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PONE-D-22-09651Hormetic and Transgenerational Effects in Spotted-wing Drosophila (Diptera: Drosophilidae) in Response to Three Commonly-Used InsecticidesPLOS ONE

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Reviewers' comments:

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Comments to the Author

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Reviewer #1: Yes

Reviewer #2: Partly

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #1: General comments

This is a good paper. Good hypotheses, good methods, good interpretation, although things get unwieldy in places (too much into the weeds on minor results that distract from key findings). I have offered some minor edits and a few papers that may or may not be of interest related to the topics discussed.

Specific comments

1. L80. It is usually advisable to avoid the word “beneficial” and instead use the word “stimulatory” when referring to hormetic effects. Beneficial effects of one endpoint measure can only be assessed in the context of other effects over the long-term, which are rarely measured in published paper. The word “stimulatory” will be more precise and accurate in most instances.

2. L95-99. Yes, under-explored but potential impacts in agriculture are becoming more appreciated, e.g. Sci Total Environ, 2022, 825, 153899

3. L100-112. Links of insecticide induced hormesis with transgenerational effects, epigenetics, resistance, and enzyme induction have been studied, e.g. papers on hormesis by Rix, Ayyanath, Cutler

4. L162-164. Modify. You are not calculating exact concentrations. You are using the data to generate a linear model that estimates LC values, some of which you hypothesize will induce stress responses reflective of hormesis

5. L170. You are exposing insects to prescribed concentrations, not doses

6. L171. Suggest using different terminology. Acute vs chronic is not differentiated one exposure vs two exposures, but rather short vs long-term exposure. Just call this what it is, being more precise and accurate: one exposure vs two exposures.

7. L171. Why was the two-time exposure not done with zeta-cypermethrin?

8. L176. Clarify. In the one-time exposure, flies were exposed for 4 h, and thereafter unexposed to insecticide for the remainder of their lives. In two-time exposure, flies were immediately exposed for 4 h, then unexposed for 116 h following, and then exposed for another 4 hours – correct? Were the “new vials” untreated with insecticide.

9. L183. Parental (P) generation adult flies?

10. L186. New untreated vials?

11. L191-192. Why do you say approximate LC25 or LC50 DD concentration. Had you not previously determined ‘exact’ estimates of LC25 and LC50 values?

12. L193. … pyrethrin).

13. L219. More complete results of the probit analyses should be presented: Chi-square, confidence intervals, slopes, df, etc. These data may be informative in interpreting hormesis results, e.g. lower slope is indicative of a more heterogeneous response to the insecticide, which in turn might correspond to a more pronounced hormetic response in the population, particularly across generations where interactive tolerance/resistance factors might be more likely to come into play. Even as is table 1 lack proper headings and is presented bass-ackwards. (‘Insecticide’ should be the left-most column, thereafter with sequential presentation of lethal concentrations)

14. L243. The very low control survival in spinetoram treatments relative to the other insecticides is striking. So low that I wonder if these data should even be included. I assume this is discussed later in the Discussion.

15. L311. Section 3.1 of this paper talks about some the experimental considerations for detecting hormesis. Perhaps more important in the number of doses/concentrations is replication within treatments to reduce type 2 error: Calabrese EJ. 2005. Paradigm lost, paradigm found…. Environ Poll 138, 378–411

16. L351-353. Seems a more likely and parsimonious explanation is that something unknown and external to the fly population, in and of itself, affected the fly population. It is doubtful genetic variability in a lab colony is so great to account for this. I bet if you did the experiment again today you’d get very different (better) survival for spinetoram treatments.

17. L391. Yes. I suspect hormetic responses, though subtle in many (not all) cases, are ubiquitous in the field and are simply not realized due to lack of study.

