Combined Effect of Sterile Insect Technique and Augmentative Biological Control Use for Ceratitis capitata Control Under Field Cage Conditions

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

See attached file.

Comments for author File: Comments.pdf

Author Response

Reviewer #1

Detailed comments are given below. The more important points that require revision include: • The information give about the number and duration of replicates (section 2.4) is confusing. This needs re-writing to improve clarity. • Much more serious is the treatment of individual sampling units in the same cage as replicates. This is a case of pseudoreplication, as the units are not independent of one another. The authors should re-analyze the data using a cage as a replicate. In my opinion, the paper is not publishable until this re-analysis is performed.

Response. Thank you for your comments, which will improve the clarity of the paper. We changed several parts of the main text. We re-analyzed the data and applied mixed models to account for the different experimental units. These models explicitly account for the repetitions of the five experimental devices within each field cage, i.e., nested models consistent with the experimental design. We have added the corresponding new descriptions and results.

-Introduction ** 2nd paragraph – sentence starting “Interestingly,…” is confusing at the end. What is meant by “accounting for 29% of both methods”? How can biological control account for any proportion of SIT? Re-write to clarify.

Response. The reviewer is right, so we decided to delete the last part of the sentence (“accounting for 29% of both methods”).

 

-Materials and Methods; Section 2.1

** Sentence starting “This rearing was…” is confusing. How about – The biparental strain was reared primarily to study biological…

Response. The reviewer's suggestion is good, but we decided to remove the sentence “This rearing was established to study biological traits of the parasitoid D. longicaudata” and replace it as follows: “The Cc_bip strain has been reared for one year at the San Juan biofactory prior to this study”.

**220 pairs per 100 cm2 – provide the approximate total numbers of wasps per cage based on this wasp/area relationship.

Response. The reviewer's suggestion is good, so the sentence was changed to the following: “The D. longicaudata colony was mass-reared inside 60 × 60 × 30-cm iron-framed, mesh-covered cage with a capacity to hold about 8,000 pairs”.

 

**Both the bisexual strain of medfly and the wasp strain were used after long-term rearing in the lab. You note that the wasps had been reared 65-67 generations in the lab when tested. I suggest giving the same information for the bisex medfly strain (a quick look ahead and I could not find the actual dates when the tests were run. I assume 2025??). Also, I suggest addressing the possibility that the use of long-adapted lab strain may have affected your results.

Response.

-The reviewer's comment led us to review the data on the number of generations of the parasitoid population line used in the trials, and also to clarify aspects of parasitoid rearing:

1) Parasitoids used in the trials were reared on the tsl genetic sexing Vienna-8 C. capitata strain (Cc_tsl strain). This was written on page 7, lines 153-154 under the subheading “2.1. Source of Insect and Rearing Procedures” in the original version submitted to the journal;

2) Instead of using parasitoids from 65-67^th generations, we used parasitoids from 60-61^th generations (there is no significant difference with the previous data, but 60-61 ^th generations is correct);

3) The C. capitata San Juan biparental strain (Cc_bip strain) used in trials had 12 generations reared under laboratory conditions;

4) The tests were carried out between late January and late March 2019 (summer); This information was written on page 11, lines 237-238 under the subheading “2.4. Experimental Procedure” in the original version submitted to the journal. However, this information was moved to section 2.2 of the new version of the manuscript.

-The text was corrected in terms of the number of parasitoid generations used in the trials, and information was added about how long the colony of the C. capitata biparental strain had been reared when the trials were conducted, as requested by the reviewer.

-As the reviewer suggests, it is possible that the prolonged parasitoid rearing time under laboratory conditions may have affected biological results. However, we cannot precisely know whether this occurred, as we do not have previous data from significantly younger generations for comparison.

 

Section 2.2

**Provide dates of your field work

Response. It was done.

 

Section 2.3

** 1 st sentence insert “cage” after “iron-framed”

Response. It was done.

 

** delete “along its middle part” – not needed

Response. It was done.

** delete “Inside and” – start sentence “In the center of each…”

Response. It was done.

 

** delete word “longitudinal” – not needed

Response. It was done.

** delete “covering the diameter of the cage” – not needed. End sentence after “from each other”

Response. It was done.

 

** should DFD really be FED?

Response. DFD was a typing error; the correct term is FED. It has been corrected.

 

** change to “…placed next to each other on the…”

Response. It was done.

