Effects of Artesunate on the Growth and Chlorophyll Fluorescence of the Cyanobacterium Microcystis aeruginosa
José Antolin Ake-Castillo
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript describes the effects of artesunate on two strains of Microcystis aeruginosa. The results are primarily based on growth (optical density) and chlorophyll fluorescence measurements. However, the manuscript is not well written. The abstract lacks sufficient detail to clearly convey the study’s scope and findings. Several sentences are unclear. The introduction should be more coherent and logically organized. The study objectives are also unclear, particularly regarding how this work addresses population structure.
The Methods section requires improvement and clarification, including the growth phase used (exponential or logarithmic), strain details, and unit consistency (mg/L vs. μmol/L). In the Discussion, the information provided is limited regarding the differing effects of artesunate on non-toxigenic and toxigenic strains. An explanation of how artemisinin selectively affects toxigenic strains of M. aeruginosa is needed. To strengthen the conclusions, additional testing on a wider range of toxigenic and non-toxigenic strains is recommended. The study also lacks comparison with Ni et al. (2015), who reported inhibition rates exceeding 90% for M. aeruginosa under certain conditions. Please clarify how your novel findings differ from those of Ni et al. (2015).
Overall, the interpretation in each section is not entirely clear, and the novel findings require further discussion. The manuscript would benefit from language editing by a native English speaker. All detailed comments are provided in the annotated PDF file.
Comments for author File: 
Comments.pdf
Author Response
Reviewer 1 General Comments:
The manuscript describes the effects of artesunate on two strains of Microcystis aeruginosa. The results are primarily based on growth (optical density) and chlorophyll fluorescence measurements. However, the manuscript is not well written. The abstract lacks sufficient detail to clearly convey the study’s scope and findings. Several sentences are unclear. The introduction should be more coherent and logically organized. The study objectives are also unclear, particularly regarding how this work addresses population structure.
Response:Thanks to the reviewer's comments. In response to the comments, we made systematic changes to the abstract and frontier.
The Methods section requires improvement and clarification, including the growth phase used (exponential or logarithmic), strain details, and unit consistency (mg/L vs. μmol/L). In the Discussion, the information provided is limited regarding the differing effects of artesunate on non-toxigenic and toxigenic strains. An explanation of how artemisinin selectively affects toxigenic strains of M. aeruginosa is needed. To strengthen the conclusions, additional testing on a wider range of toxigenic and non-toxigenic strains is recommended. The study also lacks comparison with Ni et al. (2015), who reported inhibition rates exceeding 90% for M. aeruginosa under certain conditions. Please clarify how your novel findings differ from those of Ni et al. (2015).
Response: According to your comments, we have made specific changes to the methodology and discussion.
Overall, the interpretation in each section is not entirely clear, and the novel findings require further discussion. The manuscript would benefit from language editing by a native English speaker. All detailed comments are provided in the annotated PDF file.
Response: Thank you for the comments. Based on the specific Comments throughout the manuscript, we have provided the following point-by-point responses. In view of the problem of language fluency, we have polished the language in full.
Specific Comments:
Comment1：The introduction starts with a general statement about algae, but the focus of the study is specifically on Microcystis aeruginosa, a cyanobacterium (blue-green algae). Since “algae” is a broad term that includes many unrelated groups, it would be clearer and more precise to refer directly to cyanobacteria in the opening sentence, and then narrow down to M. aeruginosa. This will help readers immediately understand the taxonomic group and ecological relevance of the organism studied.
Response：Thanks for you comments. We have now corrected the term "algae" to "cyanobacterium" in both the introduction and main text. Additionally, the description in the introduction has been logically reorganized as " cyanobacteria (blue-green algae blooms) -Microcystis aeruginosa bloom-control measures for Microcystis aeruginosa blooms". Line 30-43.
Comment 2: [How to know when the exponential growth phase from cell counting? Please clarify] in line 97-98.
Response: We agree with your advice. In the revised manuscript, the sentence has been changed to “Cells usually have a better state when they are in the exponential growth phase. Therefore, we established a standard curve for cell density by measuring the OD value at a wavelength of 680 nm using the optical density method. Then in this study, cells in the exponential growth phase were determined by counting triplicate 1 mL samples fixed with 2% (v/v) Lugol's solution.” Line 91-95.
Comment 3: [It is unclear which growth phase was used in this study—exponential or logarithmic—as these two phases represent distinct growth patterns. Please clarify to avoid confusion.] in line 105.
Response: Thank you very much for pointing out this error. This section has been modified to “the exponential growth phase”. Line 101.
Comment 4: [0, 10, 50, 100, and 200 mg∙L-1] in line 108.
Response: Thank you very much for pointing out this error. “mg∙L-1” has been modified to“μmol/L”. Line 104.
Comment 5: [What is the axenic condition? Please add information.] in line 112.
Response: This section has been modified to “The BG11 medium, pipettes and conical flasks used in the experiment were sterilized by high-pressure steam. Before using in the clean workbench, they were irradiated with ultraviolet light for 30 minutes. Algae inoculation and other operations were completed inside the ultra clean workbench to ensure that all the operations were carried out in axenic conditions.” Line 108-112.
Comment 6: [9h?] in line 116.