18. L412. These papers may be of interest as it relates to hormesis, tolerance to insecticides, resistance, etc. Pest management science 74 (2), 314-322; Journal of pest science 89 (2), 581-589; Science of The Total Environment 827, 154085

Reviewer #2: Hormetic and Transgenerational Effects in Spotted-wing Drosophila (Diptera: Drosophilidae) in Response to Three Commonly-Used Insecticides

Dear Authors:

I am glad to see more researchers examining hormesis in the context of agriculture. I have provided some specific comments to assist in editing the manuscript. By way of summary, there are areas of the introduction, methods, results, and discussion that need to be amended. The authors spend time in the introduction discussing epigenetic mechanisms of hormesis, however this is not an aspect of their paper, therefore this should be edited to be proportionate with their work and proposed potential future work. There are a number of areas in methods where not enough detail is included, and I have provided specific comments on these areas. There are a number of errors in describing the results. The results from the probit analysis are missing. This should be included. There is some confusion with mortality and survival, which confounds the findings. In addition, the figures need to be edited for consistency, and colour scheme changed. The discussion is heavily lacking in comparison and discussion of other works examining insecticide induced hormesis in insects. This is surprising as this field has grown considerably in the last 5-10 years. I have provided the authors with a number of examples of papers in this field that may be pertinent to their work, which also have works cited in this field, and I highly suggest the authors search the literature for more work on insecticide induced hormesis. Despite this, I think the authors are on solid footing with their work, and following edits, this paper should be ready for publication.

Review Comments

The abstract is well constructed and clear. I like that the authors began by discussing factors that can alter the effective exposure of pests to insecticides. This is key to understanding the legitimate implications of hormesis in agricultural systems.

Keywords

I would suggest the authors replace epigenetics with transgenerational effects, where epigenetics is often more associated with the examination of heritable effects on the genome not involving changes in nucleotide sequences, such as gene expression, gene methylation, histone modification etc, rather than strictly phenotypic changes. This could be misleading as to what was specifically studied, if interpreted as studying effects on the molecular level.

Introduction

Line 57: I think here it would be better to start this as follows:

Spotted-wing Drosophila (Drosophila suzukii Matsumura), an invasive species native to Asia, has become a serious pest in fruit-producing areas throughout the United States and Europe, following its establishment in the early 2000s.

Line 61: What is the purpose of saying “certain climates”? It is a bit too vague to provide pertinent information. Be a bit more specific, or do not include at all.

Line 63: You might want to include some specifics on regions and specific crops where D. suzukii outbreaks have been major issues.

Line 71:

“This can occur by impeding contact between the insecticide and spotted-wing Drosophila adults, especially considering that spotted-wing Drosophila are small, highly mobile, and crepuscular insects that have been documented to exhibit daily movement patterns in 74 several crops.”

Be careful of long or run-on sentences. The writing of this manuscript is very good, but when sentences are too long it confounds their meaning. I would suggest reading back through the manuscript sentence by sentence, and look for areas where you can break up your sentences, or adjust your sentences.

Line 84: “Hormetic and transgenerational effects can not only increase plant damage through

direct reductions in insecticide efficacy, they may also promote the evolution of

insecticide resistance through their impacts on phenotypic diversity”

Be careful of wording here. Any increases in plant damage that would be associated directly with hormesis would be due to stimulatory effects induced on the insect, such as stimulation in fecundity/fertility (thus increased feeding on the plant), stimulation of longevity (thus longer lifetime of feeding), stimulation of feeding (thus more feeding), etc. The reduced efficacy of the insecticide is presumably already in existence if the insect is undergoing hormesis as hormesis occurs in concentration ranges generally below the NOAEL. Hormesis and transgenerational effects in this context are a function of reduced insecticide efficacy, hormesis does not act through reducing insecticide efficacy, which is how your statement is worded.

With regard to information on hormesis and pesticide resistance and impacts on phenotypic diversity or plasticity, see works by Costantini. Some examples:

Costantini D. Hormesis Promotes Evolutionary Change. Dose Response 2019; 17: 1559325819843376.

Costantini D. Does hormesis foster organism resistance to extreme events? Frontiers in Ecology and the Environment 2014; 12: 209-210.

Lines 96: “It is particularly surprising that the effects of hormesis have not been more thoroughly addressed in agricultural pest management, considering that there are a multitude of factors in agricultural settings that may reduce effective exposure and there are significant costs associated with insecticide failures due to increases in insect tolerance or genetic resistance.”

