Trichopria anastrephae: A Promising Neotropical-Native Parasitoid for Drosophila suzukii Control

Round 1

Reviewer 1 Report

The present study investgate The Neotropical-native parasitoid Trichopria anastrephae: a potential biocontrol agent targeting the invasive pest Drosophila Suzukii

The abstract needs to be reformulated, focusing on the research outcomes and a brief summary of the abstract

Result section need to be rewritten, focusing on statistical outcome. For example, you mentioned that the performance of TaDm was significantly lower than the other three tested treatments when D. suzukii was offered as a host (Fig. 1A).   However, upon further statistical analysis, no significant differences were observed between the performance of TaDm and both TaDs-DM and TaDs-DS. Please revise all results on this basis.

What does the standard bar indicate?

In order to improve readability, it is recommended to consider changing the graph style. Additionally, including P values in statistical tables or on the graph would help provide a clear link to the corresponding averages. The keys for Table 1 and Table 2 should include reference alphabets for easy identification. For a more comprehensive discussion, it is important to provide background information related to the study area.

Lastly, the conclusion should be presented under a separate title, rather than being included within the discussion section.

Author Response

Reviewer No. 1

- Reviewer's comment. The present study investgate The Neotropical-native parasitoid Trichopria anastrephae: a potential biocontrol agent targeting the invasive pest Drosophila Suzukii.

Corresponding author's reply. It is important to point out to the reviewer that due to a high similarity index highlighted by the AGRONOMY editorial committee, the original title: “The Neotropical-native parasitoid Trichopria anastrephae: a potential biocontrol agent targeting the invasive pest Drosophila suzukii”was modified by the new title: “Trichopria anastrephae: a promising Neotropical-native parasitoid for Drosophila Suzukii control”.

- Reviewer's comment. The abstract needs to be reformulated, focusing on the research outcomes and a brief summary of the abstract.

Corresponding author's reply. The reviewer's comment is correct. We write q new abstract as follows:  Abstract: Drosophila suzukii (Matsumura) is an invasive pest mainly affecting berry and stone fruit crops worldwide. In Argentina, it inhabits throughout fruit-growing regions. An eco-friendly management strategy involves biological control by using resident natural enemies, such as the Neotropical‐native pupal parasitoid Trichopria anastrephae Lima (=Ta). The study compares the host-killing effectiveness and the offspring reproductive success of two Ta lineages on puparia of both D. suzukii (=Ds) and D. melanogaster (=Dm) in no-choice and choice tests under lab-conditions. The host preference and switching are also assessed. One parasitoid lineage was reared on Ds (=Ta_Ds), whereas the second on Dm (=Ta_Dm). In no-choice tests, both Ta lineages performed similarly on both hosts regarding the percentage of killed hosts and parasitoid offspring survival. The host-killing ability of Ta_Dm was only significantly lower when Ds was offered as host relative to Dm. In choice tests, Ta attacked mainly Ds at a 4-9 times Ds to Dm ratio, but at a 1.5-2 times Ds to Dm ratio, the host-killing ability was equal between both drosophilids. At equal host ratio or higher Dm ratios, Ta preferred native host. However, Ta killing capacity over Ds was highly relevant, showing potential to control the recently-introduced pest.

- Reviewer's comment. Result section need to be rewritten, focusing on statistical outcome. For example, you mentioned that the performance of TaDm was significantly lower than the other three tested treatments when D. suzukii was offered as a host (Fig. 1A).   However, upon further statistical analysis, no significant differences were observed between the performance of TaDm and both TaDs-DM and TaDs-DS. Please revise all results on this basis.

