Sublethal Effects of Insecticides on the Parasitism of Acerophagus flavidulus (Hymenoptera: Encyrtidae) Parasitoid of the Obscure Mealybug, Pseudococcus viburni (Hemiptera: Pseudococcidae)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The paper is presented in a clear manner with sufficient data to understand the problem to be solved and possible reproduction of the research results. There is a sufficient review and information on the relevance of the problem under study. However, it is unclear what problems exist in existing research, what problems exist in current related research, and what is the significance of this study. 

The experimental conditions and design discussion are not specific.How are defined the test conditions? 

 

Author Response

For Research article

Sublethal effects of insecticides on the parasitism of Acerophagus flavidulus (Hymenoptera: Encyrtidae) parasitoid of the obscure mealybug, Pseudococcus viburni (Hemiptera: Pseudococcidae)

 

Response to Reviewer 1 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Thanks for your suggestions and comments, which have been a real contribution to its improvement. Please find the detailed responses below and the corresponding revisions/corrections in red in the re-submitted files.

2. Point-by-point response to Comments and Suggestions for Authors

Comments 1: The paper is presented in a clear manner with sufficient data to understand the problem to be solved and possible reproduction of the research results. There is a sufficient review and information on the relevance of the problem under study. However, it is unclear what problems exist in existing research, what problems exist in current related research, and what is the significance of this study. The experimental conditions and design discussion are not specific. How are defined the test conditions?

Response 1:  Thank you for pointing this out. We agree with your comment, so answering your question about the problem and the significance of this work, it is very unlikely that biological control would be compatible with existing chemical control in obscure mealybug on deciduous fruit trees Thus, it is relevant to establish how long it would be necessary to wait for inundative releases of parasitoids after insecticide sprays. You can find this change in the page number 2, lines 76 to 85).

As you have asked about the experimental conditions and how we had defined the test conditions, new information was added concerning the route of exposure (page 3, lines 113-115); the surface used: leaf vs glass (page 3, lines 119-120), mealybug density (page 3, lines 127-129), and duration of the experiments (page 3, lines 135-136).

• The residual route of exposure tries to simulate the conditions at the field, where the parasitoid is in contact with the residues of the insecticides after an application.
• The substrate used for the residual bioassay was an apple leaf because it has been reported that glass substrate may overestimate toxicological impact of insecticides.
• The duration of the bioassay ranged from 4 to 5 days, based on longevity of adults under laboratory conditions.

The mealybug densities used in our bioassays (2, 4 and 6) were defined according to Karamaouna and Copland, (2000, 2009). In these studies, the mean number of parasitized mealybugs by Pseudaphycus = Acerophagus flavidulus was between 2 to 5.05 in hosts (Pseudococcus viburni) from different sizes.

Reviewer 2 Report

Comments and Suggestions for Authors

 

Manuscript Title

Sublethal effects of neonicotinoid and insect growth regulator insecticides on Acerophagus flavidulus (Hymenoptera: Encyrtidae) parasitoid of the obscure mealybug, Pseudococcus viburni (Hemiptera: Pseudococcidae)

 

General comment

This article investigated the sublethal effects of buprofezin, pyriproxyfen and acetamiprid, on adults of the parasitoid wasp Acerophagus flavidulus that parasites the obscure mealybug, Pseudococcus viburni. To do so, authors exposed A. flavidulus to insecticide residues on apple leaves for 24 h under laboratory conditions and allowed parasitoid females to parasitize three mealybug densities (two, four, and six nymphs) per parasitoid for 24 h. They found that buprofezin and pyriproxyfen induced less sublethal effects than acetamiprid on A. flavidulus. Moreover, pyriproxyfen and acetamiprid reduced parasitism but did not affect emergence rate, clutch size, development time, longevity, and secondary sex ratio. Author concludes that buprofezin and pyriproxyfen are more compatible with A. flavidulus than acetamiprid. The manuscript is well-written, the experiment is well-designed and I think that the results are of interest for pest control management. However, I have minor comments before acceptance.

 

 Minor comments

-        L107: Did you prepare your working solutions by diluting the three molecules in distilled water or did you use a solvent? Please clarify as you used distilled water as control.

 -        Results section: I suggest to write the exact P-values instead of "P < 0.05" throughout the results section to clarify the strength of the significance.