Response: Thank you very much for pointing out this error. This section has been modified to “96h” in Line 117.
Comment 7: [In my opinion, this table may be unnecessary as the information it contains has already been adequately interpreted in the Methods section. Consider removing it to avoid redundancy.] in line 134.
Response: Table 2 has been deleted.
Comment 8: [Observation times or sampling time ? ] in line 136.
Response: Observation times is better, and Processing time represents observation time. In addition, considering the repetition between Materials and Methods and Table 2, it has been deleted from the paper.
Comment 9: [Did the authors perform mean comparisons after obtaining significant results? If so, please provide details about the statistical analysis methods used.] in line 137.
Response: Details about the statistical analysis methods used were added in revised draft, that is “Detailed methods: Analyze the data obtained from all groups within 0, 12, 24, 48, 72, and 96 h. Firstly, input variables by column in SPSS software, perform one-way ANOVA in comparison of means, and output the mean and standard deviation in descriptive statistics. Then set the significance level to 0.01 in Options. Subsequently, save the mean and error bar data output by SPSS to Excel. Import the data to Origin 9.0, select the chart type for plotting and add error bars.” Line 139-145.
Comment 10: [each concentration group of artesunate] in line 144.
Response: The “group”has been modified to “level”. Line 148.
Comment 11: [What is the accuratal unit used in this study? umol/L or mg/L ] in line 152.
Response: Thank you very much for pointing out this error. In this paper, the accuratal unit used in this study is umol/L, the entire text has been modified.
Comment 12: [It is unclear which strains are toxigenic (toxic) and which are non-toxigenic (non-toxic) in the current description. Please clarify this distinction to improve the clarity of the manuscript.] in line 154.
Response: It is indeed very necessary to indicate which strains are toxigenic (toxic) and which are nontoxigenic (non-toxic) here. Therefore, this sentence has been modified to “Non-toxigenic strains FACHB-927 are more resistant to artesunate than toxigenic strains FACHB-315”. Line 158-159.
Comment 13: [What do author mean? Please rewrite] in line 158.
Response: We would like to use “Change in Fv/Fm” represent the effect of artesunate on Fv/FM of Microcystis aeruginosate. For accuracy, we have modified to “Effects of artesunate on Fv/FM”. Line 165.
Comment 14: [Only one strian? please check and clarify the sentence.] in line 173.
Response: This sentence has been modified to “Concentrations of artesunate between 50~100 μmol/L have similar inhibitory effects to Fv/Fm in FACHB-315 and FACHB-927”. Line 181-182.
Comment 15: [To facilitate comparison with Figure 1, this figure would be clearer if presented as a line plot rather than the current format. Please consider revising the figure style for consistency and better interpretability.] in line 174.
Response: We also considered using a line plot, but here, in addition to showing the trend of changes, we also need to reflect the significant differences between groups, so the bar chart may be more suitable. Line 183.
Comment 16: [Why do all treatments, including the control group, show a decrease in φPSII at 24 hours? Please provide a possible explanation for this observation.] in line 181.
Response: In this section, treatments refer to the experimental group that treated with artesunate, excluding the control group. We can also find that φPS â…¡ of the control group has been almost no change within 24 hours. To avoid ambiguity, we has modified this section to “As seen in Fig. 3(a) and 3(b), the φPS â…¡ of groups treated with artesunate is rapidly decreased after exposure to artesunate within 24 hours, and then gradually levels up with increasing exposure time from 24h to 72h. At 96h, the value of φPSII decreases with the increase of artesunate concentration. The minimum φPSII of experimental groups appeared at 24h both in FACHB-315 and FACHB-927.” Line 190-195.
Comment 17: [This claim appears to be an overstatement, as the study was conducted on only two strains of Microcystis, not on all algae. Please revise the statement to better reflect the scope of the research.] in line 183-186.
Response: The statement has been revised to “indicating that artesunate had an impact on Cyanobacterium Microcystis aeruginosa, and the higher the concentration, the more obvious the inhibition effect. Although the value of φPSII of treatments was smaller than that of the control in all groups, it increased slowly with the increase of culture time. Whether artesunate works on other microalgae remains to be further studied through experiments.” Line 196-200.
Comment 18: [The provided information is quite limited regarding the differing effects of artesunate on the non-toxigenic and toxigenic strains. Please consider elaborating further on the potential mechanisms, such as the role of secondary metabolites like microcystins and allelopathic interactions, as these details are important to support and strengthen your findings.] in line 195.
Response: Thank you very much for your advice. The sentence “This difference may stem from the release of secondary metabolites (e.g., microcystins) by toxigenic strains, which could interact with artesunate through allelopathic mechanisms (Babica et al., 2006). Microcystins are known to influence interspecies competition and stress responses, potentially modulating the sensitivity of cyanobacteria to external inhibitors.” has been instead of “Cyanobacteria can produce a variety of secondary metabolites, of which peptides account for approximately 65% (He et al., 2025). Microcystins produced by toxigenic strains of Microcystis aeruginosa are important secondary metabolites, and their chemical nature is cyclic heptapeptides. Microcystins are known to influence interspecies competition and stress responses, potentially modulating the sensitivity of cyanobacteria to external inhibitors. Previous studies have shown that algal toxins may be involved in regulating the activity of antioxidant enzymes such as superoxide dismutase (Babica et al., 2006; Fu et al., 2025). When Microcystis aeruginosa comes into contact with artesunate, artesunate may trigger the production of ROS inside microalgae, such as superoxide anions, hydroxyl radicals, hydrogen peroxide, and nitric oxide, thereby inducing oxidative stress, leading to membrane lipid peroxidation, protein oxidation, enzyme inactivation and nucleic acid destruction. At the same time, artesunate may also interfere with the cell division process, energy conversion and signal transduction of Microcystis aeruginosa, thereby inhibiting cell growth rate. In the future, it is very necessary to further study the toxicity mechanism of microalgae exposed to artesunate using omics techniques.” Line 208-222.