 

** I feel that the use/purpose of the figs needs a bit more explanation, especially for readers not familiar with fruit fly/parasitoid biology. The term “trap fruit” requires more explanation – what is being trapped? Why?.

Response. A more detailed explanation was provided on the selection of figs for use in oviposition devices. It was decided to remove the term “trap fruit” because it leads to confusion. Therefore, the original sentence “The fig was used as a “trap fruit” because it is one of the most important medfly hosts throughout Argentina [52]; likewise, its suitable size and weight favors handling for use in the FED”, was changed by “The fig was used in the FEDs because it is one of the most important medfly hosts throughout Argentina [52]. The fig is a medfly host fruit with advantageous qualities for the oviposition by larval parasitoids, such as small size, thin skin, and shallow pulp [23]. These fig traits allow the D. longicaudata female to easily drill through to the pulp while the medfly larvae feed [20]”.

 

Section 2.4 ** why was the 4:1 wasp:fly ratio chosen? Does this ratio correspond to ratios used/achieved in Argentina?

Response. This ratio was used based on previous trials conducted under field cages conditions at an experimental level (unpublished data) in the ground of the Plant Health Directorate of the province of San Juan. A parasitoid-to-fly ratio of 4:1 was chosen because no significant differences were observed with ratios of 5:1 and 6:1. The release values of D. longicaudata under open field conditions carried out in fruit-growing areas of San Juan were higher than 4:1 (Sánchez et al. 2016. http://dx.doi.org/10.1016/j.biocontrol.2016.08.002).

** why was the 100:1 sterile:fertile male ratio chosen? Does this ratio correspond to ratios used/achieved in Argentina?.

Response. A sterile-to-fertile ratio of 100:1 was chosen for two reasons: 1) Proposed release rates according to working area and aimed ratio sterile to wild for Ceratitis capitata recommended by the IAEA manual (Sterile Insect Release Density Calculations Spreadsheet. FAO/IAEA, Programme of Nuclear Techniques, Vienna, Austria, 2019, p. 30) for suppression in a releasing area, where the sterile/wild ratio varies between 50:1 and 150:1; 2) the sterile/wild ratio used for SIT by the Fruit Fly Control and Eradication Program in the province of San Juan (unpublished data).

** the sentence “Only fruits located at height within the field cage were used” is confusing. What is meant by “at height”? I thought all figs were placed at the same height (more or less).

Response. The sentence was confusing to the reviewer, so we rewrote it as follows: In this study, only fruits located high up in the field cage and not located on the ground were used. This was done because D. longicaudata females have shown a similar ability for foraging on fallen infested fruit or fruit still located in the tree canopy [53].

** 24 replicates per treatment and control? Do you mean 24 replicates that include all 3 treatments and the control? As stated, with each replicate lasting 12 days, a total of 288 days (= 24 replicates x 12 days per replicate) would be required to complete testing per treatment.

Response. Yes, 24 replicates were performed, but this did not involve a total of 288 days; this is because four replicates were performed for each treatment and for the control on each testing date. Therefore, the study lasted 72 days. A new sentence was added to the text stating that four replicas were performed for each treatment and for the control on each testing date.

** As written the word “These” appears to refer to “new non-infected fruits and wheat bran”. Re-word to clarify.

Response. The reviewer is correct, so the sentence was modified as follows: “Once the week was over, the figs were then dissected to recover live larvae…”

 

-Section 2.5 ** As written, you computed benefit and effectiveness both for (i) each FED within a cage and the (ii) entire cage, is that correct? If so, what was the purpose of making calculations for each FED? It seems the cage itself would be the experimental unit of interest.

Response. Thank you very much for your comment, which will allow us to improve the repeatability of the study. To fully address your concerns, we have made substantial changes to sections 2.4 and 2.5. Indeed, the food dispensing devices (FEDs) were exposed within each cage, and the cage is considered a sampling unit (the basic unit from which data are collected). Maintaining the data per FED allows us to estimate variance components within the cages, thus enabling quantitative diagnoses of the control techniques. Understanding the magnitude of variation in the variables involved in the management of living organisms is crucial. This enables accurate predictions of production processes expected to result from the experimentation. This point is especially important in human-mediated biotic interaction systems, where we can measure our experimental actions precisely. Therefore, the data for each response descriptor are used at their lowest level. The proportion of benefit was calculated per device. This generates the necessary repetitions to determine variation within each cage, which is the unit of application for each treatment. Alternatives such as using an arithmetic mean or a median flatten the variation and focus on a measure of central tendency. However, it is equally or even more important to consider the potential degree of uncertainty in the natural system, which depends on explicitly considering the variation in the statistical model since an operational field design. We hope that the changes made will allow the cage to be recognized as an experimental unit, as we interpret the reviewer's suggestion, and clarify any doubts about the methods.

