The Relationship Between Respiration Rates and Electron Transport System Activity in Fish

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

I have read through the manuscript entitled "The Relationship between Respiration Rates and Electron Transport System Activity in Fish". The authors measured two key metrics related to fish energy metabolism: oxygen consumption rate, which serves as a proxy for aerobic respiration, and electron transport system (ETS) activity, across a gradient of swimming speeds. Two experimental treatments were designed for this study: (1) a single-measurement group, where each individual was tested at only one of the six swimming speeds (N = 3 per speed); and (2) a repeated-measurement group, where each individual was subjected to sequential trials at different swimming speeds, similar to those employed during the critical swimming speed (Uₙₐₓ) assay.

With a total sample size of only 21 individuals (N = 3 per treatment-speed combination), the sample size appears somewhat limited, particularly given the typically high inter-individual variability inherent in both oxygen consumption rate (MO₂) and ETS activity measurements. Nevertheless, I recommend conditional acceptance of this manuscript pending adequate revisions. Its experimental design provides a valuable first approximation of R/ETS ratios under defined activity levels, and establishes a promising methodological framework for future assessments of active carbon flux in fish.

Specific Comments

Lines 91 and 110: The descriptions of the acclimation protocol are overly concise. Critical details including the duration of the acclimation period and acclimation conditions (e.g., water temperature, dissolved oxygen concentration, photoperiod, feeding regime) must be explicitly reported, as these factors directly influence fish metabolic performance and data interpretability.

Line 141: Inclusion of a schematic diagram would substantially improve the clarity of the experimental setup or workflow described in this section.

Lines 168–172: The rationale for selecting the three sampling sites is not adequately justified. Please provide a detailed explanation for the choice of these sites.

Line 186: A relevant reference must be cited to validate the model used in this analysis.

Data Analysis (Figures 2–4 and associated tables): The current design with only three replicates per treatment limits the statistical power to adequately address inter-individual variation. It is recommended that the authors re-analyze the dataset using the mean value of each individual across repeated ramps at each swimming speed, and apply a two-way analysis of variance (ANOVA) with treatment and swimming speed as the two fixed main effects.

Discussion Section: The discussion should be substantially revised to align with the results of the re-analysis. Specifically, the authors are advised to focus on two core issues:

(1) How the findings of the present study advance the methodological framework for future assessments of active carbon flux mediated by vertically migrating micronekton;

(2) The underlying mechanisms explaining why R/ETS ratios remained stable across swimming speeds in Treatment 1 but increased with speed in Treatment 2, and the implications of this divergent pattern for the methodological framework proposed herein.

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted in the re-submitted files.

Comments 1: Lines 91 and 110: The descriptions of the acclimation protocol are overly concise. Critical details including the duration of the acclimation period and acclimation conditions (e.g., water temperature, dissolved oxygen concentration, photoperiod, feeding regime) must be explicitly reported, as these factors directly influence fish metabolic performance and data interpretability.

Response 1: To our view, you refer to lines 91 and 110 for an overly concise description of the acclimation protocol. From line 91 to 93 (lines 109-111 in the new version modified considering the reviewer comments) we refer to the work performed by Ikeda and we consider which is not necessary to indicate the acclimation protocol related to the study of this author. We agree that acclimation details are important. We have expanded the description as follows:

• lines 178-184 “During acclimation and measurements, the system was kept in dim light by covering the respirometer with a black cover avoiding visual contact with researchers but allowing indirect light to pass downstream to keep fish faced upstream and swimming against the stream. Also, the light of the room was off, and noise was avoided as much as possible. All the processes were performed during daytime, although some experiments finished in the limit of daylight period due to experimental constrains.”.

Other details of the acclimation protocol were reported previously:

• We indicated that fish were fasted for 24 h (line 132 and 172).
• Transported from aquaculture group ECOAQUA (lines 644-645, Acknowledgments) raise tank in their experimental unit located in the same facility where we performed our respirometry experiment in a closed bucket with seawater taken form their holder tank (lines 127-130); However, now we have mentioned that animal belonged to the ”same farm raised cohort” at lines 128-129.
• Acclimated for 5 hours at 0.5 BL·s^-1 (lines 176-178);
• The water temperature during acclimatation and experiments was 19.5 ºC (lines 146-147);
• Oxygen saturation levels where keeps above 95 % when no measurements were performed (line 152-153).

 

Comments 2: Line 141: Inclusion of a schematic diagram would substantially improve the clarity of the experimental setup or workflow described in this section.

Response 2: Thank you for pointing this out. We have added two new figures (Figure 2 and 3) and adjusted the text for clarity.

The first Figure is shown between lines 153 and 161 (pages 4-5) and represent the respirometry system. Modifications of the manuscript related with this image are:

• Line 140 “(Figure 2)”;
• Lines 155-161, “Figure 2. Intermittent‑flow swimming‑tunnel respirometry setup. The swimming tunnel was submerged into the respirometer. Aeration kept ≥95% O₂ saturation; water was renewed by a flushing pump and discharged by ~1 m free‑fall to a reservoir tank from which it was pumped back to the respirometer. Temperature was held at 19.5 °C via an internal cooling coil connected to a thermostatic bath (hot/cold lines). O₂ (optode) and temperature sensor were logged by Witrox  Motor and pumps were controlled via DAQ‑M on a PC running AutoResp™. Indirect downstream lighting was used to promote upstream orientation.”.

