Impact of Dietary Potassium Nitrate on the Life Span of Drosophila melanogaster

Lines 53-55 should be listed in 2 places. In this way they can make the reading less fluid.

Thank you. Indeed, after splitting to separate lines makes it visually easier to comprehend. Corrected as advised.

Lines 78-79 a research hypothesis is exposed. Have you asked yourself any other research questions? if they should be exposed and listed in order to better interface with the subsequent paragraphs of the paper.

Dully noted and absolutely right – adding experiment expectations and questions eventually make much more sense to the reader – since they are echoed in the results. Please see updated paragraph: “We hypothesized that stimulating Drosophila via potassium nitrate supplementation could extend the lifespan of this species. Testing this hypothesis and observing food flies in these conditions would also give us more understanding of the safe dosing and evaluate any possible synergetic effect in relationship with other ingredients used in the medium from the point of view of oxidative stress. To test this hypothesis, we carried out a 39 days lasting experiment where fruit flies were supplemented with different potassium nitrate concentrate solutions added to their medium.”

Lines 122-129: In all the other methodological parts there is references, in the statistical part instead it is not present. Try to include some bibliography about it.

Corrected as advised.

Discussion and conclusion What limitations does this study have? It would be better to integrate the conclusions or make the Discussion and Conclusions paragraph unique.

Corrected. Merged into one. A “Limitations” paragraph was added in the end.

Only some references are up to date. I suggest to integrate the bibliographic part and as suggested before, I would add some references to the statistical part, which in this version of the paper I do not find

As advised – references to statistical part added. As well as, updated the list of literature.

We absolutely agree with you on the dating of references – but, as mentioned in the manuscript, not much of the research is being done in this field. The mere concentration leans towards “organic nitrates” in relationship to sports and performance and are mostly based on plant extracted and similar, but very little (besides the “health concerning” ones) are in regard to benefits from the point of view of salts.

Although the paper is well written, a moderate English language revision should be necessary. In fact, in my opinion, to facilitate the reader's understanding of the text it would be better to structure the sentences in the  passive form.

Language has been revised as advised.

However, in the introduction should be better explained the originality of this paper, that in this version, result a little lacking. To improve the originality of the paper, I suggest to the Authors to stress, in the introduction, the differences among this paper and the previous ones present in literature.

Thank you for this insightful note. As advised - the Introduction part has been expanded to include more information about the novelty of this research in relationship to other papers that could seem quite similar to ours from the first glance.  

The experimental design is well structured and adequate to the aim.

Nevertheless, to improve the reading by the readers, in this section, the number of tests performed in total and for each thesis could be reported in a table.

Thank you for your remark. We added additional explanatory sentence in order to make it more clear to future readers to understand the setting of this experiment.

The results is well presented and well discussed, also, in relation to the references reported.

However, this paragraph should be only dedicated to the data presentation

For this reason, in the results paragraph the sentence from line 131 to line 132 should be delete or shift in the aim of the paper.

Moreover, for the same reason, the sentence from line 132 to line 133 should be delete or shift in the Materials and methods paragraph

Thank you for your comment. Indeed, it makes much more sense to delete these lines - since the same information has been multiple times mentioned before. Hence, corrected as advised.

Furthermore, always in the paragraph of results, the text from line 155 to line 161 should be aligned to the margins

Thank you.  Corrected as advised.

Finally, the conclusions of the paper result adequate to the results showed and they satisfy the aim of the research.

Nevertheless, the section of the conclusions should not be limited only to reporting a summary of the data already reported, but should also include some personal comments of the Authors.

In this regard, I would suggest that the authors report their opinion on the impact that the results of their paper could have on the future research.

Yes indeed. Your remark coincides with opinion of other reviewers. For this reason, we extended the section finalizing it with personal point of view. Thank you very much for your suggestion. We also believe it makes the paper more “personal” and appeals to future readers.

- Figures need to be uploaded in higher resolution.

Corrected as advised.

- Conclusion should be more appealing and theoretical, explaining what this paper explore about further studies.

Thank you. Dully noted. As well as based on recommendation from another reviewer – Conclusion and Discussion sections has been merged and expanded including “Limitations” and also include some personal comments from the Authors and opinion on the impact that the results of their paper could have on the future research.

 Line 89 – Check the space before the bracket.

Done. Thank you.

Line 91 – It is not clear the number of groups. Did the authors mean the 5 experimental groups? Are 100 or 50 flies in each experimental group? The paragraph needs to be clearer.

Thank you. Updated as following: “Each tube contained 10 food flies and each group consisted of 10 vials, totalling 100 food flies in each (a total of 4 experimental + 1 control group) groups. All flies were randomly divided into one of 5 groups:”

Line 46 – Reference is required. E. g. Jones A. M. (2014). Dietary nitrate supplementation and exercise performance. Sports medicine (Auckland, N.Z.), 44 Suppl 1(Suppl 1), S35–S45. https://doi.org/10.1007/s40279-014-0149-y

Thank you for your suggestion. Included as reference Nr. 7 [7]

Line 51 – Check the punctuation

Corrected

Table 1 – It is not clear to this reviewer the “max” survival days. Please define this value. Do the authors mean the day when the last fly die? If so, how is possible that the median value is higher than max value? Moreover, how is it possible that the lifespan of your control flies is too short? In Chhabra et al. (2013) a similar longevity was found only with subjects fed soybean diet and “Due to the extremely short life spans of flies raised on soy diets, flies raised on soy diets were excluded from all subsequent assays”. In my opinion, this data should be more closely reasoned.

Thank you for your comments. As advised, we made the following changes:

-           “Max” survival days – defined;

The median value presents the average days of survival by the whole group, whereas the “max” value presents the longest living fly (one) within the group.

Indeed, a very good point, thank you. We also questioned this phenomenon and included the following: “It is also essential to note that the medium used in our experiment was rather sweet and high in carbohydrates (due to raisins as one of the main ingredients). Sweet medium has also shown to result in shorter life of fruit flies [30, 31]” However, we are not sure how to interpret this result at this stage (carbohydrate overdose, oxidative stress, contaminated products, etc.)? Hence more analysis is advised - based on that we believe that this data should be included and presented “as is”, since as you also noticed it brings up attention to the sweet medium/raisin point of view.

Figure 2 – This figure needs to be improved or splitted. The bar between “control” and “1%” is not centered. Moreover, I do not think that the red arrows are necessary, the authors could replace the arrows with bars under the X axis caption (7 – 14 – 21 Days). Finally, just to be sure, is the locomotion of the control group on day 7 significantly higher than the locomotion on day 21? If all 5 groups are significantly different, the authors should draw only one bar “between” day 7 and day 21.

Thank you. The figure quality has been improved as follows:

-          The bars cantered

-          Red arrows removed; bar added under X axis.

As for the clarification – yes, the flies were more active in the beginning of experiment (first week) and rather vague during last days.