Pyriproxyfen Contamination in Daphnia magna: Identifying Early Warning Biomarkers

Round 1

Reviewer 1 Report

This manuscript (jox-2787164) explored on the physiological responses of Daphnia magna individuals when exposed to chronic exposure to pyriproxyfen. The results showed that LDH activity increased significantly and cholesterol levels decreased significantly in those daphnids exposed to 14.02 μg/L pyriproxufen, while glucose, total proteins and triglycerides remained unaffected. The expression of genes linked to oxidative stress responses, lipid metabolism, haemoglobin, and vitellogenin was significantly affected in expose daphnids. This study provides valuable information for for assessing the well-being of aquatic invertebrates following pyriproxyfen spray operations. The manuscript is written generally. And the data is sufficient to support its main conclusion. It meets in the present form the criteria to be published in the journal. However, minor revision should be addressed prior to publication.

The manuscript needs careful editing by someone with expertise in technical English so that the goals of the results of this study are clear to a reader.

Line 35, which indicator could be used as the early biomarker?

Line 72-80, the reference 26 was not cited, please revise,

Line 80, delete “.” after “assessment”,

Line 119, there should be a space between “90” and “%”,

Line 119, in “CaCO₃”, “3” should be subscripted,

Line 120, “Nannochloris oculata” should be italic,

Line 120 and 143, in “105”, “5” should be superscripted,

Line 142, “N. oculata” should be italic,

Line 152, why choose to process the samples for 21 days?

Line 198 and 201, “Daphnia magna” should be italic, same as below,

Line 202, there should be a space between “<” and “0.05 (p)”,

Line 205-206, no data for these indicators (glucose, total proteins, and triglycerides) have been seen, please provide data,

Line 226, in figure 1, there was only figure 1A and figure 1B, is (C) figure 1C? please revise,

In figure 3, does the relative expression level of more than 3 times indicate a significant difference?

I don't know if it is my PDF version, figure 2 and figure 6 cannot be displayed,

The titles of figure 5 and figure 6 were the same, please revise,

Line 294-296, Why not test more GST gene expression levels? The expression level of only one GST gene could not indicate that the activity of GST was unchanged, let alone that it had no effect on Daphnia,

The manuscript needs careful editing by someone with expertise in technical English so that the goals of the results of this study are clear to a reader.

Author Response

Dear Reviewer, thank you very much for your time and comments. We have improved the manuscript following your instructions:

Line 35, which indicator could be used as the early biomarker? In our opinion Vitellogenin could be used as early biomarker. See conclusion chapter

Line 72-80, the reference 26 was not cited, please revise, OK

Line 80, delete “.” after “assessment”, OK

Line 119, there should be a space between “90” and “%”, OK

Line 119, in “CaCO₃”, “3” should be subscripted, OK

Line 120, “Nannochloris oculata” should be italic: We did in our original manuscript however something happened in your version and that was changed ……

Line 120 and 143, in “105”, “5” should be superscripted, OK

Line 142, “N. oculata” should be italic: We did in our original manuscript however something happened in your version and that was changed ……

Line 152, why choose to process the samples for 21 days?  This study was done together with another study in which a chronic test on the effect of pyriproxifen on Daphnia magna reproduction was evaluated during 21 days.

Line 198 and 201, “Daphnia magna” should be italic, same as below,: We did in our original manuscript however something happened in your version and that was changed ……

Line 202, there should be a space between “<” and “0.05 (p)”, OK

Line 205-206, no data for these indicators (glucose, total proteins, and triglycerides) have been seen, please provide data: They are already included (Figure 2).

Line 226, in figure 1, there was only figure 1A and figure 1B, is (C) figure 1C? please revise: Figure 1 included only 1(A) and 1(B). In that Figure C means control.

In figure 3, does the relative expression level of more than 3 times indicate a significant difference? : Results showed in the Figure depend on the statistical analysis.

I don't know if it is my PDF version, figure 2 and figure 6 cannot be displayed:  I don’t know why? But we inserted them again.