Corresponding author's reply. The reviewer's comment is correct, so the paragraph was rewritten to be consistent with Figure 1A as follow: There were slightly significant differences when the host-killing effectiveness of T. anastrephae was evaluated under no-choice conditions (H_{(3, n = 40)} = 13.384, p = 0.004). The performance of Ta_Dm when D. suzukii was offered as a host was significantly lower than that of Ta_Dm when D. melanogaster was the host (Fig. 1A). There were no significant differences between T. anastrephae population lineages regarding their performance parasitizing either D. suzukii or D. melanogaster (Fig. 1A). The host-killing capacity of T. anastrephae was >80% in all four treatments. There were no significant differences between the reproductive success of the two T. anastrephae population lineages or when both flies species were exposed to parasitoid females (H_{(3, n = 40)} = 3.811, p = 0.283). The mean offspring survival in both parasitoid lineages varied between 50% and 70% (Fig. 1B).

- Reviewer's comment. What does the standard bar indicate?

Corresponding author's reply. The explanation of the descriptive statistical data shown in the graphs (Box Plot) was carried out in Materials and Methods. More precisely in the subheading Statistical analysis, as follows: The box plots were plotted to show the resulting data, which involve median (horizontal line inside the box), mean (X inside the box), interquartile range Q1–Q3 (bottom and top ends of the box), range (minimum: Q0, maximum: Q4; both ends of the whisker on the vertical line outside the box), and raw data dispersal (colored circles).

- Reviewer's comment. In order to improve readability, it is recommended to consider changing the graph style.

Corresponding author's reply. We do not understand the reason for changing the style of the graphs, as in our opinion they are highly descriptive and show a cluster of important statistical data. We prefer to keep the graphics as they were designed in the original version of the manuscript.

- Reviewer's comment. Additionally, including P values in statistical tables or on the graph would help provide a clear link to the corresponding averages.

Corresponding author's reply. Tables 1 and 2 include the results of the applied statistic, among which is the p value. We believe that the reconfiguration of the original manuscript deformed both tables and therefore the statistical data could not be displayed correctly. We now proceeded to reformat the tables and with this action it is now possible to see all the statistical values accurately. The p-value of the applied statistic is included in the caption of all figures.

- Reviewer's comment. The keys for Table 1 and Table 2 should include reference alphabets for easy identification.

Corresponding author's reply. It is not clear for us to understand this reviewer's comment. Tables 1 and 2 have all their acronyms explained in the respective title. We consider that both tables are self-explanatory and clearly show results of statistics applied for data analysis.

- Reviewer's comment. For a more comprehensive discussion, it is important to provide background information related to the study area.

Corresponding author's reply. At the reviewer's request, we have included a new sentence in the discussion paragraph referring to the area of study, After the original sentence “The preference for D. melanogaster displayed by any of the T. anastrephae population lines in the current lab studies matches with field survey data recorded from non-crop fruits in wild environments from northwestern Argentina [30-31]” we have includes the sentence: “Those wilderness areas involved patches of secondary structure Yungas subtropical rainforest with a mix of feral exotic fruits plus native fruit species”.

- Reviewer's comment. Lastly, the conclusion should be presented under a separate title, rather than being included within the discussion section.

Corresponding author's reply. We agree with the reviewer's suggestion and proceed to make the respective modification in the text.

Reviewer 2 Report

Biancheri et al. report the results of a laboratory study on host selection and reproduction of a pupal parasitoid of Drosophila suzukii, an important pest of soft fruit. The manuscript requires revision before it would be suitable for publication in Agronomy.

MAJOR POINTS

The text is wordy and repetitive. The Introduction needs to be focused on the key issues of the study and reducing in length by about 50%. The Discussion is also rather repetitive and uninformative and could be reduced in length by 40% easily. The conclusions likewise. For example, we are told that D. suzukii is an invasive pest at 14 different points in the text (!).

The Discussion also needs to consider two issues that are not presently covered: (i) the potential impact of superparasitism, given that 5 wasps were confined in a cage with 50 hosts for 2 days. I suspect that superparasitism had a significant role to play in the results; (ii) density dependence, is critical to the efficacy of natural enemies as agents of biological control but is not even mentioned. The functional response of the wasp is also overlooked.

I have written suggestions and a series of numbered points on a scanned copy of the manuscript.