The second Figure is between lines 204 and 209 (page 6) and represents the experimental workflow. It has modified the manuscript at:

• Line 185 “(Figure 3)”;
• Lines 206-209, “Figure 3. Overview of the experimental workflow and the standardized intermittent‑flow cycle used in the swim‑tunnel respirometry trials: pre-treatment precedes a repeated flush-resting-measurement cycle. Treatment 1 varied speed between fish (each fish swam at one fixed speed), whereas Treatment 2 varied speed within fish (each fish swam the six speeds in sequence).”.

The modifications done for clarity improvement are:

• Lines 142-145, “The swimming tunnel was submerged in a respirometer tank, which served as a temperature-controlled, high-oxygen water reservoir. From here water is flushed by a pump to the inner of the swimming tunnel during flushing times.” instead of “used as water reservoir during flushing times.
• Line 163, we renamed the “recover tank pump” as “reservoir tank pump”

 

Comments 3: Lines 168–172: The rationale for selecting the three sampling sites is not adequately justified. Please provide a detailed explanation for the choice of these sites.

Response 3: Agree, we have clarified the rationale and standardize the wording. All muscle samples were taken above the lateral line to avoid organs and to ensure muscle tissue only. We sampled three positions to test for location-dependent differences. These edits are at:

• Line 212: “with the aim of studying differences along the fish body”;
• Line 214: ", avoiding organs and ensuring full muscle tissue”.

 

Comments 4: Line 186: A relevant reference must be cited to validate the model used in this analysis.

Response 4: We appreciate the helpful suggestion. To our knowledge, no single study validates exactly the same four‑parameter sigmoidal model for the MO₂–speed relationship. However, classical literature documents the nonlinear increase of MO₂ with swimming speed, which justifies the use of flexible nonlinear models (e.g., Brett, 1964. The respiratory metabolism and swimming performance of young sockeye salmon. DOI: https://doi.org/10.1139/f64-103).

In addition, recent literature support for using sigmoidal models when metabolic responses are physiologically bounded by lower and upper limits (i.e., standard metabolic rate, SMR and maximal metabolic rate, MMR). In particular, Wood (2018. The fallacy of the P_crit – are there more useful alternatives? Doi: https://doi.org/10.1242/jeb.163717) recommends fitting the full curve with allosteric sigmoidal model formulations rather than relying on threshold approaches (his analysis concerns MO₂–P_O₂, but the same rationale applies to MO₂–speed because both are bounded by SMR and MMR). Alfonso et al (2021. Mapping the energetic costs of free-swimming gilthead sea bream (Sparus aurata), a key species in European marine aquaculture. DOI: https://doi.org/10.3390/biology10121357) used a three‑parameter sigmoidal model, supporting the appropriateness of a sigmoidal shape for bounded activity–metabolism relationships.

On this physiological and empirical basis, we chose a four-parameter model instead of the three-parameter used by Alfonso et al. (2021). In plain terms, the usual three‑parameter logistic has no “baseline” parameter, which means its lowest possible level is fixed at zero in the mathematics of the curve (this does not force MO₂ at zero speed to be zero; rather, the curve only approaches zero in the unrealistic limit as swimming speed → −∞, which has no biological meaning in our context). The four‑parameter logistic adds that baseline as a parameter, so both the lower and upper limits are learned from the data and can be interpreted as SMR and MMR, respectively.

 

Comments 5: Data Analysis (Figures 2–4 and associated tables): The current design with only three replicates per treatment limits the statistical power to adequately address inter-individual variation. It is recommended that the authors re-analyze the dataset using the mean value of each individual across repeated ramps at each swimming speed and apply a two-way analysis of variance (ANOVA) with treatment and swimming speed as the two fixed main effects.

Response 5: Thank you for the suggestion. We explored a two-way ANOVA (treatment and swimming speed as fixed main effects), but it proved suboptimal due to (i) heteroscedasticity (Leven’s test not satisfied) and (ii) in T2, speed is a within-subject factor (each fish contributes observations at six speeds), compromising the independence required by classical ANOVA.

We therefore fitted a linear mixed-effects model with a random intercept per fish, which properly accounts for within-subject correlation and realistic variance structures. The model detected a clear effect of swimming speed (p<0.001) and no main effect of treatment (p=0.119). Estimated marginal means were higher in T1 than in T2, but not significantly. We note that the limited number of individuals per treatment reduces statistical power to detect small treatment differences. The results of the mixed model supported the unification of the T1 and T2 dataset to develop a unified sigmoidal model across treatments, so no substantial changes were required as a result of this re-analysis.

The edits related with the linear mixed effects model are in:

• Line 261-263: “MO₂ was analysed using a REML linear mixed‑effects model with treatment and swimming speed as fixed effects, and a random intercept for fish. ”
• Line 331-332: “We assessed the influence of treatment on MO₂ using a linear mixed‑effects model (Table S8) finding no significant effect (p=0.119). Accordingly,”
• Line 434: ", nor there was influence of treatment on MO₂ (Table S8)”
• Lines 601-604: “Table S8: Type III tests of fixed effects from the linear mixed‑effects model (REML; random intercept for Fish). The table reports numerator and denominator degrees of freedom (Satterthwaite), F statistics, and p‑values (Sig.) for the intercept, treatment, and swimming speed on mean oxygen consumption”

 

Comments 6: Discussion Section: The discussion should be substantially revised to align with the results of the re-analysis. Specifically, the authors are advised to focus on two core issues:

(1) How the findings of the present study advance the methodological framework for future assessments of active carbon flux mediated by vertically migrating micronekton;

(2) The underlying mechanisms explaining why R/ETS ratios remained stable across swimming speeds in Treatment 1 but increased with speed in Treatment 2, and the implications of this divergent pattern for the methodological framework proposed herein.

