

 

242 -244 The sentence starting with “This highly oscillating …” is not clear

 

276 - 279 “The copepod abundance results revealed a significant (p <0.01) positive (R2 = 0.45) correlation between the abundance estimated by conventional optical microscopy and FC (Fig. 6), though the densities estimated by FC were one magnitude order below those expected according to microscopy.”  The Figure 6 does not show this correlation, at least there are no explanations for it. What data is Figure 6 actually showing?

Why is Table 1 refered when explaining FC estimations in Line 281, as Table 1 contains only microscopy results?

284 - 286 Despite the underestimation of copepods’ total abundance made by FC, the overall seasonal distribution coincided during most of the time series with the abundance of dominant P. quasimodo (Fig. 6). This cannot be deducted from Figure 6 - either the Figure should have additions in data or additional explanations to the symbols.

 

307 -310 Style of the paragraph should be changed according to the principles of sentence construction in English.

Also, measurements of three year data are showing the natural variability but no the trend! At least 10 yrs are needed to calculate the trend (White, 2019; BioScience 69: 40–46). Please rephrase here!

 

Discussion 

Paragraph starting at 398 - if the aim is to assess also the biomass it is not recommended to use damaged organisms for further calculations. If the animal is damaged , i.e., missing a body part, then it will classify as a detritus in the food-web but not as a food resource for fish as it is not swimming any more. Please consider it when describing the additions of body shape by image analysis software.

 

Conclusions

The last paragraph actually repeats already published findings, cited in the Introduction (Line 51-63). Could you formulate more clear your original conclusions?

The content of the abstract is not supported fully with the content of conclusions. 

 

 

Author Response

Dear reviewer, thank you for your valuable suggestions, which have contributed greatly to the improvement of our study. So, below are the indications and changes according to your suggestions. The comments of the reviewer indicated in the text lines, and my revisions in Answer.

 Abstrair

Line 28 -29 Isn’t a noun missing after “planktonic”?

Answer: abundance

Line 37 – Abbreviation CF is not explained anywhere in the abstract

Answer: Explanation of what CF is in line 30.

The last sentence of the abstract is not supported by conclusions.

Answer: I agree and with that we put the last sentence to penultimate and the penultimate to last, thus finalizing the conclusion at the end of the summary.

Introduction

Line 79 - 80 Please rephrase: It is true that smaller sized copepods inhabit the upper layers, but the statement "large copepods can perform vertical diel migration efficiently" does not explain that larger copepods are dominating in deeper layers. In addition, small copepods are also performing display migrations.

Answer: Small copepods usually dominate the superficial warm waters, while large copepods can perform vertical diel migration efficiently

Line 83-92 If the seasonal variation in body size is known, then it is possible to apply the seasonal values of individual body mass to total biomass calculations without regular size measurements. This approach has been used in the Baltic Sea for many years already. https://helcom.fi/wp-content/uploads/2019/08/Guidelines-for-monitoring-of-mesozooplankton.pdf

Answer: Yes. Indeed this will be the goal of further studies, thank you very much for the relevant suggestion.

Starting at 126 - please rephrase, the sentence is difficult to capture - "They both were considered proxies for shifts in the ecosystem status, being upwelling (<20°C) [37] and bloom of phytoplankton (>2.0 mg.m-3)." 

Answer: They both were considered proxies for shifts in the ecosystem status, being upwelling <20°C [37] that provides a bloomof phytoplankton >2.0 mg.m^-3.

At 129 - "while zooplankton samples were analyzed only on full moon days in the morning." I assume that is "sampled" rather than "analyzed"? And if so, why only on full moon days?

Answer (Line 130-135): Because higher zooplankton biomass is found in this lunar phase.

What was the speed of the horizontal conveyance and what was the type of network? Was a flow meter used during the transport? Were the samples fixed (preserved)? What is the total depth of the sampling station?

Answer (Line 130-135): On each sampling date, three sub-surface (1 m depth, a 2 nós) horizontal hauls of three minutes each were performed in sequence, giving a total of 180 samples using a 100 μm mesh 40 cm diameter WP2 plankton net in water of about 10 m deep. Immediately after collection, samples were fixed in a 4% formalin solution diluted with seawater and previously buff-ered with sodium tetraborate.

Results

The question arises whether the estimation of the mean values is so important if you were looking for seasonal differences. Could you reflect on that?

Answer: I understand that it is very interesting, but I confess that I had not thought about making this kind of estimate. I will think about it, thank you very much for the suggestion.

Line 242-244 The sentence starting with “This highly oscillating …” is not clear

Answer (Line 245-246): The Chl-a over the weeks in the resulted from occasional peaks that significantly deviated from the global average.

Line 276-279 “The copepod abundance results revealed a significant (p <0.01) positive (R2 = 0.45) correlation between the abundance estimated by conventional optical microscopy and FC (Fig. 6), though the densities estimated by FC were one magnitude order below those expected according to microscopy.”  The Figure 6 does not show this correlation, at least there are no explanations for it. What data is Figure 6 actually showing?

Why is Table 1 refered when explaining FC estimations in Line 281, as Table 1 contains only microscopy results?

Answer (Line 272-275): Realmente não tinha necessidade de chamar a tabela se informar o que ela estava passando. Com isso chamei a tabela 1 depois desta frase: The dominant species most measured were: Onychocorycaeus giesbrechti, Onychocorycaeus ovalis, Oncaea media, Paracalanus quasimodo, Temora stylifera and Temora turbinata (Table 1).

Line 284 - 286 Despite the underestimation of copepods’ total abundance made by FC, the overall seasonal distribution coincided during most of the time series with the abundance of dominant P. quasimodo (Fig. 6). This cannot be deducted from Figure 6 - either the Figure should have additions in data or additional explanations to the symbols.

Answer: Really this information is not from this study. The sentence has been removed.

Discussion 

Paragraph starting at 398 - if the aim is to assess also the biomass it is not recommended to use damaged organisms for further calculations. If the animal is damaged , i.e., missing a body part, then it will classify as a detritus in the food-web but not as a food resource for fish as it is not swimming any more. Please consider it when describing the additions of body shape by image analysis software.

Answer (Line 395-397): Your explanation is actually correct and the FlowCam took several photos and when we reported a lateral, frontal position that it was not possible to measure the copepods correctly, damaged copepods were also included. With this, the denified copepods were discarded and these were not counted or measured.

Conclusions

The last paragraph actually repeats already published findings, cited in the Introduction (Line 51-63). Could you formulate more clear your original conclusions?

The content of the abstract is not supported fully with the content of conclusions. 

Answer (Line 494-490): Actually, the last paragraph was repetitive and similar to the introductory part that you indicated, but as the study focuses on size classes, I removed the entire part of the phytoplankton cycle that was published by Gonzalez-Rodrigueze et al., 1992 and left it the size classes of copepods in the upwelling cycle.

Reviewer 4 Report

This paper is based on Benchtop FlowCAM (FC) method to analyze copepods size and abundance change at a station in upwelling region. The coupling between IJ software and FC semi-automatic imaging system proved to be an effective tool in ecological development monitoring of variations in copepod populations. Results found that small copepods were more correlated to the seasonal upwelling (spring) and the following spring bloom of phytoplankton. These provide effective and fast methods for long-term monitoring of ecosystem. But there are still some questions and adviecs that need to be considered.

1.      Title. Effects of coastal upwelling on the Seasonal changes in the size distribution of copepods

2.      Figures. Figure2, from 2010 to 2014 could be combined one picture.

3.      Fig 3 add the unit um

4.      Figure 4 and , the line colors and corresponding methods should be the same

5.      The results of the MS need to be condensed again. They are all the results of some descriptions and are too tedious.

6.      Language expression is not rigorous, need to polish the language by native speakers.

7.      Others. L37 is FC, not CF L342 Figure23? Pay attention to small handwriting errors in the MS

Author Response

Dear reviewer, thank you for your valuable suggestions, which have contributed greatly to the improvement of our study. So, below are the indications and changes according to your suggestions. The comments of the reviewer, and my revisions in Answer.

1. Title. Effects of coastal upwelling on the Seasonal changes in the size distribution of copepods

Answer: We changed the title in accordance with your recommendations.

2. Números. A Figura 2, de 2010 a 2014, pôde ser combinada com uma figura.

Answer: Figure 2 shows a lot of data, temperature and Chlorophyll information for 5 years and I believe it does not need more information.

3. Fig 3 add the unit um

Answer: Answer: Ok, it was added.

4. Figure 4 and, the line colors and corresponding methods should be the same

Answer: In fact, figure 4 was confusing and, in addition, we are showing information that is also present in figure 5, so we decided to remove figure 4. As well as figure 7, requested by reviewer 3.

5. The results of the MS need to be condensed again. They are all the results of some descriptions and are too tedious.

Answer: Same answer to question 4.

6. Language expression is not rigorous, need to polish the language by native speakers.

Answer: The text will be proofread again by a native speaker.

7. Others. L37 is FC, not CF L342 Figure23? Pay attention to small handwriting errors in the MS

Answer: A typo occurred, figure 23 changed to figure 8 and the small error in Chlorophyll in the graph was corrected.

Round 2

Reviewer 4 Report

MS could be aceepted for publishcation.

Author Response

Sinto muito, mas não entendo por que essa nova abordagem, se eu já respondi e nenhuma nova pergunta apareceu, então enviarei as últimas respostas novamente.

Caro revisor, obrigado por suas valiosas sugestões, que contribuíram muito para a melhoria do nosso estudo. Então, abaixo estão as indicações e alterações de acordo com suas sugestões. Os comentários do revisor e minhas revisões em Resposta.

 

1. Título. Efeitos da ressurgência costeira sobre as mudanças sazonais na distribuição de tamanho de copépodes

Resposta: Mudamos o título de acordo com suas recomendações.

 

2. Números. A Figura 2, de 2010 a 2014, pôde ser combinada com uma figura.

Resposta: A Figura 2 mostra muitos dados, temperatura e informações de clorofila por 5 anos e acredito que não precisa de mais informações.

 

3. Fig. 3 adicione a unidade um

Responder: Resposta: Ok, foi adicionado.

 

4. Figura 4 e, as cores de linha e os métodos correspondentes devem ser os mesmos

Resposta: Na verdade, a figura 4 era confusa e, além disso, estamos mostrando informações que também estão presentes na figura 5, então decidimos remover a figura 4. Assim como a figura 7, solicitada pelo revisor 3.

 

5. Os resultados do MS precisam ser condensados novamente. Eles são todos os resultados de algumas descrições e são muito tediosos.

Resposta: Mesma resposta à pergunta 4.

 

6. A expressão da língua não é rigorosa, precisa polir a língua por falantes nativos.

Resposta: O texto será revisado novamente por um falante nativo.

 

7. Outros. L37 é FC, não CF L342 Figura23? Preste atenção aos pequenos erros de caligrafia no MS

Resposta: Ocorreu um erro de digitação, a figura 23 mudou para a figura 8 e o pequeno erro na clorofila no gráfico foi corrigido.

Author Response File: Author Response.docx