This has been examined and discussed by authors such as Cutler, Guedes, Rix, Ullah, amongst some others. Transgenerational effects are also addressed in these. For some examples see:

Cutler, G.C., Amichot, M., Benelli, G., Guedes, R.N.C., Qu, Y., Rix, R.R., Ullah, F., Desneux, N. (2022). Hormesis and insects: Effects and interactions in agroecosystems. Science of the Total Environment. doi: 10.1016/j.scitotenv.2022.153899

Ullah F, Gul HN, Desneux N, Gao XW, Song DL. Imidacloprid-induced hormesis effects on demographic traits of the melon aphid, Aphis gossypii. Entomologia Generalis 2019; 39: 325-337.

Ullah F, Gul H, Tariq K, Desneux N, Gao XW, Song DL. Thiamethoxam induces transgenerational hormesis effects and alteration of genes expression in Aphis gossypii. Pesticide Biochemistry and Physiology 2020; 165: 11.

Rix, R.R., Cutler, G.C. (2018). Does multigenerational exposure to hormetic concentrations of imidacloprid precondition aphids for increased insecticide tolerance? Pest Management Science. 74: 314-322.

Cutler GC, Guedes RNC. Occurrence and Significance of Insecticide-Induced Hormesis in Insects. Pesticide Dose: Effects on the Environment and Target and Non-Target Organisms. 1249. American Chemical Society, 2017, pp. 101-119.

Rix, R.R., Ayyanath, M.M., Cutler, G.C. (2016). Sublethal concentrations of imidacloprid increase reproduction, alter expression of detoxification genes, and prime Myzus persicae for subsequent stress. Journal of Pest Science. 89: 581-589.

Guedes RN, Cutler GC. Insecticide-induced hormesis and arthropod pest management. Pest Manag Sci 2014; 70: 690-7.

Ayyanath MM, Cutler GC, Scott-Dupree CD, Sibley PK. Transgenerational shifts in reproduction hormesis in green peach aphid exposed to low concentrations of imidacloprid. PLoS One 2013; 8: e74532

Cutler GC. Insects, insecticides and hormesis: evidence and considerations for study. Dose Response 2013; 11: 154-77.

Line 101: Transgenerational effects can also refer to hormetic stimulatory effects (such as increased fecundity) occurring across generations, not just increased tolerance in offspring. The papers cited above also have examples of this.

Lines 100-122: These paragraphs include some excellent information on potential epigenetic mechanisms of hormesis. As mentioned above regarding the abstract, the authors are not examining epigentic mehcnaisms, thus the information might be more suited to a small area of the discussion or conclusion as something that might need to be focused on in the future. Mentioning it at the outset of the paper and in a large section is misleading the reader into thinking it is part of your experimental focus. Your focus should be on the phenotypic effects observed from other studies. Given the fact the authors have missed a number of key studies/authors in this field, I would suggest perhaps searching through the agricultural hormesis literature further and focus there, rather than brining in the extraneous epigenetic work.

Methods

Fly Culture

Can the authors provide more details on the fly rearing? Were they kept in containers? Approximately how many flies per container? What are the specific details of the diet? Etc. My suggestion would be to describe it in as much detail as you would if you were giving someone an SOP (Standard Operating Procedure).

Insecticide Exposure

Make sure you include any necessary trademarks and company/manufacturer names and locations when using the brand names of the insecticides. Examples for how this may look can be found in the publications suggested in previous comments.

Dose-response assays

Can the authors list the concentrations used for each insecticide?

Hormesis protocol

The authors refer to their two-time exposure as a chronic exposure. I would suggest just calling it two-time exposure. Chronic exposure can more often be interpreted as being a constant exposure or constantly reoccurring exposure. I would not consider a two-time exposure to be chronic.

Transgenerational protocol

Were the F1 adults of the same age or close in age when exposed to the diagnostic/discriminating dose?

Also, some punctuation is missing throughout the paragraph. The copy editor may pick up on this as well.

Results

Dose-response assays

The authors should include the statistics associated with a probit analysis, ex: chi-square, fiducial limits for endpoints of interest (LC50, hormetic LC, etc).