The titles of figure 5 and figure 6 were the same, please revise: OK

Line 294-296, Why not test more GST gene expression levels? The expression level of only one GST gene could not indicate that the activity of GST was unchanged, let alone that it had no effect on Daphnia: Yes, you are right, but it is a big study, we measured many parameters and probably if we continue with this subject we will measure more genes.

Author Response File: Author Response.pdf

Reviewer 2 Report

The authors of the manuscript (JoX-2787164) analyze the possible effects of 5 sublethal concentrations of pyriproxyfen for 21 days on certain biochemical parameters as well as gene expression of certain genes related to oxidative stress, reproduction, lipid metabolism, .......

Although the results obtained and the subsequent discussion have been efficiently presented, the present paper needs revision. For this reason, I consider that the manuscript is not suitable for publication in its present form.

Next, I have some suggestions and questions for authors:

1.    Although I am not an English language expert, the authors should make a thorough revision in this regard. Here are some suggestions

in line 15 "conducting research", change it to "researching".

in line 22 "over the course of 21 days" change it to "over or for".

in line 28 "in expose daphnids" change it to "exposed".

in line 195 (materials and methods) "Dunnett's multiple test", change it to "Dunnett's multiple tests"

in line 196 “among", change it to "between".

line 98 (introduction) "they do adverse effects to the" changes it for "they have adverse effects on the".

in line 201 (results and discussion) "figure 1 showed", change it to "figure 1 shows".

in line 323 (results and discussion) " was significantly and strongly inhibited in Dapnia magna", change it to " was strongly inhibited in Dapnia magna (p<0.05).

2.       If the authors must choose between the term "litre" (British English) or liter (US English), but never use both in the manuscript.

3.       Use superscripts in the formulas of organic compounds.

4.       The name of the algae used as food for Dapnia is misspelled: line 120 change "Nannochloris oculata" to "Nannochloropsis oculata" and in italics always.

5.       The species names should always appear in italics. This is not done in most of the main text, neither in the abstract nor in the figure legends.

6.       The concentration of Nannochloropsis oculata is 5 x 105 cells/mL or it is really 5 x 10⁵ cells/mL. PLEASE USE SUPERINDEXES IN NUMERICAL DATA

7.       Please specify the acronyms of BBM medium (Bold's Basal Medium). In this sense, it would be convenient to indicate the acronyms used for the genes analyzed both in the text and the legend of Table 1.

8.       The format of the figures is not maintained in all of them. Please unify the presentation format.

9.        Figures 2 and 6 are not visible, and the legend of Figure 6 does not correspond to its content.

10.   use ml or mL but not both forms of expression.

11.   Change the writing of the following paragraph (lines 294-297) to avoid redundancies, moreover, the genes as such are not involved in metabolic pathways, but rather the enzymes they encode:

12.   In section 2.3, the authors do not specify the number of replicates used for the control group as well as for the 5 insecticide groups to be tested. However, in section 2.4 of materials and methods, they indicate that they used a total of 6 individuals to measure biochemical parameters and gene expression (2 individuals/replica). Therefore, I assume that the authors have carried out 3 replications of each experiment. Please specify this in the text.

13.   Change rpm to "RCF (relative centrifugal force or G force") (line 161). Remember that the rpm depends on the maximum radius of the rotor used.

14.   The authors have used commercial kits to measure biochemical parameters and LDH activity. However, the kits used are designed to measure these parameters in mammals, specifically in humans and not an invertebrate (planktonic crustaceans). My question is: do the levels of glucose, cholesterol, and triglycerides capable of detecting these kits fall within the levels present in Daphnia?

Enzymes must be measured at saturation to their substrate and coenzyme to obtain their reaction Vmax. Does the LDH enzyme of this crustacean have a value of S0.5 similar to that of human LDH? If the value of the kinetic constant is not the same in both species, the measurement of its activity could be underestimating or even inhibiting it.

Therefore, the authors would be making a serious mistake, especially when they want to propose it as a biomarker of contamination by the insecticide pyriproxyfen.

15.   In the results, the authors indicate that exposure to the pyriproxyfen did not affect glucose, total proteins, and triglycerides compared to the control group. Suggesting that so the insecticide does not alter the energy reserves of Dapnia.

However, levels of glycogen, a molecule of reserve present in this species (Peters, 1987; Klumpen et al., 2021), have not been measured. Therefore, it will be necessary to rewrite the paragraph (lines 205-208).

Eva Klumpen, Nadine Hoffschröer, Andrea Schwalb, Ulrike Gigengack, Marita Koch, Rüdiger J. Paul, Bettina Zeis. Metabolic adjustments during starvation in Daphnia pulex. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology. Volume 255, 2021, 110591.

Peters, R.H. Metabolism in Daphnia. R.H. Peters, R. de Bernhardi (Eds.), Daphnia, Istituto italiano di idrobiologia, Verbania Pallanza (1987)

16.   On the other hand, in lines 218-220, the authors indicate a reduction in lipid reserves, that is, triacylglycerides. However, in lines 205-206, it is specified that TAG levels have not been altered by exposure to the insecticide (although the data obtained is not shown in the text or a figure). Does something not match? The authors have observed a significant drop in cholesterol levels, but this type of lipid cannot be classified as an energy reserve.

17.       The FabD gene encodes the protein that transports acyl groups during fatty acid synthesis (malonyl-CoA: ACP transacylase), so a drop in its gene expression would lead to an alteration in the storage of lipid reserves. The following sentence (lines 229-230) is not understood, considering the role of the FabD gene. Please consider rewriting it.

Author Response

Dear Reviewer, thank you very much for your time and comments. We have improved the manuscript following your instructions:

1. Although I am not an English language expert, the authors should make a thorough revision in this regard. Here are some suggestions

in line 15 "conducting research", change it to "researching". OK

in line 22 "over the course of 21 days" change it to "over or for". OK

in line 28 "in expose daphnids" change it to "exposed". OK

in line 195 (materials and methods) "Dunnett's multiple test", change it to "Dunnett's multiple tests" OK

in line 196 “among", change it to "between". OK

line 98 (introduction) "they do adverse effects to the" changes it for "they have adverse effects on the". OK

in line 201 (results and discussion) "figure 1 showed", change it to "figure 1 shows". OK

in line 323 (results and discussion) " was significantly and strongly inhibited in Dapnia magna", change it to " was strongly inhibited in Dapnia magna (p<0.05). OK

2. If the authors must choose between the term "litre" (British English) or liter (US English), but never use both in the manuscript. OK
3. Use superscripts in the formulas of organic compounds. OK
4. The name of the algae used as food for Dapnia is misspelled: line 120 change "Nannochloris oculata" to "Nannochloropsis oculata" and in italics always. OK
5. The species names should always appear in italics. This is not done in most of the main text, neither in the abstract nor in the figure legends: We did in our original manuscript however something happened in your version and that was changed ……
6. The concentration of Nannochloropsis oculatais 5 x 105 cells/mL or it is really 5 x 10⁵ cells/mL. PLEASE USE SUPERINDEXES IN NUMERICAL DATA: We did in our original manuscript however something happened in your version and that was changed ……
7. Please specify the acronyms of BBM medium (Bold's Basal Medium). In this sense, it would be convenient to indicate the acronyms used for the genes analyzed both in the text and the legend of Table 1. They have been included in the text.
8. The format of the figures is not maintained in all of them. Please unify the presentation format. OK
9. Figures 2 and 6 are not visible, and the legend of Figure 6 does not correspond to its content. OK
10. use ml or mL but not both forms of expression. OK
11. Change the writing of the following paragraph (lines 294-297) to avoid redundancies, moreover, the genes as such are not involved in metabolic pathways, but rather the enzymes they encode: OK
12. In section 2.3, the authors do not specify the number of replicates used for the control group as well as for the 5 insecticide groups to be tested. However, in section 2.4 of materials and methods, they indicate that they used a total of 6 individuals to measure biochemical parameters and gene expression (2 individuals/replica). Therefore, I assume that the authors have carried out 3 replications of each experiment. Please specify this in the text. OK
13. Change rpm to "RCF (relative centrifugal force or G force") (line 161). Remember that the rpm depends on the maximum radius of the rotor used. OK
14. The authors have used commercial kits to measure biochemical parameters and LDH activity. However, the kits used are designed to measure these parameters in mammals, specifically in humans and not an invertebrate (planktonic crustaceans). My question is: do the levels of glucose, cholesterol, and triglycerides capable of detecting these kits fall within the levels present in Daphnia? These type of kits even they are for mammals, they are used also for fish as well as invertebrates. We have many papers published on aquatic organisms exposed to pesticides and we measured these parameters without any problem. Other authors also used them.