Numbered points (see scanned file)

1a. Simple summary. 70% of the summary is preamble. Please focus on the RESULTS and their interpretation/conclusions.

1b. Meaning unclear, please reword.

2. Indicate that female biased sex ratio favors parasitoid population growth and improved biocontrol.

3. The Abstract is mostly background with just 4 lines of Results. Suggest 30% background, 60% results, 10% conclusions.

4. The Introduction is much too long. Please summarize this paragraph on the value of berry and soft fruit crops in 2 or 3 lines.

5. Again a long text on parasitoid diversity – please focus on main issues to be addressed in this study.

6. I was confused, for me switching is when the parasitoid decides to attack the most abundant host available, not the least abundant.  Please check.

7a. Change "cc" to mL or cm3 throughout manuscript.

7b. Please provide a reference for the composition of the Drosophila diet.

8. What concentration of benzoate was used?

9. I have never heard the phrase "significantly equal", do you mean "similar to"?

10. This text was unclear. Please reword.

11. The layout of Table 1 was cluttered and difficult to read. I suggest using landscape format for large tables.

12. Same for Table 2.

13. Effective control? To achieve control the parasitoid must act in a density-dependent manner. Do you have evidence for density dependent parasitism in T. anastrephae?

14. Delete repetitive text. Reduce the length of the entire Discussion (too wordy).

15. Please upload your raw data as a supplemental file to meet the requirements of the journal for transparent access to data.

16. Some typos in the References; I only reviewed the first page.

Comments for author File: Comments.pdf

Minor edits (see scanned file)

Author Response

Reviewer No. 2

- Reviewer's comment. Biancheri et al. report the results of a laboratory study on host selection and reproduction of a pupal parasitoid of Drosophila suzukii, an important pest of soft fruit. The manuscript requires revision before it would be suitable for publication in Agronomy.

-Corresponding author's reply. It is important to point out to the reviewer that due to a high similarity index highlighted by the AGRONOMY editorial committee, the original title: “The Neotropical-native parasitoid Trichopria anastrephae: a potential biocontrol agent targeting the invasive pest Drosophila suzukii”was modified by the new title: “Trichopria anastrephae: a promising Neotropical-native parasitoid for Drosophila Suzukii control”. We thank reviewer No. 2 as well as reviewer No. 1 for their time and dedication to help us achieve a better paper for publication.

- Reviewer's comment. The text is wordy and repetitive. The Introduction needs to be focused on the key issues of the study and reducing in length by about 50%. The Discussion is also rather repetitive and uninformative and could be reduced in length by 40% easily. The conclusions likewise. For example, we are told that D. suzukii is an invasive pest at 14 different points in the text (!).
-Corresponding author's reply. The text has been reduced and restructured, although we consider that there is valuable information in the introduction that we prefer to keep; nevertheless, we consider that the text of the Introduction, Discussion and Conclusion was generally rather reduced.

- Reviewer's comment. The Discussion also needs to consider two issues that are not presently covered: (i) the potential impact of superparasitism, given that 5 wasps were confined in a cage with 50 hosts for 2 days. I suspect that superparasitism had a significant role to play in the results; (ii) density dependence, is critical to the efficacy of natural enemies as agents of biological control but is not even mentioned. The functional response of the wasp is also overlooked.

-Corresponding author's reply. Superparasitism and functional response are two very important issues to analyze in a biological control agent. We agree on this point with the reviewer. However, we did not evaluate superparasitism in the current study nor did we perform a functional response analysis, as these were not the original objectives. However, we are conducting a second set of laboratory trials with T. anastrephae and will now consider both issues, superparasitism and functional response. We thank the reviewer for this observation.

- Reviewer's comment. I have written suggestions and a series of numbered points on a scanned copy of the manuscript. Numbered points (see scanned file).

-Corresponding author's reply. The reviewer's suggestions indicated in the text were taken into account and therefore the original text was modified.