Response 6: We thank the reviewer for this helpful suggestion.

For point (1), we fully agree that it is central to our study. In the current revision, rather than redistributing material across the Discussion section, we have consolidated the methodological framework into the second paragraph of the Conclusions section. This paragraph synthesizes how activity-dependent R/ETS ratios may be used to obtain more realistic remineralization rates at depth (below the mixed layer). We have added lines 565-569: “Operationally, future active‑flux estimates could be obtained by (i) measuring ETS, (ii) using R/ETS ratios to determine the specific R value at each activity level, (iii) weighting by residence time below the mixed layer, and (iv) applying temperature corrections to obtain a more realistic remineralization rate at depth.”. Our intention is that the Conclusions section summarizes the main focus of the work.

For point (2), we clarify the complementary roles of T1 and T2. In T1, given the limited number of individuals, we cannot affirm that R/ETS is constant; our analyses indicate no dependence on speed. In this treatment, each fish was measured at a single, specific speed. Because different speeds were assessed in different individuals, any change across speeds cannot be separated from differences among fish. After normalizing MO₂ by each fish’s ETS, R/ETS expresses the fraction of potential aerobic capacity used at that specific speed. In T1 we are therefore comparing these fractions across different fish measured at different speeds, which can vary idiosyncratically and show no residual speed effect, unlike in T2.

T1 is useful to determine whether ETS is correlates with swimming speed (it does not), which supports using ETS as a short‑term invariant capacity and motivates normalization. However, because R/ETS is speed‑independent in T1, we do not pool T1 with T2 to fit the activity‑dependent R/ETS model. We have revised the Discussion to note this limitation and make the complementary roles of T1 and T2 explicit:

• Line 439: “Based on T1, we observed that”,
• Lines 442-443: “ETS activity results in a stable reference capacity to normalize oxygen consumption.”,
• Lines 471-472: ", and therefore invalidates to pool T1 and T2 for modelling an activity-dependent R/ETS”.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The Relationship between Respiration Rates and Electron Transport System Activity in Fish - fishes-4121827 - by Medina-Suárez and Henández-León

 

The study deals with the relationship between swimming activity, oxygen consumption, and electron transport system activity in juvenile Sparus aurata using swimming-tunnel respirometry. The paper covers an interesting issue and is informative on link between oxygen consumption and electron transport system activity in actively swimming fish, which results provide a methodological basis for estimating respiration rates in mesopelagic species and improving carbon flux models.

In my opinion, this paper is well written. Its hypotheses, methods, results, discussion, and conclusion are well addressed. It deserves to be published. I only have a few comments, listed below.

 

L69"... after the seminal paper by [24]..." = "... after the seminal paper by Longhurst et al. [24]..."

L263-265 "... , but marginal differences between 1.25 and 3.5 and 4.25 BL s-1 (p=0.08)." This is confusing. Please check and rewrite.

L242 In Fig. 2C, please use more contrasted colors between predicted curves.

Authors stated : L378-380 "We observed no relationship between ETS activity and physical traits (Table S11), although a larger dataset including fish at different maturity stages is needed to confirm this observation." and L110-112 "A total of 21 gilthead sea breams (Sparus aurata) of unknown sex were used in the experiments. Mean wet mass (WM) was 47.11 ± 9.81 g, length (L) 12.09 ± 0.80 cm, height (H) 4.43 ± 0.47 cm, and width (W) 1.78 ± 0.32 cm (mean ± standard deviation)." The authors should clearly specify the origin of these fish and also detail their individual physical characteristics. If they are farm-raised fish, do they come from the same cohort? This could influence the trends observed in the results. The means and their standard deviations would show some variability between individuals.

Please use the abbreviated journal names for references [13], [24], [28], [32], [34], [46], [56], [57], [59] and [60].

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted in the re-submitted files.

Comment 1: L69"... after the seminal paper by [24]..." = "... after the seminal paper by Longhurst et al. [24]..."

Response 1: Thank you for noting it. We have corrected the citation at line 82 to “Longhurst et al.”.

 

Comment 2: L263-265 "... , but marginal differences between 1.25 and 3.5 and 4.25 BL s-1 (p=0.08)." This is confusing. Please check and rewrite.

Response 2: Thank you for pointing this out. We have rewritten the sentence for clarity at lines 317-319: “For Ramp 2, there were no differences between 0.5 and 1.25 BL·s⁻¹, between 2.0 and 2.75 BL·s⁻¹, or between 1.25 and 2.0/2.75 BL·s⁻¹ (all p > 0.05), but we observed a borderline difference for 1.25 vs 3.5 BL·s⁻¹ (p = 0.08) and 1.25 vs 4.25 BL·s⁻¹ (p = 0.08).”

 

Comment 3: L242 In Fig. 2C, please use more contrasted colors between predicted curves.

