Effects of Temperature on Competition Between Toxic and Non-Toxic Raphidiopsis raciborskii and Cylindrospermopsin Production

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

see attached

Comments for author File: Comments.pdf

Author Response

Comments 1: Line 3. Species name has to be in italics.
Response 1: We have revised the title and the species name in line 3 to italicize “raciborskii” in accordance with standard scientific nomenclature.
Line 1-3: “Effects of temperature on competition between toxic and non-toxic Raphidiopsis raciborskii and cylindrospermopsin production”

Comments 2: Line 49. Add atmospheric N2 to the sources this organism can utilize.
Response 2: We have revised the sentence in lines 49–50 to include atmospheric N₂ as one of the nitrogen sources utilized by this organism.
Line 49-50: “This cyanobacteria is able to utilize diverse nitrogen sources, including ammonia, nitrate, urea, and atmospheric N₂, making it a nitrogen generalist”

Comments 3: Line 64/65. The “in contrast” is not a comparison to the previous sentence. Season dynamics is not equal to the cite work in the previous sentence.
Response 3: We agree with the reviewer’s comment. In the resubmitted manuscript, we have revised the sentence and replaced “in contrast” with “however” in line 69.
Line 69: “However, no such correlation has been observed in Lake Taihu (China) and…”

Comments 4: Line 69: English usage-“only several”, - this is not logical. Only implies a few…not several.
Response 4:  We agree with the reviewer’s comment. In the resubmitted manuscript, we have revised the sentence in line 74 to improve clarity and accuracy.
Line 74: “For R. raciborskii, only a few studies have examined the population dynamics of toxic and non-toxic genotypes”

Comments 5: Line 76: since it is the author’s own work, why be non-specific? ( “nutrient availability” ) when the cited study prior sentence says it was phosphorus. What were your findings in the prior work?Response 5: In accordance with the reviewer’s comment, we have revised the sentence in line 81 to clearly state that both nitrogen and phosphorus availability were involved.
Line 81: “Our recent study, combining field investigation with competition experiments, further demonstrated that nitrogen and phosphorus availability modulates the relative dominance of toxic versus non-toxic R. raciborskii strains and the CYN production”

Comments 6: Line 76: since it is the author’s own work, why be non-specific? ( “nutrient availability” ) when the cited study prior sentence says it was phosphorus. What were your findings in the prior work?Response 6: We agree that the phrase “environmental factors” was too general. To improve clarity, we have revised the sentence in lines 83–84 to specify key environmental variables of interest.
Line 83-84: “Despite these, how environmental factors such as nitrogen, phosphorus, and temperature regulate the relative dominance of toxic and non-toxic genotypes remains poorly understood”

Comments 7: Line 84: “As a tropical species”, - this is not a true statement given the locations it is found.
Response 7: We agree that R. raciborskii is no longer confined to tropical regions, although it originated there. To avoid misleading implications, we have revised the sentence in lines 90.
Line 90: “As a species of tropical origin, R. raciborskii is expected to benefit more from rising temperatures driven by climate change”

Comments 8: Line 87-90. It would be good to distinguish between what is happening in the population…. Selection for toxin producers, compared to what is happening to amount of CYN made per cell. It could lead to different concepts and understandings about the role of toxins in a cell.
Response 8: We fully agree that distinguishing between the selection for toxin-producing strains and changes in toxin production per cell is important, as they reflect different ecological processes. In our study, we focused on the population-level dynamics, particularly how rising temperatures affect the relative abundance of toxic genotypes and overall CYN concentrations. This focus is in line with the objective of our work, as indicated by the sentence “However, little is known about how its toxic and non-toxic genotypes respond to temperature changes.” Our aim was to explore how temperature influences the competitive interactions between genotypes and the resulting toxin levels at the population scale, which directly addresses the research question posed in the manuscript title.

Comments 9: 105: is microscopy sufficient to see microbial contamination?
Response 9: In accordance with many published studies [Rippka, R.; Deruelles, J.; Waterbury, J.B.; Herdman, M.; Stanier, R.Y. Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J. Gen. Microbiol. 1979, 111, 1–61] [Walther, J.; Schwarz, A.; Witthohn, M.; Strieth, D.; Muffler, K.; Ulber, R. A qPCR method for distinguishing biomass from non-axenic terrestrial cyanobacteria cultures in hetero- or mixotrophic cultivations. J. Appl. Phycol. 2020, 32, 3767–3774], regular light microscopic examination (400–1000×) is a widely used and accepted method for monitoring bacterial contamination in non-axenic cyanobacterial cultures. Therefore, we also used microscopic inspection to assess contamination in our experiments. In the resubmitted manuscript, we have added a reference as follow:
Line 115: “These R. raciborskii strains were non-axenic and regular microscopic inspection was used for confirming low bacterial contamination [40]”

Comments 10: 113: Stating 10⁵ cells per mL when this organism is a filament of cells, difficult to decern individual cells, make the reader wonder if you were truly able to do this accurately.
Response 10: We can decern individual cells of R. raciborskii filaments at 400× magnification, and some photos are showed as follow:

