General comments:

N2 fixation plays important role in food-web process, carbon sequestration and export of organic carbon to the deep ocean. Kuroshio intrusion and associated environmental alteration may profoundly affect biogeography and N2 fixation rate of diazotrophs. This study demonstrated changes in diazotrophic community structure and N2 fixation because of Kuroshio intrusion in the northern South China Sea (nSCS) based on two cruises in 2017 and 2018. The authors found that Trichodesmium was more abundant and N2 fixation rate was higher at stations strongly affected by Kuroshio intrusion, whereas UCYN-B were more abundant at stations least affected by Kuroshio intrusion. UCYN-C and γ-proteobacteria were mainly distributed at stations moderately affected by the Kuroshio. These results suggested that diazotrophic community composition and nitrogen fixation rate in nSCS are highly regulated by Kuroshio intrusion, which will contribute to our understanding of how Kuroshio affect diazotrophic diversity and nitrogen fixation. Overall, I appreciate their excellent work. However, the presentation of manuscript (particularly Results and Discussion) needs to be improved. I will recommend consideration of its acceptance for potential publication after a minor revision.

1. Introduction should review what is already known about effects of Kuroshio intrusion on diazotrophs (particularly Trichodesmium) and nitrogen fixation in the in marginal seas of NW Pacific. Also, there is a lack of hypothese on the effects of Kuroshio intrusion on the diazotrophic community composition and N2 fixation in the nSCS as well as the intrusion intensity.
2. Prefilteration using a 100μm pore-size nylon mesh can remove large zooplankton, but probably remove Trichodesmium, particularly large-sized colonial trichomes, resulting in a potential underestimation of Trichodesmium abundance.
3. The authors don’t describe how to collect qPCR samples for daytime and nighttime in Materials and Methods, is it to take parallel samples at the same time, one of which is filtered during the daytime (nighttime) to obtain the samples, and the other is put into the nighttime (daytime) to be filtered again? Discussion also does not address in detail why the nifH gene abundance of the diazotrophic groups differed largely between daytime and nighttime. In general, the nifH gene expression in diazotrophic groups should be determined using RT-qPCR rather than qPCR.
4. Nitrogen fixation rate and primary production are missing in Results. The authors can compare nitrogen fixation rates and primary production in the Kuroshio water, mixed water and SCS water. This analysis should be useful in further exploring the influences of changes in diazotrophic composition and nitrogen fixation induced by the Kuroshio intrusion on the carbon and nitrogen biogeochemical cycling of the nSCS as well as the implications.
5. Conclusions are not simple repetitions of the results, please revise them.

Specific comments:

1. I suggest delete some unnecessary connecting adverbs in the text, such as “Moreover” and “Collectively” in Abstract.
2. L45: Please replace “UCYN-B is mostly free-living with Crocosphaera watsonii being a cultivated representative” with “UCYN-B (Crocosphaera watsonii) is mostly free-living, being a cultivated representative”.
3. L124-125: Light gradient should be provided.
4. L207: Please delete “Du et al., 2013”.
5. L295: This paragraph is not directly related to nitrogen fixation. I suggest the authors emphasize the changes in physical parameters and nutrients in nSCS caused by Kuroshio intrusion, in addition the description of dynamic process. How and why these changes affect N2 fixation?
6. L314-315: In addition to the results of Kao et al., 2012, how diazotrophs contributed nitrogen budget and primary/new production.
7. L318-319: These values of 75.98 ±48.77 μmol N m−2 d−1 and 74.98 ± 26.55 μmol N m−2 d−1 were the average NFR in nSCS during 2017 and 2018, respectively?
8. Table S4: I suggest remove Table S4 from supplementary materials to the text.
9. Table S5: Why are there two sampling times for the same station in Table S5, is it to compare the effects of daytime and nighttime on diazotrophs? If so, RT-qPCR should have been used to determine nifH gene expression in different diazotrophic groups.
10. Figure 1: Please give the full names of nSCS an KI.
11. Figure 2: “(b)” is lacking.
12. Figure 6: Please give the full name of RDA. In addition, the relative contribution of different parameters to variation in diazotrophic community composition needs to be quantified.
13. The station numbers in Fig. 3 and Table S5 did not match. Figure 3 and Table S5 showed that the abundance of Richelia in Kuroshio water was higher than that in SCS water, which is similar to the findings of Tuo et al (2014). Chen et al. (2014) has showed that nitrogen fixation rate was much higher in Kusoshio than in nSCS during warm seasons. Also, they found that relative contribution (59%) of filamentous diazotrophs (>10 or 20 mm, mostly Trichodesmium and Richelia) to nitrogen fixation was higher than that (41%) of unicellular filamentous during warm seasons. Similarly, result of Jiang et al. (2023) suggested that Kuroshio intrusion stimulated growth of Trichodesmium and enhanced nitrogen fixation in the East China Sea during summer. Therefore, the present study on diazotrophic abundance and nitrogen fixation was highly consistent with earlier studies in marginal seas of NW Pacific suffered from intrusion of Kuroshio. I suggest the authors analyze more deeply about the mechanism of Kuroshio intrusion on the community composition of diazotrophs and nitrogen and carbon fixation.

Tuo SH, Chen YLL, Chen HY (2014) Low nitrate availability promotes diatom diazotroph associations in the marginal seas of the western Pacific. Aquat Microb Ecol 73:135-150. https://doi.org/10.3354/ame01715

Chen, Y. L. L., Chen, H. Y., Lin, Y. H., Yong, T. C., Taniuchi, Y., and Tuo, S. H.: The relative contributions of unicellular and filamentous diazotrophs to N2 fixation in the South China Sea and the upstream Kuroshio, Deep-Sea Res. Pt. I, 85, 56-71, https://doi.org/10.1016/j.dsr.2013.11.006, 2014.