Comment 19: [Please consider comparing your experimental results with those reported by Ni et al. (2015), who observed inhibition rates exceeding 90% for M. aeruginosa under certain conditions. Discussing similarities or differences may help to better contextualize and strengthen your findings.] in line 200.
Response: Thank you very much for your suggestion. After the sentence“Ni et al. (2015) observed that M. aeruginosa exhibited varying susceptibility to artemisinin, with inhibition rates exceeding 90% under certain conditions.”, the following sentence was added "In Ni et al.'s (2015) article, in addition to using pure artemisinin, they also used artemisinin sustained-release granules, with the concentration range from 1 to 20 g/L. In our study, the maximum concentration of artesunate was 200 μ mol/L. Their concentration is significantly higher than that of this experiment, so their inhibition rate is higher.”Line 226-230.
Comment 20: [As the authors mentioned above, what differences exist in the effect of DHA between toxigenic and non-toxigenic strains? Additionally, why does artemisinin have a stronger effect on the toxigenic strain compared to the non-toxic one? Please provide clarification. Please provide a Clarifying the possible mechanisms would greatly enhance the understanding of your study.] in line 239-240.
Response: Thank you very much for your suggestion. This part have been carefully supplemented and improved. The sentence “This suggests that artemisinin could selectively regulate algal community composition,favoring non-toxic species over bloom-forming cyanobacteria.” in line 239-240 now has been replaced by the following content “ Dihydroartemisinin (DHA) mainly induces oxidative stress by producing reactive oxygen species (ROS), thereby interfering with the structure and function of cell membranes. Theoretically, compared with the non-toxigenic strain FACHB-927, the toxigenic strain FACHB-315 of Microcystis aeruginosa may have a stronger antioxidant defense system and higher tolerance to DHA induced oxidative stress. non-toxigenic strains may be more susceptible to oxidative damage from DHA, leading to more pronounced growth inhibition. However, similar to other environmental stress, artesunate may stimulate the production of strains to increase the synthesis and release of microcystins, and this response may consume a large amount of energy and metabolic resources. Non-toxigenic strain do not exhibit this metabolic redistribution due to their lack of toxin producing ability. In addition, DHA may also affect photosynthesis by inhibiting photosystem II function. From Figure 2, we can also find that the decrease in photosynthetic activity (Fv/Fm) of toxigenic strain is smaller than that of non-toxigenic strain, indicating that the expression of photosynthesis related genes in non-toxigenic strain is more easily affected.”Line 274-287.
Comment 21: [What do the finding differ from Ni et al. 2015?] in line 244
Response: No references were cited here, it have been deleted now.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Comments and Suggestions for AuthorsThe work presents several errors and weaknesses:
1. Conceptual errors:
M. aeroginosa is not an algae but a cyanobacterium. The use of the term algae is incorrect, and a phycology journal establishes that the terms algae and cyanobacteria should be separated.
Phycotoxin is a term used for toxins produced by algae (e.g., dinoflagellates). For cyanobacteria, the correct term is cyanotoxins.
2. Methodology:
They do not describe which of the two strains is toxic and which is not, either in the methodology or in the results. It is only mentioned in the discussion, when the objective of the research should be explained from the outset.
In the description of the measurement of chlorophyll fluorescence variables, they do not explain the dark adaptation time of the strains. Did they create a curve to determine the minimum appropriate time? This value varies between species and sometimes between strains.
What adapter did you use to measure water samples in the FMS 2 chlorophyll fluorimeter? Did you use an adapter?
3. Results:
It is recommended that the y-axis scale in Figure 2 be the same to compare the two strains. Furthermore, no units are assigned to FV/Fm; this is an index.
Figure 2 (b) shows that the control Fv/Fm values are equal to or close to 1; this is not possible for that variable. Possibly, there was mishandling of the equipment or a measurement error. If an adapter was not used to measure fluorescence in liquid samples, this could be the cause.
The φPSII results in Figure 3 (a) show a rapid recovery in the treatment over time, with values very close to the control. This does not correlate with the results achieved in Fv/Fm, which do show a decrease over time. Is there a possible error in the measurement conditions for the chlorophyll fluorescence variables?
4. Discussion and Conclusions
This work does not involve experiments that would allow them to understand the mode of action of artesunate in the cell; therefore, it can only be concluded that its effect is reflected in a decrease in the Fv/Fm value.
Nor can it be said that there is an inhibition of photosystem II; the authors should express this under the concept of the Fv/Fm variable.