Response 3: Thank you for the suggestion. We have revised the lines colour and type to improve the contrast between them. We have modified the caption of the Figure 5C, page 10, lines 297-300 to note the changes “predicted curve for T1 (solid light blue line), predicted individual curves for T2 (dashed, dot-dashed and two-dashed lines in blue, green and magenta for fish labelled Ramp 1, 2, and 3 respectively), predicted curve for T2 (solid orange line), and predicted curve for T1+T2 (solid black line)”.

 

Comment 4: Authors stated: L378-380 "We observed no relationship between ETS activity and physical traits (Table S11), although a larger dataset including fish at different maturity stages is needed to confirm this observation." and L110-112 "A total of 21 gilthead sea breams (Sparus aurata) of unknown sex were used in the experiments. Mean wet mass (WM) was 47.11 ± 9.81 g, length (L) 12.09 ± 0.80 cm, height (H) 4.43 ± 0.47 cm, and width (W) 1.78 ± 0.32 cm (mean ± standard deviation)." The authors should clearly specify the origin of these fish and also detail their individual physical characteristics. If they are farm-raised fish, do they come from the same cohort? This could influence the trends observed in the results. The means and their standard deviations would show some variability between individuals.

Response 4: Agree. Now, we have included the following information in the text:

• Lines 128-129: “…from the same farm raised cohort of unknown sex (immature gonads) were used in the experiments”.
• Lines 131-132: “Detailed individual morphometrics are shown in Table S1”
• Lines 572-575: “Table S1: Individual morphometric data for the 21 juvenile gilthead sea breams (Sparus aurata) used in this study. Variables reported for each fish include Treatment (T1 and T2), Wet mass (g), Length (cm), Width (cm), and Height (cm). Fish 1–18 correspond to T1; Fish 19–21 correspond to T2.”

We also added a table with individual physical traits in the Supplementary Material labelled as the new Table S1.

 

Comment 4: Please use the abbreviated journal names for references [13], [24], [28], [32], [34], [46], [56], [57], [59] and [60].

Response 4: Thank you for noting it. We have abbreviated the journal names in references [13] “J. Mar. Syst.”at line 686, [24] “Deep-Sea Res. Part A. Oceanogr. Res. Pap.” at line 712, [28] “ICES J. Mar. Sci.” at line 721, [32] “S. Afr. J. Mar. Sci.” at line 731, [34] “J. Oceanogr. Soc. Jpn” at lines 735-736, [46] “J. Biol. Chem.” at line 761, [56] “Philos. Trans. R. Soc. B” at lines 783-784 , [57] “ICES J. Mar. Sci.” at line 787, [59] “ICES J. Mar. Sci.” at line 793, and [60] “J. Anim. Ecol.” at line 794.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

General comments:

 

This is an interesting and useful study of respiration rates and ETS activity in juvenile sea bream.  However, I have three major concerns that I feel that the authors should address.

 

1) I appreciate the importance of fish energetics in marine carbon cycles.   However, is a long discussion of the marine carbon cycle needed at the beginning of this paper, especially given that this cycle is not directly studied?  Perhaps it would be better if the Introduction focused on the authors’ specific purpose of analyzing micronekton fish energetics, while only briefing mentioning its value in relation to understanding the carbon cycle.  Later in the Discussion section, the carbon cycle and its components could be more fully described.  In that section, a diagram of how micronekton fit into the carbon cycle may be helpful. 

 

2) Greater clarification of the terms “respiration rate”, “ETS activity”, and “(maximal) aerobic capacity” and their interrelationships is needed.  Other terms such as Ramp 1, 2 and 3 should be explained, as well.  This would help the reader to understand better many sections of this manuscript.

 

3) Improvement of word choice and syntax is needed in many parts of the manuscript.  See also specific comments.

 

Specific comments:

 

L 12: Should “constrained” be replaced with “understood”?

 

L 26: Replace “zones” with “parts”?

 

L 38: Replace “export” with “transport” here and elsewhere?  The word “export” implies that carbon is being moved beyond (outside) the ocean, when the authors really mean that it is being transported to different parts (depths) of the ocean.

 

L 55: Change “by” to “of”.

 

L 57: Change “relevant” to “significant” or “important”?

 

L 58: Change “metabolized” to “metabolize”.

 

L 75-76: Plankton are smaller but more numerous than micronekton.  Therefore, do you mean “population biomass” rather than individual biomass here?

 

L 81: Change “playing” to “play”.

 

L 88: Does “R” refer to “respiration?

 

L 110: Please indicate that juveniles were studied. 

 

L 122-123: Perhaps change “tank called respirometer” to “respirometer tank”?

 

L 154: Please explain “Ramp 1, 2 or 3”.

 

L 181-183: Does the dry mass to wet mass ratio apply to juvenile fish?  Water content can vary with age in fish and other animals. 

 

L 199-200: Perhaps reword “was used for temperature corrections through the Arrhenius equation” as “was used in the Arrhenius equation to make temperature corrections”?

 

L 234: Change “oxygen consumptions” to “the rate of oxygen consumption”.

 

L 236: Change “indicating” to “indicated”.

 

L 237, 245: Change “parameters” to “parameter”.

 

L 239-240: Change “56.3% to 14.5%” to “14.5% to 56.3%”?  And consider similar reordering elsewhere (e.g., L 271, 282).

 

L 242-247: Please explain all features of the box plots.  Please explain Ramp 1, 2 and 3. It is difficult to distinguish the curves for T2 and T1+2.  The curve for T1+2 seems to be steeper than the curves for T1 and T2.  How can this be?

 

L 303-305, 312-317: Please explain all aspects of box plots. Each figure should stand on its own.

 

L 345-351: Please explain what the ramps are.

 

L 400: Please remind readers that MS-222 is an anesthetic. 

 

L 405: Change RETS to R/ETS.

 

L 418: Change “parameters” to “parameter”.

 

L 425: Change “Moreover, However” to “However”.

 

L 433: Change “was” to “has been”.

 

L 443: Change “and” to “to”.

 

L 446: Change “individual” to “individuals”.

 

L 474-475: Please explain why the independence of ETS & swimming activity shows that ETS activity is a good measure of maximum aerobic capacity.  Maximum aerobic capacity constrains maximum swimming effort and therefore if ETS activity reflects maximum aerobic capacity, it should be highest during maximal swimming activity.   

 

L 485-487: Consider rewording “If this model were applicable to micronekton fish, aerobic metabolism would range from 17% to 71% of their ETS activity depending on swimming speed.” as “According to this model, aerobic metabolism should range from 17% to 71% of their ETS activity depending on swimming speed.”

 

L 488: Change “traditionally used” to “traditional use”.

 

L 490: I suggest changing “could” to “may”.

Comments on the Quality of English Language

See my previous comments to the authors.

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted in the re-submitted files.

Comment 1: I appreciate the importance of fish energetics in marine carbon cycles. However, is a long discussion of the marine carbon cycle needed at the beginning of this paper, especially given that this cycle is not directly studied? Perhaps it would be better if the Introduction focused on the authors’ specific purpose of analyzing micronekton fish energetics, while only briefing mentioning its value in relation to understanding the carbon cycle. Later in the Discussion section, the carbon cycle and its components could be more fully described. In that section, a diagram of how micronekton fit into the carbon cycle may be helpful.

Response 1: Thank you for the constructive suggestion. We agree that the Introduction dedicates substantial space to the biogeochemical context. Our intention, however, is to ensure that readers who are not specialists in ocean biogeochemistry can follow the rationale of the study and appreciate why micronektonic fish are relevant to the ocean carbon cycle and why studying their respiration is necessary. In particular, the Introduction briefly defines the specific components of the cycle in which vertically migrating micronekton fish participate once the carbon enters the trophic web (the biological pump, active carbon flux, diel vertical migration, and respiration/excretion at dept), so that the link between organismal respiration and active carbon transport is clear. Given the limited availability of in vivo respiration measurements in micronekton fish, we confine the Introduction to the essential energetics concepts required for our R/ETS focus. Accordingly, we have retained the Introduction as written, while we remain open to minor revisions if the reviewer considers them necessary. But we modified the introduction to add a diagram to improve the comprehension of the role of micronekton fish into the carbon cycle by biological pump at lines 53-65: “Figure 1. Conceptual diagram of the marine carbon cycle highlighting the role of diel vertical migrating micronekton fish. Atmospheric CO₂ dissolves into surface waters as dissolved inorganic carbon (DIC) and is fixed by autotrophic organisms, producing organic carbon. A fraction is exported below the mixed layer as particulate organic carbon (POC) via gravitational sinking of senescent phytoplankton, mucous feeding webs, aggregates, faecal pellets and molts, and through physical mixing. Night-time ascent and daytime descent of zooplankton and micronekton actively transport ingested carbon to the mesopelagic zone, where respiration, excretion and gut flux, return organic carbon to DIC. Dissolved organic matter (DOM) is also transferred below the mixed layer by physical mixing and gravitational export. Symbols: orange dots, POC; orange clusters, particle aggregates; blue silhouettes, migratory zooplankton; black silhouettes, migratory micronektonic fish.” A mention to the figure was inserted at line 53: “(Figure 1)”

 

Comment 2: Greater clarification of the terms “respiration rate”, “ETS activity”, and “(maximal) aerobic capacity” and their interrelationships is needed. Other terms such as Ramp 1, 2 and 3 should be explained, as well. This would help the reader to understand better many sections of this manuscript.

Response 2: Thank you for pointing this out. Agree, we have, accordingly, clarified terminology and linkages as follows:

• For respiration rate. Lines 88-89: “, that is, how much oxygen is consumed to produce energy,”
• For ETS activity. Lines 101-103: “Because the assay is performed in vitro under saturating (non-limiting) substrate and cofactor concentrations, ETS activity serves as a proxy for the maximal mitochondrial oxygen-processing capacity for energy production.” An “aerobic” at line 100 to indicate that ETS is and approach related with oxygen metabolism.
• For maximal aerobic capacity. Lines 239-240: “we refer to this limit as the maximal aerobic capacity.” To indicate that it is the upper asymptote and the point in which aerobic metabolism cannot longer support the increase in activity.
• For interrelationships. We added lines 488-492: “Accordingly, the R/ETS ratio could be interpreted as the fraction of the maximal mitochondrial oxygen-processing capacity being used at a given activity level. Since the respiration rate ranges from the vital-maintenance levels to values approaching the maximal aerobic capacity, it follows that 0<R/ETS<1. The upper bound reflects that physiological conditions cannot match the ETS non-limiting conditions.”
• Additionally, we have introduced a definition for carbon remineralization as is could not be clear in the text. Line 86: ", which produce CO₂ as a by-product (or remineralize carbon)”

Ramp 1–3 are simply identifiers for the three individual fish used in Treatment 2 (T2). We use “Ramp” to indicate that, unlike T1, each T2 fish was subjected to a progressive sequence of swimming speeds (i.e., a speed ramp). We modified the text at lines 190-191: “Every fish in treatment T2 was labelled as Ramp 1, 2 or 3 to indicate that it underwent a progressive sequence of swimming speeds.” to clarify why are they named with this code.