 

Based on the reviewer's comment, we have decided to make a substantial change to the text of Section 2.4: Experimental procedure, as follows (New version): “The experimental design considered a cage as the application unit of one of the following treatment levels: (1) treatment BC, involved only the Biological Control through the release of the parasitoid D. longicaudata; (2) treatment SIT, involved only the SIT through the release of radiation-sterilized males from Cc_tsl strain; (3) treatment SITBC, involved both previous techniques; and (4) Control, in which neither parasitoids or sterile male flies were released (S4). Each experimental level was randomly applied to a cage. Six trials (dates) were run, and in each trial, four cages were used to apply each treatment level. In summary, five FEDs were used as sampling units within each cage, four cages per treatment level were run within each trial, and each trial was repeated six times. Therefore, 24 repetitions were performed for both treatment and control levels. On the first day of each trial, one hundred pairs of 2-d-old non-mated fertile females and males from the Cc_bip strain were released into all cages (N = 16). Except in the Control, in both SIT and SITBC, sterile males of the Cc_tsl strain were released simultaneously with fertile adults. The number of medfly sterile males released was 10,000 per cage, and per release date, i.e., 100 sterile males were used per 1 fertile male. In the BC and SITBC, D. longicaudata individuals were released five days after the previous fertile fly release. Four hundred pairs of 3-d-old adult parasitoids were released per cage. Parasitoid-to-fly ratio was 4 D. longicaudata females per 1 fertile female medfly. Parasitoids foraged during 7 days”.

** delete sentence “Effectiveness was directly…” This seems like a result, not a method.

Response. The sentence was removed.

** “Calculation was performed for each experimental repetition”. This is confusing – do you mean a separate GLM was run those replicates (across treatments) that were performed at the same time? This doesn’t make sense as it would seem a GLM would include final data (= all replicates) from all treatments. Indeed, I just saw that a few lines later you write “All data were used for these analyses..”. This is confusing; clarification is needed.

Response. Based on the reviewer's comment, we have decided to make a substantial change to the text of Section 2.5: Data Analysis, as follows (New version): “A statistical summary was performed for each of the following parameters: number of medfly puparia recovered, number of non-hatching puparia, and number of emerged flies and parasitoids recovered from the fruit. The minimal calculation unit was each of five FEDs within each cage. For practical reasons, the data analysis was focused on two response parameters of the fruit-fly management portfolio: (1) the proportion of benefit, and (2) the effectiveness of Abbott. The following formula calculates the proportion of benefit for each FED = (number of non-hatched puparia + number of emerged parasitoids ) / ( total number of puparia recovered ). The proportion of benefit estimates the expected utilities of each applied technique in terms of pest suppression. Also, considering the calculus at the FED level allows accounting for the variation in the response variables within each field cage (variance components). For Abbott's effectiveness, we used the corrected mortality formula = C – T/C × 100% = (1 – T/C ) × 100%, in which C is the observed number of surviving insects in the Control level, and T is the observed number of surviving insects in the treatment level. The correction is justified because C – T is the observed number of insects killed due to treatment (= T), and this can be stated as a proportion of the natural population survival (= C) [54]. Given that Abbott’s effectiveness is a measure of the effect size of a given treatment level with respect to the control one, we used each cage as the minimal sampling unit of effectiveness. Thus, in this case, it is not possible to account for the variation within cages, as the experimental procedure allowed for the proportion of benefit, but only for the variation between treatments. Untransformed data for statistical analyses were used. To analyze the variation in the proportion of benefit, we ran a general linear mixed model with normal error fitted by REML [55]. In the model, FED sampling units were nested within each cage, and each cage was nested within each treatment within each trial. To analyze the variation in effectiveness, we ran a general linear mixed model with normal error. In the model, cages were nested within each treatment within each trial. Tukey's HSD test was used for post hoc comparisons once each global model was performed. Data analysis was performed using R-software version 4.5.1 [56] via RStudio 2025.09.2+418. RMarkdown scripts are available at https://github.com/maordano/Ceratitis-field-experiment.”

 

Results Section 3.2 ** 1st paragraph – given the df of 3, 476 it seems that each FED was treated as a replicate (24 replicates per treatment [n = 4] with 5 FED per relicate = 24 x 4 x 5 = 480; 480-4 = 476). However, it seems that treating each FED as a replicate is a case of pseudo-replication, since the 5 FED in the same cage were not independent units from a statistical sense. The FED in the same cage are multiple measurements (sub-samples) in the same experimental unit. This inflates the sample size and may lead to incorrect conclusions. The authors should run the test with data pooled across FEDs in the same cage. Also, again, it seems impossible to conduct 24 replicates per treatment with each lasting 12 days in the span of 2 months or so. Am I missing something here?.

Response. As mentioned in other responses in this review, we have completely changed the description of the experimental design and analysis (sections 2.4 and 2.5). Regarding the specific point about the degrees of freedom, we have addressed this by presenting a new analysis and have also changed the results, including those in section 3.2. 

-** 2nd paragraph – it is not obvious to me why the denominator in the F test is 690. Could you explain that?.

Response. We have changed the text of this section and reported the corresponding degrees of freedom of the new analysis.

Reviewer 2 Report

Comments and Suggestions for Authors

Abstract:

The article entitled “Combined Effect of Sterile Insect Technique and Augmentative Biological Control Use for Ceratitis capitata Control under Field Cage Conditions” highlights the importance of integrating the Sterile Insect Technique (SIT) and the solitary larvopupal endoparasitoid Diachasmimorpha longicaudata under semi-field conditions in San Juan, Argentina. The authors compared four treatments: (I) control, (II) parasitoid alone, (III) sterile males alone, and (IV) the combination of both techniques. The trials were conducted from January to April 2019 in field cages using figs as an oviposition substrate. The number of pupae recovered, unemerged pupae, emerged adults, and Abbott's efficacy were recorded. The combined strategy showed an additive effect, achieving 96 % population suppression, a value considered close to near eradication. These findings support the integrated use of both techniques in large-area management programs against the Mediterranean fruit fly.

 However, some issues identified in the article need to be addressed and are described as minor (including formatting issues previously submitted through the platform) and major. The following are the major issues:

 1. Clarity and statistical interpretation in the Results section:

 In the Results section, the interpretation of Table 1 presents substantial deficiencies from a statistical and editorial point of view. First, the separation of means using superscript letters (a, b, c, etc.) derived from a multiple comparison test (e.g., Tukey, Duncan, LSD, or another appropriate post hoc test) is not specified. The omission of this feature makes it impossible to accurately determine which treatments differ significantly from each other and at what significance level (α).

 In experimental studies with multiple treatments, simply presenting means and measures of dispersion (± standard error or standard deviation) is insufficient to support comparative conclusions. It is essential to explicitly state the statistical procedure used, the value of α considered, and how the letters indicating homogeneous groupings were assigned. Without this information, the interpretation of the results may be ambiguous or even lead to erroneous conclusions that do not reflect the true potential of the study.

 Likewise, the presentation of the maximum values generates conceptual and statistical confusion. It is not specified whether these values correspond to individual replicates, outliers, the maximum observed within a confidence interval, or estimates derived from the statistical model used. The inclusion of maximum values without a clear methodological explanation can distort the perception of the actual variability of the data and affect the biological interpretation of the results, confusing readers.

 2. Need for explanatory notes and greater clarity in Tables 2 and 3:

 Tables 2 and 3 require the addition of explanatory footnotes to facilitate their independent interpretation. For the Journal Agronomy (Q1), tables should be self-explanatory, without requiring the reader to constantly refer to the Materials and Methods section to understand abbreviations, units of measurement, statistical analysis criteria, or operational definitions. Therefore, it is necessary to specify:

 

The number of replicates (n), even if these have already been described in the Materials and Methods section; the type of statistical analysis applied; the significance level used; and the meaning of any added abbreviations.

 Additionally, the column organization should be reviewed to ensure coherence and terminological consistency. When analyzing the results, it is necessary to include other studies, for example, a variable that determines the combined potential of using the Sterile Insect Technique (SIT) and the parasitoid D. longicaudata. For this, I suggest a study that measures its impact, such as herbivory, by calculating the percentage of crop damage using the same treatments described in this article. This will allow for more precise conclusions and an estimation of the true impact of combining these techniques.

 3. General character of the Discussion:

 The Discussion section presents a broad argumentative development but lacks sufficient depth from an analytical point of view. A general exposition of background information predominates, without a detailed critical analysis of the findings obtained in the study. To minimize this shortcoming, I suggest: I) Specifically comparing the data obtained with previous studies, pointing out similarities, discrepancies, and possible causes; II) Acknowledging the methodological limitations of the study and their potential impact on the interpretation of the results; III) Outlining practical implications and future research directions, especially in Argentina where the study was conducted; V) Discuss with sufficient rigor the possible ecological interactions between the combined techniques and the environmental factors that could modulate their effectiveness under real field conditions.

 4. Incorporation of line numbering:

 The absence of line numbering considerably hindered the peer review process. Numbering is a very important element in manuscripts submitted for scientific evaluation, as it allows us, as reviewers, to precisely indicate observations, corrections, or suggestions.

 

Author Response

Reviewer #2

The article entitled “Combined Effect of Sterile Insect Technique and Augmentative Biological Control Use for Ceratitis capitata Control under Field Cage Conditions” highlights the importance of integrating the Sterile Insect Technique (SIT) and the solitary larvopupal endoparasitoid Diachasmimorpha longicaudata under semi-field conditions in San Juan, Argentina. The authors compared four treatments: (I) control, (II) parasitoid alone, (III) sterile males alone, and (IV) the combination of both techniques. The trials were conducted from January to April 2019 in field cages using figs as an oviposition substrate. The number of pupae recovered, unemerged pupae, emerged adults, and Abbott's efficacy were recorded. The combined strategy showed an additive effect, achieving 96 % population suppression, a value considered close to near eradication. These findings support the integrated use of both techniques in large-area management programs against the Mediterranean fruit fly.  However, some issues identified in the article need to be addressed and are described as minor (including formatting issues previously submitted through the platform) and major. The following are the major issues:

1. Clarity and statistical interpretation in the Results section. In the Results section, the interpretation of Table 1 presents substantial deficiencies from a statistical and editorial point of view. First, the separation of means using superscript letters (a, b, c, etc.) derived from a multiple comparison test (e.g., Tukey, Duncan, LSD, or another appropriate post hoc test) is not specified. The omission of this feature makes it impossible to accurately determine which treatments differ significantly from each other and at what significance level (α). In experimental studies with multiple treatments, simply presenting means and measures of dispersion (± standard error or standard deviation) is insufficient to support comparative conclusions. It is essential to explicitly state the statistical procedure used, the value of α considered, and how the letters indicating homogeneous groupings were assigned. Without this information, the interpretation of the results may be ambiguous or even lead to erroneous conclusions that do not reflect the true potential of the study. Likewise, the presentation of the maximum values generates conceptual and statistical confusion. It is not specified whether these values correspond to individual replicates, outliers, the maximum observed within a confidence interval, or estimates derived from the statistical model used. The inclusion of maximum values without a clear methodological explanation can distort the perception of the actual variability of the data and affect the biological interpretation of the results, confusing readers.

Response. We have updated the Experimental Procedure, Data Analysis, and corresponding Results, including the tables, based on the review suggestions. A new explanatory title was provided for Table 1 as follows:

Table 1. Summary of descriptive statistics (mean ± SD, median, and maximum value) of the number of medfly puparia, non-hatching puparia, emerged flies and parasitoids from the fruit-bearing sentinel devices (FED, N = 480 = 5 FEDs x 4 cages x 4 treatment levels x 6 trials) used in the following treatments: Biological Control alone (BC), Sterile Insect Technique alone (SIT), both methods combined (SITBC), and Control (N = 120 per treatment level). NA, not available (parasitoids were released only in BC treatments).

 

2. Need for explanatory notes and greater clarity in Tables 2 and 3. Tables 2 and 3 require the addition of explanatory footnotes to facilitate their independent interpretation. For the Journal Agronomy (Q1), tables should be self-explanatory, without requiring the reader to constantly refer to the Materials and Methods section to understand abbreviations, units of measurement, statistical analysis criteria, or operational definitions. Therefore, it is necessary to specify: The number of replicates (n), even if these have already been described in the Materials and Methods section; the type of statistical analysis applied; the significance level used; and the meaning of any added abbreviations. Additionally, the column organization should be reviewed to ensure coherence and terminological consistency. When analyzing the results, it is necessary to include other studies, for example, a variable that determines the combined potential of using the Sterile Insect Technique (SIT) and the parasitoid D. longicaudata. For this, I suggest a study that measures its impact, such as herbivory, by calculating the percentage of crop damage using the same treatments described in this article. This will allow for more precise conclusions and an estimation of the true impact of combining these techniques.

Response. We have updated the Experimental Procedure, Data Analysis, and corresponding Results, involving tables and figures, based on the review suggestions. New explanatory titles were provided for Table 2 and 3 as follows:

Table 2. Tukey's HSD post-hoc test comparisons of the benefit proportion between experimental and control groups. Post-hoc comparisons were applied after fitting the mixed-effects model by REML. Adjusted p-values considered spurious probabilities (N = 120 FEDs per experimental group (N = 5 FEDs x 4 cages x 6 trials).

Table 3. Tukey's HSD post-hoc test comparisons of Abbott’s effectiveness between experimental and control groups. Post-hoc comparisons were applied after fitting the mixed-effects model by REML. Adjusted p-values considered spurious probabilities (N = 120 FEDs per experimental group (N = 5 FEDs x 4 cages x 6 trials).

 

 -3. General character of the Discussion: The Discussion section presents a broad argumentative development but lacks sufficient depth from an analytical point of view. A general exposition of background information predominates, without a detailed critical analysis of the findings obtained in the study. To minimize this shortcoming, I suggest: I) Specifically comparing the data obtained with previous studies, pointing out similarities, discrepancies, and possible causes; II) Acknowledging the methodological limitations of the study and their potential impact on the interpretation of the results; III) Outlining practical implications and future research directions, especially in Argentina where the study was conducted; V) Discuss with sufficient rigor the possible ecological interactions between the combined techniques and the environmental factors that could modulate their effectiveness under real field conditions.

Response. We believe that the reviewer made a useful critique to improve the discussion, so we have restructured it based on the reviewer's suggestions. Therefore, the discussion was rewritten as it appears in the new version.

 

4. Incorporation of line numbering: The absence of line numbering considerably hindered the peer review process. Numbering is a very important element in manuscripts submitted for scientific evaluation, as it allows us, as reviewers, to precisely indicate observations, corrections, or suggestions.

Response. The original version of the manuscript submitted to the journal had line numbering; the system probably removed the numbering when editing the version in journal format. I think that is how it happened, but we complied with the rule of submitting a manuscript with continuous line numbers throughout the text.

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript evaluated the combined application of the Sterile Insect Technique and augmentative releases of D. longicaudata for the suppression of C. capitata under controlled conditions. The study is well described and demonstrates that integrating both control techniques results in higher reduction in medfly occurrence. Nevertheless, the experimental contribution is limited, as the observed additive effect could be anticipated when two established control methods are applied concurrently. Although the findings support ongoing area-wide management programs in Argentina, the manuscript would be strengthened by a more thorough discussion of its novelty, scalability, and implications for operational implementation.

The figures and tables presented are confusing because the authors mention performing Tukey's multiple-comparisons test, yet this is not reflected in the data. Table 2 shows the statistics obtained from this test; however, it would be better to present the average values and other relevant statistics, along with their standard deviations and the groups identified in Tukey's test. Similarly, the figures are confusing because they contain letters that possibly represent statistical groups derived from Tukey's test, but it is not specified whether the lines represent error bars, standard deviations, etc., nor is the meaning of the letters explained.

Author Response

Reviewer #3

-This manuscript evaluated the combined application of the Sterile Insect Technique and augmentative releases of D. longicaudata for the suppression of C. capitata under controlled conditions. The study is well described and demonstrates that integrating both control techniques results in higher reduction in medfly occurrence. Nevertheless, the experimental contribution is limited, as the observed additive effect could be anticipated when two established control methods are applied concurrently. Although the findings support ongoing area-wide management programs in Argentina, the manuscript would be strengthened by a more thorough discussion of its novelty, scalability, and implications for operational implementation.

Response. The reviewer made a useful critique to improve the manuscript; so the Results and Discussion sections were rewritten as they appear in the new version of the manuscript. The reviewer stated that “the experimental contribution is limited, as the observed additive effect could be anticipated when two established control methods are applied concurrently”. In the discussion of this manuscript, reference is made to two previously published field cage studies in which all or part of the trials did not provide an additive effect when combining the release of parasitoids and sterile flies. A discussion of possible causes was held.

-The figures and tables presented are confusing because the authors mention performing Tukey's multiple-comparisons test, yet this is not reflected in the data. Table 2 shows the statistics obtained from this test; however, it would be better to present the average values and other relevant statistics, along with their standard deviations and the groups identified in Tukey's test. Similarly, the figures are confusing because they contain letters that possibly represent statistical groups derived from Tukey's test, but it is not specified whether the lines represent error bars, standard deviations, etc., nor is the meaning of the letters explained.

Response. Titles of the figures and tables were improved to be more explanatory and contain more data as follows:

Figure 1. Variation in the Proportion of Benefit recorded from experimental field cage conditions in Tulum fruit-growing valley, San Juan, central-western Argentina. BC: Biological Control alone, SIT: Sterile Insect Technique alone, SITBC: both methods combined, and Control (any previous treatment applied). Box-plots depict the median (horizontal line inside the box), interquartile range Q1–Q3 (bottom and top ends of the box), dispersion (whiskers depicting 1.5 times the IQ range), and extreme values (points). Different letters depict significant differences between experimental groups (Tukey post-hoc comparisons, p < 0.05, N = 120 per group) after fitting a mixed-effects model conditional on fruit-bearing device variation within cage, within treatment, within trial (F _{(3, 87)} = 252.46, p < 0.00001, conditional R² = 0.756).

Figure 2. Variation in Abbott’s effectiveness from experimental field cage conditions in Tulum fruit-growing valley, San Juan, central-western Argentina. BC: Biological Control alone, SIT: Sterile Insect Technique alone, SITBC: both methods combined, and Control (any previous treatment applied). Box-plots depict the median (horizontal line inside the box), interquartile range Q1–Q3 (bottom and top ends of the box), dispersion (whiskers depicting 1.5 times the IQ range), and extreme values (points). Different letters depict significant differences between experimental groups (Tukey post-hoc comparisons, p < 0.05, N = 24 per group) after fitting a mixed-effects model conditional on field cage variation, within treatment, within trial (F _{(2, 64)} = 49.671, p < 0.00001, conditional R² = 0.676).

Table 1. Summary of descriptive statistics (mean ± SD, median, and maximum value) of the number of medfly puparia, non-hatching puparia, emerged flies and parasitoids from the fruit-bearing sentinel devices (FED, N = 480 = 5 FEDs x 4 cages x 4 treatment levels x 6 trials) used in the following treatments: Biological Control alone (BC), Sterile Insect Technique alone (SIT), both methods combined (SITBC), and Control (N = 120 per treatment level). NA, not available (parasitoids were released only in BC treatments).

Table 2. Tukey's HSD post-hoc test comparisons of the benefit proportion between experimental and control groups. Post-hoc comparisons were applied after fitting the mixed-effects model by REML. Adjusted p-values considered spurious probabilities (N = 120 FEDs per experimental group (N = 5 FEDs x 4 cages x 6 trials).

Table 3. Tukey's HSD post-hoc test comparisons of Abbott’s effectiveness between experimental and control groups. Post-hoc comparisons were applied after fitting the mixed-effects model by REML. Adjusted p-values considered spurious probabilities (N = 120 FEDs per experimental group (N = 5 FEDs x 4 cages x 6 trials).

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The article entitled “Combined Effect of Sterile Insect Technique and Augmentative Biological Control Use for Ceratitis capitata Control under Field Cage Conditions” has substantially improved based on the previously provided comments. However, several additional modifications are still required:

 

In the Abstract section (line 1), please remove the capital letters and include the acronym so that it reads: “sterile insect technique (SIT)”.

 

On page 3 of the Introduction, in the second line, the text appears as “control[36]”. Please add a space so that it reads “control [36]”.

 

In the Materials and Methods section, subsection “2.3. Field cage setup”, the scientific name "Fraxinus americana" appears without italics. Please format it in italics.

 

In line 17 of subsection “2.3. Field cage setup” (Materials and Methods), the unit appears as 2000-ml. Please capitalize the letter “L” so that it reads “2000-mL”.

 

In line 10 on page 5, within subsection “2.4. Experimental Procedure”, the phrase “On the” appears in blue font; please change it to black.

 

On page 7, subsection “3.2. Treatment effects”, Figure 1 is positioned too far from the corresponding text. The same issue occurs with Figure 2 on page 8.

 

In the Discussion section (page 9), the scientific name Anastrepha ludens (Loew) should be written in its complete form as: Anastrepha ludens (Loew, 1873) (Diptera: Tephritidae).

 

In the Discussion section (page 10), the scientific name Psyttalia fletcheri (Silvestri) (Hymenoptera: Braconidae) should include the year of description: Psyttalia fletcheri (Silvestri, 1916) (Hymenoptera: Braconidae).

