From Factory to Field: Sex Pheromone of Plutella xylostella Produced in Yeast Cell-Factories Validated in Laboratory and Field Trials

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors of this manuscript describe the effectivity of yeast-derived pheromone blends against DBM population. The manuscript is interesting and in my opinion, the main concern about the study is the novelty of the use of sex pheromone synthetized by biotechnological production.

 I believe it is a valuable paper, and it represents a relevant scientific contribution within the scope of Insects journal. Nevertheless, there are few questions and corrections before the manuscript should be accepted for publications. I therefore recommend its publication with a few minor corrections.

The issues that need to be addressed are listed below:

1. The Materials and Methods section should be expanded with a “2.1 Chemicals” which to include a detailed description of the chemicals used. Currently, there is no information regarding the specific compounds and formulations employed in either the laboratory or field tests that should be critical to get reproducible results.
2. The manuscript lacks a detailed section on the GC-MS characterization of the pheromone components. Without this data, it is unclear how the elution orders, retention times or the correct pheromone mixture ratios were established. The authors should provide the specific GC-MS conditions to validate the identity of each compound.
3. The figure notes (Figure 1, figure 3, Figure 6……) do not provide sufficient information about the statistics used or the level of significance. It would be advisable to include this information.
4. Figure 5. “Mean time (sec) spent by DBM males….. ” No time information appears on the X axis.
5. In Figure 3, please include the standard deviations, which give us an idea about the bioassay test variability. Results should be displayed as the mean ± SD

1. Line 327-328:  “However, the male catches in BIO baited traps were 327 significantly lower than those …..”  This reported finding are not supported by statistical (e.g., p-values, ANOVA, …..) tests on Figure 6.

Author Response

Reviewer #1:
The authors of this manuscript describe the effectivity of yeast-derived pheromone blends against DBM population. The manuscript is interesting and in my opinion, the main concern about the study is the novelty of the use of sex pheromone synthetized by biotechnological production.

 I believe it is a valuable paper, and it represents a relevant scientific contribution within the scope of Insects journal. Nevertheless, there are few questions and corrections before the manuscript should be accepted for publications. I therefore recommend its publication with a few minor corrections.

The issues that need to be addressed are listed below:

1. The Materials and Methods section should be expanded with a “2.1 Chemicals” which to include a detailed description of the chemicals used. Currently, there is no information regarding the specific compounds and formulations employed in either the laboratory or field tests that should be critical to get reproducible results.

The chemicals tested are listed in Materials and Methods, section 2.2, electrophysiological responses, which also details their origin, preparation, and how they were presented to the male antennae. We opted to include the chemicals in this section rather than adding a separate one, believing that this will help readers better connect the chemicals with the methodology.

2. The manuscript lacks a detailed section on the GC-MS characterization of the pheromone components. Without this data, it is unclear how the elution orders, retention times or the correct pheromone mixture ratios were established. The authors should provide the specific GC-MS conditions to validate the identity of each compound.

The yeast-derived compounds tested in this work were provided by BioPhero, a partner in the two HORIZON 2020 projects (OLEFINE and PHERA) which funded the research. Identification of minor compounds was carried out by BioPhero as part of a five-batch analysis required for Active Ingredient registration. In the present study, we performed GC analyses comparing the yeast-derived compounds with their chemically synthesized standards that were also tested. We did not investigate the physiological or behavioral effects of individual minor compounds, as we saw that at the concentrations tested, the mixture does not influence insect behavior—contrary to findings we have reported for Ostrinia nubilalis using yeast-derived pheromones (see penultimate paragraph of the Discussion in the current ms).

3. The figure notes (Figure 1, figure 3, Figure 6……) do not provide sufficient information about the statistics used or the level of significance. It would be advisable to include this information.

Added. The statistics referred in the text included in the legend

4. Figure 5. “Mean time (sec) spent by DBM males….. ” No time information appears on the X axis.

Added.

5. In Figure 3, please include the standard deviations, which give us an idea about the bioassay test variability. Results should be displayed as the mean ± SD

We included the statistics in the legend

6. Line 327-328:  “However, the male catches in BIO baited traps were 327 significantly lower than those …..”  This reported finding are not supported by statistical (e.g., p-values, ANOVA, …..) tests on Figure 6.

