The Resistance of Drosophila melanogaster to Oxidative, Genotoxic, Proteotoxic, Osmotic Stress, Infection, and Starvation Depends on Age According to the Stress Factor

Round 1

Reviewer 1 Report

General comments

The Authors studied the resistance of D. melanogaster to oxidative and genotoxic (separately paraquat, Fe3+, Cu2+, and Zn2+ ions), proteotoxic (hyperthermia, Cd2+ ions) and osmotic (NaCl) stresses, starvation and infection with the pathological Beauveria bassiana fungus at different ages.

The analysis of the effect of each stress factors alone showed that the most powerful impacts are cadmium ions (5 mM), starvation and hyperthermia, and the least dangerous - infection by B. bassiana, as well as the impact of ferric (10 mM) and zinc ions (5 mM).

Interaction of factors "stress" and "age" was further analysed; the analysis showed that for both sexes age is an important risk factor that worsens the prognosis of survival time in all studied stress conditions (Fig. 3). The most significant decrease in stress resistance with age occurred under the oxidative and genotoxic stress induced by paraquat (20 mM), and at a lesser extent under the osmotic stress.

Overall, the paper is clear and well written, in particular the elaborated mathematical model is of interest. However, in my opinion, the strength and relevance of the study should be improved, and new analyses should be performed.

Specific Comments

The study allowed the Authors to estimate changes of stress resistance with age to a very wide range of factors, with the covering various metabolic pathways that regulate stress responses. Although certain processes during aging can be specific to insects and flies, their investigation by using D. melanogaster provides an understanding of aging mechanisms common to all living organisms. However, I think it would be important to test the power of this mathematical model on a relevant biological system different from Drosophila such as primary cultures cells from human donors of different ages.

Minor comments

• the readability of the article would benefit from shortening the discussion

Author Response

Comments and Suggestions for Authors

General comments

The Authors studied the resistance of D. melanogaster to oxidative and genotoxic (separately paraquat, Fe3+, Cu2+, and Zn2+ ions), proteotoxic (hyperthermia, Cd2+ ions) and osmotic (NaCl) stresses, starvation and infection with the pathological Beauveria bassiana fungus at different ages.

The analysis of the effect of each stress factors alone showed that the most powerful impacts are cadmium ions (5 mM), starvation and hyperthermia, and the least dangerous - infection by B. bassiana, as well as the impact of ferric (10 mM) and zinc ions (5 mM).

Interaction of factors "stress" and "age" was further analysed; the analysis showed that for both sexes age is an important risk factor that worsens the prognosis of survival time in all studied stress conditions (Fig. 3). The most significant decrease in stress resistance with age occurred under the oxidative and genotoxic stress induced by paraquat (20 mM), and at a lesser extent under the osmotic stress.

Overall, the paper is clear and well written, in particular the elaborated mathematical model is of interest. However, in my opinion, the strength and relevance of the study should be improved, and new analyses should be performed.

Specific Comments

The study allowed the Authors to estimate changes of stress resistance with age to a very wide range of factors, with the covering various metabolic pathways that regulate stress responses. Although certain processes during aging can be specific to insects and flies, their investigation by using D. melanogaster provides an understanding of aging mechanisms common to all living organisms. However, I think it would be important to test the power of this mathematical model on a relevant biological system different from Drosophila such as primary cultures cells from human donors of different ages.

Thank you for this comment. We discussed this.

It would be important to test the advantages of the described mathematical model on a relevant biological system different from Drosophila. For example, taking into account that the replicative lifespan of human fibroblasts in culture correlates with donor age {Kaji, 2009 #144}, we may expect that cell stress resistance estimated by survival will also predict the biological age of humans. However, further studies are required to test this suggestion.

Minor comments

• the readability of the article would benefit from shortening the discussion

We have taken into account this comment and reduced the discussion section.

Reviewer 2 Report

In the manuscript “The resistance of Drosophila Melanogaster to Oxidative, Genotoxic, Proteotoxic, Osmotic Stress, Infection and Starvation Depends on Age According to the Stress Factor” by Alexei et al., the authors studied how aging affects the ability to tolerate stress factors in D. melanogaster. Since aging and stress tolerance are negative correlated, the authors propose to use the stress tolerance parameter to predict the biological age of flies.