 

Comment 3: Improvement of word choice and syntax is needed in many parts of the manuscript. See also specific comments.

Response 3: Thank you for these detailed suggestions. Detailed modifications related with each comment are:

• L 12: Should “constrained” be replaced with “understood”?

We appreciate the suggestion. In this sentence we intended to emphasize the quantitative uncertainty in current estimates; therefore, we have retained the concept but improved readability by changing “insufficiently constrained” to “poorly constrained” at line 11-12.

• L 26: Replace “zones” with “parts”?

We thank the reviewer. We have replaced “zones” with “body regions” in the abstract (lines 25-26) to use standard anatomical terminology and to be consistent with the sampling description (head, mid‑body, tail) provided in Methods and Figure 3.

• L 38: Replace “export” with “transport” here and elsewhere?  The word “export” implies that carbon is being moved beyond (outside) the ocean, when the authors really mean that it is being transported to different parts (depths) of the ocean.

We thank the reviewer for this helpful suggestion. To avoid ambiguity, we now use “transport to depth” when describing within‑ocean movement of carbon and retain “export” only when referring explicitly to export from the surface layer to the ocean interior (export flux). We have revised the relevant sentences accordingly and changes appear at lines 47 “transported”; and 51 "transported”.

• L 55: Change “by” to “of”

Thank you for the suggestion. We agree and have implemented the change. Line 68 “of”.

• L 57: Change “relevant” to “significant” or “important”?

Thank you for the suggestion. We agree that “relevant” is not idiomatic here. To avoid any ambiguity with statistical significance, we have adopted “important” rather than “significant.” Line 70 “an important”.

• L 58: Change “metabolized” to “metabolize”.

Thank you for the suggestion. We agree and have corrected the verb to present tense. Line 71 “metabolize”.

• L 75-76: Plankton are smaller but more numerous than micronekton.  Therefore, do you mean “population biomass” rather than individual biomass here?

Thank you for pointing this out. Yes, we intended population biomass (stock) rather than individual body mass. We have modified the text to clarify it at line 90 “population biomass”.

• L 81: Change “playing” to “play”.

Thank you for the suggestion. We agree and have changed “playing” to “play” (line 96) to improve verb form and clarity.

• L 88: Does “R” refer to “respiration?

Yes. R refers to respiration (measured as oxygen consumption, MO₂). It was defined in the abstract, but to avoid any ambiguity in the main text, we have added a parenthetical definition at line 107, “respiration (R)”.

• L 110: Please indicate that juveniles were studied.

Thank you for the suggestion. We now explicitly state in the Animals subsection that the study used juvenile Sparus aurata at line 128 “juvenile”.

• L 122-123: Perhaps change “tank called respirometer” to “respirometer tank”?

Thank you for the suggestion. We agree and have revised the phrase to “respirometer tank” (line 143). In addition, in response to other reviewers, we have made changes in these tenses (lines 142-145) and included a schematic illustration of the intermittent-flow swim-tunnel respirometry (Figure 2, lines 154-161).

• L 154: Please explain “Ramp 1, 2 or 3”.

Thank you. As noted in our response to your second comment, we have clarified this term at lines 190-191.

• L 181-183: Does the dry mass to wet mass ratio apply to juvenile fish? Water content can vary with age in fish and other animals.

Agreed. The paper cited here studied this relationship in a large range of fish size accounting for small to large fish. This relationship was also in accordance to another paper by Childress et al. (1973) ([43], mentioned at line 228). Therefore, we assume juvenile fish is covered by the used ratio.

• L 199-200: Perhaps reword “was used for temperature corrections through the Arrhenius equation” as “was used in the Arrhenius equation to make temperature corrections”?

Thank you for the suggestion. We agree and have revised the sentence to read: “was used in the Arrhenius equation to make temperature corrections” (lines 245-246).

• L 234: Change “oxygen consumptions” to “the rate of oxygen consumption”.

Thank you. We agree and have modified the sentence at line 282 to “the rate of oxygen consumption”

• L 236: Change “indicating” to “indicated”.

Thank you for the suggestion. We respectfully prefer to retain the participial construction because this part of the sentence states our interpretation of the preceding result rather than a second, independent result. We have clarified this by writing “thus indicating” (line 284).

• L 237, 245: Change “parameters” to “parameter”.

Thank you. We agree and have changed “four‑parameters” to “four‑parameter” at the requested location and across the manuscript: lines 230-231, 297, 376, 391, and 467

• L 239-240: Change “56.3% to 14.5%” to “14.5% to 56.3%”? And consider similar reordering elsewhere (e.g., L 271, 282)

Thank you. We agree. We have reordered the percentage ranges to run from lower to higher values for clarity and style consistency at the requested lines and anywhere else applicable in the manuscript. Lines.287-288 “14.5 % to 56.3%”; line 330 “5.3% to 26.7%”, line 338 “8.3% to 44.3%”, and line 401 “7.94% to 17.47%”.

