Nonlinear Responses and Population-Level Coupling of Growth and MC-LR Production in Microcystis aeruginosa Under Multifactorial Conditions

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article is devoted to a very interesting topic, namely, the identification of aspects of multifactorial impact on Microcystis aeruginosa, which leads to a change in the level of toxin synthesis in this cyanobacterium, causing harmful algal blooms in water bodies.

Undoubtedly, the merit of this work is the well-organized experimental study of the impact on growth and toxin synthesis in Microcystis aeruginosa.

Thus, the authors used a full-factorial design with three environmental variables—temperature (26, 30, and 86 36°C), irradiance (30, 50, and 70 μmol photons m⁻² s⁻¹), and N:P ratio (10, 100, and 150), resulting in 27 experimental conditions.

In addition, growth parameters (µ, lag phase duration, and maximum cell density), chlorophyll-a production, and MC-LR synthesis were modeled using Gompertz, linear, and dynamic approaches.

The reviewer has no questions about the design of this experimental work and the data obtained. However, there are some questions about the presented discussion of the problem under consideration.

In their discussion and analysis, the authors missed and did not consider one important reason that can lead to a change in the level of toxin synthesis (from virtually zero to very high) in Microcystis aeruginosa (as well as in other cyanobacteria). This factor is the allelopathic interactions of Microcystis aeruginosa with other plankton representatives, as well as macrophytes. Such literature is available. It is clear that the authors did not conduct joint cultivation of Microcystis aeruginosa with other organisms. However, at the discussion level, this aspect should be considered and included in the article.

This is all the more important to consider, since the authors came to the conclusion that, from an ecological point of view, the obtained results confirm the hypothesis that microcystins can act as adaptive molecules in suboptimal conditions, potentially increasing the survival or competitive advantage of Microcystis aeruginosa. At the same time, allelopathic interactions are characteristic of non-optimal conditions for the development of Microcystis aeruginosa, when other algae and cyanobacteria can create a competitive environment that limits the growth of the Microcystis aeruginosa population. It is under such conditions that toxin synthesis can increase as an adaptive response aimed at suppressing competitors. In this case, the synthesized toxins act as allelochemical molecules. In this regard, the conclusion made by the authors that microcystin production is not a passive consequence of cell proliferation but a regulated physiological trait with potentially adaptive value is very important.

There is an unfortunate carelessness in the design of the article. Thus, after Figure 2 there is Figure 5 and only then Figure 2.

The article can be published after making an appropriate addition to the discussion of the results of this work.

Author Response

Dear Reviewer,

First of all, we thank the reviewer for their thorough and encouraging evaluation of our manuscript. We are especially grateful for the positive remarks regarding the experimental design and the ecological relevance of our study.

Comment 1:

As suggested, we have incorporated a short paragraph in the Discussion section addressing potential allelopathic interactions. While our experimental design did not include co-cultivation, we agree that this ecological mechanism is relevant when interpreting the adaptive role of microcystins. To provide context, we have cited key literature that proposes the role of microcystins as allelochemicals in interspecific interactions.

The following paragraph has been added to the Discussion (in red), and the corresponding references have been included in the bibliography:

"Although this study was conducted under controlled laboratory conditions using a monoculture, it is important to consider that in natural environments, Microcystis aeruginosa interacts with a variety of organisms, including other phytoplankton and macrophytes, which may compete for resources. Such biotic interactions can trigger chemical signaling or resource competition that influences the regulation of microcystin production. Several studies have proposed that, under certain conditions, microcystins may act as allelochemicals—that is, compounds involved in interspecific chemical interference—modulating competitive dynamics within the community (Babica et al., 2006; Leão et al., 2009). Incorporating this ecological perspective reinforces the interpretation of microcystins as adaptive molecules whose expression may be shaped not only by abiotic stressors but also by complex ecological signals."

Comment 2:

After carefully reviewing our submitted draft, we confirmed that all figures were correctly numbered and positioned at the end of the manuscript, following the journal’s submission guidelines. The apparent misplacement of Figure 5 before Figure 2 in the peer-review PDF seems to be due to automatic formatting by the submission system. We would be happy to assist the editorial team in correcting this during the final layout stage, to ensure a coherent flow of figure citations.

We appreciate the reviewer’s constructive comments and believe that these improvements enhance the manuscript.

Sincerely,
The Authors

 

Reviewer 2 Report

Comments and Suggestions for Authors

This is a useful contribution to the knowledge of Microcystis ecology. One point that would assist in relating this work to the literature is to quote the actual concentrations of N and P in the cultures. Ratios are valid, but do not help to relate this work to the environmental conditions in lakes and rivers in which microcystis blooms.

Author Response

Dear Reviewer,

We thank you for your positive evaluation and insightful suggestion. We agree that reporting the absolute concentrations of nitrogen and phosphorus enhances the ecological relevance of our results. Accordingly, we have added this information to the Materials and Methods section. Specifically, we now indicate that the base medium contained 1.76 mM nitrate-N and 0.175 mM phosphate-P (corresponding to a molar N:P ratio of 10:1), and that the higher N:P ratios (100 and 150) were obtained by proportionally adjusting these concentrations. This addition will help readers better relate our experimental conditions to nutrient levels observed in natural ecosystems.

Kind regards,

The authors

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have made the required changes to the article. This work can be published.

Author Response

Dear Reviewer,

We sincerely thank the reviewer for the positive evaluation and support for publication.

Sincerely,
The Authors