The main results are:

1) a reduction in stress resistence with age for all the stressors analyzed

2) females have a higher resistance to stress than males

3) significant differences in response to different stress relative to chronological age

4) creation of an algorithm for the assessment of biological age using a two-parameter model of survival curve.

The first two results are not new but the numerical data is used to create the algotithm for assessment of biological age. The proposed method for predicting a priori unknown age of the flies is new and it might have some practical use for experimental purposes with Drosophila. For example, it could be useful for predicting the life expectancy of flies subjected to a stress. The authors analyze several biotic and abiotic stressors, but not psychological stress, such as sleep deprivation and social isolation. Since it might be interesting to see if this type of stress induces tha same changes in flies with age, the authors could at least explain the reasons for this lack.

Furthermore, I struggle to understand how this method could be used for other stressors in Drosophila and/or other organisms. The authors expect their method to also be used in studying the survival of primary of human cell cultures. They should provide a brief explanation on how this method can be exploited for human cells and other organisms.

In general, the authors should better explain the objectives of their work. The too long discussion, in some points difficult to read, makes you lose the main meaning.

Author Response

Comments and Suggestions for Authors

In the manuscript “The resistance of Drosophila Melanogaster to Oxidative, Genotoxic, Proteotoxic, Osmotic Stress, Infection and Starvation Depends on Age According to the Stress Factor” by Alexei et al., the authors studied how aging affects the ability to tolerate stress factors in D. melanogaster. Since aging and stress tolerance are negative correlated, the authors propose to use the stress tolerance parameter to predict the biological age of flies.

The main results are:

1) a reduction in stress resistence with age for all the stressors analyzed

2) females have a higher resistance to stress than males

3) significant differences in response to different stress relative to chronological age

4) creation of an algorithm for the assessment of biological age using a two-parameter model of survival curve.

The first two results are not new but the numerical data is used to create the algotithm for assessment of biological age. The proposed method for predicting a priori unknown age of the flies is new and it might have some practical use for experimental purposes with Drosophila. For example, it could be useful for predicting the life expectancy of flies subjected to a stress. The authors analyze several biotic and abiotic stressors, but not psychological stress, such as sleep deprivation and social isolation. Since it might be interesting to see if this type of stress induces tha same changes in flies with age, the authors could at least explain the reasons for this lack.

Unfortunately, the method proposed by us is limited to the use of rather acute stress factors. We need the intensity of mortality to be high enough to be sure that the stress factor was the main cause of individual lethality in the population. If the stress factor is more moderate, the time of death increases significantly and other random factors start to influence. A variety of psychological factors will thus only show a decrease in longevity, which may be only slightly different from the natural mortality of the population.

We added the following explanation to the ms text:

In this study, we did not analyze age-related changes in tolerance to psychological stresses, such as sleep deprivation and social isolation. This lack is due to weakly pronounced effects of these stresses on survival and lifespan {Geissmann, 2019 #142}{Leech, 2017 #143}.

Furthermore, I struggle to understand how this method could be used for other stressors in Drosophila and/or other organisms. The authors expect their method to also be used in studying the survival of primary of human cell cultures. They should provide a brief explanation on how this method can be exploited for human cells and other organisms.

Many stresses share a common signaling pathway and biological effects on cells and the whole organism. In many cases, this common stress response will result in oxidative stress. Such mechanisms are often conservative and common to different groups of organisms.

In general, the authors should better explain the objectives of their work. The too-long discussion, in some points difficult to read, makes you lose the main meaning.

Studies of stress effects are quite common and well-established for various model organisms and cell cultures. In order to apply our approach, in this case, it would require the preliminary generation of a model for populations with known chronological age. In our study, we did not aim to test this approach for other organisms but only experimentally confirmed the possibility of its use for flies.