Author Response
-
The work presents several errors and weaknesses:
- Conceptual errors:
- aeroginosais not an algae but a cyanobacterium. The use of the term algae is incorrect, and a phycology journal establishes that the terms algae and cyanobacteria should be separated.
Phycotoxin is a term used for toxins produced by algae (e.g., dinoflagellates). For cyanobacteria, the correct term is cyanotoxins.
Response: Thank you very much for pointing out this error. We have now corrected the term "algae" to "cyanobacterium" in both the introduction and main text.
- Methodology:
They do not describe which of the two strains is toxic and which is not, either in the methodology or in the results. It is only mentioned in the discussion, when the objective of the research should be explained from the outset.
In the description of the measurement of chlorophyll fluorescence variables, they do not explain the dark adaptation time of the strains. Did they create a curve to determine the minimum appropriate time? This value varies between species and sometimes between strains.
What adapter did you use to measure water samples in the FMS 2 chlorophyll fluorimeter? Did you use an adapter?
Response: Thank you very much for your suggestions. (1) Based on your advices, we have added the toxigenic strain FACHB-315 and non-toxigenic strain FACHB-927 of Microcystis aeruginosa in the Materials and Methods and Discussion of the paper. (2) In the measurement of chlorophyll fluorescence variables, dark adaptation is indeed an important pretreatment step. However, compared with other algae, Microcystis aeruginosa shows some particularity in chlorophyll fluorescence measurement, that is, the photosynthetic system of Microcystis aeruginosa can quickly adapt to changes in light, so a relatively short adaptation time (1~5 minutes) is sufficient. (3) In this study, the handheld water chlorophyll fluorescence meter (AquaPen-C 100, The Czech Republic) was used for measurement, which is particularly suitable for the physiological research of Microcystis aeruginosa and measures its key photosynthesis parameters accurately.
- Results:
It is recommended that the y-axis scale in Figure 2 be the same to compare the two strains. Furthermore, no units are assigned to FV/Fm; this is an index.
Figure 2 (b) shows that the control Fv/Fm values are equal to or close to 1; this is not possible for that variable. Possibly, there was mishandling of the equipment or a measurement error. If an adapter was not used to measure fluorescence in liquid samples, this could be the cause.
The φPSII results in Figure 3 (a) show a rapid recovery in the treatment over time, with values very close to the control. This does not correlate with the results achieved in Fv/Fm, which do show a decrease over time. Is there a possible error in the measurement conditions for the chlorophyll fluorescence variables?
Response: (1) According to your suggestion, Figure 2 has been modified to unify the y-axis scale in two strains, and remove the Fv/Fm units. (2) In this study, the handheld water chlorophyll fluorescence meter (AquaPen-C 100, The Czech Republic) was used for measurement. In addition to setting three replicates for each group, we also conducted multiple measurements for each sample in the experiment to avoid possible errors. (3) In Figure 3 (a), although the PSII value was very close to the control group at 72 h, it was still lower than the control group, and concentrations of 50 μ mol/L and 100 μ mol/L subsequently decreased at 96 h. Considering that each group has three replicates, the likelihood of an error is low, but the specific reason is not yet clear.
- Discussion and Conclusions
This work does not involve experiments that would allow them to understand the mode of action of artesunate in the cell; therefore, it can only be concluded that its effect is reflected in a decrease in the Fv/Fm value.
Nor can it be said that there is an inhibition of photosystem II; the authors should express this under the concept of the Fv/Fm variable.
Response: We have revised the discussion and conclusions. The title of 4.3 will be changed to "may mechanisms" line 260, and "inhibit photosystem II" will be changed to "decrease the Fv/Fm value" in the conclusion, line 293.
Author Response File: Author Response.docx
Reviewer 3 Report
Comments and Suggestions for AuthorsThe research has a good experimental design. However, the methods need more information as well as a good statistical approach:
In table 2, you declared 5 variables measured: cell density, chlorophyll a, dissolved oxygen, Fv/Fm, and φPSII as indicators of the response of the different treatment. So, procedures for measuring chlorophyll “a” and dissolved oxygen are missing.
Statistical analyses: you must detail factors and variables selected for analyses and how many of them you executed. ANOVA for each response variable? is treatment the only Factor? Strain is not a factor? I recommend use a factorial ANOVA for each response variable.
Results: the only ANOVA result is indicated (indirectly) only for the effect of treatments on Fv/Fm (in figure 2).
The other results of the effect on optical density and φPSII are merely descriptive. I recommend executing ANOVA´s and showing statistical differences.
There are not results for chlorophyll “a” nor Dissolved oxygen.
Statistical results are important part of the experimental design, so I recommend use tables to resume results with data resulting from ANOVA´s.
The last part of the discussion is bibliographic. It must be resumed and addressed with the facts you demonstrated experimentally.
Author Response
The research has a good experimental design. However, the methods need more information as well as a good statistical approach:
In table 2, you declared 5 variables measured: cell density, chlorophyll a, dissolved oxygen, Fv/Fm, and φPSII as indicators of the response of the different treatment. So, procedures for measuring chlorophyll “a” and dissolved oxygen are missing.
Response: Thank you very much for pointing out the mistake. The initial experimental design included measurements of chlorophyll a and dissolved oxygen, but for some reason it was not carried out. The content mentioning chlorophyll a and dissolved oxygen in the text has been removed, such as the "chlorophyll a, and dissolved oxygen" in line 113 in the PDF file. In addition, considering the repetition between Materials and Methods and Table 2, it has been deleted from the paper.
Statistical analyses: you must detail factors and variables selected for analyses and how many of them you executed. ANOVA for each response variable? is treatment the only Factor? Strain is not a factor? I recommend use a factorial ANOVA for each response variable.
Response: In this experiment, we aimed to explore whether artesunate would affect the growth and photosynthesis system of Microcystis aeruginosa. Therefore, culture medium containing different concentrations of artesunate were set up in the experiment. Meanwhile, the volume of algae added and the final volume of the culture medium in each flasks were the same. Considering that Microcystella aeruginosa has both toxigenic strains and non-toxigenic strains, FACHB-315 (toxigenic strain) and FACHB-927 (non-toxigenic strain) were chosed to explore the changes in different strains under the same concentration of artesunate. In the result, we conducted a variance analysis on the data of different groups (0, 10, 50, 100, and 200μmol/L) of each strains, and obtained the trend of parameters with the variation of artesunate concentration. In addition, we also discussed the results of different strains at the same concentration.
Results: the only ANOVA result is indicated (indirectly) only for the effect of treatments on Fv/Fm (in figure 2).
Response: The main purpose of choosing a line chart is to display the continuous trend of various indicators over time, so the bar chart format in Figure 2 was not chosen. Meanwhile, in order to reflect the degree of data variability, error bars are used in the graph.
The other results of the effect on optical density and φPSII are merely descriptive. I recommend executing ANOVA´s and showing statistical differences.
Response: In addition to descriptive statements, through line charts Figure 1 and Figure 3 that visually and quickly display the changing trends of each group at different concentrations, as well as the differences between different groups .
There are not results for chlorophyll “a” nor Dissolved oxygen.
Response: Thank you very much for pointing out the mistake. The initial experimental design included measurements of chlorophyll a and dissolved oxygen, but for some reason it was not carried out. The content mentioning chlorophyll a and dissolved oxygen in the text has been removed, such as the "chlorophyll a, and dissolved oxygen" in line 113 in the PDF file.
Statistical results are important part of the experimental design, so I recommend use tables to resume results with data resulting from ANOVA´s.
Response: First of all, we agree with the reviewers' opinion that the data can be clearly seen in the table, but we think it is better to present the experimental data with time control in statistical charts, because the trend of the experimental data over time can be clearly seen.
The last part of the discussion is bibliographic. It must be resumed and addressed with the facts you demonstrated experimentally.
Response: Based on our research results and references, we have revised the discussion. Line 207-289.
Author Response File: Author Response.docx
Reviewer 4 Report
Comments and Suggestions for AuthorsThis study examined the effects of artesunate on growth and photosystem II (Fv/Fm, φPSII) parameters in two different Microcystis aeruginosa strains (FACHB-315 and FACHB-927). The results indicate that artesunate inhibits algal growth and photosynthetic efficiency in a concentration- and time-dependent manner.
The introduction provides a comprehensive overview of the literature, addressing both the medicinal use and ecotoxicological effects of artemisinin. However, a research gap needs to be highlighted more clearly. A lengthy overview is already provided, but the novelty of the article (e.g., “the differential effects of artesunate on toxigenic and non-toxigenic M. aeruginosa strains have not been examined”) should be stated. The authors should emphasize what is missing from the existing literature and the hypothesis tested by this study.
The study was conducted using only two strains and limited parameters (OD680, Fv/Fm, φPSII). The data is sufficient to demonstrate that artesunate suppresses growth, but:
The measured parameters are very few (e.g., cell morphology, toxin levels, ROS levels, membrane damage, etc. are missing). Therefore, the mechanistic data supporting the article are insufficient and remain at a proof-of-concept level. There is an inconsistency in the units: μmol/L is used in the abstract and mg/L in the methods section (needs clarification).
The methods used in the article are technically correct, but the data coverage is very limited. Looking only at growth and two photosynthetic parameters does not suffice to present a strong biological narrative.
Author Response
This study examined the effects of artesunate on growth and photosystem II (Fv/Fm, φPSII) parameters in two different Microcystis aeruginosa strains (FACHB-315 and FACHB-927). The results indicate that artesunate inhibits algal growth and photosynthetic efficiency in a concentration- and time-dependent manner.
The introduction provides a comprehensive overview of the literature, addressing both the medicinal use and ecotoxicological effects of artemisinin. However, a research gap needs to be highlighted more clearly. A lengthy overview is already provided, but the novelty of the article (e.g., “the differential effects of artesunate on toxigenic and non-toxigenic M. aeruginosa strains have not been examined”) should be stated. The authors should emphasize what is missing from the existing literature and the hypothesis tested by this study.
Response: According to the reviewer's opinion，the description in the introduction has been logically reorganized as " cyanobacteria (blue-green algae blooms) -Microcystis aeruginosa bloom-control measures for Microcystis aeruginosa blooms" (line 30-43). The main innovation of this study is to verify the growth inhibition of artesunate on the main species of harmful cyanobacteria-Microcystis aeruginosa, and to demonstrate the differential effects of artesunate on toxigenic and non-toxigenic M. aeruginosa strains (line 74-78).