• L 242-247: Please explain all features of the box plots. Please explain Ramp 1, 2 and 3. It is difficult to distinguish the curves for T2 and T1+2. The curve for T1+2 seems to be steeper than the curves for T1 and T2. How can this be?

Thank you for the helpful comment. We have clarified what the box plots summarize and refined the styling to avoid visualization issues. Now, we indicate that each box represents independent respirometry cycles per fish at each speed (T1: 8 cycles per box; T2: 5 cycles per box). This makes clear that the boxes capture within‑fish, within‑speed variability and how it evolves with increasing speed (low dispersion at low speeds, broader dispersion at high speeds). For Figure 5A, lines 290-302 “Figure 5. Relationship between oxygen consumption and swimming speed. (A) Treatment T1 in which oxygen consumption was measured in each fish at a single swimming speed. Three boxes per speed are shown (one per fish; 8 independent cycles per box) summarizing MO2 at a fixed speed. We found an increase in oxygen consumption at increasing speed. Here, we observed a high dispersion between individuals at higher speeds. (B) Treatment T2, in which each fish (labelled Ramp 1, 2, and 3) was forced to a progressive increase in swimming speed. Three boxes per speed (5 cycles per box) show within-individual increases of MO2 with speed; dispersion is low at low speeds and widens at higher speeds. (C) Four-parameter sigmoidal model fits: predicted curve for T1 (solid light blue line), predicted individual curves for T2 (dashed, dot-dashed and two-dashed lines in blue, green and magenta for fish labelled Ramp 1, 2, and 3 respectively), predicted curve for T2 (solid orange line), and predicted curve for T1+T2 (solid black line). Box‑plot convention: central line = median; box = IQR (Q1–Q3); whiskers = most extreme points within 1.5×IQR (Tukey); points beyond whiskers are outliers.”.

We state that Ramp 1, 2, and 3 are the three individual fish in T2 at line 294-295 “(labelled Ramp 1, 2, and 3)”

In the Figure 5C, we modified the colours and the line type of the curves to improve the identification. Now T1 predicted curve is represented as “solid light blue line” (line 298), T2 as “solid orange line” (line 300), T1+T2 as “solid black line” (line 300), and individuals curves for T2 as “dashed, dot-dashed and two-dashed lines in blue, green and magenta for fish labelled Ramp 1, 2, and 3 respectively” (lines 298-299).

The steepness in a four-parameter logistic model is governed jointly by the vertical amplitude (a-d) and the slope parameter b. The maximum slope at the inflection point is proportional to (a-d)b/4. In our fits (Table 1), The T1+T2 model inherits a large amplitude close to T1 (2.683 vs 2.872) while retaining the higher slope parameter characteristic of T2 (b=1.443). Consequently, (a-d)b/4 results largest for T1+T2 (0.97) compared with T1 (0.80) and T2 (0.47). Thus, T1+T2 curve is visibly steeper than T1 and T2.

• L 303-305, 312-317: Please explain all aspects of box plots. Each figure should stand on its own.

We thank the reviewer for this helpful suggestion. We have revised the captions of Figures 6 and 7 to fully describe the box‑plot elements and to make each figure self‑contained. We clarify what each point represents (e.g., one value per fish) and provide the sample size per box (n), which is now labelled above each box. Where applicable, we indicate whether data are pooled across treatments, how colours/styles map to sample types, and how panels differ in data aggregation. Thus, caption for Figure 6 (lines 359-365) results “Figure 6. ETS activity in whole-body homogenates (blue) and partial samples (samples 1, 2 and 3; green, purple and red). (A) ETS activity across swimming speeds for treatment T1. Each box plot summarizes the values across fish at the specific speed (3 values per speed per box) (B) Overall comparison of ETS activity across sample types. Each box plot pools whole body or partial samples across treatments (21 values per sample type per box). Box‑plot convention: central line = median; box = IQR (Q1–Q3); whiskers = most extreme points within 1.5×IQR (Tukey); points beyond whiskers are outliers.”, and for Figure 7 (lines 372-380) results “Figure 7. Comparison of R/ETS ratios across swimming speeds. (A) Treatment T1, where each fish was measured at a single swimming speed; boxplots show pooled R/ETS across individuals at each speed (3 values per speed per box). (B) Treatment T2, where each fish was subjected to a progressive increase in swimming speed; boxplots display per-fish R/ETS values at each speed for three fish (labelled as Ramp 1, 2, and 3). (C) Four-parameter sigmoidal model fits for R/ETS -speed relationship in T2: dashed lines represent predicted individual curves for T2 (for fish Ramp 1, 2, and 3), and the solid line corresponds to predicted curve for T2. Box‑plot convention: central line = median; box = IQR (Q1–Q3); whiskers = most extreme points within 1.5×IQR (Tukey); points beyond whiskers are outliers.”.

• L 345-351: Please explain what the ramps are

We appreciate the reviewer’s request. We have modified the text to explain that ramp is how we identified T2 fish at lines 408-409 as “for T2 fishes labelled Ramp 1–3”. We have also revised the rest of the captions for consistence and modified the line 308, “labelled as Ramp 1-3”.

• L 400: Please remind readers that MS-222 is an anesthetic.

We thank the reviewer for this useful suggestion. We have amended the text to explicitly remind readers that MS‑222 is an anesthetic, line 465 “anesthetic”.

• L 405: Change RETS to R/ETS.