Reviewer 3 Report

The authors presented an interesting approach to analyzing the lifespan of the fruit fly. While reading the manuscript, I tried to find a connection between the Antioxidants journal and the content of this work. Unfortunately, I failed. Therefore, unfortunately, I cannot accept this publication in this journal. However, I do recommend Insects.
If the Authors decide to submit to another journal, they should supplement the content with some information:
1. The origin of fruit flies. Were they checked for homozygosity and how long they were kept at the same temperature?
2. Has the appropriate humidity been maintained, if so, please write down the value.
3. What was used to anesthesia.
4. There is no information on the composition of the medium in the materials and methods section.
5. Is the temperature of 35oC too high for a fruit fly? The stress conditions are obtained at 30 ° C. Therefore, the authors should discuss the context of this temperature more closely, for example based on earlier work by Shaposhnikov, M. et al. Lifespan and Stress Resistance in Drosophila with
Overexpressed DNA Repair Genes. Sci. Rep. 5, 15299; doi: 10.1038 / srep15299 (2015). There is also no precise information on how the flies were prepared for this experiment. They should be raised from parents who lived at this temperature.
6. An additional attribute of this study could be the assessment of reproductive success expressed in the number of eggs laid, the number of hatched eggs and the resulting imago in selected analyzed stress factors

Author Response

Comments and Suggestions for Authors

The authors presented an interesting approach to analyzing the lifespan of the fruit fly. While reading the manuscript, I tried to find a connection between the Antioxidants journal and the content of this work. Unfortunately, I failed. Therefore, unfortunately, I cannot accept this publication in this journal. However, I do recommend Insects.
If the Authors decide to submit to another journal, they should supplement the content with some information:
1. The origin of fruit flies. Were they checked for homozygosity and how long they were kept at the same temperature?
2. Has the appropriate humidity been maintained, if so, please write down the value.
3. What was used to anesthesia.
4. There is no information on the composition of the medium in the materials and methods section.

To take into account all these comments we expanded the Materials and methods section.

2.1. Drosophila rearing

The wild type D. melanogaster strain Canton-S (#64349, Bloomington Drosophila Stock Center, USA) was maintained for more than two years by mass transfer at  25 °C, 60% relative humidity and 12-hour lighting regime on food medium containing 1000 mL water, 7 g agar,  8 g dry yeast, 30 g sugar, 30 g semolina, and 3 mL propionic acid. Experimental animals were kept in a climate chamber Panasonic MIR-554-PE on the same food medium. To reduce condensation, the chamber was running in low humidity mode (relative humidity varied between 60-50%).

To avoid overpopulation in the experimental groups, 10 pairs of males and females per vial were used for 24 hours of egg-laying. After the imago hatching, we collected flies from the vials using diethyl ether anesthesia and separated them by the sex. For each experiment, the flies were kept in 45 ml vials with 5 ml food medium.

5. Is the temperature of 35oC too high for a fruit fly? The stress conditions are obtained at 30 ° C. Therefore, the authors should discuss the context of this temperature more closely, for example based on earlier work by Shaposhnikov, M. et al. Lifespan and Stress Resistance in Drosophila with
   Overexpressed DNA Repair Genes. Sci. Rep. 5, 15299; doi: 10.1038 / srep15299 (2015). There is also no precise information on how the flies were prepared for this experiment. They should be raised from parents who lived at this temperature.

We have taken into account this comment and discussed the hyperthermia treatment more closely.

Hyperthermia treatment was performed using a temperature-controlled chamber. Dead flies were counted at room temperature. Our previous experiments demonstrated that at 35 °C flies can survive for 24-72 hours (depending on the genotype) {Shaposhnikov, 2015 #141}, which corresponds to our requirements in this study.

However, we did not select parents for heat resistance.

6. An additional attribute of this study could be the assessment of reproductive success expressed in the number of eggs laid, the number of hatched eggs and the resulting imago in selected analyzed stress factors

Thank you for this comment. The effects of studied stress factors on age-related changes in reproductive potential may be the topic of future study.

Round 2

Reviewer 2 Report

The manuscript “The resistance of Drosophila Melanogaster to Oxidative, Genotoxic, Proteotoxic, Osmotic Stress, Infection and Starvation Depends on Age According to the Stress Factor” by Alexei et al. has been revised according to the reviewers' commentsand the responses appear satisfactory. Therefore, I agree with its publication in Antioxidants journal

Reviewer 3 Report

I have no more objections