Shiozaki, T., Takeda, S., Itoh, S., Kodama, T., Liu, X., Hashihama, F., and Furuya, K.: Why is Trichodesmium abundant in the Kuroshio? Biogeosciences, 12, 6931-6943, https://doi.org/10.5194/bg-12-6931-2015, 2015b.

Jiang, Z., Zhu, Y., Sun, Z., Zhai, H., Zhou, F., Yan, X., Zeng J., Chen J., Chen Q. Enhancement of summer nitrogen fixation by the Kuroshio intrusion in the East China Sea and southern Yellow Sea. Journal of Geophysical Research: Biogeosciences, 128(3), e2022JG007287. https://doi.org/10.1029/2022JG007287, 2023.

General comments

The study explores the impact of interannual Kuroshio intrusions (KIs) on diazotrophic communities in the northern South China Sea. The research identifies a correlation between diazotroph variations and KI-induced changes in environmental factors. Notably, Trichodesmium abundance and N2 rate increases in strongly affected stations, while unicellular N2-fixing cyanobacteria (UCYN-B) thrive in less affected stations. UCYN-C and non-cyanobacterial γ-proteobacteria dominate moderately affected stations. The findings suggest KI succession shapes diazotrophic communities as a stochastic process, potentially influencing region-scale redistribution of diazotrophs and nitrogen budgets in a warming ocean.

I find the work excellent and should be considered for acceptance, however some minor revision is needed. 

Introduction: As there is some execellent work done with non-cyanobacteria diazotrophs I recommend a deeper introduction into those. As a start, look into "Non-cyanobacterial diazotrophs: global diversity, distribution, ecophysiology, and activity in marine waters" by Kendra A. Turk-Kubo to get an overview.  

Method: 

-- Regarding N2 fixation and productivity rate. What elemental analyzer were used (i.e. model).

-- You used nifH ARB database from john zehr. However, this is from 2017 and has not been updated. I suggest that you use the new nifH database, based on ARB and John zehr but updated in 2023 according to the NCBI. See link https://github.com/moyn413/nifHdada2

-- It would be helpfull to know if you did qPCR on all stations and if you did sequence of all stations or only some.

Discussion:

-- At section 4.1, I think that the paper could benefit from a deeper discussion about non-cyanobacterial diazotrophs. 

Specific comments:

Title: Should it not be "changes in diazotrophic community....."

In line 18 at the abstract, you write "..non-cyanobacterial gamme-proteobacteria..", you can delete either non-cyanobacteria or gamma-proteobactera. As it is evident that a gamma-proteobacteria is a NCD.

Line 20 - remove "more".

Line 20-21 - Rephrase "..in 2017 when KI was strong than in 2018 when KI appeared to have retreated." or perhaps just say "..when KI was stronger comapared to 2018 where KI retreated". If I understood it correctly

Line 50-52 - Perhaps expand the importance of these new groups? e.g., they has been models and measurement of NCDs contribution to N2 fixation. 

Line 180 - were relative abundance of OTU determined from UPARSE? Or how was this determinde

Line 184 - you mention "some samples". What is the threshold? How many samples? below or above e.g. 10?

Line 191 - why not transform pH?

Line 268-269 - I would recommend to include station number, to help the reader.

Line 275 - Regarding sinking trichodesmium. Check this references "Sinking Trichodesmium fixes nitrogen in the dark ocean" by Mar Benavdies. 

Line 343 - Regarding salinity affecting diazotrophs. Look into "Salinity as a keycontrol on the diazotrophic community composition in the southern Baltic Sea" by Christian Reeder and "Diversity, structure, and distribution of bacterioplankton and diazotroph communities in the Bay of Bengal during the winter monsoon" by Chao Wu.

Figure 2 - "b" is needed. Which figures belong to which year? and secondly, legend title is needed. E.g. depths for b,c and tempeature for d,e. Also legend title and unit for a

References

Benavides, M., Bonnet, S., Le Moigne, F.A.C. et al. Sinking Trichodesmium fixes nitrogen in the dark ocean. ISME J 16, 2398–2405 (2022). https://doi.org/10.1038/s41396-022-01289-6

Wu C, Narale DD, Cui Z, Wang X, Liu H, Xu W, Zhang G, Sun J. Diversity, structure, and distribution of bacterioplankton and diazotroph communities in the Bay of Bengal during the winter monsoon. Front Microbiol. 2022 Nov 30;13:987462. doi: 10.3389/fmicb.2022.987462. PMID: 36532434; PMCID: PMC9748438.

Reeder, C. F., Stoltenberg, I., Javidpour, J., and Löscher, C. R.: Salinity as a key control on the diazotrophic community composition in the southern Baltic Sea, Ocean Sci., 18, 401–417, https://doi.org/10.5194/os-18-401-2022, 2022.

Kendra A Turk-Kubo, Mary R Gradoville, Shunyan Cheung, Francisco M Cornejo-Castillo, Katie J Harding, Michael Morando, Matthew Mills, Jonathan P Zehr, Non-cyanobacterial diazotrophs: global diversity, distribution, ecophysiology, and activity in marine waters, FEMS Microbiology Reviews, 2022;, fuac046, https://doi.org/10.1093/femsre/fuac046