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Food Web Responses to a Cyanobacterial Bloom in a Freshwater Eutrophic Lake

Water 2021, 13(9), 1296; https://doi.org/10.3390/w13091296

by Mengqi Han, Chenchen Dong, Siqi Ma, Cui Feng, Chengqiang Lei, Zemao Gu and Xiangjiang Liu^*

Reviewer 1: Anonymous

Reviewer 2: Anonymous

Reviewer 3:

Barry Rosen

Reviewer 4: Anonymous

Water 2021, 13(9), 1296; https://doi.org/10.3390/w13091296

Submission received: 7 February 2021 / Revised: 21 April 2021 / Accepted: 28 April 2021 / Published: 5 May 2021

(This article belongs to the Special Issue Algae: Indices of Water and Ecological Quality)

Round 1

Reviewer 1 Report

Food Web Responses to a Cyanobacterial Bloom Period in a Freshwater Eutrophic Lake, by Mengqi Han

Introduction needs to be more general.

There are many species included. And statistics was made. But at the end one would expect a simple model to explain the results?

What about the application?

There is no clear conclusion. What about application?

One way to get read of microorganism is to use the effect of bacterial adhesion to the surfaces (Biofouling of stainless steel surfaces by four common pathogens : the effects of glucose concentration, temperature and surface roughness. Biofouling, 2019, vol. 35, 3, 273-282.). Do the authors see any possibility to use this effect?

Author Response

Response to Reviewer 1#

Food Web Responses to a Cyanobacterial Bloom Period in a Freshwater Eutrophic Lake, by Mengqi Han

Q1. Introduction needs to be more general.

Response：

Thank you very much for your suggestions. As you suggested, we have revised the introduction again (Line 53-56, 63-66, 78-81).

“Bacteria respond rapidly to environmental changes, so the change of community structure can be used to indicate the water ecological environment [19-20]. Bacterial communities in water are affected by complex biological and abiotic processes, such as dissolved oxygen, pH, temperature, water nutritional status, and plankton interactions [21-22].” (Line 52-56)

“Planktonic virus can cause the lysis of the bacteria, transforming particulate organic matter into dissolved organic matter utilized by bacteria. At the same time, as the hosts of viruses, bacteria have a great impact on the abundance of planktonic viruses [28].” (Line 63-66)

“This study will help us to understand the role of microbial loop during algal blooms occurred in eutrophic lakes, and provide theoretical basis for improving water quality of eutrophic lakes according to biomanipulation and sustainable development.” (Line 78-81)

Q2. There are many species included. And statistics was made. But at the end one would expect a simple model to explain the results?

Response:

Thank you very much for your comments. The conclusions of this study are as follows: Cyanophyta, especially A. circinalis and M. flosaquae, were mainly dominant species during bloom period. Secondly, rotifers were the main components of zooplankton in Lake Nanhu, followed by cladocerans. B. calyciflorus and M. macrocopa were the most dominant zooplankton in the non-bloom and bloom period, respectively. Thirdly, Bacteroidetes showed significantly higher mean proportion in bloom period than that in non-bloom period. Moreover, microzooplankton grazing could respond quickly to the increase in phytoplankton abundance. Finally, microzooplankton preference during bloom period was mainly affected by the community composition of phytoplankton. However, microzooplankton grazing has little effect during the outbreak period.

In addition, the main content of this study can be summarized as the following model figure:

Q3. What about the application? There is no clear conclusion. What about application?

Response:

Thank you very much for your comments. This study will help us to understand the role of microbial loop during algal blooms occurred in eutrophic lakes, and provide theoretical basis for improving water quality of eutrophic lakes according to biomanipulation and sustainable development.

Response:

Thank you very much for your suggestion. This article has a wide range of applications and flexible experimental methods. We will learn from this reference in future study.

Author Response File: Author Response.pdf

Reviewer 2 Report

Major Comments

This manuscript presented data on the community changes of phytoplankton, zooplankton and bacteria between the bloom and non-bloom period, and microzooplankton grazing experiments were also conducted to measure the grazing pressure and selectivity of microzooplankton on phytoplankton community in eutrophic Lake Nanhu. I must to say this manuscript would not make any advancement in microbial ecology and food-web studies because of the inadequacy of the novelty of findings. Furthermore, to me, I think this paper has not been well characterized as of yet, so I strongly encourage the authors to reanalyze their data and make the appropriate modifications to the manuscript. This manuscript needs to be addressed and discuss more deeply. With the above points in mind, at present, I cannot recommend its publication in WATER.

Specific Comments

Introduction

As for authors showed that interaction between phytoplankton and bacteria could affect the dynamics of bacterial communities on lines 55-57, however, other biological factors, such as grazing and viral lysis also would affect the variations of bacterial communities, the authors should add these points in the Introduction section. Please reword this paragraph in the Introduction.

In this study, the authors focused on two questions, firstly, they wanted to study the changes in the community of phytoplankton, zooplankton and bacteria…, however, the relationship between phytoplankton and zooplankton, phytoplankton and bacteria is different trophic levels in grazing food chain and microbial loop, respectively. It is not clear to me for studying what kind of important issue or findings in these large ranges of food chain from bacteria to zooplankton. Secondly, it is also not clear to me what is the interesting issue that authors have to do the dilution method for measuring the grazing rates of microzooplankton between non- and bloom period. Anyway, what is the hypothesis in this study? Please add some sentences to show your reasons and good hypothesis in the Introduction section.

Methods

In the Method section on lines 162-164, the dilution experiments obtain three kinds of phytoplankton with different sizes, what is the important topic the authors want to study? They did not explain any reasons or why they choose these three kinds of phytoplankton in the dilution experiments?

In the Results section, the authors did not show any statistical analysis for comparing the values of non- with bloom period. Such as on lines 225-227, the concentration of TN………in non-bloom period is higher than that…. (what kind of statistical analysis was used). On lines 282-285, the authors did not show any statistical analysis in these data. …….

As for Figure 7B, what is the meaning of negative values of grazing rates? The authors should consider these negative values of grazing rates, are they appropriate for calculating the PI and PP in this study? Furthermore, Fig. 6, the authors showed the data of different sites (S1, S2 and S3). However, I did not find the spatial variations of growth and grazing rates in three sampling stations in Figure 7.

At present, I cannot give any specific comments about Discussion section, this manuscript needs to be addressed and the results and discussion rewritten to focus on the new analysis.

Author Response

Response to Reviewer 2#

Major Comments

Response:

Thank you very much for your help with the paper review along the way. As you suggested, we have tried our best to revised the manuscript again (Line 52-56, 63-66, 78-81, 86-90, 274-277, 313-315, 317-318, 345 348-350, 411-412, 433-434, 469-480, 529-534, 536-545).

“There are generally two views on the definition of algal bloom: (1) the cell density of algae is 0.5×10⁶ ~ 15×10⁶ cells/L [32]; (2) some algae multiply and gather in large numbers, forming algae floating on the water surface [33]. Taken together, we define algal blooms in the Lake Nanhu as algal cell density of 15 × 10⁶ cells/L and floating algae on the water surface.” (Line 86-90)

“Quantitative analysis of microzooplankton showed that the density abundance of rotifers was higher bloom period (523.89 ind. /L) than that in non-bloom period (mean of 471.27 ind. /L), especially Brachionus and Polyarthra (Fig. 4B).” （Line 274-277）

“Actinobacteria was significantly more abundant in March and May (mean of 32.34% and 28.66% respectively) than it in November.” (Line 313-315)

“Moreover, Chloroflexi (mean of 22.76%) and Acidobacteria (mean of 10.26%) were more abundant in November than those in March and May (Fig. 6B).” (Line 317-318)

“Meanwhile, the size-specific microzooplankton grazing rates (g) were also higher in bloom period (0.61 d^-1 ~ 1.05 d^-1) than in non-bloom period (-1.15 d^-1 ~ -0.17 d^-1) (Fig. 7B), which were positively correlated with phytoplankton growth rates significantly (p =0.03, r = 0.81).” (Line 345)

“In May, microzooplankton grazing rates for < 5 μm groups were higher than other groups (p =0.04), and phytoplankton growth rates were lower than other groups (p =0.04).” (Line 348-350)

“As a primary producer in water, phytoplankton species composition can objectively reflect the changing law of water environment and play an important indicator role.” (Line 411-412)

“Zooplankton, as the primary consumers of aquatic ecosystems, play an important role in the food chain and affect the quality of water environment.” (Line 433-434)

“Temperature can indirectly affect the community composition of bacteria by affecting the community structure of phytoplankton and zooplankton [86]. The organic matter produced by phytoplankton and zooplankton provides energy for the growth of bacteria [87-89]. In addition, zooplankton can also directly graze bacteria, which affects the number and distribution of bacteria [90]. In lake ecosystems with different nutrient levels, nutrients have different effects on bacteria. In oligotrophic lakes, nutrients can become a limiting factor for the growth of bacteria [91-92]. In mesotrophic lakes, nutrients have different limiting effects on bacteria in different seasons [93]. In eutrophic lakes, the effect of nutrients on bacteria is less than that of phytoplankton [94]. Studies have also found that during the lake bloom period, the number and species of bacteria have changed significantly, and this change could be used to predict the algal bloom [95]. Therefore, planktonic bacteria in water are regarded as important environmental indicators of water ecology.” (Line 469-480)

“In the natural environment, due to the lack or slow proliferation of original zooplankton, the process of improving water quality by biomanipulation is relatively slow or inefficient [100]. It was also confirmed in this study that microzooplankton played little role during the outbreak period. Nevertheless, this study provides some basic information for biomanipulation to improve water quality in freshwater eutrophic lakes.” (Line 529-534)

“The community of phytoplankton, zooplankton, and bacteria changed between bloom and non-bloom periods: firstly, Cyanophyta, especially A. circinalis and M. flos-aquae, were mainly dominant species during bloom period; secondly, B. calyciflorus and M. macrocopa were the most dominant zooplankton in the non-bloom and bloom period, respectively; thirdly, Bacteroidetes showed significantly higher mean proportion in bloom period than that in non-bloom period. Moreover, microzooplankton grazing could respond quickly to the increase in phytoplankton abundance. In the natural environment, microzooplankton grazing has little effect during the outbreak period. This study will help to understand the role of microbial webs during algal blooms in fresh-water eutrophication lakes and provide basic data for the application of biomanipulation in the future.” (Line 536-545)

Specific Comments

Introduction

Q1. As for authors showed that interaction between phytoplankton and bacteria could affect the dynamics of bacterial communities on lines 55-57, however, other biological factors, such as grazing and viral lysis also would affect the variations of bacterial communities, the authors should add these points in the Introduction section. Please reword this paragraph in the Introduction.

Response:

Thank you very much for your suggestions. As you suggested, we have reworded this paragraph in manuscript (Line 51-69).