Added in the legend of Figure 6

Reviewer 2 Report

Comments and Suggestions for Authors

This study demonstrated the effectiveness of yeast-derived binary pheromone blend comprising (Z)-11-hexadecenal and (Z)-11-hexadecenyl acetate was as good as conventional synthetic ternary blend (with (Z)-11-hexadecen-1-ol as extra component) by field monitoring trials, in terms of efficiency and specificity for diamondback moth. The yeast-derived mixture contained small amounts of unoxidized (Z)-11-hexadecen1-ol due to incomplete oxidation, which is advantageous as this compound serves as synergist. When it is higher in percentage, as in the BIO blend in 2021, Z11-16:OH raise to over 13%, lead to reduced trap-catch efficacy. 

The methods and experimental design are straightforward and sound. The results are presented in a proper way and provided valuable information to understand the value of biotechnological derived pheromones, which provide theoretical basis for using these pheromones to control many pest species. 

I suggest the paper can be accepted after the authors taking care of the following minor comments.

Line 163-169, the reference stimulus, reference compound, test stimuli, need to be defied in more details. As it is stand now, they are confusing. Perhaps, more detailed descriptions can be added to Figure legend of Fig. 1.

Line 228-234, different statistical tests were used for analyzing different data, the authors may add some descriptions to help the readers to understand the reasons behind.

In Fig. 2, the authors are urged to label the impurities in the GC trace of yeast-derived pheromone blend, there are at least 8 additional peaks need to be labeled, in order to let the reader know and think about how to improve.

Author Response

Reviewer #2:
This study demonstrated the effectiveness of yeast-derived binary pheromone blend comprising (Z)-11-hexadecenal and (Z)-11-hexadecenyl acetate was as good as conventional synthetic ternary blend (with (Z)-11-hexadecen-1-ol as extra component) by field monitoring trials, in terms of efficiency and specificity for diamondback moth. The yeast-derived mixture contained small amounts of unoxidized (Z)-11-hexadecen1-ol due to incomplete oxidation, which is advantageous as this compound serves as synergist. When it is higher in percentage, as in the BIO blend in 2021, Z11-16:OH raise to over 13%, lead to reduced trap-catch efficacy. 

The methods and experimental design are straightforward and sound. The results are presented in a proper way and provided valuable information to understand the value of biotechnological derived pheromones, which provide theoretical basis for using these pheromones to control many pest species. 

I suggest the paper can be accepted after the authors taking care of the following minor comments.

Line 163-169, the reference stimulus, reference compound, test stimuli, need to be defied in more details. As it is stand now, they are confusing. Perhaps, more detailed descriptions can be added to Figure legend of Fig. 1.

We changed the phrase “A reference stimulus” to “A stimulus”, and the “The EAG response to each reference stimulus” to “The EAG response to each stimulus of the reference compound”.

Z7-12:OAc was used solely to normalize the antennal response data obtained from stimulation with the test compounds and their combinations. It is not included in Figure 1, as the aim was to compare the chemically synthesized compounds and their blend with the yeast-derived pheromone blend. However, it is shown in Figure 2 (GC-EAD of DBM).

Line 228-234, different statistical tests were used for analyzing different data, the authors may add some descriptions to help the readers to understand the reasons behind.

Added in the text

In Fig. 2, the authors are urged to label the impurities in the GC trace of yeast-derived pheromone blend, there are at least 8 additional peaks need to be labeled, in order to let the reader know and think about how to improve.

The aim of the present study was to determine whether the mixture of yeast-produced pheromone compounds elicits a behavioral response in DBM males comparable to that of chemically synthesized compounds. The most prominent minor compounds present in the original fermented and purified yeast broth are hexadecanol (16:OH) and Z9-16:OH, which are subsequently oxidized or acetylated, respectively, and are present in their converted forms in the BIO mixture presented to the insects.

Reviewer 3 Report

Comments and Suggestions for Authors

The article by Betsi et al. reports the production of insect pheromones in engineered yeast cell factories as a cost-effective alternative to the chemical synthesis of these lipid molecules. The authors compare chemically synthesized and purified pheromones with those produced via engineered yeast.

The manuscript evaluates the performance of yeast-derived sex pheromone blends for Plutella xylostella using electrophysiological, behavioral, GC-EAD, and field assays. The topic is timely and relevant to sustainable pest management. The multi-level validation approach is a major strength, and the work has clear applied potential. The authors conclude that yeast-derived pheromones elicit responses comparable to chemically synthesized pheromones in the diamondback moth (DBM). However, several concerns should be addressed.