The study was conducted using only two strains and limited parameters (OD680, Fv/Fm, φPSII). The data is sufficient to demonstrate that artesunate suppresses growth, but:
The measured parameters are very few (e.g., cell morphology, toxin levels, ROS levels, membrane damage, etc. are missing). Therefore, the mechanistic data supporting the article are insufficient and remain at a proof-of-concept level. There is an inconsistency in the units: μmol/L is used in the abstract and mg/L in the methods section (needs clarification).
Response: Thank you very much for pointing this out. At the beginning of this study, the aim was to investigate whether artemether affects Microcystis aeruginosa by measuring several representative indices, whether it promotes, inhibits, or has no effect, and the differences in its effects on toxigenic and non-toxigenic strains. Next, we will conduct detailed research on cell morphology, membrane damage, toxin levels, ROS, and other aspects that you mentioned. Due to negligence in writing, there were inconsistencies in the unit. We have conducted a full text check and unified the concentration unit to "μmol/L".
The methods used in the article are technically correct, but the data coverage is very limited. Looking only at growth and two photosynthetic parameters does not suffice to present a strong biological narrative.
Response: Thanks for the comments from the reviewers. The insufficient data on mechanism validation will be made up for in following studies. Additionly，We have revised the discussion and conclusions.
Author Response File: Author Response.docx
Reviewer 5 Report
Comments and Suggestions for AuthorsAuthors investigate the possibility of using the artemisinin derivate artesunate for controlling cyanobacterial blooms by exposing two strains of Microcystis aeruginosa, one toxic an one non-toxic, to artesunate concentrations ranging from 0 to 200 micromol/L and measuring the culture optical density and two fluorescence parameters, the maximum photosynthesis efficiency Fv/Fm and the photosynthesis quantum efficiency ΦPSII, during 96 hours of cultivation. Reported results show a difference between the toxic and the non-toxic strain, the artesunate concentration necessary to lower the cyanobacteria growth rate and the restoration of the quantum efficiency of cyanobacteria photosynthesis after the initial inhibition.
Comments:
Abstract: L15: ... artesunate, a more water soluble derivative of artemisinin, on ...
Keywords: please add artemisinin
L85: ... artesunate, a more water soluble derivative of artemisinin, was ...
L98, L105: use consistently either exponential growth or logarithmic growth. Generally, please avoid using two different terms for the same attribute.
L116: 96h (a typo)
L130: how exactly was ΦPSII measured? Please describe or insert a reference.
Table 2/Heading: Reagent concentration units should be micromol/L as used in figures
Table 2: you measured also chlorophyll a and DO concentration. Could you please add a table with these measured values (and discuss them), or explain why they are not shown?
L154: you state that phycotoxin producing species (plural) are more resistant to artesunate than non-producing species (plural) but you have investigated only one specie of either type. Please add a reference to support this plural or reformulate this sentence to reflect reality.
L160-163: add a reference or measured data after "under stress conditions", or reformulate (you say "obviously" so support it, please)
L163: please replace "know" by "measure" or "ascertain" or a similar appropriate synonym.
L164: please replace "function" by "values" (a kind of typo)
L171-172: please reformulate to be more clear, e.g., "... to 0.424 in Strain 315 and 1.03 to 0.459 in Strain 927."
Figure 2: why mixed p<0.05 and p<0.01? Please explain.
L192-193: non-toxigenic Strain 315 and toxigenic Strain 927 - please state this difference earlier, e.g., in 2.1.
L209-210: at concentrations 100 and 200 (S315) and 200 (S927) it does not weaken, at least for 96h as shown in your data. Please reformulate (e.g., add "at lower artesunate concentrations" at the end of the sentence).
L226: ... artemisinin derivatives as artesunate ...
L234-235: allelopathic interaction (with lower case "a" and "i")
L251: please reformulate slightly: "the appropriate addition of artesunate" or "the addition of appropriate artesunate concentration"
L252-253: the "Moreover ..." sentence says the opposite of what show the data in Fig. 1 where the lower artesunate concentration show the same effect for both species but the nontox-specie was already inhibited at 100 where the tox-specie was not. The inhibition depth seems to be about the same in both species.
For me, one of the conclusions from the data in Fig. 1 would be (formulated very concisely): With the tox-specie, 2x higher artesunate concentration is necessary than with the nontox-specie to achieve the same OD suppression. The effect on lowering the quantum efficiency fades after 96 hours for both species.
What seems important is the phycotoxin concentration produced by the algae. Could it not be measured as well?
Author Response
Authors investigate the possibility of using the artemisinin derivate artesunate for controlling cyanobacterial blooms by exposing two strains of Microcystis aeruginosa, one toxic an one non-toxic, to artesunate concentrations ranging from 0 to 200 micromol/L and measuring the culture optical density and two fluorescence parameters, the maximum photosynthesis efficiency Fv/Fm and the photosynthesis quantum efficiency ΦPSII, during 96 hours of cultivation. Reported results show a difference between the toxic and the non-toxic strain, the artesunate concentration necessary to lower the cyanobacteria growth rate and the restoration of the quantum efficiency of cyanobacteria photosynthesis after the initial inhibition.