“In aquatic ecosystem, bacteria are an important part of the microbial food web, which are mainly responsible for the mineralization and recycling of organic matter [16-18]. Bacteria respond rapidly to environmental changes, so the change of community structure can be used to indicate the water ecological environment [19-20]. Bacterial communities in water are affected by complex biological and abiotic processes, such as dissolved oxygen, pH, temperature, water nutritional status, and plankton interactions [21-22]. It has been found that the interaction between phytoplankton and bacteria could affect the dynamics of bacterial communities [23-24]. Bacteria can make use of secretions released by phytoplankton, as well as the debris of algal cells [23-25]. Phytoplankton also adversely affect bacterial communities through nutrient competition and antibiotic release [26]. In addition, previous study found that microzooplankton grazing in shallow eutrophic lakes eliminated 90% to 99% of the potential single-celled cyanobacteria production and 46% of the potential heterotrophic bacteria production [27]. Planktonic virus can cause the lysis of the bacteria, transforming particulate organic matter into dissolved organic matter utilized by bacteria. At the same time, as the hosts of viruses, bacteria have a great impact on the abundance of planktonic viruses [28]. Moreover, some studies have shown that some bacteria play an important role in the occurrence of eutrophic lake blooms [29-31]. Therefore, further study on the variation of bacterial community structure could help us to clarify the functional role of microbial food web in eutrophic lake.” (Line 51-69)

Q2. In this study, the authors focused on two questions, firstly, they wanted to study the changes in the community of phytoplankton, zooplankton and bacteria…, however, the relationship between phytoplankton and zooplankton, phytoplankton and bacteria is different trophic levels in grazing food chain and microbial loop, respectively. It is not clear to me for studying what kind of important issue or findings in these large ranges of food chain from bacteria to zooplankton. Secondly, it is also not clear to me what is the interesting issue that authors have to do the dilution method for measuring the grazing rates of microzooplankton between non- and bloom period. Anyway, what is the hypothesis in this study? Please add some sentences to show your reasons and good hypothesis in the Introduction section.

Response:

Thank you very much for your comments and suggestions. Firstly, the study focused on the changes in the community structure of bacteria between the bloom and non-bloom periods, and did not study the grazing relationship between microzooplankton and bacteria in the food chain. Secondly, the purpose of dilution method is to study whether microzooplankton can control the outbreak of cyanobacterial blooms by grazing, and then determine whether microzooplankton can inhibit cyanobacterial blooms by biological manipulation. This study will help us to understand the role of microbial loop during algal blooms occurred in eutrophic lakes, and provide theoretical basis for improving water quality of eutrophic lakes according to biomanipulation and sustainable development.

Methods

Q3. In the Method section on lines 162-164, the dilution experiments obtain three kinds of phytoplankton with different sizes, what is the important topic the authors want to study? They did not explain any reasons or why they choose these three kinds of phytoplankton in the dilution experiments?

Response:

Thank you very much for your comments. Firstly, phytoplankton is generally divided into three categories according to particle size: picophytoplankton (<5 μm), nanophytoplankton (5-20 μm) and microphytoplankton (20-200 μm). Secondly, dilution experiment, combined with HPLC pigment analysis, were conducted to measure microzooplankton selective grazing on phytoplankton. In summary, the purpose of this study is to explore the selective grazing of microzooplankton on phytoplankton (particle size and pigment) by grazing experiments.

Q4. In the Results section, the authors did not show any statistical analysis for comparing the values of non- with bloom period. Such as on lines 225-227, the concentration of TN………in non-bloom period is higher than that…. (what kind of statistical analysis was used). On lines 282-285, the authors did not show any statistical analysis in these data. …….

Response:

Thank you very much for your comments. On Line 232-234 and Line 315-318, we compared the average between groups of data.

Q5. As for Figure 7B, what is the meaning of negative values of grazing rates? The authors should consider these negative values of grazing rates, are they appropriate for calculating the PI and PP in this study? Furthermore, Fig. 6, the authors showed the data of different sites (S1, S2 and S3). However, I did not find the spatial variations of growth and grazing rates in three sampling stations in Figure 7.

Response:

Thank you very much for your comments. This is a good question. When the temperature is too low, microzooplankton hardly eat phytoplankton, and the grazing rates measured by dilution method will be negative. However, the negative values of grazing rate do not affect the calculation of grazing pressure. In addition, Lake Nanhu covers an area of 5.50 km², with a maximum depth of 3.2 m and an average depth of 1.6 m. Therefore, we believe that there is basically no spatial variation among the three sampling stations.

Q6. At present, I cannot give any specific comments about Discussion section, this manuscript needs to be addressed and the results and discussion rewritten to focus on the new analysis.

Response:

Thank you very much for your suggestions. As you suggested, we have reworded this paragraph in manuscript (Line 348-350, 411-412, 433-434, 469-480, 529-534, 536-545).

“In May, microzooplankton grazing rates for < 5 μm groups were higher than other groups (p =0.04), and phytoplankton growth rates were lower than other groups (p =0.04).” (Line 348-350)

“As a primary producer in water, phytoplankton species composition can objectively reflect the changing law of water environment and play an important indicator role.” (Line 411-412)

“Zooplankton, as the primary consumers of aquatic ecosystems, play an important role in the food chain and affect the quality of water environment.” (Line 433-434)

Author Response File: Author Response.pdf

Reviewer 3 Report

Very well written and the material is well documented.

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 3#

Very well written and the material is well documented.

Response:

Thank you very much for your help with the paper review along the way.

Author Response File: Author Response.pdf

Reviewer 4 Report

General Comments:

This manuscript is well-written and includes a valuable dataset in examining the responses of food web to a cyanobacterial bloom in a relatively small-sized eutrophic lake (Lake Nanhu, China). Authors investigated grazing pressure and selectivity of microzooplankton by conducting grazing experiments as well as the dynamics of phytoplankton, zooplankton and bacteria. However, authors need to address what are the major findings and the significance of their study in local and global scale clearly in discussion or conclusion.

Specific Comments:

Line 1-3: Authors need to check the tile of manuscript. “Period” may not be needed.

Line 70: Any references? And what are the base lines for the blooms defined here?

Line 79: samples were collected monthly? and at surface or/and bottom? I recommend authors to describe how they defined the bloom and non-bloom periods here.

Line 81: One of important physical parameter is turbidity. Was it measured?

Line 228: positively? Authors may need to insert R value.

Line 266-267: No significant difference between bloom and non-bloom periods was shown. Why? What this indicates? These should be addressed in discussion.

Line 309: more than what periods?

Line 338: “< 0.05” is not needed here and in other lines. “r²” is correct instead of r?.

Line 340-344: The correlation coefficient r may be added.

Line 341: months instead of mouths?.

Figure 7: What are the units of P_i in panels C and D of Fig. 7?

In the discussion section, authors need to describe the response of food webs to the cyanobacteria blooms in consortium by synthesize all the results.

Line 520-523: I do not understand this sentence. There are logical jumps here.

The conclusion section is needed to be revised so that the major findings including significance of study locally and/or globally are addressed.

Author Response

Response to Reviewer 4#

General Comments:

Q1. This manuscript is well-written and includes a valuable dataset in examining the responses of food web to a cyanobacterial bloom in a relatively small-sized eutrophic lake (Lake Nanhu, China). Authors investigated grazing pressure and selectivity of microzooplankton by conducting grazing experiments as well as the dynamics of phytoplankton, zooplankton and bacteria. However, authors need to address what are the major findings and the significance of their study in local and global scale clearly in discussion or conclusion.

Response:

Thank you very much for your advice and help with the paper review. As you suggested, we have modified it in the manuscript (Line 78-81, 529-534, 536-545).

Specific Comments:

Q2. Line 1-3: Authors need to check the tile of manuscript. “Period” may not be needed.

Response:

Thank you very much for your suggestions. As you suggested, we have modified it in the manuscript (Line 2-3).

“Food Web Responses to a Cyanobacterial Bloom in a Freshwater Eutrophic Lake” （Line 2-3）

Q3. Line 70: Any references? And what are the base lines for the blooms defined here?

Response:

Thanks a lot for your comments. This is a good question. Firstly, there are generally two views on the definition of algal bloom: (1) the cell density of algae is 0.5×10⁶ ~ 15×10⁶ cells/L [32]; (2) some algae multiply and gather in large numbers, forming algae floating on the water surface [33]. Taken together, we define algal blooms in the Lake Nanhu as algal cell density of 15 × 10⁶ cells/L and floating algae on the water surface. Therefore, the bloom period of Lake Nanhu is from May to September each year.

Lu, D., Liu, P., Fan, T., Peng, H., Zhang, Z. The investigation of “water bloom” in the downstream of the Han River. Res. Environ. Sci. 2000, 13(2): 29-31.
Zheng, J., Zhong, C., Deng, C. Discussion on definition of algal bloom. Water Resources Protection 2006, 22(5): 45-47, 80.

Q4. Line 79: samples were collected monthly? and at surface or/and bottom? I recommend authors to describe how they defined the bloom and non-bloom periods here.

Response:

Thanks for your comments and suggestions. Firstly, we collected surface water samples and plankton in the middle of each month. Moreover, the sampling time was in the sunny morning, and the same sampling location was guaranteed. As you suggested, we have added the division of bloom and non- bloom period in the manuscript (Line 86-90).

Lu, D., Liu, P., Fan, T., Peng, H., Zhang, Z. The investigation of “water bloom” in the downstream of the Han River. Res. Environ. Sci. 2000, 13(2): 29-31.
Zheng, J., Zhong, C., Deng, C. Discussion on definition of algal bloom. Water Resources Protection 2006, 22(5): 45-47, 80.

Q5. Line 81: One of important physical parameter is turbidity. Was it measured?

Response:

Thanks for your comments. In the present study, we define algal blooms in the Lake Nanhu as algal cell density of 15 × 10⁶ cells/L and floating algae on the water surface. Son we did not measure the turbidity in this study, and we'll add this physical parameter in future studies.

Q6. Line 228: positively? Authors may need to insert R value.

Response:

Thank you very much for your suggestions. As you suggested, we have added the r value in the manuscript (Line 238).

“And the concentration of TN is significantly positively related to the concentration of NH₄⁺ (r = 0.93)”. (Line 238)

Q7. Line 266-267: No significant difference between bloom and non-bloom periods was shown. Why? What this indicates? These should be addressed in discussion.

Response:

Thank you very much for your comments. Rotifers were the major microzooplankton in microbial food web, and the density abundance of rotifers was higher in algal bloom (mean of 523.89 ind. /L) than that in non-bloom (mean of 471.27 ind. /L). Therefore, we forced on it in discussion.

Q8. Line 309: more than what periods?

Response:

Thank you very much for your suggestions. As you suggested, we have modified it in the manuscript (Line 317, 318).

“Actinobacteria was significantly more abundant in March and May (mean of 32.34% and 28.66% respectively) than it in November.” (Line 317)

“Moreover, Chloroflexi (mean of 22.76%) and Acidobacteria (mean of 10.26%) were more abundant in November than those in March and May (Fig. 6B).” (Line 318)

Q9. Line 338: “< 0.05” is not needed here and in other lines. “r²” is correct instead of r?.

Response:

Thank you very much for your suggestions. As you suggested, we have modified it in the manuscript (Line 342-345, 348-350).

“In May, microzooplankton grazing rates for < 5 μm groups were higher than other groups (p =0.04), and phytoplankton growth rates were lower than other groups (p =0.04).” (Line 348-350)

Q10. Line 340-344: The correlation coefficient r may be added.

Response:

Thank you very much for your comments. Significance levels of differences in phytoplankton growth rates and microzooplankton grazing rates were measured using one-way ANOVA analysis. Therefore, we only wrote the p value in the manuscript.

Q11. Line 341: months instead of mouths?.

Response:

Thank you very much for your suggestions. As you suggested, we have modified it in the manuscript (Line 348).

