This study examined the ETS (electron transport system) method, which has gained traction as a tool for estimating respiration in marine plankton communities, with a focus on prokaryoplankton. The authors measured the INT_R and oxygen consumption (Winkler titrations and optodes) simultaneously on a wide range of relevant marine prokaryoplankton to establish the empirical equations between O₂C and INT_R. They examined whether it is constant within species and whether it can be extrapolated to natural plankton communities. Overall, this study is of significant necessity, serving as an essential reference for refining the ETS method. Also, the manuscript is well-written. I have several comments for further improving this manuscript.

Line 15: Spell out the full name for “prokaryo-, zoo- and phytoplankton”.

Line 31: Also, use “prokaryoplankton, zooplankton” instead of “bacterio-, zoo-”.

Line 37: add reference for 0.8 μm. Some studies used 1 μm for prokaryoplankton.

Line 137: How are the incubation times “between 5 and 20 minutes” determined?

Line 191: use “FC” instead of flowcytometry to be consistent with previous text.

Table 1: It is better to add one column to show the experiment number to distinguish the two experiments.

Fig. 2: Add mean values to the boxplot to illustrate the data distribution more visually. Also, I suggest putting all the figures of single-cell respiration, including O2C, into Fig. 2. All the figures support the data grouping into “copiotrophs” and “oligotrophs”.

Line 249: Pooling the data from both methods (i.e., Winkler titrations and optodes) to build a linear regression requires the assumption that there are no significant differences between the two methods. Otherwise, it is preferable to use a single dataset.

Fig. 3: Add p-value to each sub-figure.

Line 266: Which statistical method was used for the covariance analysis? Please specify it.

Fig. 5 and Fig. 4 are similar. It seems not necessary to use two figures. I suggest removing Fig. 4 and moving Fig. 5 forward.

Line 424: Which equation was used for this calculation? Please specify it.

Conclusion: Can you draw more specific findings from your study or suggestions for the experiments in natural waters? For instance, within what time frame can safety be guaranteed without triggering toxicity?

1) Summary

The study demonstrates that all tested prokaryoplankton species import and reduce INT. Still, the O₂ consumed and INT reduced ratio is species-dependent, making it impossible to assume a single universal conversion factor.

The authors quantify species-specific toxicity and adjust incubation times (5–20 min) to avoid bias, also showing good agreement between O₂ measured by optodes and by Winkler titration.

In copiotrophs (Halomonas, Ruegeria, Agrococcus) the INTR–O2C relationship has a strong linear fit; in oligotrophs and cyanobacteria (SAR11, Synechococcus, Prochlorococcus), the fit is significantly weaker, but the linear model lies within the model for natural plankton communities (Fig. 4).

The conclusion is that in situ studies must derive the O2C/INTR relationship locally and check for toxicity in the studied community.

2) Scientific questions

The study addresses the quantification of prokaryotic plankton respiration and the validity of the in vivo INT method.

Novelty

• Comparative multi-taxa dataset under controlled conditions.
• Derivation and comparison of slopes/intercepts per species,
• An operational framework to define toxicity and optimal incubation times.

Conclusions

Conclusions are well supported: no single O2C/INTR factor exists, and studies must derive species-specific relationships (especially in eutrophic systems dominated by copiotrophs).

Methods and assumptions

• Robust, O₂ measured by optodes and Winkler titration, avoiding hypoxia.
• INTR with INT 0.2 mM, killed and media controls, propanol extraction, and calibration curve.
• Quantitative toxicity criterion and choice of incubation.
• Per-cell rates calculated from FC/CFU counts.

Support for results

• Toxicity curves (Appendix A), per-cell rates (Appendix B with Winkler and optodes), and species-specific relationships are presented.
• Table 1 with toxicity, abundance, and O2C/INTR values from replicates per species.

Traceability and reproducibility

Solutions, equipment, calibrations, and regression models.

The authors declare the availability of the data in the public BODC repository and provide a DOI. However, at the time of this review, the dataset could not be accessed through the provided link. Authors are requested to verify the DOI and ensure the data are fully accessible to the public before final publication.

Credit and originality

Context and limitations of the method (classic ETS, constant O2C/INTR assumption) are well referenced and contrasted; the authors’ contribution (species test and comparative analysis) is clearly stated.

Title

Clear. Respiration rates of marine prokaryotes and implications for the in vivo INT method.

Abstract

Complete. States the problem, approach, organisms, main finding (O2C/INTR variability), and methodological implications.

Structure and clarity

Introduction–Methods–Results–Discussion–Conclusions–Appendices. Figures and tables are well integrated with clear cross-referencing.

Language

Technical English is fluent and precise.

Mathematical formulation and symbols

Units are consistent, equations and parameters are defined.

References

Appropriate in number and quality, covering the state of the art.

Supplementary material

Pertinent:

Appendix A (toxicity curves) and Appendix B (per-cell O₂ rates).

3) Major comments

Ecological generalization and growth media

Although the discussion notes possible effects of growth medium and respiratory chain diversity, extrapolation to natural communities could be strengthened with an analysis showing how much slopes/intercepts vary across media.

Underlying physiological mechanisms

The discussion proposes several hypotheses (cell wall, AOX, etc.) to explain the observed variability. To unify these ideas, the creation of a conceptual diagram is recommended. This figure would serve as a visual summary, linking the proposed mechanisms with their theoretical effects on the O₂C and INTR.

4)  Editorial recommendation

• Accept with minor revisions. The manuscript provides new and relevant evidence for the in vivo INT method, and the conclusions are well-supported and do not require new experiments.