- Reviewer's comment. 1a. Simple summary. 70% of the summary is preamble. Please focus on the RESULTS and their interpretation/conclusions. 1b. Meaning unclear, please reword.

-Corresponding author's reply. The reviewer's comment is correct. We write a new abstract as follows:

Simple summuary. Drosophila suzukii (=Ds), or the spotted-wing drosophila, is an invasive global pest that mainly causes economic damage to healthy, fresh, soft, and stone fruit crops. This fly, native to Southeast Asia, has rapidly spread worldwide. In the fruit-growing region of northwestern Argentina, a Neotropical‐native parasitoid assemblage is associated with Ds. Trichopria anastrephae (=Ta), a pupal parasitoid, stands out from all other species. Therefore, the study provides information about the host-killing parasitoid capacity and the subsequent reproductive success in terms of the parasitoid offspring survival when Ds and the native Drosophila melanogaster (=Dm) are exposed in choice and non-choice conditions. In addition, the study reports on host preference and host-switching behavior. Results shows that  the host-killing capacity of Ta is >80% in no-choice tests. However, in choice tests  Ta prefers Dm at an equal host ratio or at higher ratios of Dm over Ds, and the effectiveness to kill Ds is 40-50%. However, at a 4-9 times Ds to Dm ratio, the Ta killing effectiveness over Ds and the offspring survival are highly relevant. Ta has a female-biased offspring sex ratio, which favors parasitoid population growth. Such data highlight Ta as an important Ds natural enemy and its potential use in biocontrol

- Reviewer's comment. 2. Indicate that female biased sex ratio favors parasitoid population growth and improved biocontrol.

-Corresponding author's reply. That's right, it was added to the simple summary text

- Reviewer's comment. 3. The Abstract is mostly background with just 4 lines of Results. Suggest 30% background, 60% results, 10% conclusions.

-Corresponding author's reply. The abstract was modified following the recommendations of reviewer 2, which were consistent with the suggestions of reviewer 1. The new Abstract is as follows:

Abstract: Drosophila suzukii (Matsumura) is an invasive pest mainly affecting berry and stone fruit crops worldwide. In Argentina, it inhabits fruit-growing regions. An eco-friendly management strategy involves biological control by using resident natural enemies, such as the Neotropical‐native pupal parasitoid Trichopria anastrephae Lima (=Ta). The study compares the host-killing effectiveness and the offspring reproductive success of two Ta lineages on puparia of both D. suzukii (=Ds) and D. melanogaster (=Dm) in no-choice and choice tests under laboratory-conditions. The host preference and host-switching behaviors are also assessed. One parasitoid lineage was reared on Ds (=Ta_Ds), whereas the second on Dm (=Ta_Dm). In no-choice tests, both Ta lineages performed similarly on both hosts regarding the percentage of killed hosts and parasitoid offspring survival. The host-killing ability of Ta_Dm was only significantly lower when Ds was offered as host relative to Dm. In choice tests, Ta attacked mainly Ds at a 4-9 times Ds to Dm ratio, but at a 1.5-2 times Ds to Dm ratio, the host-killing ability was equal between both drosophilids. At equal host ratio or higher Dm ratios, Ta preferred native host. However, Ta killing capacity over Ds was highly relevant, showing potential to control the recently-introduced pest.

- Reviewer's comment. 4. The Introduction is much too long. Please summarize this paragraph on the value of berry and soft fruit crops in 2 or 3 lines.

-Corresponding author's reply. We have made a thorough and extensive reduction and restructuring of the paragraphs referred to the reviewer; however, we believe that there is valuable information that we would like to remain in the text.

- Reviewer's comment. 5. Again a long text on parasitoid diversity, please focus on main issues to be addressed in this study.

-Corresponding author's reply. The paragraph was reduced in length as suggested by the reviewer.

- Reviewer's comment. 6. I was confused, for me switching is when the parasitoid decides to attack the most abundant host available, not the least abundant.  Please check.

-Corresponding author's reply. The reviewer's observation is right, there was an error by us in the drafting. We thank the reviewer for such a detailed review.