“Phytoplankton growth rates and microzooplankton grazing rates for phytoplankton < 5 μm groups were both significantly lower than other groups in January and November (p=0.03), and did not differ significantly among the three size fractions in other months (p > 0.05).” (Line 348)

Q12. Figure 7: What are the units of P_i in panels C and D of Fig. 7?

Response:

Thank you very much for your comments. The units of P_i and P_p are “%”, and we have modified the Fig.7C&D.

Q13. In the discussion section, authors need to describe the response of food webs to the cyanobacteria blooms in consortium by synthesize all the results.

Response:

Thank you very much for your suggestions. As you suggested, we have modified it in the manuscript (Line 411-412, 433-434, 469-480, 529-534).

“As a primary producer in water, phytoplankton species composition can objectively reflect the changing law of water environment and play an important indicator role.” (Line 411-412)

“Zooplankton, as the primary consumers of aquatic ecosystems, play an important role in the food chain and affect the quality of water environment.” (Line 433-434)

Q14. Line 520-523: I do not understand this sentence. There are logical jumps here.

Response:

Thank you very much for your suggestions. As you suggested, we have deleted this sentence in the manuscript.

Q15. The conclusion section is needed to be revised so that the major findings including significance of study locally and/or globally are addressed.

Response:

Thank you very much for your suggestions. As you suggested, we have modified it in the manuscript (Line 536-545).

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Introduction needs to be more general.

There are many species included. And statistics was made. But at the end one would expect a simple model to explain the results?

What about the application?

There is no clear conclusion. What about application?

Author should not exclude possibilities to remove bacteria by bacterial adhesion to surfaces. Biofouling should be considered (Biofouling, 2019, vol. 35, 3, 273-282.)

Author Response

Response to Reviewer 1#

Comments and Suggestions for Authors

Q1. Introduction needs to be more general.

Response：

Thank you very much for your suggestions. As you suggested, we have revised the introduction again (Line 34-36, 41-42, 67-70, 74-76).

“According to previous studies, the microbial food web was related to the classic food chain in multiple ways instead of a single "loop" [3].” (Line 34-36)

“Previous studies have shown that microzooplankton played an important role in the grazing on phytoplankton [10-12].” (Line 41-42)

“Therefore, further studies on the changes of bacterial community structure between algal bloom and non-bloom period could help people to clarify the functional role of microbial food web in freshwater eutrophic lakes.” (Line 67-70)

“Due to the discharge of a large amount of domestic sewage and the introduction of aquaculture feed, the water of Lake Nanhu has long-term eutrophication, and algal blooms usually occur more than three times from May to September every year. Using Lake Nanhu as the study object, this study is intended to explore the following two questions.” (Line 74-76)

Q2. There are many species included. And statistics was made. But at the end one would expect a simple model to explain the results?

Response:

Thank you very much for your comments. The conclusions of this study are as follows: The community of phytoplankton, zooplankton, and bacteria changed between bloom and non-bloom periods. And microzooplankton grazing could respond quickly to the increase in phytoplankton abundance. However, microzooplankton grazing has little effect during the outbreak period in the natural environment. This study will help to understand the role of microbial webs during algal blooms in freshwater eutrophication lakes and provide basic data for the application of biomanipulation in the future.

In addition, the main content of this study can be summarized as the following model figure:

Q3. What about the application? There is no clear conclusion. What about application?

Response:

Thank you very much for your comments. This study will help us to understand the role of microbial web during algal blooms occurred in freshwater eutrophic lakes, and provide theoretical basis for improving water quality of freshwater eutrophic lakes according to biomanipulation and sustainable development.

Q4. One way to get read of microorganism is to use the effect of bacterial adhesion to the surfaces (Biofouling of stainless steel surfaces by four common pathogens: the effects of glucose concentration, temperature and surface roughness. Biofouling, 2019, vol. 35, 3, 273-282.). Do the authors see any possibility to use this effect? Author should not exclude possibilities to remove bacteria by bacterial adhesion to surfaces. Biofouling should be considered (Biofouling, 2019, vol. 35, 3, 273-282.)

Response:

Thank you very much for your suggestions. As you suggested, we have added it in the

manuscript (Line 492-493).

“Thus, it is also an effective method to remove cyanobacteria by bacterial adhesion to surface [102].” (Line 492-493)

[102] Bezek, K.D., Nipič, D., Torkar, K.G., Oder, M., Dražić, G., Abram, A., Žibert, Janez, Raspor, P., Bohinc, K. Biofouling of stainless steel surfaces by four common pathogens: the effects of glucose concentration, temperature and surface roughness. Biofouling 2019, 35(3): 273-283

Author Response File: Author Response.pdf

Reviewer 4 Report

General Comments:

I appreciate the authors' attention to my comments in their revision of the manuscript and the manuscript was greatly improved although their conclusion is not sufficient yet. I also recommend authors to have the English of the revised manuscript especially the conclusion section checked by professional editors.

Specific Comments:

Line 418-419 & 440-441: Authors need to add references here.

Author Response

Response to Reviewer 4#

General Comments:

Response:

Thank you very much for your help with the paper review along the way. As you suggested, we have revised the manuscript again (Line 419421, 530-531, 532-534, 534-535, 543-544).

“In this study, when the temperature increased from April to May, the abundance of Cyanophyta increased rapidly and became the dominant species in water (Fig. 4A).” (Line 419-421)

“Due to the lack or slow proliferation of original microzooplankton in the natural environment.” (Line 530-531)

“In this study, it was also found that microzooplankton played little role during algal blooms occurred in freshwater eutrophic lakes.” (Line 532-534)

“Nevertheless, this study still provides some basic information for improving water environment quality according to biomanipulation.” (Line 534-535)

“However, microzooplankton grazing has little effect during the outbreak period in the natural environment.” (Line 543-544)

Specific Comments:

Q1. Line 418-419 & 440-441: Authors need to add references here.

Response:

Thank you very much for your suggestions. As you suggested, we have added the explanations and references in the manuscript (Line419-421, 442).

“In this study, when the temperature increased from April to May, the abundance of Cyanophyta increased rapidly and became the dominant species in water (Fig. 4A).” (Line 419-421)

“Within the appropriate range of temperature and food density, the population density of rotifers and cladocerans increase with the increase of temperature and food density [57-63].” (Line 442)

[62] Wu, J., Yan, B., Feng, Z., Li, Y., Shen, X. Zooplankton ecology near the tianwan nuclear power station. Acta Ecologica Sinica. 2011, 31(22): 6902-6911

[63] Wang, Y., He, Z.H. Effect of temperature and salinity on intrinsic increasing rate of Moina mongolica Daddy (Cladocera: Moinidae) population. J. Appl. Ecol. 2011, 12(1): 91-94

Author Response File: Author Response.pdf

Round 3

Reviewer 1 Report

The paper can be accepted

Author Response

Response to Reviewer 1

Q1. The paper can be accepted.

Thank you very much for your help with the paper review along the way.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

I have examined the paper by Han et al "The responses of microbial food web o algal bloom in freshwater eutrophic lake." The work appears to be of good quality but there is a lack of clarity regarding the details of the work. Additionally, the English needs extensive adjustment and redaction; there are such serious issues that they interfere with the interpretation of the work. I will examine some of the highlights but the fundamental issue is that the paper must be rewritten for it to be understandable. The work seems to be of good quality and it reflects much effort, the work is relevant to the readership, and eventually, after much adjustment should be published.

Firstly the strongest part of the paper is the well-characterized Materials and Methods. Even so, there are problems:

94-95; does this mean the authors did the work after the methodology of [30]? if so please clearly state this; if the reference means something else, please clarify.

106 'according to the different sampling time' - what does this mean? not time I think; season perhaps?

113: I see that this is a dilution but to what actual end? and please clarify the use of samples...what precisely they were...where they filtered samples or the in situ water sample?

115-116: 'fixed with a rope' what does this mean? and experimental pool near Lake Nanhu...to what end? what is the purpose...please explain. in these experiments, which are the controls?

125 filters containing phytoplankton...Which samples are these? are they from the growth experiments?

128 In 40 years of research I've never heard of an ultrasonic vortex. please explain and identify the instrument

129 4000 rpm is not sufficient; we need the rcf.

138 probably this is clear to people who work with HPLC but why run for 30 min? I don't understand but thank you for the partial explanation.

I would like to see a more complete explanation of the interpretation of the results. that should be provided in the results section and could be expanded on in the Conclusions section.

in many places, the results and/or descriptions are reported in a conclusive manner and also with potentially mixed meaning, such as line 333: 'dechlorination which can participate....' such phrasing here and throughout needs to be adjusted so that there is a commitment to a result.

In the conclusion, there is no indication of future plans or how the work could be used to improve water resource quality or guidance for water resource management. This should be part of the author's discussion and conclusions.

The work seems to be quite good but the authors need to help the reader put the work in context and indicate the value of such work for future studies in aquatics; they should also explain why this work is remarkable, especially in view of the use of methods that individually are not that unusual, but which in the place created an interesting new series of prodcuts.

Accept for publication only aftre careful redaction and adjustment of the wording to make the work more meaningful to the reader.

Author Response

Response to Reviewer 1:

Comments and Suggestions for Authors

I have examined the paper by Han et al "The responses of microbial food web to algal bloom in freshwater eutrophic lake." The work appears to be of good quality but there is a lack of clarity regarding the details of the work. Additionally, the English needs extensive adjustment and redaction; there are such serious issues that they interfere with the interpretation of the work. I will examine some of the highlights but the fundamental issue is that the paper must be rewritten for it to be understandable. The work seems to be of good quality and it reflects much effort, the work is relevant to the readership, and eventually, after much adjustment should be published.

Response:

Thank you very much for your help with the paper review. As you suggested, we have revised the manuscript.

Q1. Firstly， the strongest part of the paper is the well-characterized Materials and Methods. Even so, there are problems: 94-95; does this mean the authors did the work after the methodology of [30]? if so please clearly state this; if the reference means something else, please clarify.

Response:

Thank you very much for your comments. The reference means that the specific methods of DNA extraction and PCR amplification are identical to [30]. And we added detailed experimental methods in the modified version (Line 98-112).

“Bacterial DNA was extracted from all water and sediment samples using the E.Z.N.A.fi soil DNA Kit (Omega Bio-tek, Norcross, GA, USA) according to the manufacturer’s protocols. The final DNA concentration and purification were determined using a NanoDrop 2000 UV–vis spectrophotometer (Thermo Scientific, Wilmington, NC, USA), and the DNA quality was checked via 1% agarose gel electrophoresis. The V3–V4 hypervariable regions of the bacteria 16S rRNA gene were amplified with primers 338F (50-ACTCCTACGGGAGGCAGCAG-30) and 806R (50-GGACTACHVGGGTWTCTAAT-30) using a thermocycler PCR system (GeneAmp 9700, ABI, Foster City, CA, USA). The PCR reactions were conducted using the following program: 3 min of denaturation at 95 ◦C; 27 cycles of 30 s at 95 ◦C, 30 s for annealing at 55 ◦C, and 45 s for elongation at 72 ◦C; and a final extension at 72 ◦C for 10 min. PCR reactions were performed in triplicate in a 20 µL mixture containing 4 µL of 5× FastPfu Buffer 2 µL of 2.5 mM dNTPs, 0.8 µL of each primer (5 µM), 0.4 µL of FastPfu Polymerase, and 10 ng of template DNA. The PCR products were extracted from a 2% agarose gel, further purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA), and quantified using QuantiFluor™-ST (Promega, Madison, WI, USA) according to the manufacturer’s protocol.” (Line 97-111)

Q2. 106 'according to the different sampling time' - what does this mean? not time I think; season perhaps?