Comments 11: 119: Major research design flaw. In vivo fluorescence is a function of cell health and can not accurately be used to estimate biomass. The values are relative units and vary widely with cell health.
Response 11: Many previous studies have showed that chlorophyll-a (Chl-a) fluorescence can be used to assess phytoplankton growth under different environmental conditions. This approach is also widely adopted in cyanobacterial research as a practical, rapid, and non-destructive proxy for biomass estimation. Thus, we used the TD-700 laboratory fluorometer (Turner Designs, California, United States) to measure Chl-a concentration as a proxy for Raphidiopsis raciborskii biomass. [Hamdhani, H.; E.Eppehimer, D.; Walker, D.; T.Bogan, M. Performance of a Handheld Chlorophyll-a Fluorometer: Potential Use for Rapid Algae Monitoring. Water 2021, 13, 1409] [Bordignon, G.; Sexton, S.; Van Cise, A. Phytoplankton Studies in the South Shetland Islands. In 2010/11 Field Season Report, U.S. Antarctic Marine Living Resources Program; Chapter 2; NOAA-TM-NMFS-SWFSC-524, 2011] [Pérez, P.; Estévez-Blanco, P.; Beiras, R.; Fernández, E. Effect of copper on the photochemical efficiency, growth, and chlorophyll a biomass of natural phytoplankton assemblages. Environ. Toxicol. Chem. 2006, 25, 137–143].

Comments 12: 130: Two items of concern. Sedwick rafter cells typically cannot be used at 400x, rather 200x. Also, cells typically cannot be decerned at this power. Were the filaments preserved so they settled to the bottom to get an accurate count?
Response 12: The Sedgwick-Rafter chamber is commonly used at 200× magnification, but it is also applicable at 400× under suitable conditions. For example, Lee et al. (2013) [Lee, Y.C.; Jin, E.; Jung, S.W.; Kim, Y.M.; Chang, K.S.; Yang, J.W.; Kim, S.W.; Kim, Y.O.; Shin, H.-J. Utilizing the algicidal activity of aminoclay as a practical treatment for toxic red tides. Scientific Reports, 2013, 3, 1292] employed a Sedgwick-Rafter chamber with an optical microscope (Axioplan; Zeiss, Germany) at 400× magnification to accurately count phytoplankton cells. As we have showed above, individual cells at 400× magnification are clearly visible.
To ensure accurate enumeration in our study, the samples were allowed to settle before counting so that the filaments could be evenly distributed at the bottom of the chamber. In the resubmitted manuscript, we have added the corresponding description as follows:
Line 140-142: “Cell concentrations of four Raphidiopsis raciborskii strains were counted using a Sedgwick-Rafter chamber under an Olympus microscope at 400× magnification. Prior to counting, the samples were allowed to settle to ensure even distribution of filaments at the bottom of the chamber”

Comments 13: 132: Major research design flaw. It is highly irregular to fill a flask so full as to reduce surface area. Placing 400 mL in a 500 mL flask is stressful and reduces gas exchange to the narrow neck of the flask. A 500 mL flask should not have more than 250 mL of medium.
Response 13: We agree with your comment that, under standard cultivation practices, a 500 mL flask is typically filled with approximately 200–250 mL of medium to ensure sufficient surface area and gas exchange. However, under specific experimental conditions where larger sampling volumes are required, it is feasible to increase the culture volume appropriately while still maintaining adequate aeration through proper shaking. For instance, in the study by Adam et al. (2022) [Adam, B.S.; Abubakar, B.M.; Garba, L.; Hassan, I. Effect of growth media and pH on microalgal biomass of Chlorella vulgaris for biodiesel production. Bioremediat. Sci. Technol. Res. 2022, 10(1), 22–25.], 400 mL of medium was added to a 500 mL flask, and gentle shaking was used to ensure sufficient oxygen transfer. In the resubmitted manuscript, we have added the corresponding description as follows:
Line 144-145: “Toxic (CS506 or QDH7) and non-toxic (CS510 or N8) strains were inoculated at a cell ratio of 1:1 in 500 mL Pyrex Erlenmeyer flasks containing 400 mL of BG11 medium. The 400 mL volume was used to ensure sufficient sample availability. To maintain adequate gas exchange, the flasks were gently shaken twice daily. Each treatment was conducted in triplicate.”

Comments 14: 322: The logic that ample nutrients in the lab allowed for more toxin production is sound. But that ample nutrient condition is transient in the natural environment.
Response 14: We agree that nutrient-rich conditions used in laboratory experiments are often transient in natural environments. To clarify this point, we have revised the manuscript to explicitly acknowledge this limitation and highlight the ecological relevance of our findings under fluctuating nutrient conditions. In the resubmitted manuscript, we have added the corresponding description as follows:
Line 344–345: “It is worth noting that while ample nutrients in the laboratory may promote toxin-producing strains, such conditions are often transient in natural environments”