- Reviewer's comment. 7a. Change "cc" to mL or cm3 throughout manuscript.

-Corresponding author's reply. Change was made.

- Reviewer's comment. 7b. Please provide a reference for the composition of the Drosophila diet.

-Corresponding author's reply. The combination of dietary ingredients and the respective proportions were performed in our laboratory and is not a diet cited in other papers.

- Reviewer's comment. 8. What concentration of benzoate was used?.

-Corresponding author's reply.It was used 10% benzoate. This data was added in the text.

- Reviewer's comment.  9. I have never heard the phrase "significantly equal", do you mean "similar to"?

-Corresponding author's reply. Yes, “similar to” is best to use in the sentence

- Reviewer's comment.  10. This text was unclear. Please reword.

-Corresponding author's reply. The sentence was rewritten as follows: Offspring sex ratio of the two T. anastrephae lineages was significantly similar in both choice and non-choice tests (Table 1).

- Reviewer's comment.  11. The layout of Table 1 was cluttered and difficult to read. I suggest using landscape format for large tables.

-Corresponding author's reply. Tables 1 and 2 include the results of the applied statistic. We believe that the reconfiguration of the original manuscript deformed both tables and therefore the da and the statistical results could not be displayed correctly. We now proceeded to reformat the tables and with this action it is now possible to see all the statistical values accurately.

- Reviewer's comment.  12. Same for Table 2.

-Corresponding author's reply. The same as written above.

- Reviewer's comment.  13. Effective control? To achieve control the parasitoid must act in a density-dependent manner. Do you have evidence for density dependent parasitism in T. anastrephae?.

-Corresponding author's reply. The reviewer is right, it is better to write in the text that the parasitoid causes mortality in the pest. We have no evidence of a density dependence between parasitism and the pest.

- Reviewer's comment.  14. Delete repetitive text. Reduce the length of the entire Discussion (too wordy).

-Corresponding author's reply. The discussion and conclusion were reduced according to the reviewer's suggestions. The conclusion was separated from the discussion (reviewer 1's suggestion).

- Reviewer's comment.  15. Please upload your raw data as a supplemental file to meet the requirements of the journal for transparent access to data.

-Corresponding author's reply. The raw and analyzed data were attached as supplementary files.

- Reviewer's comment.  16. Some typos in the References; I only reviewed the first page.

-Corresponding author's reply. Corrections were made to the list of references highlighted by the reviewer.

Round 2

Reviewer 1 Report

It is preferable to delete references from the conclusion and focus only on the research outcomes

Author Response

Reviewer's Comment: It is preferable to delete references from the conclusion and focus only on the research outcomes.

Response. The reviewer's suggestion was made, the conclusion was modified and the references were removed from it. The new conclusion text is as follow:

5. Conclusions

Results suggest that T. anastrephae was effective in killing D. suzukii based on its performance relative to the pest and producing reproductively successful offspring. Given the preference of T. anastrephae for resident saprophytic drosophilids, the concurrent presence of those hosts in equal or higher ratios to that of D. suzukii in the same microhabitat influenced the effectiveness of the parasitoid to kill this pest. However, the mortality of D. suzukii by T. anastrephae under such conditions was significant. Optimizing important biological traits can be explored through the native parasitoids selective rearing in studies based on the experimental adaptation of resident parasitoids on D. suzukii. This would provide a more specific biological control agent targeted at the pest.

Reviewer 2 Report

The authors have improved their manuscript, but some issues still require attention.

Numbered points on manuscript (see scanned copy).

1. The term "killing effectiveness" is used throughout the manuscript. This is not a suitable term for the efficacy of a natural enemy. The authors use better terms such as "parasitoid performance" and "host-killing capacity" or "parasitoid-induced mortality" or simply "parasitism". Please delete "effectiveness" throughout the manuscript and figures.

2. Meaning of "highly relevant"? This is not the correct term in English. Do you mean that the parasitoid is an effective natural enemy?

3. Ditto (line 390) in the Discussion.

Other issues.

I) The authors state that the Drosophila diet has not been published, so please provide the recipe (ingredients + quantities and preparation details) as Supplementary Material, so that other authors can replicate your study.

II) I looked at the original data files, which were impossible to understand as PDF files. Please upload these as Excel files and label appropriately.

III) The authors state that they are preparing to examine superparasitism in future studies, but the possibility that the results of the present study may have involved superparasitism given the density of wasps and the period of exposure of hosts is something that the authors should point out in the Discussion, at least.

IV) I have suggested additional improvements to the English (see scanned manuscript). The authors should be aware that you cannot state that two values were "significantly equal" or "significantly similar" because statistical tests are designed to test for DIFFERENCES (i.e., reject the null hypothesis). If values were not significantly different, just state that they were similar (not significantly similar).

Comments for author File: Comments.pdf

Requires editing (see comments on manuscript).

Author Response

Reviewer's Comment: The authors have improved their manuscript, but some issues still require attention.

Response. Once again I must express my gratitude to reviewer 2 for his dedication to thoroughly review the manuscript and his valuable suggestions and corrections. I find all his comments very pertinent. Thank you for proofreading the text so thoroughly. The manuscript has greatly improved with respect to the original version, due to the important contribution of both reviewers.

Numbered points on manuscript (see scanned copy).

1. The term "killing effectiveness" is used throughout the manuscript. This is not a suitable term for the efficacy of a natural enemy. The authors use better terms such as "parasitoid performance" and "host-killing capacity" or "parasitoid-induced mortality" or simply "parasitism". Please delete "effectiveness" throughout the manuscript and figures.

Response. Changes in the text according to the reviewer's suggestion were made.

2. Meaning of "highly relevant"? This is not the correct term in English. Do you mean that the parasitoid is an effective natural enemy?

Response. As the reviewer says - highly relevant - means it is an effective natural enemy. We made the respective change in the text.

3. Ditto (line 390) in the Discussion.

Response. Sentence of the line 390 was modified.

Other issues.

1. I) The authors state that the Drosophila diet has not been published, so please provide the recipe (ingredients + quantities and preparation details) as Supplementary Material, so that other authors can replicate your study.

Response: The correct larval diet with its ingredients and proportions was included in the text. Larval diet was made of corn flour (32.5 g), brewer's yeast (18.5 g), cane sugar (52.5 g), agar-agar (8 g), absolute ethyl alcohol 99.5% (15 mL), vitamin C (1 g), vitamin E (1 g), Nipagin (methyl 4-hydroxybenzoate) (1.6 g). Agar-agar and corn flour were dissolved in 200 mL and 800 mL of water, respectively. Such ingredient proportions are enough for 500 g of diet.

1. II) I looked at the original data files, which were impossible to understand as PDF files. Please upload these as Excel files and label appropriately.

Response: Raw Data were  uploaded as Excel files.

III) The authors state that they are preparing to examine superparasitism in future studies, but the possibility that the results of the present study may have involved superparasitism given the density of wasps and the period of exposure of hosts is something that the authors should point out in the Discussion, at least.

Response. As suggested by the reviewer, a paragraph related to superparasitism has been included in Discussion: In both Drosophila species studied, the host mortality caused by T. anastrephae was high. An additional mortality factor might be due to superparasitism, particularly when considering the relationship between the low density of exposed hosts and the long host exposure time in the tests. For the current study, verifying whether the host mortality was also caused by superparasitism was not achievable. However, a second step of the study has been planned to ascertain whether superparasitism influenced high host mortality levels reported herein.

1. IV) I have suggested additional improvements to the English (see scanned manuscript). The authors should be aware that you cannot state that two values were "significantly equal" or "significantly similar" because statistical tests are designed to test for DIFFERENCES (i.e., reject the null hypothesis). If values were not significantly different, just state that they were similar (not significantly similar).

Response: Changes in the text according to the reviewer's suggestion were made.