Response:

Thank you very much for your suggestions. As you suggested, I have replaced the “time” with “month” (Line 128-129).

“One-way ANOVA was used to test for significant differences. in bacteria structure among different months.” (Line 128-29)

Q3. 113: I see that this is a dilution but to what actual end? and please clarify the use of samples...what precisely they were...where they filtered samples or the in situ water sample?

Response:

Thanks a lot for your comments. Firstly, the purpose of the dilution experiment is to measure phytoplankton growth rate and microzooplankton grazing rate. The raw water of Lake Nanhu was filtered with 0.22 μm filter membrane to obtain particle-free water. Then, the raw water was diluted into different proportions with particle-free water and cultured. Phytoplankton growth rate and microzooplankton grazing rate can be calculated according to the change of chlorophyll-a concentration before and after culture. Secondly, the filtration process of raw water was carried out in the laboratory

Q4. 115-116: 'fixed with a rope' what does this mean? and experimental pool near Lake Nanhu...to what end? what is the purpose...please explain. in these experiments, which are the controls?0:1

Response:

Thank you very much for your comments. Firstly, I'm very sorry that "fixed with a rope" is a language problem. We have reedited the manuscript (Line 138-141). Secondly, the proportion of 0:1 is the control group.

“All bottles were incubated at ambient light levels for 24 h at the surface (~0.5 m) in an experimental tank (~10 m diameter; ~2 m deep) cooled by running natural water from Lake Nanhu.” (Line 137-140)

Q5. 25 filters containing phytoplankton...Which samples are these? are they from the growth experiments?

Response:

Thank you very much for your comments. These samples are from microzooplankton grazing experiments. In the grazing experiment, 3 × 300mL water samples were taken from each proportion before and after culture and filtered through 0.7 μm Whatman GF/F filters to analyze phytoplankton pigments.

Q6. 128 In 40 years of research I've never heard of an ultrasonic vortex. please explain and identify the instrument

Response:

Thank you very much for your comments. I am very sorry that “ultrasonic vortex” is a language problem. We originally wanted to express the “ultrasonic cleaner”. The mistake was corrected (Line 154).

“Wrapping the centrifuge tubes with aluminum foil and treating it with sonicated for 5 min.” (Line 154)

Q7. 129 4000 rpm is not sufficient; we need the rcf.

Response:

Thank you very much for your suggestions. As you suggested, we have added “2325 ×g” with “4000 rpm” (Line 154-155).

“The samples were extracted overnight at 4°C, and centrifuged at 4000 rpm (2325 ×g) for 20 min at 4°C after extraction.” (Line 154-155)

Q8. 138 probably this is clear to people who work with HPLC but why run for 30 min? I don't understand but thank you for the partial explanation.

Response:

Thanks a lot for your comments. According to the results of preliminary experiments, running for 30 minutes can ensure that all pigments can be retained.

Q9. I would like to see a more complete explanation of the interpretation of the results. that should be provided in the results section and could be expanded on in the Conclusions section. in many places, the results and/or descriptions are reported in a conclusive manner and also with potentially mixed meaning, such as Line 333: 'dechlorination which can participate....' such phrasing here and throughout needs to be adjusted so that there is a commitment to a result.

Response:

Thanks a lot for your suggestions. As you suggested, we have revised the manuscript (Line 374-376).

“Chloroflexi can participate in the degradation of organic matter. In this study, the results showed that the abundance of the Chloroflexi was relatively higher after algal bloom (November).” (Line 374-376)

Q10. In the conclusion, there is no indication of future plans or how the work could be used to improve water resource quality or guidance for water resource management. This should be part of the author's discussion and conclusions. The work seems to be quite good but the authors need to help the reader put the work in context and indicate the value of such work for future studies in aquatics; they should also explain why this work is remarkable, especially in view of the use of methods that individually are not that unusual, but which in the place created an interesting new series of products. Accept for publication only after careful redaction and adjustment of the wording to make the work more meaningful to the reader.

Response:

Thanks a lot for your suggestions. This study investigated the changes in the community structure of plankton and planktonic bacteria between the bloom and non-bloom period, and the grazing of microplankton on phytoplankton. The results showed that plankton and bacteria can respond to blooms, so the time of blooms can be estimated by observing changes in their community structure. Our present study has also found that microplankton can grazing on algae, but they have little effect during the outbreak period. The treatment of water blooms should still reduce sewage discharge from the source and pay attention to ecological protection.

Author Response File: Author Response.pdf

Reviewer 2 Report

Review for microorganisms-994681

Han et al provides food web data on a eutrophic lake ecosystem in China by studying the development of the bacterial community composition and grazing activities in response to a bloom of two cyanobacterial species. The manuscript contains interesting and extensive data, which could however be discussed more efficiently. Here I provide some suggestions on how to improve the manuscript and especially the discussion, which should be reworked. The underlying sequencing data is not accessible.

General comments:

The manuscript needs English editing. Lots of articles are missing, e.g. in the abstract, line 12 “the” microbial food web and many others

Check that all taxa names are in italics, e.g. in line 16 Anabaena circinalis and Microcystis flos-aquae

Title

The title does not really fit, because you also study grazing (top-down control) effects on the food web, not only responses of microbes. How about:

Food web responses to a cyanobacterial bloom period in a freshwater eutrophic lake (just a suggestion)

Abstract

-very little new insights presented. The take-home message and implications for the “bigger picture” could be presented in more details. That something is “more abundant” than something else is not very detailed either. Please try to add numbers where possible.

-Please add where Lake Nanhu is located (city, country), also in the methods

Introduction:

Needs English editing.

Line 8: microbial loop not microbial food loop

Line 8: material circulation sounds technical, better “matter” or “organic matter” circulation

Line 34-36 “However, little is known about the microbial food web in freshwater ecosystem, especially in freshwater eutrophic lakes.”

I do not agree with this very general knowledge gap presented here. There is lots of work out there:

Kiersztyn, B., Chróst, R., Kaliński, T., Siuda, W., Bukowska, A., Kowalczyk, G., & Grabowska, K. (2019). Structural and functional microbial diversity along a eutrophication

gradient of interconnected lakes undergoing anthropopressure. Scientific reports, 9(1), 1-14.

Kalinowska, K., & Grabowska, M. (2016). Autotrophic and heterotrophic plankton under ice in a eutrophic temperate lake. Hydrobiologia, 777(1), 111-118.

Velimirov, B., Fischer, U. R., Kirschner, A. K., & Wieltschnig, C. (2019). Microbial and Viral Loop in Alte Donau. The Alte Donau: Successful Restoration and Sustainable Management: An Ecosystem Case Study of a Shallow Urban Lake, 10, 227.

Maybe it’s worth to explain more what is special about the lake you investigated compared to other freshwater ecosystems (if there is anything special).

L37: Microzooplankton consist of flagellates (will not comment on English any further)

L39/40: The growth, metabolism and grazing rate of microzooplankton are very fast

>What does it mean? Please make it more quantitative.

L49/50: Please write: Phytoplankton organisms are primary producers in the microbial food web

L53: algal cells

L54: Please provide reference for antibiotic release and nutrient competition by phytoplankton

L58 to clarify not to clear

L68/69: sentence has no verb

Material & Methods

L74: every month over which period of time? How many samples in total? Any controls?

L75/76: how was the sample for bacterial composition exactly taken? From a boat? Which device?

Figure 1: Please show within a bigger map where Hubei is located and what Hubei is (city, region…?)

L90: sentence has no verb

Please explain the rationale behind the filtration procedure.

L94: Please mention details on DNA extraction kit, primers used and PCR conditions

Please mention earlier that you target the 16S rRNA gene.

L98: The bioproject PRJNA673947 does not exist in SRA.

L104: How (using which programs) were these parameters checked?

L106/107: one-way ANOVA was used to test for significant differences.

Significant differences between which variables? Were prerequisites for a parametric test fulfilled?

L109/110: please add more details on how grazing was studied rather than just referring to the literature

L118 provide city, country for manufacturers (here and everywhere else)

Results:

L.183 (and in other lines): with “density” of organisms, you refer to their (relative) abundance?

L.185: according to the species table, there is only one species of copepod. Wouldn’t it make sense to mention that here instead of just “copepod”.

L204: Anabaena circinalis (1.45 × 107 cells/L) and Microcystis flos-aquae (1.10 × 107 cells/L) were considered to be the causative organisms

This is rather discussion than results

L224-226: The results revealed that Bacillariophyta biomass was high both in non-bloom and bloom period, while Pyrrophyta biomass was high in non-bloom period, Cyanophyta biomass was high in bloom period.

Please provide numbers. This is more informative than stating 3x that something is high.

Figure 5: please remove two decimal places on y axis, since it’s all zero. Caption: the term is “accessory”; please add some more details to the caption (which pigments were assayed etc.)

Table 1: please write the sampling month next to S2. Please describe terms and abbreviations in the header (Ace, Chao1, OTUs) and upon first use in the main text.

L242: were the most abundant bacteria

L244: proportions (plural) > English editing please

Figure 6: It’s ANOVA, not ANOVE; please use scientific notation for very small p-values;

Sample IDs need to be explained or changed: previously S1-S3 in Table 1, now changed to A1-A3, etc. Legends look squeezed. Better use term “bacterial community composition” instead of classification structure.

L.270 Correlation statistics were not explained in the methods. Always state the test used next to the p-value, e.g. (Spearman rank correlation, p < 0.05, r2 = 0.81).

L.276 standing stocks (Pi) in the main text, but in the caption of Fig 7, Pi is phytoplankton biomass. Please use consistent terms throughout the manuscript

Figure 7: the label of the y axis in D) is cut off. Is this mean or median presented? Is the error bar standard deviation or standard error? Is this from n=3? Please mention filter sizes in the caption as well.

Discussion

I feel the discussion is quite superficial (considering the amount of results presented) and should be much extended.

e.g. L 333: Rotifers could respond to Microcystis bloom by a variety of mechanisms. > What are these mechanisms? How would other zooplankton species cope?

You described Anabaena and Microcystis and that rotifers feed on Microcystis (which is long know). Why should a reader care, what are the implications of this, e.g. for the food web or status of the Lake ecosystem? Is there something special about this Lake compared to others? What can induce such blooms (high nutrient levels, anthropogenic input close by?)

Same in 4.2

e.g. l345 the results showed that the bacterial community structure could respond to the occurrence of algal blooms

> This is a very general statement and nothing really new.

Also, I would not consider the different relationships in isolated paragraphs (cyanobacteria-rotifers, cyanobacteria-heterotrophic bacteria). It is a food web and the interactions of organisms are tightly interwoven.

L343 sounds like dechlorination participates in organic matter degradation, please re-phrase.

Please rework the discussion as suggested and include more literature. You have many nice results, but they are not optimally used in the discussion and the overall ecological context. As it is presented now, the story is very descriptive, and I unfortunately do not see so much novelty in the study.

Author Response

Response to Reviewer 2:

Comments and Suggestions for Authors

Review for microorganisms-994681

Response:

Thank you very much for your suggestions. We have revised the manuscript carefully according to your suggestion. Previously, due to lack of time, only the Biological ID was provided in the precious manuscript. We have edited the Accession Numbers of the raw reads in the SRA database in this revision (Line 113-115).