Comments:
Abstract: L15: ... artesunate, a more water soluble derivative of artemisinin, on ...
Response:Thanks for the reviewer’s suggestion. We have added “Artesunate is a water-soluble derivative of artemisinin” in line 16.
Keywords: please add artemisinin
Response: We have added "artemisinin" to the keywords.
L85: ... artesunate, a more water soluble derivative of artemisinin, was ...
Response: We adjusted the research purpose at the end of the introduction so that the content and significance of the research were clearer.
L98, L105: use consistently either exponential growth or logarithmic growth. Generally, please avoid using two different terms for the same attribute.
Response:Thank you very much for pointing out this error. This section has been modified to “the exponential growth phase”. Line 101.
L116: 96h (a typo)
Response:Thank you very much for pointing out this error. This section has been modified to “96h”. Line 117.
L130: how exactly was ΦPSII measured? Please describe or insert a reference.
Response:In this study, the handheld water chlorophyll fluorescence meter (AquaPen-C 100, The Czech Republic) was used for measurement. In addition to setting three replicates for each group, we also conducted multiple measurements for each sample in the experiment to avoid possible errors. And we have indicated the instrument model used in the paper for the convenience of others to check. Line 113-114.
Table 2/Heading: Reagent concentration units should be micromol/L as used in figures
Response: Considering the repetition between Materials and Methods and Table 2, Table 2 has been deleted from the paper. We have conducted a full text check and unified the concentration unit to "μmol/L".
Table 2: you measured also chlorophyll a and DO concentration. Could you please add a table with these measured values (and discuss them), or explain why they are not shown?
Response:Thank you very much for pointing out the mistake. The initial experimental design included measurements of chlorophyll a and dissolved oxygen, but for some reasons they were not carried out. The content mentioning chlorophyll a and dissolved oxygen in line 113 in the PDF file has been removed.
L154: you state that phycotoxin producing species (plural) are more resistant to artesunate than non-producing species (plural) but you have investigated only one specie of either type. Please add a reference to support this plural or reformulate this sentence to reflect reality.
Response: Similarly studies exist on other factors such as amoxicillin, sulfonamide antibiotics that affect Microcystis aeruginosa, such as stuies in Yisa et al,(2023), results shown amoxicillin was more toxic to the toxin-producing strains than the non-toxin-producing strains in M. aeruginosa. The study also have investigated only one specie of either type. Here, we added references to support this plural in line 159-162.
L160-163: add a reference or measured data after "under stress conditions", or reformulate (you say "obviously" so support it, please)
Response:Thank you for your suggestions. We have selected two articles as support, respectively discussing the impact of short-term waterlogging stress and irradiance levels on the Fv/Fm of plants and algae. In line 169.
L163: please replace "know" by "measure" or "ascertain" or a similar appropriate synonym.
Response:Thanks. "know" has been replaced by "measure". In line 191.
L164: please replace "function" by "values" (a kind of typo)
Response: Thanks."function" has been replaced by "values". In line 170.
L171-172: please reformulate to be more clear, e.g., "... to 0.424 in Strain 315 and 1.03 to 0.459 in Strain 927."
Response: The sentence has been reformulate to "The Fv/Fm significantly decreased (p<0.01) in 200 μmol/L after 96 h exposure from 0.654 to 0.424 in FACHB-315, 1.03 to 0.459 in FACHB-927, respectively." in line 181-182.
Figure 2: why mixed p<0.05 and p<0.01? Please explain.
Response: To better reflect the gradient difference in effect intensity, we simultaneously chose p<0.05 and p<0.01 in Figure 2.
L192-193: non-toxigenic Strain 315 and toxigenic Strain 927 - please state this difference earlier, e.g., in 2.1.
Response: We have revised the description of toxic and non-toxic strains in 2.1, line 88.
L209-210: at concentrations 100 and 200 (S315) and 200 (S927) it does not weaken, at least for 96h as shown in your data. Please reformulate (e.g., add "at lower artesunate concentrations" at the end of the sentence).
Response: We agree with this advice. In the revised manuscript, the sentence has been changed to “the φPS â…¡ of groups treated with artesunate is rapidly decreased after exposure to artesunate within 24 hours, and then gradually levels up with increasing exposure time from 24 h to 72 h.” in Line 237-243.
L226: ... artemisinin derivatives as artesunate ...
Response: We have changed “artemisinin derivatives” to “artesunate” in Line 261.
L234-235: allelopathic interaction (with lower case "a" and "i")
Response: We have changed to lower case "a" and "i" in Line 270.
L251: please reformulate slightly: "the appropriate addition of artesunate" or "the addition of appropriate artesunate concentration"
Response: We have added " the appropriate addition of artesunate" in line 291.
L252-253: the "Moreover ..." sentence says the opposite of what show the data in Fig. 1 where the lower artesunate concentration show the same effect for both species but the nontox-specie was already inhibited at 100 where the tox-specie was not. The inhibition depth seems to be about the same in both species.
Moreover, the inhibition effects on toxigenic species were stronger than those on non-toxigenic species with 72h.