-        L150-151: Did you try performing post hoc Dunn’s tests for the GLM to find significant differences between combinations of treatments? It may be an alternative to Tukey test

-        Table 1: I suggest to highlight the letters that represent statistically significant differences in bold font

-        Table 2: As you did not indicate statistically significant differences in the table like in Table 1, I recommend to indicate the P-values of the post hoc Tukey test in the corresponding text

 

Author Response

For Research article

Sublethal effects of insecticides on the parasitism of Acerophagus flavidulus (Hymenoptera: Encyrtidae) parasitoid of the obscure mealybug, Pseudococcus viburni (Hemiptera: Pseudococcidae)

 

Response to Reviewer 2 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Thanks for your suggestions and comments, which have been a real contribution to its improvement. Please find the detailed responses below and the corresponding revisions/corrections in red in the re-submitted files.

2. Point-by-point response to Comments and Suggestions for Authors

Manuscript Title

Sublethal effects of neonicotinoid and insect growth regulator insecticides on Acerophagus flavidulus (Hymenoptera: Encyrtidae) parasitoid of the obscure mealybug, Pseudococcus viburni (Hemiptera: Pseudococcidae)

General comment

This article investigated the sublethal effects of buprofezin, pyriproxyfen and acetamiprid, on adults of the parasitoid wasp Acerophagus flavidulus that parasites the obscure mealybug, Pseudococcus viburni. To do so, authors exposed A. flavidulus to insecticide residues on apple leaves for 24 h under laboratory conditions and allowed parasitoid females to parasitize three mealybug densities (two, four, and six nymphs) per parasitoid for 24 h. They found that buprofezin and pyriproxyfen induced less sublethal effects than acetamiprid on A. flavidulus. Moreover, pyriproxyfen and acetamiprid reduced parasitism but did not affect emergence rate, clutch size, development time, longevity, and secondary sex ratio. Author concludes that buprofezin and pyriproxyfen are more compatible with A. flavidulus than acetamiprid. The manuscript is well-written, the experiment is well-designed and I think that the results are of interest for pest control management. However, I have minor comments before acceptance.

Comments 1: L107: Did you prepare your working solutions by diluting the three molecules in distilled water or did you use a solvent? Please clarify as you used distilled water as control.

Response 1: Thank you for pointing this out. We prepared insecticide treatments by diluting each commercial insecticide in distilled water, so we use distilled water as a control. We did not use any solvent, but distilled water. This change can be found in page number 3, lines 118-119.

Comments 2: Results section: I suggest to write the exact P-values instead of "P < 0.05" throughout the results section to clarify the strength of the significance.

Response 2: Thank you for your suggestion. We have revised and the exact values for the interaction in parasitism and emergence rates; for the density in clutch size and sex ratio and for the treatment in development time are given. The values that were described as P≤ 0.001 were found to be very small (smaller than 0.001) and were therefore, reported as such in the article. You can find this values in the section Results, pages number 4 and 5, lines 166 to 206.

Comments 3: L150-151: Did you try performing post hoc Dunn’s tests for the GLM to find significant differences between combinations of treatments? It may be an alternative to Tukey test

Response 3: Thank you for pointing this out. According to the literature, the post-hoc Dunn's test is suggested to be performed after a significant non-parametric test (e.g. Kruskal-Wallis). Therefore, we perform a Tukey post-hoc test after significant effects in the GLM for parasitism rate. Tukey test is the most used and cited in literature for GLM analyses. Supporting literature can be found below.

Dinno, Alexis. Nonparametric pairwise multiple comparisons in independent groups using Dunn’s test. The Stata Journal (2015) 15, 1, 292–300

Dinno, Alexis. Dunn Test of Multiple comparisons using Rank Sums. Package “dunn.test”. Version 1.3.5. 26.10.2017. http://r.meteo.uni.wroc.pl/web/packages/dunn.test/dunn.test.pdf -

Elliott, Alan C., Hynan Linda, S. A SAS® macro implementation of a multiple comparison post hoc test for a Kruskal–Wallis analysis. Computer methods and programs in biomedicine (2011) 102, 75-80.

 

Comments 4: Table 1: I suggest to highlight the letters that represent statistically significant differences in bold font.

Response 4: Thank you for pointing this out. We have highlighted the letters in bold font in Table 1 as you suggested (page 5).

Comments 5: Table 2: As you did not indicate statistically significant differences in the table like in Table 1, I recommend to indicate the P-values of the post hoc Tukey test in the corresponding text.

Response 5: As you have suggested, P values have been written in each parameter of Table 1 and Table 2 (pages 5 and 6).

Reviewer 3 Report

Comments and Suggestions for Authors

This contribution mainly explaining the impact of pesticides on the growth, development, and reproduction of parasitic wasps. However, the explanation of the experimental results is not sufficient. In terms of experimental design, the pest density used was relatively low, without expanding the density for comparison.  the reference value of this data may be insufficient. Overall,  thedata analysis was reasonable, and the conclusions were relatively credible. but authors have to explain why such a density gradient was chosen， and discuss the result further, to clarify the relationship among Insecticide, Mealybug density and parasitoid wasp。

Author Response

For Research article

Sublethal effects of insecticides on the parasitism of Acerophagus flavidulus (Hymenoptera: Encyrtidae) parasitoid of the obscure mealybug, Pseudococcus viburni (Hemiptera: Pseudococcidae)

 

Response to Reviewer 3 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Thanks for your suggestions and comments, which have been a real contribution to its improvement. Please find the detailed responses below and the corresponding revisions/corrections in red in the re-submitted files.

2. Point-by-point response to Comments and Suggestions for Authors

Comments 1: This contribution mainly explaining the impact of pesticides on the growth, development, and reproduction of parasitic wasps. However, the explanation of the experimental results is not sufficient. In terms of experimental design, the pest density used was relatively low, without expanding the density for comparison. The reference value of this data may be insufficient. Overall, the data analysis was reasonable, and the conclusions were relatively credible, but authors have to explain why such a density gradient was chosen and discuss the result further, to clarify the relationship among Insecticide, Mealybug density and parasitoid wasp.

Response 1: Thank you for pointing this out. We have, accordingly, revised the text to emphasize this point. The mealybug densities selected in our bioassay were used since Karamaouna and Copland (2000, 2009) reported that the oviposition capacity of Acerophagus flavidulus reaches a saturation point close to 2-5 mealybugs (nymphs) per female. Thus, we used two, four and six mealybugs per female. This change can be found in page number 3, lines 127-129.

In section Discussion, it is explained the relation among the effects of insecticides, mealybug density and A. flavidulus, while the parasitism was always lower as host density increased, which may be because at low densities of P. viburni, the parasitoid did not have to make great efforts to parasitize it. The insecticides pyriproxyfen and acetamiprid decreased A. flavidulus parasitism on P.viburni at the highest density of mealybugs offered (six). If the number of hosts offered to each parasitoid was increased, parasitism could decrease further, which would be interesting to evaluate, since at a density of six mealybugs, A. flavidulus is close to the maximum oviposition capacity (Karamaouna and Copland 2000, 2009). This change can be found in page number 7, lines 242- 246.

Reviewer 4 Report

Comments and Suggestions for Authors

The article investigated an important aspect of plant protection: the effect of Insecticides used to control agricultural pests on beneficial arthropods. Results of the study revealed the compatibility of the two insect growth regulators with the parasitoid of the obscure mealybug, Pseudococcus viburni applied at label rates. They also showed the compatibility of the neonicotinoid insecticide with the parasitoid, under the condition that very low residue levels are reached in the field.

The obtained results will contribute to improving the integration of chemical and biological control, as the basis for integrated pest management (IPM) strategies in crops.

I have only one minor suggestion for the authors, which is to provide complete address information including city, zip code, state/province, and country to comply with the journal's instructions for authors.

Author Response

For Research article

Sublethal effects of insecticides on the parasitism of Acerophagus flavidulus (Hymenoptera: Encyrtidae) parasitoid of the obscure mealybug, Pseudococcus viburni (Hemiptera: Pseudococcidae)

 

Response to Reviewer 4 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Thanks for your suggestions and comments, which have been a real contribution to its improvement. Please find the detailed responses below and the corresponding revisions/corrections in red in the re-submitted files.

2. Point-by-point response to Comments and Suggestions for Authors

Comments 1: The article investigated an important aspect of plant protection: the effect of Insecticides used to control agricultural pests on beneficial arthropods. Results of the study revealed the compatibility of the two insect growth regulators with the parasitoid of the obscure mealybug, Pseudococcus viburni applied at label rates. They also showed the compatibility of the neonicotinoid insecticide with the parasitoid, under the condition that very low residue levels are reached in the field.

The obtained results will contribute to improving the integration of chemical and biological control, as the basis for integrated pest management (IPM) strategies in crops.

I have only one minor suggestion for the authors, which is to provide complete address information including city, zip code, state/province, and country to comply with the journal's instructions for authors.

Response 1: Thank you for pointing this out. We have added the requested information in the correspondence author: 2 Norte 685, Talca, Chile, P.O. Box 747, Talca, Chile. This change can be found in page number 1, line 8.

Reviewer 5 Report

Comments and Suggestions for Authors

the manuscript is very clear and the methodology supports the results; I would suggest just mentioning in the title what was evaluated (the current title is written in a very general way "sublethal effect"). sublethal effects can be various, here were evaluated specifics. 

Comments for author File: Comments.pdf

Author Response

For Research article

Sublethal effects of insecticides on the parasitism of Acerophagus flavidulus (Hymenoptera: Encyrtidae) parasitoid of the obscure mealybug, Pseudococcus viburni (Hemiptera: Pseudococcidae)

 

Response to Reviewer 5 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Thanks for your suggestions and comments, which have been a real contribution to its improvement. Please find the detailed responses below and the corresponding revisions/corrections in red in the re-submitted files.

2. Point-by-point response to Comments and Suggestions for Authors

Comments 1: the manuscript is very clear and the methodology supports the results; I would suggest just mentioning in the title what was evaluated (the current title is written in a very general way "sublethal effect"). sublethal effects can be various, here were evaluated specifics. 

Response 1: We agree with your comment. We have, accordingly, changed the title for the sublethal effects on parasitism, because the significant effects were seen on this variable.

The original title was: “Sublethal effects of neonicotinoid and insect growth regulator insecticides on Acerophagus flavidulus (Hymenoptera: Encyrtidae) parasitoid of the obscure mealybug, Pseudococcus viburni (Hemiptera: Pseudococcidae)” and has been changed for:

“Sublethal effects of insecticides on the parasitism of Acerophagus flavidulus (Hymenoptera: Encyrtidae) parasitoid of the obscure mealybug, Pseudococcus viburni (Hemiptera: Pseudococcidae)”

 

Reviewer 6 Report

Comments and Suggestions for Authors

The manuscript titled "Sublethal effects of neonicotinoid and insect growth regulator insecticides on Acerophagus flavidulus (Hymenoptera: Encyrtidae) parasitoid of the obscure mealybug, Pseudococcus viburni (Hemiptera: Pseudococcidae)" offers an insightful exploration into the sublethal impacts of three insecticides—buprofezin, pyriproxyfen, and acetamiprid—on Acerophagus flavidulus, a key natural antagonist of the obscure mealybug. This study is methodically designed, and the findings contribute significantly to the sphere of integrated pest management. Nevertheless, the manuscript would considerably gain in credibility and depth by incorporating more meticulous details about the experimental conditions, an enriched discussion relating it to pre-existing literature, and a section on ecological ramifications stemming from the study's findings.

1) The existing title appears prolix. A suggested revision for brevity and clarity could be "Insecticide Sublethal Effects on Acerophagus flavidulus, Parasitoid of Obscure Mealybug."

2) There is a paucity of information regarding the experimental setup specifics, such as replicates per treatment and the statistical methods applied to each parameter. Elaborating on these would substantiate the study's reproducibility.

3) A thorough discourse that juxtaposes the results with antecedent research on insecticide sublethal effects on parasitoids would fortify the manuscript.

4) An omission in the current analysis is the discussion of potential ecological consequences of sublethal effects observed. Elucidating on this would amplify the manuscript's relevance to the readership.

5) The manuscript's reliance on two tables without accompanying figures results in a presentation that may be perceived as somewhat lackluster. Incorporating illustrative figures could enliven the presentation of results and enhance reader engagement.

 

There are several questions:

1) Could the authors possibly provide additional details on the experimental framework, such as replication counts for each treatment and the specific statistical methods utilized for evaluating each parameter?

2) May the authors contextualize the observed sublethal effects within the ambit of extant studies examining similar impacts on parasitoids?

3) Would the authors be willing to delve into the potential ecological implications of the sublethal effects noted in the study, with particular attention to the implications for population dynamics of A. flavidulus and its biological control efficacy against P. viburni?

Comments on the Quality of English Language

The English Language is okay

Author Response

For Research article

Sublethal effects of insecticides on the parasitism of Acerophagus flavidulus (Hymenoptera: Encyrtidae) parasitoid of the obscure mealybug, Pseudococcus viburni (Hemiptera: Pseudococcidae)

 

Response to Reviewer 6 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Thanks for your suggestions and comments, which have been a real contribution to its improvement. Please find the detailed responses below and the corresponding revisions/corrections in red in the re-submitted files.