 

In the Discussion section (page 10), the scientific name Bactrocera cucurbitae (Coquillet) should be written in full as: Bactrocera cucurbitae (Coquillett, 1899) (Diptera: Tephritidae).

 

Finally, in the Discussion section (page 10), the scientific name Fopius arisanus (Sonan) should include the year of description: Fopius arisanus (Sonan, 1932), as well as for Diachasmimorpha kraussii (Fullaway, 1951) and Diachasmimorpha tryoni (Cameron, 1911). In addition, the last two species names are misspelled in the manuscript; the correct forms are provided here.

Comments for author File: Comments.pdf

Author Response

Reviewer # 2.

The article entitled “Combined Effect of Sterile Insect Technique and Augmentative Biological Control Use for Ceratitis capitata Control under Field Cage Conditions” has substantially improved based on the previously provided comments. However, several additional modifications are still required:

Response: I am deeply grateful to the reviewer for their valuable comments and corrections, which have helped to produce a clearer, higher-quality manuscript.

-The English could be improved to more clearly express the research

Response: It was done.

-In the Abstract section (line 1), please remove the capital letters and include the acronym so that it reads: “sterile insect technique (SIT)”.

Response: It was done.

-On page 3 of the Introduction, in the second line, the text appears as “control[36]”. Please add a space so that it reads “control [36]”.

Response: It was done.

In the Materials and Methods section, subsection “2.3. Field cage setup”, the scientific name "Fraxinus americana" appears without italics. Please format it in italics.

Response: It was done. Note: the species Fraxinus americana was originally written in italics, but there is an editing issue in the journal format that changes the entire paragraph in subsection “2.3. Field cage setup” to italics, leaving the species in regular font.

-In line 17 of subsection “2.3. Field cage setup” (Materials and Methods), the unit appears as 2000-ml. Please capitalize the letter “L” so that it reads “2000-mL”.

Response: It is correct to change ml to mL, given that mL (with a capital L) is  preferred in scientific contexts. Therefore, I have reviewed the text and made required changes. I appreciate the reviewer's useful comment. Changes were made as follows: “125-mL plastic cups…”, “2000-mL plastic bottle…”, “in 500-mL plastic containers with…”, “250-mL plastic cups with fresh…”

-In line 10 on page 5, within subsection “2.4. Experimental Procedure”, the phrase “On the” appears in blue font; please change it to black.

Response: It was done. It was my mistake when correcting the original manuscript.

-On page 7, subsection “3.2. Treatment effects”, Figure 1 is positioned too far from the corresponding text. The same issue occurs with Figure 2 on page 8.

Response: The gaps between the text and both figures (1 and 2) were corrected; spacing was adjusted based on manuscript format requirements.

-In the Discussion section (page 9), the scientific name Anastrepha ludens (Loew) should be written in its complete form as: Anastrepha ludens (Loew, 1873) (Diptera: Tephritidae).

Response: It was done.  The reviewer is right; it is more complete to provide the scientific name with its author and year of original description. For this reason, the years of authorship for C. capitata and D. longicaudata were also included in the Introduction section: Ceratitis capitata (Wiedemann, 1824), Diachasmimorpha longicaudata (Ashmead, 1905). In addition, the parasitoid Coptera haywardi (Ogloblin, 1944) was written with the author's name corrected and the year of original description.

-In the Discussion section (page 10), the scientific name Psyttalia fletcheri (Silvestri) (Hymenoptera: Braconidae) should include the year of description: Psyttalia fletcheri (Silvestri, 1916) (Hymenoptera: Braconidae).

Response: It was done. Thank you for reviewing the species names.

-In the Discussion section (page 10), the scientific name Bactrocera cucurbitae (Coquillet) should be written in full as: Bactrocera cucurbitae (Coquillett, 1899) (Diptera: Tephritidae).

Response: It was done. Thank you for reviewing the species names.

Finally, in the Discussion section (page 10), the scientific name Fopius arisanus (Sonan) should include the year of description: Fopius arisanus (Sonan, 1932), as well as for Diachasmimorpha kraussii (Fullaway, 1951) and Diachasmimorpha tryoni (Cameron, 1911). In addition, the last two species names are misspelled in the manuscript; the correct forms are provided here.

Response: It was done. I would like to thank the reviewer for correcting the names of the braconid species and authors; this is a very important detail, which I did not check in the previous version.

In addition, general corrections were made to the text and the list of references (year of publication of each paper in bold and species names in italics).