Response: Thank you very much for pointing out this. In Figure 1a, when the concentrations of 100 and 200, the OD value of FACHB-315 gradually decreased over time within 96 hours. However, in Figure 2b, it only continued to decline at concentration 200, while the OD values at other concentrations all showed varying degrees of increase after 48 hours. Therefore, we believe that the inhibition effect on toxigenic strain FACHB-315 is stronger than non-toxigenic strain FACHB-927 under short-term artesunate stress. This section has been modified to “Moreover, the inhibition effect on toxigenic strain FACHB-315 is stronger than non-toxigenic strain FACHB-927 under short-term artesunate stress.” in line 293-296.
For me, one of the conclusions from the data in Fig. 1 would be (formulated very concisely): With the tox-specie, 2x higher artesunate concentration is necessary than with the nontox-specie to achieve the same OD suppression. The effect on lowering the quantum efficiency fades after 96 hours for both species.
Response: Thanks for your suggestions. We have also considered adding the above conclusions, but from Figure 1, we can also see that the inhibitory effect of artesunate varies over time and is not a constant value. Thus, increasing the concentration ratio of artesunate such as within 48 hours or 72 hours to achieve the same OD suppression has theoretical significance, but there are certain difficulties in application.
What seems important is the phycotoxin concentration produced by the algae. Could it not be measured as well?
Response: Algal toxins can be detected by various methods such as high-performance liquid chromatography (HPLC). While, this experiment focuses on the differences between toxigenic and non-toxigenic strain of M. aeruginosa under artesunate stress, and requires comparison of data with non-toxigenic strains. Therefore, toxins were not extracted and measured in toxigenic strain. In the future, we will pay attention to the measurement of algal toxins concentration in the research.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThank you very much for your careful revisions and for your efforts in addressing the reviewers’ comments.
Author Response
We sincerely appreciate your constructive suggestions on the manuscript and the language quality. In response, we have revised the manuscript and conducted a comprehensive language refinement. All modifications have been carefully implemented throughout the manuscript. We believe these revisions have substantially improved the overall linguistic quality while maintaining the integrity of our research findings.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors welcomed the referees' recommendations.
The document expands the description of the methodology and provides a more thorough analysis of the results.
Conclusions should be more limited to the results, without speculation, since this study cannot conclude its use as an algaecide; further studies would be needed. The algaecide function should remain a recommendation.
Author Response
Comments 2：The authors welcomed the referees' recommendations.
The document expands the description of the methodology and provides a more thorough analysis of the results.
Conclusions should be more limited to the results, without speculation, since this study cannot conclude its use as an algaecide; further studies would be needed. The algaecide function should remain a recommendation.
Response: Thanks for pointing out this error. We completely agree with you. The conclusions has been modified.
Reviewer 3 Report
Comments and Suggestions for AuthorsThe document has improved. However, statistical section of methods can be improved. Altough results clarify the techniques applied, it must be clearly specified how many ANOVA s were performed and clearly indicated the factor tested with its levels.
Comments on the Quality of English LanguageThe grammar and spelling are correct. English is not my native language, but I believe that the style can be improved.
Author Response
Comment 3：The document has improved. However, statistical section of methods can be improved. Altough results clarify the techniques applied, it must be clearly specified how many ANOVA s were performed and clearly indicated the factor tested with its levels.
Response:Thank you very much for your constructive suggestions. In this study, the toxigenic and non-toxigenic strains of Microcystis aeruginosathe were used to study the effect of artesunate on them. Therefore, one-way ANOVA is used in data analysis. The statistical analysis part has been modified in the manuscript.
Comments on the Quality of English Language
The grammar and spelling are correct. English is not my native language, but I believe that the style can be improved.
Response: We sincerely appreciate your valuable feedback on the manuscript's language quality. In response, we have conducted a comprehensive language refinement. All modifications have been carefully implemented throughout the manuscript. We believe these revisions have substantially improved the overall linguistic quality while maintaining the integrity of our research findings.
Reviewer 4 Report
Comments and Suggestions for AuthorsThe authors carefully considered the points:
The introduction has been logically reorganized (lines 30-43), and the significance of the study has been emphasized. The novelty of the study, namely the differential effects of artesunate on toxigenic and non-toxigenic Microcystis aeruginosa strains, has been clearly stated (lines 74-78).
Inconsistencies between μmol/L and mg/L have been corrected. A full text revision has been made, and all concentrations are now uniformly expressed as "μmol/L."
The study design and methods are technically sound. Although only surrogate parameters (OD680, Fv/Fm, φPSII) were measured during this proof-of-concept phase, the authors explicitly acknowledge this limitation and recommend follow-up studies such as toxin levels, ROS, cell morphology, and membrane damage.
The discussion has been revised to reflect limitations, emphasize the proof-of-concept nature of the study, and offer recommendations for future directions. The conclusion has been strengthened to highlight both the observed inhibitory effects and the need for further research before environmental application.
These revisions have significantly improved the article. The key issues raised have been addressed, and the article is now suitable for publication.
Author Response
Response: We sincerely appreciate your constructive suggestions on the manuscript and the language quality. In response, we have revised the manuscript and conducted a comprehensive language refinement. All modifications have been carefully implemented throughout the manuscript. We believe these revisions have substantially improved the overall linguistic quality while maintaining the integrity of our research findings.