“The raw reads were deposited into the NCBI Sequence Read Archive (SRA) database (Accession Number: SRR12968935~ SRR12968943 (9 objects)) “(Line 113-115)

General comments:

Q1. The manuscript needs English editing. Lots of articles are missing, e.g. in the abstract, Line 12 “the” microbial food web and many others

Response:

Thanks a lot for your suggestion. We have edited the language of the manuscript (Line 12).

“The microbial food web, including phytoplankton, microzooplankton, bacteria, and planktonic viruses.” (Line 12)

Q2. Check that all taxa names are in italics, e.g. in Line 16 Anabaena circinalis and Microcystis flos-aquae

Response:

Thank you very much for your comments. These mistakes have been corrected (Line 16, 19, 20).

Q3. The title does not really fit, because you also study grazing (top-down control) effects on the food web, not only responses of microbes. How about:

Food web responses to a cyanobacterial bloom period in a freshwater eutrophic lake (just a suggestion)

Response:

Thanks a lot for your suggestion. In this study, we investigated the changes in the community structure of phytoplankton, zooplankton, and bacteria between the bloom and non-bloom periods, and studied the grazing of microzooplankton on phytoplankton. Therefore, we believe that the "microbial food web" is more appropriate than the "food web".

Abstract

Q4. -very little new insights presented. The take-home message and implications for the “bigger picture” could be presented in more details. That something is “more abundant” than something else is not very detailed either. Please try to add numbers where possible.

Response:

Thank you very much for your comments. Firstly, the study has the following highlights: (1) Both Anabaena circinalis and Microcystis flos-aquae were dominant species in bloom period; (2) The density of rotifers in algal bloom was higher than that in non-bloom; (3) Bacteroidetes were more abundant in bloom period than that in non-bloom; (4) Microzooplankton showed a higher grazing preference on Cyanophyta in bloom period compared to non-bloom period. Secondly, as you suggested, we have added number in the manuscript (Line 16-22).

“Firstly, Anabaena circinalis and Microcystis flos-aquae were both dominant species in bloom period. Secondly, rotifers were the major microzooplankton in microbial food web, and the density abundance of rotifers was higher in algal bloom (mean of 523.89 ind. /L) than that in non-bloom (mean of 471.27 ind. /L). Thirdly, Bacteroidetes were more abundant in bloom period (mean of 15.93%) than that in non-bloom (mean of 7.36%), while Chloroflexi and Acidobacteria were more abundant in non-bloom period (mean of 12.32% and 10.31%, respectively) compared to algal bloom period (mean of 0.78% and 0.22%, respectively).” (Line 16-22)

Q5. -Please add where Lake Nanhu is located (city, country), also in the methods.

Response:

Thank you very much for your comments. We supplemented the description of relevant information of Lake Nanhu in the article, and modified the map of Lake Nanhu in the method (Line 68-70).

“Lake Nanhu is located in Wuhan, Hubei Province, on the middle and lower reaches of the Yangtze River in China. It covers an area of 5.50 km², with a maximum depth of 3.2 m and an average depth of 1.6 m.” (Line 68-70)

Introduction:

Needs English editing.

Q6. Line 8: microbial loop not microbial food loop

Response:

Thank you very much for your suggestion. As you suggested, the mistake has been corrected in the revised manuscript (Line 30).

“Microbial food loop plays an important role in the material matter circulation and energy transfer of water ecosystem, which is an effective supplement to the classic food chain.” (Line 30)

Q7. Line 8: material circulation sounds technical, better “matter” or “organic matter” circulation

Response:

Thank you very much for your suggestion. As you suggested, we have replaced “material” with “matter” (Line 30).

“Microbial food loop plays an important role in the material matter circulation and energy transfer of water ecosystem, which is an effective supplement to the classic food chain.” (Line 30)

Q8. Line 34-36 “However, little is known about the microbial food web in freshwater ecosystem, especially in freshwater eutrophic lakes.” I do not agree with this very general knowledge gap presented here. There is lots of work out there:

Kiersztyn, B., Chróst, R., Kaliński, T., Siuda, W., Bukowska, A., Kowalczyk, G., & Grabowska, K. (2019). Structural and functional microbial diversity along a eutrophication gradient of interconnected lakes undergoing anthropopressure. Scientific reports, 9(1), 1-14.

Kalinowska, K., & Grabowska, M. (2016). Autotrophic and heterotrophic plankton under ice in a eutrophic temperate lake. Hydrobiologia, 777(1), 111-118.

Response:

Thanks a lot for your suggestion. We have revised the manuscript according to your suggestions (Line 36-37).

“However, more basic research is needed on the microbial food web in freshwater ecosystem, especially in freshwater eutrophic lakes.” Line 36-37

Q9. Maybe it’s worth to explain more what is special about the lake you investigated compared to other freshwater ecosystems (if there is anything special).

Response:

Thank you very much for your comments. Firstly, Lake Nanhu is a typical eutrophic lake in the city. The eutrophication of water body has been serious in the past 30 years due to the influence of human activities. Secondly, our laboratory has set up a monitoring station near Lake Nanhu, which is very convenient for long-term observation and experiment. Therefore, we chose Lake Nanhu as the investigation object.

Q10. L37: Microzooplankton consist of flagellates (will not comment on English any further)

Response:

Thank you very much for your suggestion. As you suggested, the mistake has been corrected (Line 38).

“Microzooplankton consist of flagellates, ciliates, heterotrophic dinoflagellates, microcrustaceans and small metazoans.” (Line 38)

Q11. L39/40: The growth, metabolism and grazing rate of microzooplankton are very fast. >What does it mean? Please make it more quantitative.

Response:

Thanks a lot for your comments. As you suggested, we have deleted this sentence and revised the language (Line 40).

“According to previous studies, microzooplankton played a vital role in the grazing on phytoplankton” (Line 40)

Q12. L49/50: Please write: Phytoplankton organisms are primary producers in the microbial food web

Response:

Thank you very much for your suggestion. As you suggested, the mistake has been corrected in the revised manuscript (Line 49-50).

“Phytoplankton organisms are major producers in the microbial food web and can respond quickly to environmental changes.” (Line 49-50)

Q13. L53: algal cells

Response:

Thank you very much for your suggestion. As you suggested, the mistake has been corrected in the revised manuscript (Line 53).

“Bacteria can make use of secretions released by phytoplankton, as well as the debris of algal cells.” (Line 53)

Q14. L54: Please provide reference for antibiotic release and nutrient competition by phytoplankton

Response:

Thank you very much for your suggestion. We have added references to the manuscript [22] (Line 54).

“Nygaard, K., Tobiesen, A. Bacterivory in Algae - a Survival Strategy during Nutrient Limitation. Limnol. Oceanogr. 1993, 38, 273–279.”

Q15. L58 to clarify not to clear

Response:

Thank you very much for your suggestion. As you suggested, the mistake has been corrected in the revised manuscript (Line 58-59).

“Therefore, further study on the variation of bacterial community structure could help us to clarify the functional role of microbial food web in eutrophic lake.” (Line 58-59)

Q16. L68/69: sentence has no verb

Response:

Thank you very much for your comments. As you suggested, the mistake has been corrected in the revised manuscript (Line 70-71).

“From October 2016 to September 2017, field surveys and sample collections were conducted at three different sites in Lake Nanhu every month.” (Line 70-71)

Material & Methods

Q17. L74: every month over which period of time? How many samples in total? Any controls?

Response:

Thank you very much for your comments. Firstly, we collected surface water samples and plankton in the middle of each month. Secondly, in this study, we have a total of 36 water samples and 144 plankton samples, respectively. Finally, the sampling time was in the sunny morning, and the same sampling location was guaranteed.

Q18. L75/76: how was the sample for bacterial composition exactly taken? From a boat? Which device?

Response:

Thank you very much for your comments. Firstly, we collected water samples at three different sites and quickly brought it back to the laborator. Then the samples were firstly through a 20 µm membraneand then filtered onto 0.22 µm polycarbonate filters. The filters containing bacteria were placed into 2 mL sterile tubes and immediately frozen in liquid nitrogen, and transferred to a −80 ◦C refrigerator for storage until further procedures. Our laboratory has a monitoring station by the Lake Nanhu, so all operations can be completed within 1-2 hours.

Q19. Figure 1: Please show within a bigger map where Hubei is located and what Hubei is (city, region…?)

Response:

Thanks a lot for your suggestion. As you suggested, we have modified the map of Lake Nanhu in the method.

Q20. L90: sentence has no verb

Response:

Thank you very much for your suggestion. As you suggested, the mistake has been corrected (Line 92).

“Water samples for bacteria community analysis were firstly filtered through a 20 µm membrane (Millipore, Carrigtwohill, Co, Cork, Ireland) and then filtered onto 0.22 µm polycarbonate filters (Millipore, Carrigtwohill, Co, Cork, Ireland).” (Line 92)

Q21. Please explain the rationale behind the filtration procedure.

Response:

Thanks a lot for your comments. The filtration procedure uses the principle that the pore size of the filter membrane cannot allow bacteria to pass through to keep the bacteria on the filter membrane. Firstly, using 20 µm filter membrane to filter the water samples to remove impurities and retain bacteria. Then filter the filtered water with a 0.22 µm filter membrane to keep the bacteria on the filter membrane.

Q22. L94: Please mention details on DNA extraction kit, primers used and PCR conditions

Please mention earlier that you target the 16S rRNA gene.

Response:

Thank you very much for your suggestion. As you suggested, we have added detailed experimental methods in the modified version (Line 97-111).

“Bacterial DNA was extracted from all water samples using the E.Z.N.A.fi soil DNA Kit (Omega Bio-tek, Norcross, GA, USA) according to the manufacturer’s protocols. The final DNA concentration and purification were determined using a NanoDrop 2000 UV–vis spectrophotometer (Thermo Scientific, Wilmington, NC, USA), and the DNA quality was checked via 1% agarose gel electrophoresis. The V3–V4 hypervariable regions of the bacteria 16S rRNA gene were amplified with primers 338F (50-ACTCCTACGGGAGGCAGCAG-30) and 806R (50-GGACTACHVGGGTWTCTAAT-30) using a thermocycler PCR system (GeneAmp 9700, ABI, Foster City, CA, USA). The PCR reactions were conducted using the following program: 3 min of denaturation at 95 ◦C; 27 cycles of 30 s at 95 ◦C, 30 s for annealing at 55 ◦C, and 45 s for elongation at 72 ◦C; and a final extension at 72 ◦C for 10 min. PCR reactions were performed in triplicate in a 20 µL mixture containing 4 µL of 5× FastPfu Buffer 2 µL of 2.5 mM dNTPs, 0.8 µL of each primer (5 µM), 0.4 µL of FastPfu Polymerase, and 10 ng of template DNA. The PCR products were extracted from a 2% agarose gel, further purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA), and quantified using QuantiFluor™-ST (Promega, Madison, WI, USA) according to the manufacturer’s protocol. “(Line 97-111)

Q23. L98: The bioproject PRJNA673947 does not exist in SRA.

Response:

Thank you very much for your comments. Previously, due to lack of time, only the Biological ID was provided in the article. We have edited the Accession Numbers of the raw reads in the SRA database (Line 113-115).

“The raw reads were deposited into the NCBI Sequence Read Archive (SRA) database (Accession Number: SRR12968935~ SRR12968943 (9 objects)).” (Line 113-115)

Q24. L104: How (using which programs) were these parameters checked?