Comments 15: Why is no mention of the nitrogen-fixing capabilities of this organism mentioned? By providing the abundance of N in the experiment, the authors have seeming dismissed this potentially important aspect of the physiological changes. This may change their perspective in these kinds of studies.
Response 15: Thank you for your comment. We have added a discussion on nitrogen fixation in the revised manuscript, as shown in the newly added section (Lines 50–55):
Line 50-55: “Among these, nitrogen fixation allows R. raciborskii to convert atmospheric N₂ into bioavailable forms. This ability is an important mechanism enabling it to outcompete non-N₂-fixing species under nitrogen-limited conditions [8]. However, nitrogen fixation is a highly energy-consuming process [9], and many studies have shown that the nitrogen obtained through nitrogen fixation is often insufficient to support the development of cyanobacterial blooms [10,11]”

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The authors of this study expand our understanding of the toxic and non-toxic strains of Raphidiopsis raciborskii, and specifically examine how temperature affects the relative dominance of the toxic strains and the production of cylindrospermopsin (CYN). This is a well-designed and well-written manuscript that will provide valuable insights to researchers and practitioners dealing with toxic cyanobacterial blooms and potentially toxic cyanobacteria.

Title: Please put „raciborskii“ in italics
Line 106: What was the pH of the BG11 medium?
Line 189: Are the data tested for normality and homogeneity of variance before ANOVA?
Lines 228-291: This should be mentioned together with the objectives at the end of the introduction
Lines 298-300: Does the geographical origin possibly influence the preference of a strain for a certain optimal temperature?
Line 304: Please delete „etc.“. Indicate other strain-specific variations, or if there are none, simply remove „etc.“
Line 341: Can you name some of these substances?

Author Response

Comments 1: Title: Please put "raciborskii" in italics.
Response 1: We have revised the title and the species name in line 3 to italicize “raciborskii”.
Line 1-3: “Effects of temperature on competition between toxic and non-toxic Raphidiopsis raciborskii and cylindrospermopsin production”

Comments 2: Line 106: What was the pH of the BG11 medium?
Response 2: We have added the pH of the BG11 medium to the Methods section. The pH was adjusted to 7.4 before autoclaving. This information has been included in line 115 of the revised manuscript.
Line 115: “The strains were maintained in BG11 medium (pH 7.4)…”

Comments 3: Are the data tested for normality and homogeneity of variance before ANOVA?
Response 3: We appreciate your suggestion to clarify the statistical assumptions prior to ANOVA. We have added a description in lines 205–207 indicating that the data were tested for normality using the Shapiro–Wilk test and for homogeneity of variance using Levene’s test, with a significance level of P < 0.05 used to assess whether the assumptions were met.
Line 205-207: “…across three temperature conditions. Prior to ANOVA, the data were tested for normality using the Shapiro–Wilk test and homogeneity of variance using Levene’s test. A significance level of P < 0.05 was used to determine whether the assumptions were met. In the co-culture experiments”

Comments 4: Lines 228-291: This should be mentioned together with the objectives at the end of the introduction.
Response 4: We have added a statement at the end of the introduction (lines 103–106) to clarify the use of the RCD model and linear regression analysis as part of the study objectives.
Line 103-106: “To test this hypothesis, we conducted monoculture and co-culture experiments under different temperatures, using toxic and non-toxic R. raciborskii strains isolated from China and Australia respectively. We analyzed the influence of temperature on the dominance of CYN-producing strains by applying the rate of competitive displacement (RCD) model and performing linear regression analysis between cell growth rate (μ_C) and toxin production rate (μ_TOX). This study aims to provide new insights into how future climate warming may alter the toxicity of R. raciborskii blooms”

Comments 5: Lines 298-300: Does the geographical origin possibly influence the preference of a strain for a certain optimal temperature?
Response 5: We agree that geographical origin could play an important role in shaping the optimal temperature preference of different R. raciborskii strains. In response, we have added a clarifying sentence in lines 316–318 to explicitly address this point and enhance the discussion of our findings.
Line 316-318: “Specifically, the Chinese strains grew well at 16°C but the Australian strains didn’t. These findings imply that the optimal growth temperature of R. raciborskii may be shaped by the geographical origin of the strains. In terms of…”

Comments 6: Line 304: Please delete „etc.“. Indicate other strain-specific variations, or if there are none, simply remove „etc.“
Response 6: We have removed “etc.” from the sentence to avoid ambiguity. Instead, we clarified the strain-specific variations observed by specifying growth, toxin production, and morphology, in line with previous studies. The revision can be found in line 321-322.
Line 321-322: “Our results were consistent with previous studies showing R. raciborskii presented strain-specific variations in growth, toxin production, and morphology”

Comments 7: Line 341: Can you name some of these substances?
Response 7: Thank you for this important question. According to Figueredo et al. (2007) [Figueredo, C.C.; Giani, A.; Bird, D.F. Does allelopathy contribute to Cylindrospermopsis raciborskii (cyanobacteria) bloom occurrence and geographic expansion? Journal of Phycology, 2007, 43, 256-265], the authors did not specify which exact substances are produced by R. raciborskii. Their results showed that most target species were sensitive to C. raciborskii exudates, which strongly inhibited their photosynthesis. This suggests allelopathy helps C. raciborskii compete and maintain dominance in Lagoa Santa. Therefore, we did not specify the substances.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

 

 

 

 

 

 

 

thank you for clarifying the questions