Differential Effects of the Allelochemical Juglone on Growth of Harmful and Non-Target Freshwater Algae

Round 1

Reviewer 1 Report

A general observation is that the study contains interesting new information on the effects of the allelochemical juglone on the bloom-forming, harmful algae Microcystis aeruginosa and Stephanodiscus hantzschii and on several non-target algal species, including cyanobacteria, diatoms and green algae, in laboratory and field enclosure bioassays. Few parts of the manuscript however need some improvement in order to better present the information provided and to make more comprehensive and reader-friendly. Detailed comments follow:

1. Introduction:

- Page 2, lines 43-44: Please provide examples (namely) of physical and chemical methods that have been applied to control HABs, according to references 11-14, in order to facilitate understanding of the readers.

- Page 2, lines 44-45: Please provide some more details on the shortcomings of these methods.

- Page 2, lines 50-51: Please indicate some of the various allelochemicals that have been so far successfully applied.

3. Results:

- Pages 7-8, Table 1: Please fix the symbols in the same row – they are not in all cases at the same height which confuses the reader (e.g. the row for Merismopedium glaucum, the row for Microcystis aeruginosa, the row for Ankistrodesmus falcatus, the row for Scenedesmus quadricauda, etc).

4. Discussion

- Page 10, lines 221-223: Please provide (indicatively) some details on the allelochemicals used in these studies (references 19, 22, 39, 40).

Author Response

Authors are very grateful to the reviewer’s valuable comments and suggestions. Please check the attached file.

Author Response File: Author Response.pdf

Reviewer 2 Report

• Introduction – despite the Introduction briefly mentions the existence of previous studies on effects of juglone on the growth of algae, the authors avoided to disclose any details and simply claimed that the studies “have not examined its specific effects on a broad spectrum of algal species”. This is unacceptable. The outcomes of previous studies should be clearly provided and interpreted in the Introduction with respect to the study subject. The whole Introduction needs to be rewritten and focused on this specific topic instead of covering the field in superficial sentences that have little information value for the reader.
• 63 – “Pure juglone” – specify what means “pure” and how was it assessed
• 86 – “experiments were conducted in triplicate.” – this is not acceptable. You need to perform three separate experiments (biological replicates); each of them can be performed in triplicates, if needed.
• 89 – “Lake Ilgam” – coordinates needed.
• 96 – “after mixing its entire water column” – how did you mix the entire water column?
• 102 – “×200” should be “200×”
• 104 – “analyzed by ANOVA” – did you check for data normality and variance equality?
• 111 – “significantly inhibited” – the outcomes of statistical tests need to be reported with each such claim.
• 115 – “cellular growth of M. aeruginosa compared to that in the control, but the cell growth was sustained in a range very similar to the initial” – unclear claim, rephrase.
• The way how the cells were counted is undisclosed. It is not sufficient to state that you stained the cells before counting.
• 122 – “66.2% and 75.1%,” – uncertainty needs to be reported with all such values – you may use CI, SE or SD for this purpose…
• 2 – a large number of one-point increases is reported. Therefore, these data cannot be trusted and more concentrations need to be tested each of the claims. Some claims are suspicious because they were unexpected (although possible). To avoid any misinterpretations, additional concentrations (e.g., 0.03, 0.3 mg/L; the more, the better – I personally would prefer some 10 concentrations to be tested for each sub-figure) need to be tested and the data interpreted only if the results are consistent across multiple concentrations.
• 3 – different visualization needs to be used for figs B-D. Currently, the differences are poorly visible and the variability is not modelled at all.
• 4 – what is a “cell density”? I did not find any definition in the methods.
• 4 – replace stripes with some colors or shades of grey.
• Table 1 – provide real data (numbers). Instead of the “plus” symbols. Or convert the table to the heatmap.
• Table 2 – I do not see support for, e.g., Fragillaria crotonensis values. Should the graph 2B be considered the source of these evaluations? Where is the control? How comes that Chlorella was thriving in all three concentrations? If so, you would need to go down to the concentrations, which would turn to be ineffective. However, it seems to me like that these data are compared relative to the number of cells in the inoculum (that is why Chlorella was that high). However, then it would be impossible to claim where the growth was supported and where it simply followed the standard growth curve of the respective species.
• Recent references are lacking. There is only one paper cited from 2019 and none from 2018.
• pH of the water is undisclosed. Juglone is unstable in basic pH.
• Initial cell density is not indicated for most of the results presented in the manuscript.
• Juglone is poorly soluble in water. Therefore, I assume you used some organic solvent (DMSO, ethanol). However, it is not indicated that this vehicle was added to the control groups. Therefore, all the performed experiments are questioned despite small amounts of these compounds may not necessarily have any effects on the tested organisms. It is simply unacceptable to perform the experiments without proper controls.

Author Response

Authors are very grateful to the reviewer’s valuable comments and suggestions. Please check the attached file.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The manuscript was improved but many issues remain to be solved. Below please find comments that require further attention:

• The authors newly claim that they dissolved juglone in methanol and that 0.1% methanol (=final concentration) did not affect the growth of tested algae. However, the opposite results were reported by other authors, e.g., in https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5535920/ for the same methanol concentrations.
• Scenedesmus quadricauda lives mostly in coenobia. Some other tested algae also form colonies. Please provide more details on how you ensured that proper cell counts were inoculated.
• L. 77 – “were isolated from Lake Paldang” – more details are needed. Where can we found these strains in case the study needs to be replicated?
• L. 86 – “Test algae were stored” – you indicate storage conditions but like mean cultivation conditions. Provide information on both.
• L. 100 – “and then juglone was added in concentrations of 0.01, 0.1, and 1 mg L-1.” – this indicates that you did not pre-dilute methanol and added 100% methanol with juglone directly to the algae. That would not be a good option.
• L. 102 – “thoroughly mixed test flasks and fixed with Lugol’s solution” – more details need to be provided on cell counting. You only indicate the volume counted and that it was stained with Lugol.
• L. 117 – “Sedgewick-Rafter counting chamber” – more details need to be disclosed. How many fields did you count. What was the uniformity among the counted fields. Ideally disclose the raw data as a supplement.
• L. 131 – Report the P values in a standard way. Anything below 0.001 should be reported as <0.001 instead of =0.000
• L. 131 – it is unclear what groups were compared for, e.g., “S. hantzschii (F(3, 20) = 10.240, p = 0.000)” – different juglone concentrations applied to S. hantzschii? Where is the Df? More details need to be provided.
• P values should never be reported alone. Always provide more details on outcomes of the respective statistical test.
• L. 147 – “66.2% and 75.1% in average” – always report the uncertainty – you have chosen SE, so report SE with every such value
• Fig. 2, axis X – concentrations of what?
• Fig. 2 – I criticized the odd settings of concentration ranges – for example for C. vulgaris, even the lowest concentration had an enormous effect. More concentrations (lower) need to be tested.

Author Response

Authors are very grateful to the reviewer’s valuable comments and suggestions. All the comments were very constructive, and we agree with all the suggestions. We considered them all carefully and revised the manuscript according to the reviewer comments.
Please check the attached file.

Author Response File: Author Response.pdf