Response:

Thank you very much for your comments. As your suggestion, we have added detailed methods in the modified version (Line 116-125).

“Raw reads were demultiplexed, quality filtered by Trimmomatic, and merged by FLASH with the following criteria: (i) The reads were truncated at any site receiving an average quality score of <20 over a 50 bp sliding window. (ii) Primers were exactly matched allowing two nucleotide mismatches, and reads containing ambiguous bases were removed. (iii) Sequences with overlap longer than 10 bp were merged according to their overlap sequence. Operational taxonomic units (OTUs) were clustered with a 97% similarity cutoff using UPARSE (version 7.1 http://drive5.com/uparse/) and chimeric sequences were identified and removed using UCHIME. The taxonomy of each 16S rRNA gene sequence was analyzed using the RDP Classifier algorithm (http://rdp.cme.msu.edu/) against the Silva (SSU123) 16S rRNA database using a confidence threshold of 70%.” (Line 116-125)

Q25. L106/107: one-way ANOVA was used to test for significant differences.

Significant differences between which variables? Were prerequisites for a parametric test fulfilled?

Response:

Thanks for your comments. One-way ANOVA was used to test for significant differences in bacteria structure among different months. The variables were months and prerequisites were fulfilled for a parametric test.

Q26. L109/110: please add more details on how grazing was studied rather than just referring to the literature

Response:

Thank you very much for your suggestion. The specific experimental methods of the grazing experiment are as follows:

Before the experiment, the culture bottle (1.2 L, Whatman) was soaked with 10% hydrochloric acid for more than 10 h, and then washed with the collected water. Subsequently, water samples were filtered with 0.22 μm (Millipore) filter membrane to obtain particle-free water. Then the particle-free water was mixed with the water samples in four proportions of 0:1, 1:3, 1:1, 3:1, and divided into culture bottles with 3 parallel samples for each proportion. Taking care to fill each culture bottle with water and avoid air bubbles. The culture bottle was fixed with a rope and then cultured for 24 hours in an experimental pool near Lake Nanhu. Before and after culture, 3 × 300mL water samples were taken from each proportion and filtered with 20 μm, 5 μm (Millipore) and GF/F (GE Healthcare) filtration membrane, respectively, to obtain three kinds of phytoplankton with different particle sizes (20-200 μm, 5-20 μm and <5 μm). The filter membranes were stored in a refrigerator at -20 °C, and the concentration of chlorophyll a was measured by spectrophotometric method to obtain the biomass of phytoplankton (Line 132-148).

Q27. L118 provide city, country for manufacturers (here and everywhere else)

Response:

Thanks for your suggestions. As you suggested, we have modified it in the manuscript (Line 93-94, 132, 134, 141-142).

Results:

Q28. L.183 (and in other Lines): with “density” of organisms, you refer to their (relative) abundance?

Response:

Thank you very much for your suggestions. As you suggested, we have modified it in the manuscript (Line 229, 231-232, 237-239).

“Secondly, rotifers were the major microzooplankton in microbial food web, and the abundance of rotifers was higher in algal bloom (mean of 523.89 ind. /L) than that in non-bloom (mean of 471.27 ind. /L).” (Line 17-19)

“As shown in Fig. 3, TN, temperature and NO^3- were the main factors affecting the plankton abundance in Lake Nanhu. The abundance of Cyanophyta and Euglenophyta were positively correlated with T, NO^2- and pH, and negatively correlated with NH⁴⁺ and TN. The abundance of Cryptophyta, Bacillariophyta and copepod (Mesocyclops leuckarti) were positively correlated with TP and negatively correlated with pH and DO, while Pyrrophyta and rotifer were positively opposite to the above three. The abundance of cladoceran were positively correlated with TP and PO₄^3-, while the abundance of protozoan was positively correlated with NO^3-.” (Line 206-212)

“The average abundance of phytoplankton during non-bloom period in Lake Nanhu was 8.01 × 10⁶ cells/L, mainly dominated by Bacillariophyta (3.47 × 10⁶ cells/L). However, the average abundance of phytoplankton during bloom period was 2.71× 10⁷ cells/L, and mainly dominated by Cyanophyta (1.64 × 10⁷ cells/L) (Fig. 4A). Moreover, the abundance of phytoplankton was highest in May (3.37 × 10⁷ cells/L) and lowest in December (1.26 × 10⁶ cells/L).” (Line 224-228)

“Variation of abundance on plankton in Lake Nanhu during non-bloom and bloom period. The density on phytoplankton (A) and zooplankton (B) in Lake Nanhu.” (Line 241)

Q29. L.185: according to the species table, there is only one species of copepod. Wouldn’t it make sense to mention that here instead of just “copepod”.

Response:

Thank you very much for your suggestions. As you suggested, we have edited it in the manuscript (Line207-208, 220, 231-233).

“The abundance of Cryptophyta, Bacillariophyta and copepod (Mesocyclops leuckarti) were positively correlated with TP and negatively correlated with pH and DO, while Pyrrophyta and rotifer were positively opposite to the above three.” (Line 207-208)

“Z2: Copepod (Mesocyclops leuckarti)” (Line 220)

“Qualitative analysis of zooplankton revealed that the main zooplankton in Lake Nanhu included protozoans (39 species), rotifers (21 species), cladocerans (8 species) and copepods (Nauplii and Mesocyclops leuckarti) (Table S2).” (Line 231-233)

Q30. L204: Anabaena circinalis (1.45 × 107 cells/L) and Microcystis flos-aquae (1.10 × 107 cells/L) were considered to be the causative organisms

This is rather discussion than results

Response:

Thank you very much for your suggestions. As you suggested, we have deleted it and removed it to discussion (Line 349-350).

“In Lake Nanhu, both A. circinalis and M. flos-aquae were considered to be the causative organisms.” (Line 349-350)

Q31. L224-226: The results revealed that Bacillariophyta biomass was high both in non-bloom and bloom period, while Pyrrophyta biomass was high in non-bloom period, Cyanophyta biomass was high in bloom period.

Please provide numbers. This is more informative than stating 3x that something is high.

Response:

Thank you very much for your suggestions. We have modified it in the manuscript (Line 249-251).

“The results revealed that Bacillariophyta biomass was high both in non-bloom and bloom period (mean of 41.26% and 21.08%, respectively), while Pyrrophyta biomass was high in non-bloom period (mean of 47.12%), Cyanophyta biomass was high in bloom period (mean of 32.67%).” (Line 249-251)

Q32. Figure 5: please remove two decimal places on y axis, since it’s all zero. Caption: the term is “accessory”; please add some more details to the caption (which pigments were assayed etc.)

Response:

Thank you very much for your comments. As you suggested, Figure 5 has been modified. In addition, we have added the pigments to the caption (Line 253-254).

“The relative abundance of different accessory pigments (Fucoxanthin, Peridinin, Zeaxanthin, Alloxanthin, Lutein and Violaxanthin) during non-bloom and bloom period in Lake Nanhu.” (Line 253-254)

Q33. Table 1: please write the sampling month next to S2. Please describe terms and abbreviations in the header (Ace, Chao1, OTUs) and upon first use in the main text.

Response:

Thank you very much for your suggestions. As you suggested, Table 1 has been modified. In addition, we have modified it in the manuscript (Line 120-122, 125-128, 265-266) and table.

“Operational taxonomic units (OTUs) were clustered with 97% similarity cutoff using UPARSE（version 7.1 http://drive5.com/uparse/) and chimeric sequences were identified and removed using UCHIME. ” (Line 120-122)

“Nonparametric indicators (the Chao 1 estimator (Chao1), the Shannon estimator (Shannon), the Ace estimator (Ace) and the Good’s coverage (coverage)) were used to evaluate the relationships between bacterial community diversity characteristics and community coverage in Lake Nanhu.” (Line 125-128)

“OTUs: Operational taxonomic units, Shannon: the Shannon estimator, Chao1: the Chao 1 estimator, Ace: the Ace estimator, Coverage: the Good’s coverage.” (Line 265-266)

Q34. L242: were the most abundant bacteria

Response:

Thank you very much for your comments. As you suggested, the mistake has been corrected (Line 268).

“Proteobacteria (32.97–52.78%) were the most abundant bacteria.” (Line 268)

Q35. L244: proportions (plural) > English editing please

Response:

Thank you very much for your suggestions. As you suggested, the mistake was corrected (Line 270).

“In the contrast, Nitrospinae, Firmicutes and Verrucomicrobia, only represented a minor proportions.” (Line 270)

Q36. Figure 6: It’s ANOVA, not ANOVE; please use scientific notation for very small p-values;

Response:

Thanks a lot for your comments. The mistake was corrected and the very small p-values have been modified to use scientific notation. We have unified the sample IDs in Figure 6 and Table 1 with S1, S2, and S3. According to your suggestion, we used “community composition” instead of “classification structure” (Line 284, 290-291).

“Community composition and relative abundance of bacteria.” (Line 284)

“S1, S2, and S3: surface water samples. A, B, and C represent March, May, and November respectively.” (Line 290-291)

Q37. L.270 Correlation statistics were not explained in the methods. Always state the test used next to the p-value, e.g. (Spearman rank correlation, p < 0.05, r2 = 0.81).

Response:

Thank you very much for your suggestions. We have modified it in the manuscript (Line 297).

Q38. L.276 standing stocks (Pi) in the main text, but in the caption of Fig 7, Pi is phytoplankton biomass. Please use consistent terms throughout the manuscript

Response:

Thanks a lot for your suggestions. As you suggested, we have unified the caption of figure and main text (Line 317, 345).

“The grazing pressure of microzooplankton on size specific phytoplankton (C) standing stocks (Pi) and (D) primary productivity (Pp).” (Line 317)

“The grazing pressure of microzooplankton on pigment specific phytoplankton (C) standing stocks (Pi) and (D) primary productivity (Pp).” (Line 345)

Q39. Figure 7: the label of the y axis in D) is cut off. Is this mean or median presented? Is the error bar standard deviation or standard error? Is this from n=3? Please mention filter sizes in the caption as well.

Response:

Thanks a lot for your suggestions. As you suggested, we have corrected the figure. Firstly, the numerical values in the figure are mean values. Secondly, the error bar is standard deviation (n=3). Finally, we have added filter sizes in the caption (Line 314).

“Variation of microzooplankton grazing on size specific phytoplankton (20-200 μm, 5-20 μm and <5 μm) during non-bloom and bloom period.” (Line 314)

Discussion

I feel the discussion is quite superficial (considering the amount of results presented) and should be much extended.

Q40. e.g. L 333: Rotifers could respond to Microcystis bloom by a variety of mechanisms. > What are these mechanisms? How would other zooplankton species cope?

Response:

Thanks a lot for your suggestions. Firstly, rotifers could adapt to algal bloom by changing their life history and grazing intensity. Secondly, we have supplemented the mechanism by which cladocerans adapt to cyanobacteria blooms (Line 362-366).

“Previous studies have proposed that small cladocerans were more resistant to cyanobacteria and ingesting bacteria than large cladocerans, so they are more adaptable to algal blooms [50-51]. This study also found that in the Lake Nanhu, small cladocerans such as Moina macrocopa and Diaphanosoma leuhtenbergianum were the main cladocerans in the bloom period.” (Line 362-366)

Q41. You described Anabaena and Microcystis and that rotifers feed on Microcystis (which is long know). Why should a reader care, what are the implications of this, e.g. for the food web or status of the Lake ecosystem? Is there something special about this Lake compared to others? What can induce such blooms (high nutrient levels, anthropogenic input close by?)