Major Comments

1. Source of pheromone compounds Used in this Study
   It is unclear whether the chemically synthesized and yeast-derived pheromones were newly produced for this study or obtained from prior work. The origin, preparation, and prior validation of these compounds should be explicitly described and properly referenced throughout the manuscript. Even if produced previously, their sourcing and characterization must be clearly stated.
2. Discussion
   The discussion is often descriptive rather than interpretive. Emphasis on interpretation such as ratio-dependent responses (see the point below) could be elaborated.
3. Z11-16:OH concentration-dependent inhibitory effect
   The proposed concentration-dependent inhibitory effect of Z11-16:OH is intriguing but insufficiently developed. Only two levels (≈3% and ≈13%) are discussed. Intermediate concentrations (e.g., 4–5%) would help substantiate a dose-response relationship. At minimum, this limitation should be acknowledged.

Minor Comments

• Correct minor grammatical issues (e.g., “resulting to” → “resulting in”).
• Avoid repeating results in the Discussion section.
• Ensure consistent formatting of chemical nomenclature.

Comments on the Quality of English Language

• Correct minor grammatical issues (e.g., “resulting to” → “resulting in”).
• Avoid repeating results in the Discussion section.
• Ensure consistent formatting of chemical nomenclature.

Author Response

Reviewer #3:
The article by Betsi et al. reports the production of insect pheromones in engineered yeast cell factories as a cost-effective alternative to the chemical synthesis of these lipid molecules. The authors compare chemically synthesized and purified pheromones with those produced via engineered yeast.

The manuscript evaluates the performance of yeast-derived sex pheromone blends for Plutella xylostella using electrophysiological, behavioral, GC-EAD, and field assays. The topic is timely and relevant to sustainable pest management. The multi-level validation approach is a major strength, and the work has clear applied potential. The authors conclude that yeast-derived pheromones elicit responses comparable to chemically synthesized pheromones in the diamondback moth (DBM). However, several concerns should be addressed.

Major Comments

1. Source of pheromone compounds Used in this Study
   It is unclear whether the chemically synthesized and yeast-derived pheromones were newly produced for this study or obtained from prior work. The origin, preparation, and prior validation of these compounds should be explicitly described and properly referenced throughout the manuscript. Even if produced previously, their sourcing and characterization must be clearly stated.

The yeast-produced pheromone compounds were generated by BioPhero as part of two Horizon 2020 projects (OLEFINE and PHERA), in which BioPhero was a partner. The compounds used in the present study originated each from a single batch, one of five produced for regulatory purposes. (A.I. registration) 

Discussion
The discussion is often descriptive rather than interpretive. Emphasis on interpretation such as ratio-dependent responses (see the point below) could be elaborated.

Our study focused on validating yeast-produced pheromones for use against yet another major agricultural pest. We believe that pheromones generated in cell factories represent a revolutionary technology with the potential to transform pest management with pheromones in row crops. Compared to conventionally synthesized pheromones, yeast-based production offers a more sustainable alternative by utilizing renewable resources in fermentation and generating a lower carbon footprint, aligning with the principles of green agriculture. The next logical step is to evaluate their efficacy in mating disruption trials, following the approach we have already implemented for Helicoverpa armigera.

 

2. Z11-16:OH concentration-dependent inhibitory effect
   The proposed concentration-dependent inhibitory effect of Z11-16:OH is intriguing but insufficiently developed. Only two levels (≈3% and ≈13%) are discussed. Intermediate concentrations (e.g., 4–5%) would help substantiate a dose-response relationship. At minimum, this limitation should be acknowledged.

We thank Reviewer #3 for raising this interesting point. The literature reports a wide range of Z11-16:OH concentrations, from 1% to 10%. However, as the aim of the present study was simply to validate the yeast-derived pheromone compounds against their chemically synthesized counterparts—as we have previously done for Helicoverpa armigera and Ostrinia nubilalis—we only offer a tentative explanation. A more detailed study, incorporating additional factors such as the geographical origin of the insects, is currently being planned to address this question more thoroughly.

Minor Comments

• Correct minor grammatical issues (e.g., “resulting to” → “resulting in”). Done
• Avoid repeating results in the Discussion section. Done
• Ensure consistent formatting of chemical nomenclature. Done

 

 

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Overall, the authors have made reasonable efforts to address the reviewer comments, and the revisions have improved the clarity of the manuscript. The study provides useful validation of yeast-derived pheromones as potential alternatives to chemically synthesized compounds. The manuscript is suitable for publication.

Comments on the Quality of English Language

• Correct minor grammatical issues (e.g., “resulting to” → “resulting in”).
• Avoid repeating results in the Discussion section.
• Ensure consistent formatting of chemical nomenclature.

Author Response

In accordance with the Reviewer 3’s suggestion, the manuscript has been thoroughly revised for language and style to improve clarity and flow.

We would like to thank the Editor and the reviewers for their assiduous revision and helpful comments on the manuscript.

Author Response File: Author Response.pdf