2. Point-by-point response to Comments and Suggestions for Authors

The manuscript titled "Sublethal effects of neonicotinoid and insect growth regulator insecticides on Acerophagus flavidulus (Hymenoptera: Encyrtidae) parasitoid of the obscure mealybug, Pseudococcus viburni (Hemiptera: Pseudococcidae)" offers an insightful exploration into the sublethal impacts of three insecticides—buprofezin, pyriproxyfen, and acetamiprid—on Acerophagus flavidulus, a key natural antagonist of the obscure mealybug. This study is methodically designed, and the findings contribute significantly to the sphere of integrated pest management. Nevertheless, the manuscript would considerably gain in credibility and depth by incorporating more meticulous details about the experimental conditions, an enriched discussion relating it to pre-existing literature, and a section on ecological ramifications stemming from the study's findings.

 

Comments 1: The existing title appears prolix. A suggested revision for brevity and clarity could be "Insecticide Sublethal Effects on Acerophagus flavidulus, Parasitoid of Obscure Mealybug."

Response 1: Agree. We have changed the title, incorporating the main significant sublethal effect, the parasitism. The new title is “Sublethal effects of insecticides on the parasitism of Acerophagus flavidulus (Hymenoptera: Encyrtidae) parasitoid of the obscure mealybug, Pseudococcus viburni (Hemiptera: Pseudococcidae)”.

Comments 2: There is a paucity of information regarding the experimental setup specifics, such as replicates per treatment and the statistical methods applied to each parameter. Elaborating on these would substantiate the study's reproducibility.

Response 2: Thank you for pointing this out. We have added the number of experimental units per treatment on each mealybug density used to be more specific: “The bioassays were replicated three times with the following number of experimental units per treatment: density two (9-14 experimental units), four (12-18 experimental units) and six (9-12 experimental units), respectively”. This change can be found in page number 4, lines 148-151.

In terms of the statistical methods applied for each parameter, they are described in the section “Statistical analysis”. We had evaluated the effects of insecticides, mealybug densities, and their interaction. If the interaction was not significant, the additive model was used, since it had lower Aikaike information criterion (AIC) value. This change can be found in page 4, lines 158-160.

Comments 3: A thorough discourse that juxtaposes the results with antecedent research on insecticide sublethal effects on parasitoids would fortify the manuscript.

Response 3: There is no further literature for the insecticides evaluated in this work on parasitoids of encyrtids and we did not want to extend the discussion by referring to other insecticides and to other parasitoids that are not strictly comparable. The literature cited in the manuscript is the most relevant (See in References 6, 36-40).

Comments 4: An omission in the current analysis is the discussion of potential ecological consequences of sublethal effects observed. Elucidating on this would amplify the manuscript's relevance to the readership.

Response 4: Thank you for pointing this out. We agree with this comment. Therefore, we have incorporated a new paragraph discussing the potential ecological consequences. From the population dynamics point of view, we found that the parasitism rate of A. flavidulus is sensitive to sublethal doses of pyriproxyfen and acetamiprid. Therefore, it could affect the biological control of P. viburni. These insecticides would be directly affecting the intrinsic growth rate (r_m) of the parasitoid, through a lower oviposition rate and the subsequent lower mortality of the mealybug. This change can be found in Discussion section, page number 8, lines 304-308.

Comments 5: The manuscript's reliance on two tables without accompanying figures results in a presentation that may be perceived as somewhat lackluster. Incorporating illustrative figures could enliven the presentation of results and enhance reader engagement.

Response 5: Unfortunately, we do not have good quality pictures to include in the manuscript.

 

There are several questions:

 

• Could the authors possibly provide additional details on the experimental framework, such as replication counts for each treatment and the specific statistical methods utilized for evaluating each parameter?

The replications counts for each treatments were answered in Response 2.

• May the authors contextualize the observed sublethal effects within the ambit of extant studies examining similar impacts on parasitoids?

This question was answered in Response 3.

• Would the authors be willing to delve into the potential ecological implications of the sublethal effects noted in the study, with particular attention to the implications for population dynamics of flavidulus and its biological control efficacy against P. viburni?

This question has been answered in Response 4.

Response to Comments on the Quality of English Language

Point 1: The English Language is okay.

Response 1: Thanks.