Same in 4.2

Response:

Thanks a lot for your suggestions. As you suggested, we have edited the manuscript (Line 400-403). Firstly, Lake Nanhu is a typical eutrophic lake in the city. The eutrophication of water body has been serious in the past 30 years due to the influence of human activities. Secondly, our laboratory has set up a monitoring station near Lake Nanhu, which is very convenient for long-term observation and experiment. Therefore, we chose Lake Nanhu as the investigation object.

“Although some previous studies have individually studied the response of phytoplankton, microzooplankton and bacteria to cyanobacteria blooms. It is a food web and the interactions of organisms are tightly interwoven. Therefore, in this study, we have studied the response of the entire microbial food web to the algal bloom.” (Line 400-403)

Q42. e.g. l345 the results showed that the bacterial community structure could respond to the occurrence of algal blooms

> This is a very general statement and nothing really new.

Response:

Thanks a lot for your suggestions. As you suggested, we have edited the manuscript (Line 400-403).

Q43. L343 sounds like dechlorination participates in organic matter degradation, please re-phrase.

Response:

Thanks a lot for your suggestions. As you suggested, we have revised the manuscript (Line 374-376).

Q44. Please rework the discussion as suggested and include more literature. You have many nice results, but they are not optimally used in the discussion and the overall ecological context. As it is presented now, the story is very descriptive, and I unfortunately do not see so much novelty in the study.

Response:

Thanks a lot for your comments. In this study, we used some simple experimental methods to investigate the response of the microbial food web in the freshwater eutrophic lake Nanhu to algal blooms. The study has the following highlights: (1) Both Anabaena circinalis and Microcystis flos-aquae were dominant species in bloom period; (2) The density of rotifers in algal bloom was higher than that in non-bloom; (3) Bacteroidetes were more abundant in bloom period than that in non-bloom; (4) Microzooplankton showed a higher grazing preference on Cyanophyta in bloom period compared to non-bloom period. Although some previous studies have individually studied the response of phytoplankton, microzooplankton and bacteria to cyanobacteria blooms. It is a food web and the interactions of organisms are tightly interwoven. Therefore, in this study, we have studied the response of the entire microbial food web to the algal bloom.

Author Response File: Author Response.pdf

Reviewer 3 Report

Journal Microorganisms (ISSN 2076-2607)

Manuscript ID microorganisms-994681

Title The responses of microbial food web to algal bloom in freshwater eutrophic lake

Authors

Mengqi Han , Chenchen Dong , Siqi Ma , Cui Feng , Chengqiang Lei , Zemao Gu , Xiangjiang Liu *

Major Comments

This manuscript showed data on the structural changes of phytoplankton and zooplankton in eutrophic Lake Nanhu, and compared these structural changes between the bloom and non-bloom period. The results demonstrated that the microbial food webs could respond to the occurrence of algal blooms in eutrophic lake.

First of all, in your literature search, the main components of the microbial food web include heterotrophic bacteria, phytoplankton, microzooplankton, and planktonic viruses. However, in your study, just focus on the phytoplankton and bacterial communities, to me, I think that picophytoplankton (including Synechococcus, Prochlorococcus, and picoeukaryotes), that are the major components in the microbial loop. However, the authors did not show any data about these. As for microzooplankton, ciliates and nanoflagellates are the important components in microzooplankton communities; however, the authors also did not show this information. In this situation, I strong suggest authors to change the “Title” to “The responses of phytoplankton communities to algal bloom…….”.

Secondly, in Methods section. To me, I am interest in the area of three different study sites, why did the authors collect water samples from three sites (S1, S2 and S3)? In addition, how to analyses the data of comparing non-bloom and bloom in phytoplankton, bacterial and zooplankton communities?

Thirdly, please discuss deeper in the Discussion section.

To me, I think this paper has not been well characterized as of yet, so I strongly encourage the authors to reanalyze their data and make the appropriate modifications to the manuscript. This manuscript needs to be addressed and the results and discussion rewritten to focus on the new analysis.

Author Response

Response to Reviewer 3:

Comments and Suggestions for Authors

Major Comments

Response:

Thank you very much for your help with the paper review.

Q1. First of all, in your literature search, the main components of the microbial food web include heterotrophic bacteria, phytoplankton, microzooplankton, and planktonic viruses. However, in your study, just focus on the phytoplankton and bacterial communities, to me, I think that picophytoplankton (including Synechococcus), Prochlorococcus, and picoeukaryotes, that are the major components in the microbial loop. However, the authors did not show any data about these. As for microzooplankton, ciliates and nanoflagellates are the important components in microzooplankton communities; however, the authors also did not show this information. In this situation, I strong suggest authors to change the “Title” to “The responses of phytoplankton communities to algal bloom…….”.

Response:

Thanks a lot for your comments. Firstly, all the algae we have identified are listed in supplemental table 1, including Synechococcus. In the main text, we have discussed phytoplankton at the phylum level, among which the Cyanophyta includes Synechococcus. Similarly, we also have mentioned a variety of ciliates (e.g. Strombidium sp, Vorticella sp, Coleps hitus) in the zooplankton in supplemental table 2. Therefore, we believe that the "microbial food wed" is more suitable than "phytoplankton community".

Q2. Secondly, in Methods section. To me, I am interest in the area of three different study sites, why did the authors collect water samples from three sites (S1, S2 and S3)? In addition, how to analyses the data of comparing non-bloom and bloom in phytoplankton, bacterial and zooplankton communities?

Response:

Thank you very much for your comments. Firstly, S1, S2 and S3 are evenly distributed throughout the Lake Nanhu, which can more comprehensively reflect the situation of the lake. And Nanhu Lake is a small shallow lake, and the entire water area will enter the bloom period at the same time. Secondly, we averaged the community abundances of phytoplankton, zooplankton and bacteria on a monthly basis to compare the bloom period and non-bloom period.

Q3. Thirdly, please discuss deeper in the Discussion section. To me, I think this paper has not been well characterized as of yet, so I strongly encourage the authors to reanalyze their data and make the appropriate modifications to the manuscript. This manuscript needs to be addressed and the results and discussion rewritten to focus on the new analysis.

Response:

Thank you very much for your suggestions. Based on the suggestions of you and other reviewers, we have revised the discussion section of the manuscript (Line 363-366, 33-377, 388-389, 401-406).

“Previous studies have proposed that small cladocerans are more resistant to cyanobacteria and ingesting bacteria than large cladocerans, so they are more adaptable to algal blooms [50-51]. This study also found that in the Lake Nanhu, small cladocerans such as Moina macrocopa and were the main cladocerans in the bloom period.” (Line 364-366)

“During algal bloom, this study also found that both cyanobacteria and Bacteroidetes were more aboundant than during non-bloom period. Chloroflexi can participate in the degradation of organic matter [54-55]. In this study, the results showed that the abundance of the Chloroflexi was relatively higher after algal bloom (November). Therefore, the results showed that the bacterial community structure could change between the bloom and non-bloom.” (Line 373-377)

“Studies have also found that although microplankton can grazing on algae, they have little effect during the outbreak period.” (Line 388-389)

“Although previous studies have individually studied the response of phytoplankton, microzooplankton and bacteria to cyanobacteria blooms. It is a food web and the interactions of organisms are tightly interwoven. Therefore, in this study, we have studied the response of the microbial food web to the algal bloom. However, microplankton grazing has little effect during the outbreak period. The treatment of algal blooms should still reduce sewage discharge from the source and pay more attention to ecological protection.” (Line 401-406)

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

the authors have carefully and exhaustively corrected the manuscript and their effort is much appreciated. They have corrected all language difficulties - again thank you.

recommendation: Publish

Author Response

Thank you very much for your help with the paper review along the way.

Reviewer 2 Report

Microorganisms-994681 – R2

The authors have worked on the manuscript and I see some improvement. However, I still have some concerns: the underlying sequence data is not accessible, p-value not adequately reported. The discussion is still very superficial and descriptive. What the authors describe as highlights in their cover letter is also not very convincing in terms of novelty, e.g. that an Anabaena species is a dominant species in a bloom is not a surprising finding. Due to missing relevance, novelty and overall substance, I cannot recommend publication of the manuscript.

-English editing still required

-abundances in abstract: is this relative or absolute abundance?

The title is not suitable, because zooplankton are simply not microbes.

Figure 1: the map should say what this country is.

l.38 more basic research

l.97: the 20 µm membrane was for pre-filtration but has not been further used in any way? This is still not clear.

l.111 the primers lack a reference

l.124: the data is still not accessible to me

l.137: it should be mentioned that the prerequisites for ANOVA were tested.

l.253: respectively

L.322: here and elsewhere: please give the actual p-value, not just <0.05. This could mask weak significance. Spearman rank correlation has not been mentioned in the methods.

L.435: microzooplankton?

Author Response

Response to Reviewer 2:

The authors have worked on the manuscript and I see some improvement. However, I still have some concerns: the underlying sequence data is not accessible, p-value not adequately reported. The discussion is still very superficial and descriptive. What the authors describe as highlights in their cover letter is also not very convincing in terms of novelty, e.g. that an Anabaena species is a dominant species in a bloom is not a surprising finding. Due to missing relevance, novelty and overall substance, I cannot recommend publication of the manuscript.

Response:

Thank you very much for your help with the paper review. As you suggested, we have revised the manuscript, especially discussion part (Line 362-466). And we have revised the reply about the highlights in the last cover letter (Q44).

“HPLC characteristic pigments also showed that Cyanophyta was the algae that caused the bloom in Lake Nanhu (Fig. 5).” Line 363-364

“In this study, the composition of the phytoplankton community in Lake Nanhu exhibited changes between bloom and non-bloom period. Bacillariophyta and Cyanophyta were the main dominant species in non-bloom and bloom period, respectively. The result of HPLC characteristic pigments also confirmed this conclusion. There are many factors that influence the change of phytoplankton community (such as physical indicators, nutrients, grazing pressure). When the temperature increased from April to May, the abundance of Cyanophyta increased rapidly and became the dominant species in water. Previous study has shown that Cyanophyta can reduce the abundance of other phytoplankton species by allelopathic mechanism [46]. Therefore, Bacillariophyta cannot continue to be dominant species in water even though their abundance increased with the increased of TP concentration. Studies have shown that Cyanophyta abundance is positively correlated with TN concentration [47-48], which is inconsistent with the results of this study. The possible explanation is that the Microcystis blooms could reduce the nitrogen in water bodies [49]. In addition, the discharge of domestic sewage and aquaculture are also important reasons for the changes in the phytoplankton community. Compared with deep lakes, shallow-water lakes are more susceptible to external changes, and the interaction between phytoplankton and its ecosystem is more significant. Therefore, the relationship between phytoplankton and environmental factors in shallow eutrophic lakes needs more in-depth research.” Line 354-381

“Zooplankton is an important part of the water environment and is crucial to maintaining the stability of freshwater ecosystems [50]. In this study, rotifers were the main components of zooplankton in Lake Nanhu, followed by cladocerans. The abundance of rotifers and cladocerans in bloom period was higher than that in non-bloom period. This may be due to the higher abundance of phytoplankton and water temperature during the bloom. Within the appropriate range of temperature and food density, the population density of rotifers and cladocerans increase with the increase of temperature and food density [51-54].” Line 383-389