Here you have two examples:

Beatriz Salesa, Encarnación Sancho, María Dolores Ferrando-Rodrigo, Javier Torres-Gavilá,(2022). The prochloraz chronic exposure to Daphnia magna derived in biochemical alterations of F0 generation daphnids and malformed F1 progeny, Chemosphere, Volume 307, Part 3,135848,

Beatriz Salesa, María D. Ferrando, María J. Villarroel & Encarna Sancho (2017) Effect of the lipid regulator Gemfibrozil in the Cladocera Daphnia magna at different temperatures, Journal of Environmental Science and Health, Part A, 52:3, 228-234

Enzymes must be measured at saturation to their substrate and coenzyme to obtain their reaction Vmax. Does the LDH enzyme of this crustacean have a value of S0.5 similar to that of human LDH? If the value of the kinetic constant is not the same in both species, the measurement of its activity could be underestimating or even inhibiting it.  Yes, it is.

15. In the results, the authors indicate that exposure to the pyriproxyfen did not affect glucose, total proteins, and triglycerides compared to the control group. Suggesting that so the insecticide does not alter the energy reserves of Dapnia. Not in the pyriproxifen concentrations tested.

However, levels of glycogen, a molecule of reserve present in this species (Peters, 1987; Klumpen et al., 2021), have not been measured. Therefore, it will be necessary to rewrite the paragraph (lines 205-208). When you studied the effect of a pesticide with fish you can measure glycogen from fish tissues for example muscle and also glucose levels from blood. However when you work with a very small invertebrate and you measure carbohydrates in the whole animal body (as in the present study) that included glycogen and glucose all together.

Eva Klumpen, Nadine Hoffschröer, Andrea Schwalb, Ulrike Gigengack, Marita Koch, Rüdiger J. Paul, Bettina Zeis. Metabolic adjustments during starvation in Daphnia pulex. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology. Volume 255, 2021, 110591.

Peters, R.H. Metabolism in Daphnia. R.H. Peters, R. de Bernhardi (Eds.), Daphnia, Istituto italiano di idrobiologia, Verbania Pallanza (1987)

16. On the other hand, in lines 218-220, the authors indicate a reduction in lipid reserves, that is, triacylglycerides. However, in lines 205-206, it is specified that TAG levels have not been altered by exposure to the insecticide (although the data obtained is not shown in the text or a figure). Does something not match? The authors have observed a significant drop in cholesterol levels, but this type of lipid cannot be classified as an energy reserve. You are right, the text has been corrected.

17. The FabD gene encodes the protein that transports acyl groups during fatty acid synthesis (malonyl-CoA: ACP transacylase), so a drop in its gene expression would lead to an alteration in the storage of lipid reserves. The following sentence (lines 229-230) is not understood, considering the role of the FabD gene. Please consider rewriting it. You are right. It has been corrected.

Author Response File: Author Response.pdf

Reviewer 3 Report

Line 147 - What is Chapter 2.1 clarify or make necessary changes.

Figure 2 & 6 are not clear, please redo the images.

Figure 1: Include the sample size (n=?)

Results: When mentioning the significant and not significant please include mean ± SEM value for easy understanding of the results and to increase the clarity.

Author Response

Dear Reviewer, thank you very much for your time and comments. We have improved the manuscript following your instructions:

Line 147 - What is Chapter 2.1 clarify or make necessary changes. OK

Figure 2 & 6 are not clear, please redo the images. OK

Figure 1: Include the sample size (n=?) The simple size is indicated in the text, in Material and Methods

Results: When mentioning the significant and not significant please include mean ± SEM value for easy understanding of the results and to increase the clarity. OK

Author Response File: Author Response.pdf