Thank you for pointing this out. We have corrected “RETS” to “R/ETS” at line 470.

• L 418: Change “parameters” to “parameter”.

Thank you for catching this typographical error. We have corrected “parameters” to “parameter” at line 484.

• L 425: Change “Moreover, However” to “However”.

Thank you for pointing this out. We have corrected the duplicated connector by replacing “Moreover, However” with “However,” at line 495 to improve clarity and readability.

• L 433: Change “was” to “has been”.

Thank you for the suggestion. We have revised the sentence to use the present perfect. Line 504 “has been”.

• L 443: Change “and” to “to”.

Thank you for catching this. We have corrected the range construction by replacing “and” with “to” (line 514).

• L 446: Change “individual” to “individuals”.

Thank you for pointing this out. We have corrected the noun to the plural form “individuals” (line 517).

• L 474-475: Please explain why the independence of ETS & swimming activity shows that ETS activity is a good measure of maximum aerobic capacity. Maximum aerobic capacity constrains maximum swimming effort and therefore if ETS activity reflects maximum aerobic capacity, it should be highest during maximal swimming activity.

We appreciate the reviewer’s opportunity to clarify this point. The ETS assay measures how much oxygen can be processed by mitochondria to produce energy. It is always a maximal measure, and it is independent of the activity level, because mitochondrial enzyme content, capacity and mitochondrial density do not change at minute to hour scales. In contrast, MO₂ increases with swimming effort in vivo up to the limit imposed by that capacity. Therefore, ETS gives us a maximal aerobic capacity while MO₂ an activity-dependent aerobic rate. Since the ETS assay is performed under non-limiting conditions, it serve as a proxy for maximal aerobic capacity. This is why an R/ETS calibration is important to adjust ETS activity to physiological conditions. As noted in our response to comment 2, we have already added explanations to the manuscript that support this statement; no further changes are required.

• L 485-487: Consider rewording “If this model were applicable to micronekton fish, aerobic metabolism would range from 17% to 71% of their ETS activity depending on swimming speed.” as “According to this model, aerobic metabolism should range from 17% to 71% of their ETS activity depending on swimming speed.”

Thank you for the helpful suggestion. We agree and have adopted the proposed wording for clarity: “According to this model, aerobic metabolism should range from 17% to 71% of their ETS activity depending on swimming speed.”, lines 556-558.

• L 488: Change “traditionally used” to “traditional use”.

Thank you for the suggestion. We agree and have revised the sentence accordingly at line 559, “traditional use”.

• L 490: I suggest changing “could” to “may”.

Thank you for the suggestion. We agree and have replaced “could” with “may” at line 560.

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors addressed all my comments well. I have no further question.

Author Response

Thank you very much for your careful assessment and constructive suggestions in the previous round. We are glad to hear that our revisions resolved your comments. We appreciate your effort and consideration.

Reviewer 3 Report

Comments and Suggestions for Authors

General comments:

The authors have diligently and carefully addressed all my comments.  At this stage I have only two minor suggestions. 

 

Specific comments:

L 11-12: The term “poorly constrained” is not the correct word choice here.  Please replace with more appropriate words such as “poorly understood”.   The first sentence has to do with our understanding not constraints.    

L 37: As I said in my original review, the word “export” implies movement of C that leads to it leaving the ocean system.  However, the authors discuss how C moves within the ocean system, which is better described as “transport”.

 

Comments on the Quality of English Language

See my previous comments to the authors.

Author Response

We appreciate your assessment and are pleased that the prior revisions satisfactorily addressed your earlier concerns. Below, we provide detailed responses to the comments. The corresponding revisions are highlighted in the resubmitted files.

 

Comment 1: L 11-12: The term “poorly constrained” is not the correct word choice here.  Please replace with more appropriate words such as “poorly understood”.   The first sentence has to do with our understanding not constraints.    

Response 1: Thank you for the helpful suggestion. Our intention was to reflect the methodological limitation that leads to knowledge gaps. To reduce ambiguity while preserving this nuance, we revised the sentence to “poorly constrained due to the difficulty of obtaining direct metabolic measurements and, consequently, poorly understood.” (lines 11-13) We hope this clarifies that methodological constraints (difficulty of direct metabolic measurements) underlie the current knowledge gap.

 

Comment 2: L 37: As I said in my original review, the word “export” implies movement of C that leads to it leaving the ocean system.  However, the authors discuss how C moves within the ocean system, which is better described as “transport”.

Response 2: Thank you for this helpful observation. In the revised text we retain the term “export” in the canonical ocean‑biogeochemical sense: the flux that leaves the surface layer (euphotic or mixed layer) and enters the ocean interior. We now make this explicit in the sentence (lines 38-39): “The ocean plays a central role in the global carbon cycle, acting as a major sink for anthropogenic carbon through export from the surface layer and subsequent sequestration that leads to long‑term storage in the deep sea.”

This usage follows standard practice in the field. Our key references defines biological‑pump efficiency in terms of the amount of carbon exported from the surface layer relative to production, and consistently refer to “export from the surface” when describing the initiation of interior sequestration (see our refs. [2], [3], [11]). Particularly, Le Moigne [11] expresses that export from the surface is mediated by different transport pathways (gravitational settling, physical mixing/subduction, and migration).

Accordingly, we believe “export … from the surface layer” is the accurate and widely adopted terminology for the process referenced here.

Author Response File: Author Response.pdf