“B. calyciflorus was the most dominant zooplankton in the non-blooming period, while the most dominant species in the blooming period was M. macrocopa, followed by B. calyciflorus. There are many factors influencing the relationship between rotifers and cladocerans, which are often the result of the combined action of biological and non-biological factors, such as temperature [55-56], food [57], individual size [58-59] and grazing [60-61]. When the individual size of cladocerans is less than 1200 μm, the competitive inhibitory effect of cladocerans on rotifers is much weaker, and rotifers can coexist with them at a higher density [62]. Previous studies reported that the individual size of M. macrocopa is mostly around 1200 μm [63-64]. Therefore, when M. macrocopa competes with rotifers, environmental factors such as temperature and food may have a greater effect.“ Line 390-398

“Zooplankton, especially rotifers and cladocerans, are very sensitive to temperature changes in shallow lakes [65]. Many studies have shown that the quantity, quality and type of food have a significant impact on the abundance, diversity and interspecific competition outcome of zooplankton [66-70]. Previous study has shown that the cyanobacteria bloom was observed together with the high abundance of small-sized zooplankton [71]. Studies have shown that B. calyciflorus could adapt to eutrophic water by changing their life history and grazing intensity [72]. Fulton and Paerl pointed out that rotifers could consume small Microcystis groups [73]. Small cladocerans have a more obvious competitive advantage than large cladocerans at higher temperatures, which is believed to be the reason why small cladocerans have higher population abundance in warm waters [74]. Previous studies have also proposed that small cladocerans were more resistant to cyanobacteria and ingesting bacteria than large cladocerans, so they are more adaptable to algal blooms [75-76]. This may be the reason why M. macrocopa become the dominant species during bloom period of the eutrophic shallow lake Nanhu.” Line 399-411

“The composition of the plankton bacterial community in water is regulated by a variety of biological and non-biological factors such as phytoplankton, zooplankton, temperature [77], and nutrients [78]. The organic matter produced by phytoplankton and zooplankton provides energy for the growth of bacteria [79-81]. Studies have found that during the lake bloom period, the number and species of bacteria have changed significantly, and this change could be used to predict the algal bloom [82]. In addition, zooplankton can also directly graze bacteria, which affects the number and distribution of bacteria [83]. Temperature can indirectly affect the community composition of bacteria by affecting the community structure of phytoplankton and zooplankton [84]. In lake ecosystems with different nutrient levels, nutrients have different effects on bacteria. In oligotrophic lakes, nutrients can become a limiting factor for the growth of bacteria [85-86]. In mesotrophic lakes, nutrients have different limiting effects on bacteria in different seasons [87]. In eutrophic lakes, the effect of nutrients on bacteria is less than that of phytoplankton [88].” Line 413-424

“In this study, bacteria communities were mainly dominated by Proteobacteria and Actinobacteria, which contributed more than 50% to relative abundance of bacteria in both bloom and non-bloom periods (Fig. 6A). Moreover, the mean proportion of Bacteroidetes in May (bloom period) was significantly higher than that in March and November (non-bloom period) (Fig. 6B). The result also supported the increase of cyanobacteria could promote the growth of Proteobacteria, Actinobacteria and Bacteroidetes [89]. Previous studies have found that the organic secretions of cyanobacteria could adsorb Bacteroidetes, and the Bacteroidetes could lyse cyanobacteria cells [90-91]. Pinhassi et al. also found that the Bacteroidetes may play a vital role in the processing of organic matter during the algal bloom [92]. Bacteroidetes were tightly associated with the algal bloom. Thus, bacteria were believed to participate in certain important activities during algae blooms, and need to be study further.” Line 425-434

“Microzooplankton grazing rates varied similarly with the growth rates of phytoplankton in different size groups during bloom period (Figure 7A, B). The result showed that microzooplankton grazing can respond quickly to the increase in phytoplankton abundance [93]. During non-bloom period, grazing pressure of microzooplankton on the standing stocks (Pi) for phytoplankton <5 μm groups were relatively higher than that for the other two size groups. In comparison, Pi were not significantly different among those three size groups of phytoplankton during bloom period (p>0.05), even though all the Pi values increased. This indicates that small size groups of phytoplankton could be effectively controlled by microzooplankton during non-bloom period.” Line 436-443

“Grazing pressure of zooplankton can be related to the type of food, body size, feeding mode, selectivity and tolerance to prey [94]. Copepods and cladocerans display selectivity on size of food particles and type of food, while rotifers display selectivity in regard to condition of algal cells as wells as type of food [95]. In this study, microzooplankton showed preference to Pyrrophyta and Bacillariophyta both in bloom and non-bloom periods, while they showed preference to Cryptophyta and Cyanophyta during non-bloom period and bloom period, respectively. During bloom period, phytoplankton community was dominated by Cyanophyta and microzooplankton community was dominated by rotifers. Although phytoplankton such as Anabaena and Microcystis were poor quality prey to rotifers, microzooplankton still showed preference on Cyanophyta. This indicates that grazing preference of microplankton during the bloom period was relatively more affected by the community composition of phytoplankton.” Line 444-454

“In the present study, microzooplankton grazing could quickly respond to phytoplankton growth. However, the improved growth conditions at the onset of a bloom allow phytoplankton to escape microzooplankton grazing pressure [96]. Therefore, the regulation of cyanobacteria blooms in Lake Nanhu requires more other methods. In the past 30 years, the main pollution sources of Lake Nanhu were the discharge of domestic sewage and the release of aquaculture feed. At present, under the management of relevant departments and policies, the sewage outlets of Lake Nanhu have been basically blocked. To further improve the water environment, more approaches need to be undertaken. Biomanipulation is an important theory first proposed by Shapiro et al to control algae in eutrophic lakes [97]. Since then, traditional and non-traditional biomanipulation has been widely used in the prevention and control of eutrophication of water bodies in Europe, North America and China [97-99]. According to the current status of Lake Nanhu, it is possible to counteract cyanobacteria blooms by protecting and re-establishing the submerged macrophytes, and increase the stocking of filter-feeding fish.” Line 455-468

“In the present study, the community of phytoplankton, zooplankton, and bacteria changed between bloom and non-bloom periods. Firstly, Cyanophyta, especially A. circinalis and M. flos-aquae, were mainly dominant species during bloom period. Secondly, rotifers were the main components of zooplankton in Lake Nanhu, followed by cladocerans. B. calyciflorus and M. macrocopa were the most dominant zooplankton in the non-bloom and bloom period, respectively. Thirdly, Bacteroidetes showed significantly higher mean proportion in bloom period than that in non-bloom period. Moreover, microzooplankton grazing could respond quickly to the increase in phytoplankton abundance. Finally, microzooplankton preference during bloom period was mainly affected by the community composition of phytoplankton.” Line 470-478

Q1. English editing still required

Response:

Thank you very much for your suggestions. As you suggested, we have modified it in the manuscript (Line 40-41, 42-43, 66-69)

“However, studies on the microbial food web in freshwater ecosystem, especially in freshwater eutrophic lakes, still need further investigation.” Line 40-41

“Microplankton is a general term for a kind of heterotrophic and polyculture zooplankton with a body length of less than 200 μm.” Line 42-43

“Due to the discharge of a large amount of domestic sewage and the introduction of aquaculture feed, the water of Lake Nanhu has long-term eutrophication, and algal blooms occur more than three times a year, usually from May to September.” Line 66-69

Q2. abundances in abstract: is this relative or absolute abundance?

Response:

Thanks for your comments. The abundance of phytoplankton and zooplankton refers to absolute abundance, and the abundance of bacteria refers to relative abundance.

Q3. The title is not suitable, because zooplankton are simply not microbes.

Response:

Thank you very much for your comments. As you suggested, we have modified it in the manuscript (Line 2-3).

“Food web responses to a cyanobacterial bloom period in a freshwater eutrophic lake” Line 2-3

Q4. Figure 1: the map should say what this country is.

Response:

Thank you very much for your suggestions. As you suggested, we have edited the caption of the map (Line 90).

“The location of three sampling sites in Lake Nanhu, Wuhan, China.” Line 90

Q5. l.38 more basic research

Response:

Thank you very much for your suggestions. As you suggested, we have modified it in the manuscript (Line 40-41).

“However, studies on the microbial food web in freshwater ecosystem, especially in freshwater eutrophic lakes, still need further investigation.” Line 40-41

Q6. l.97: the 20 µm membrane was for pre-filtration but has not been further used in any way? This is still not clear.

Response:

Thank you very much for your comments. In this study, the 20 µm membrane was used for pre-filtration to remove impurities, so it was not mentioned later. As you suggested, we have modified it in the manuscript (Line 99-103).

“Water samples for bacteria community analysis were firstly filtered through a 20 µm membrane to remove impurities (Millipore, Carrigtwohill, Co, Cork, Ireland) and then filtered onto 0.22 µm polycarbonate filters (Millipore, Carrigtwohill, Co, Cork, Ireland). The 0.22 µm filters containing bacteria were placed into 2 mL sterile tubes and immediately frozen in liquid nitrogen, and transferred to a −80 ◦C refrigerator for storage until further procedures.” Line 99-103

Q7. l.111 the primers lack a reference

Response:

Thank you very much for your suggestion. As you suggested, we have supplemented the reference [31] (Line 111).

“31. Feng, C., Jia, J., Wang, C., Han, M., Dong, C., Huo, B., Li, D., Liu X. Phytoplankton and Bacterial Community Structure in Two Chinese Lakes of Different Trophic Status. Microorganisms 2019, 7, 621.”

Q8. l.124: the data is still not accessible to me

Response:

Thank you very much for your comments. We've updated the release date for the BioProject below to 2020-12-17.

Q9. l.137: it should be mentioned that the prerequisites for ANOVA were tested.

Response:

Thank you very much for your suggestions. As you suggested, we have modified it in the manuscript (Line 135).

“The prerequisites were fulfilled for a parametric test and One-way ANOVA was used to test for significant differences in bacteria structure among different months.” Line 135

Q10. l.253: respectively

Response:

Thank you very much for your suggestion. As you suggested, the mistake has been corrected (Line 244).

“Rotifers (mean of 471.27 ind./L and 523.89 ind./L, respectively) were the main microzooplankton components in two periods.” Line 244

Q11. L.322: here and elsewhere: please give the actual p-value, not just <0.05. This could mask weak significance. Spearman rank correlation has not been mentioned in the methods.

Response:

Thank you very much for your suggestion. As you suggested, we have modified it in the manuscript (Line 305-306, 308, 309-310, 192-193).

“Phytoplankton growth rates and microzooplankton grazing rates for phytoplankton < 5 μm groups were both significantly lower than other groups in January and November (p=0.03 < 0.05).” Line 308

“In May, microzooplankton grazing rates for < 5 μm groups were higher than other groups (p =0.04< 0.05), and phytoplankton growth rates were lower than other groups (p =0.04< 0.05).” Line 309-310

“The correlation between microplankton grazing rate and phytoplankton growth rate was tested by Spearman rank correlation.” Line 192-193

Q12. L.435: microzooplankton?

Response:

Thank you very much for your reminding. We have checked and corrected the spelling mistakes.

Reviewer 3 Report

Sorry, I cannot give any comments in this round review process.

Author Response

Thank you very much for your help with the paper review along the way.

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Food Web Responses to a Cyanobacterial Bloom in a Freshwater Eutrophic Lake

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