Sato et al. conducted a field study to investigate the distribution of non-cyanobacterial diazotrophs (NCDs), especially Gamma A, around the Kuroshio region and used a combination of dilution experiments and quantitative PCR to explore the top-down control of microzooplankton grazing on Gamma A. The topic is interesting and will attract many audiences interested in marine nitrogen fixation. Overall, the manuscript is well-written. However, some issues need to be addressed prior to publication.

General comments

1. My major concern is about the calculation of the growth rate of Gamma A and the whole phytoplankton community via the dilution technique. The authors mention three kinds of growth rates: the maximum growth rate (μ_max), the net growth rate without nutrient enrichment (μ₀), and the net growth rate with nutrient enrichment (μ_en). The μ_max is the growth rate of phytoplankton (Gamma A) under nutrient-replete conditions. It is not the in situ growth rate because phytoplankton growth could be limited by in situ nutrient concentrations. It is reasonable to name it the maximum growth rate, although it is rarely seen in previous dilution papers. The μ₀ and μ_en are the apparent growth rates in the 100% seawater bottles, which involved the effects of microzooplankton grazing. They should equal the instantaneous growth rate (μ) – grazing mortality rate (m). Therefore, to calculate the in situ instantaneous growth rate, we can use the net growth rate in the bottles without nutrient enrichment and the grazing rate: μ= μ₀ + m. Please refer to Marrec, P., et al. (2021), Seasonal variability in planktonic food web structure and function of the Northeast U.S. Shelf. Limnol Oceanogr, 66: 1440-1458.https://doi.org/10.1002/lno.11696. To my understanding, comparing μ_max and μ can reveal the effects of nutrient addition on phytoplankton's growth rate and evaluate whether the in situ nutrient condition limits it. By contrast, the μ₀ and μ_en involve grazing loss, and it is hard to identify the effects of nutrients.

1. The authors would like to highlight the top-down control of microzooplankton biomass on Gamma A. However, nutrient concentration is one of the important factors. Although nutrient addition did not increase the growth rate of Gamma A in the dilution experiments, it affects the growth rate directly but not the abundance. The abundance of Gamma A is significantly correlated with nutrient concentration, which would indicate the nutrient effects on Gamma A's distribution. Therefore, I suggest discussing this part more comprehensively.

Specifical comments

Line 20: microzooplankton grazing mortality rate

Line 26: microzooplankton grazing mortality rate of Gamma A

Lines 27-28: The nutrient concentration also affects the distribution of Gamma A, as Table 2 shows a significant correlation between them. This sentence seems to negate the effect of nutrients.

Line 85: “quantify” should be better than “understand”.

Line 97: How much seawater was collected for Chl a samples? What kind of membrane is used for Chl a filtration? Please provide the details.

Line 160: I cannot understand how to calculate the net growth rates without nutrient enrichment. It should be the net growth rate of the 100% seawater without nutrient addition, i.e., the apparent growth rate estimated from these two bottles.

Line 227: The average value of the maximum growth rate of Gamma A should be mentioned.

Fig. 4: What is the optimal growth rate? It did not occur in the main text.

Line 251: the grazing mortality rate of Gamma A

Line 251: Add the result of the significance test after this statement.

Line 293: “In situ μ_max ”is strange because μ_max is the maximum growth rate with nutrient enrichment of phytoplankton. It is not the growth rate under in situ conditions. Same problem in line 297.

Line 300: not only… but also Gamma A has…

Lines 312-313: what do “ a distinct ecological strategy” mean? Please provide more details.

Line 320: change “including” to “especially” and add a comma before “remains”.

Line 340: According to Fig. 4c, some points are far from the 1:1 line, indicating lower grazing rates at some stations. Therefore, this statement is imprecise without mentioning these points.

Line 347: Delete “successfully”

Line 347: Add “around the Kuroshio region” after “Gamma A”.

Line 351-352: I cannot understand this sentence. Why fixed nitrogen can be introduced into food webs on a global scale? Please rephrase it.

Sato et al. conducted a field study to investigate the distribution of Gamma A, a non-cyanobacterial diazotroph and the effect of grazing by microzooplankton on Gamma A (and cyanobacterial diazotrophs). As someone with knowledge of the N cycle (including diazotrophy) but not necessarily in microzooplankton and/or grazing, this was a very interesting read and I think it will attract different audiences (i.e., those interested in the N cycle, those interested in NCD and those interested in grazing). The manuscript is well-written, well-prepared and I enjoyed reading it. I only have minor comments.

General comments:

1. Although it is shortly mentioned upon in the introduction, there are two top-down controls on the distribution of Gamma A: viral infections and zooplankton grazing. In the dilution experiment, the seawater was prefiltered with a 200 µm mesh whereas the particle-free seawater was prepared by filtration through a 0.2 µm filter. Thus, both filtration set-ups would allow for the presence of viral particles. How does the dilution method distinguish between viral lysis or grazing. Does the grazing mortality rate (m) calculated via the dilution method not represent the combined effect of the two top-down processes? Besides that, there also might be an interaction between the grazers and viruses. See e.g.: doi:10.1093/plankt/fbv011. I would like the authors to elaborate a bit more on the potential effect of viruses in their experimental set-up in the discussion.

    

2. Statistical analysis

   I do not think a Pearson’s correlation analysis is the right statistical method to explain the distribution of Gamma A. For instance, it is mentioned that gamma A has significant correlations with both nitrate and phosphate concentrations. However, there is a very clear correlation between [NO₃^-] and [PO₄^3-] (see figure S6 and table 2), which could confound the statistical analysis. These measurements are not independent and a mixed-effect model is required for the statistical analysis.

    

3. NifH is a biomarker for potential nitrogen fixation but since the experimental set-up did not include any transcriptomic or proteomic analysis, it remains a potential. I would like the authors to elaborate a bit more about this in the discussion (for instance in chapter 4.2). Does Gamma A have other means to utilize nitrogen-compounds (e.g., ammonium, nitrate, urea) or is nitrogen-fixation the only method to get cellular nitrogen?

    

Specific comments:

Introduction

l60: in situ in italic

l75: I do not understand this sentence. What stable isotope ratio is a proxy for diazotrophy? Everything containing N has a δ¹⁵N value, but when is this a proxy for nitrogen fixation? Elaborate.

l78-l79: These results suggest the importance of NCDs, including Gamma A, and a distinct….

l83: plays is to strong, change to “might play”

Results

Figure 4c: In l229, the significant positive correlation between µ_max and m is mentioned (r = 0.83, p < 0.01). Put these statistics in the graph.

Figure S6: Temperature in the second column (not tempereture)