Comments on “Temperature fluctuation alleviates the negative effects of warming on marine diatoms: comparison between Thalassiosira sp. and Nitzschia closterium f. minutissima” by Sheng et al.

General Comments: This manuscript addresses a compelling and timely topic by exploring how temperature fluctuations influence the physiological and biogeochemical responses of marine diatoms to ocean warming, an aspect often overlooked in studies conducted under static temperature conditions. The authors provide valuable data on two ecologically significant diatom species, Thalassiosira sp. and Nitzschia closterium f. minutissima, revealing species-specific responses in growth rate, particulate organic carbon (POC), biogenic silica (BSi), and sinking rate. These findings contribute to our understanding of how diatom-driven biogeochemical cycles may respond to future ocean conditions. The manuscript is well-structured, clearly written, and supported by robust experimental methods. However, I have several minor concerns and suggestions to enhance the manuscript’s clarity, scientific rigor, and overall impact.

Specific Comments:

1. Lines 92–94: Please provide details on the season when Thalassiosira sp. and Nitzschia closterium f. minutissima were isolated from the Yellow Sea, along with the corresponding mean water temperature at the time of collection. Additionally, clarify how long these species were maintained in the laboratory prior to the experiments, as this could influence their acclimation to culture conditions.
2. Line 102: Correct the typographical error in the cell abundance notation. The number “4” in “1 × 104 cells mL-1” should be formatted as a superscript (i.e., 1 × 10⁴ cells mL⁻¹).
3. Line 110: Clarify whether Figure 1 represents recorded temperature changes from the experiment or is a schematic diagram of the temperature treatments. If it is a schematic, consider including a supplementary figure with actual temperature data to validate the experimental setup.
4. Line 115: Specify the storage conditions for samples during chlorophyll a (Chl a) extraction. Were the samples stored in the dark, and at what temperature (e.g., 4°C)?
5. Line 118: For the growth rate calculations, confirm whether Chl a fluorescence was measured directly from the algal culture or after filtration. If measured directly, discuss any potential interference from culture medium or cell aggregation that might affect fluorescence readings.
6. Line 158: Origin 2021 is a product of OriginLab Corporation), not “Tukey”, which is a statistical method.
7. Discussion Section: I recommend adding a paragraph to discuss the differential responses between Thalassiosira sp. (a centric diatom) and Nitzschia closterium f. minutissima (a pennate diatom). Highlight how their morphological and ecological differences (e.g., cell size, silica structure, or habitat preferences) might contribute to their distinct responses to warming and temperature fluctuations. This would strengthen the manuscript’s ecological and taxonomic insights.

In this paper, Sheng et al. investigated the response of two marine diatom species, Thalassiosira sp. and Nitzschia closterium f. minutissima, to ocean warming and temperature fluctuation (± 4 °C) under low (20 °C) and high (25 °C) average temperatures. The semi-continuous incubation method was adopted. Their results demonstrate that temperature fluctuation alleviated the negative effects of elevated temperatures on the growth of both species and revealed distinct responses of the two diatoms in cellular element contents and sinking rate. This study explored the influence of temperature fluctuation on the physiology of marine diatoms and shed light on the biogeochemical feedbacks in the context of global warming.

In general, the methods and the analyses are very sound, and the interpretation of the results are overall appropriate. Moreover, the manuscript is generally well-written and referenced. I feel that this is in principle an excellent study. However, several points need to be addressed before acceptance.

Comments:

1. Line 25-30: “However, warming significantly decreased the cellular particulate organic carbon (POC) and biogenic silica (BSi) contents, and sinking rate, while increasing protein content to cope with the thermal stress under temperature fluctuation. Temperature fluctuation at low average temperatures significantly increased the cellular POC and BSi contents, as well as POC productivity and sinking rate, while at high average temperatures, these parameters were significantly decreased.” The two sentences are somewhat repetitive, which may cause confusion. Please revise for conciseness.

1. Line 40: Please clarify the time span over which the 1°C increase is expected to occur or has already occurred.

1. Line 53: It would not be appropriate to use “detrimental” here since the following sentence highlighted “the temperature fluctuation (2 days) reduced the mortality rate of Emiliania huxleyi”. Please consider using a different word to describe the complex situation. Also, please add basic descriptions for the coccolithophore Emiliania huxleyi, in case that readers are not familiar with it.

1. Line 86: Please add more details explaining why the two diatoms were selected, e.g. their ecological roles, importance, size, common and different characteristics. Some contents in Line 300-310 could be moved to the Introduction.

1. Line 95: Please provide more information for the experiments. Why “20°C” and “25°C” were chosen as the average temperature for “cold” and “warm” status? Have the optimal growth temperatures of two diatoms been determined using the temperature curves? How many days have been taken to reach the stabilized growth rate? Unless this experiments took years to complete, please use the words like “adapt” or “adaptation” with caution. “Acclimation” would be a better word for the short-term experiments.

1. Line 96: Please consider including a table of abbreviations like “LTCT”, “LTFT”,“HTCT” and “HTFT”.

1. Line 163-165: Please add more details for the formula and rephrase the sentence “When the positive and negative results of OE1 and OE2 are the same, …and vice versa, they are antagonistic interaction effects”. Are the results of OE1 and OE2 always be opposite?

1. Line 313: It would be a good idea to add the information about the temperature range and fluctuation conditions in the coastal habits where the two diatoms were collected, to interpret the results in the context of evolution.

1. Although species-specific responses to environmental factors are widely acknowledged, it would be helpful if the authors could interpret the different responses of two diatoms revealed here based on their distinct characteristics, and add this point to the Discussion section.

1. In the Discussion section, it's generally not necessary to cite figures again, especially if those figures are already presented and mentioned in the Results section.

1. There are several typos in the manuscript. For instance, there is a repeated use of 'overall' in Line 32 and Line 34, which could be streamlined. In Line 58, “Microcystis aeruginosa” should be italic. In Figure 6, the symbols of Thalassiosira in two columns are different. In Line 312, there is a space in the word “tolerance”. Hence one more round of thorough proof reading would be in order.