Hormetic effects

I think the authors are mixing up mortality and survival here. Fig 2a shows that the LC10 and LC20 treatments had the highest survival for males and females, not the highest mortality as stated in the text. And survivorship decreases significantly at the LC30 and LC40 concentrations in Fig 2a. It does not increase as was stated in the text.

I would also suggest the authors include the supplementary material as main material.

Line 243: “Despite having the highest initial LC50 of all the insecticides, the spinetoram treatment had the lowest survivorship across the sub-lethal concentrations. Survivorship in the controls only averaged ~40% for females and 13% for males.”

Given the large control mortality (40%) for females, it may not actually be that the spinetoram has the lowest survivorship, it may simply be a weak group of insects. There seems to be a vast difference between survivorship in controls with experiments corresponding to Figs 2b,c. From close to 80-100% survival to less than 10% in some cases, although it does seem proportional to the treatments. The authors should discuss and try to account for this.

Transgenerational effects

Line 287: LC15 should be LC10

Line 290: “Results showed a trend toward comparable or lower mortality for some sub-lethal parental treatments”. While there are some very small trends, if anything, there appears to be slightly increased mortality in most concentrations compared with controls. However, there are no statistically significant differences. Line 291: “For instance, Fig 6a shows that male mortality was lower than controls in the zeta-cypermethrin LC10 and LC25 treatments for female flies.” This is not what I see in this figure, although the trends mentioned in the other figures are visible. The legend is also in grey, while the bars in the figure are in orange, which may be confounding the authors findings.

Discussion

Line 310: Despite this trend, the differences between the controls and other concentrations were not statistically significant. These results, however, advocate further testing, as it is possible that the number of doses we tested limited our ability to detect a maximized hormetic response.

Yes, indeed your results warrant further testing, you did see some small trends. I would agree that your concentration ranges may have limited your ability to detect the maximized hormetic dose. The range in which the maximal hormetic responses occur can be wide ranging, thus it can be difficult to detect in a first experiment. You may want to consider next time testing a number of concentrations below the LC10 and even LC1. Calabrese indicates that frequently the hormetic concentration falls below the NOAEL, however, with insects, studies have shown that hormetic effects can occur outside the NOAEL. Looking at transgenerational effects as well could certainly impact where the hormetic concentration could fall. A concentration that is not hormetic in one generation, could in fact be hormetic in another generation (as you saw with your eclosion data).

I like the fact the authors include a lot of analysis of what they found, unwinding their findings for the reader. I would suggest the authors find more works on hormesis in insects in the literature (there are lots) and weave this into the discussion, to perhaps assist their insights. For example, the authors could discuss other works on transgenerational effects, the ranges of concentrations other studies used compared with theirs. This would be a nice addition to the authors’ existing discussion on concentration ranges. The discussion is highly lacking in examples of hormesis in insects in response to insecticides, and the connection to agriculture. There is solid work on this. I have provided a number of examples, however there are many others, and the authors should provide some analysis of their work in relation to other studies.

Tables and Figures:

Table 4: Represent the degrees of freedom with the F-value as per the previous tables.

I would suggest the authors use greyscale for their figures, rather than colour. If they want to use colour, I would suggest using darker colours. These colours are not aesthetically pleasing.

The authors should be consistent with their formatting. For example, Fig 1 has the y-axis survivorship label on all the individual graphs, whereas the other figures do not. The graphs in Fig 6 are also boxed in. The top and right side lines should be removed from each graph to be consistent with the other graphs. Only the bottom x-axis, and left y-axis are necessary for these graphs.

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Reviewer #1: No

Reviewer #2: No

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Hormetic and transgenerational effects in spotted-wing Drosophila (Diptera: Drosophilidae) in response to three commonly-used insecticides

PONE-D-22-09651R1

Dear Dr. Deans,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Giancarlo López-Martínez, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Dear authors:

I really appreciate the detailed effort put forth to address the comments brought by the reviewers. In my opinion, the MS was solid during the initial submission, but many of the changes made make aspects of this neat story clearer to the wider audience of this journal. Personally, and as a hormesis/transgenerational person, these results are really cool, and I really appreciate a thorough rebuttal letter.

Thank you,

GC

Reviewers' comments: