Volatiles Produced by Yeasts Related to Prunus avium and P. cerasus Fruits and Their Potentials to Modulate the Behaviour of the Pest Rhagoletis cerasi Fruit Flies

Round 1

Reviewer 1 Report

This is an excellent paper, which presents the results of an effect of yeast produced semiochemicals on the fruits of P. avium and P. cerasus and its interactions with important fruit fly - R. cerasi. I don't have any suggestion for improving the paper, since it is written on high scientific level. I suggest to publish the paper, however before acceptation I only suggest to make the following improvement:
p. 2, line 70: "L." after "Rhagoletis cerasi" should be written within brackets

Author Response

Responses to the first reviewer’s comments

Authors. We thank the reviewer for evaluating the manuscript. We have made the correction according to the comment.

Reviewer. This is an excellent paper, which presents the results of an effect of yeast produced semiochemicals on the fruits of P. avium and P. cerasus and its interactions with important fruit fly - R. cerasi. I don't have any suggestion for improving the paper, since it is written on high scientific level. I suggest to publish the paper, however before acceptation I only suggest to make the following improvement:
p. 2, line 70: "L." after "Rhagoletis cerasi" should be written within brackets

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript by Mozūraitis and colleagues reports a study with great potential. With this study, the authors analyzed the volatile compounds produced by yeast strains belonging to 11 yeast species isolated at various stages of cherry fruit ripening. Besides observing, as expected, differences among the volatilomes produced by different yeast species, the authors have also assessed the capability of the produced chemicals to modulate the behavior of one of the cherry pests, Rhagoletis cerasi fruit flies. Despite being of great interest, the manuscript severely suffers for lack of experimental details, and potentially for the lack of some controls, as detailed below.

How were the 11 yeast species and strains selected for the analysis? I believe the isolation resulted in more than 11 strains isolated, so I guess the authors had to make a choice on the strains to be tested?

Details on the culturing conditions used to produce the quantified volatile compounds should be provided. Which was the substrate (medium)? Were yeasts grown onto solid medium or in liquid medium? At which concentration were yeast cells inoculated? At which temperature were the yeasts grown? At which time were the cultures sampled for GC analysis?

Similarly, further information should be provided on NMDS analysis. E.g. in Figure 2, 3 points are shown for each yeast species. Are these three points biological/technical replicates or profiles obtained from three different isolates of each species? I guess three isolates, as reported in supplementary table 1, but it should be clearly stated. This information, together with the information on culturing conditions, would make a huge difference in how to interpret results.

Furthermore, details on the frequency of isolation of the different yeast species over the different ripening stages of cherries should be provided, especially considering that this characteristic is used to select the yeast species (and strains) analyzed in terms of the production of volatile compounds attracting insects.

Finally, despite the dissection of the contribution of the most abundant volatile metabolites is appreciated, considering the potential applications of the findings of this study, the evaluation of the impact of the entire set of produced metabolites on insect behavior should be evaluated. Indeed, it is likely that even poorly concentrated metabolites contribute to the flavor of the fermented fruit, hence possibly modifying the behavioral response of insects to is. It would be interesting, and surely more useful for the proposed applications, to assess the response of insects to the entire bouquet of aromas produced by the fermenting yeasts. E.g. yeast cultures, rather than isolated volatile compounds produced by them, could be used as a bite for pest biocontrol and make easier and more affordable the approach.

The title is not clear. I would suggest: “Volatiles produced by yeasts related to Prunus avium and P. cerasus fruits and their potentials to modulate the behavior of the pest Rhagoletis cerasi fruit flies”

L14: ...little is known on which yeasts…

L13-L18: this sentence should be modified to better contextualize the study, possibly reducing the technical aspects (because they are not innovative, one expects the techniques are these actually used by the authors).

L39: “insects and yeasts…”

L224-228: Further details should be provided on the generation of the cluster and the following analyses. First of all, why three volatilome profiles per species have been analyzed through NMDS analysis and only one sample per species is reported in the cluster analysis? Was clustering carried out on the average of quantified volatile compounds? In addition, were the different clusters identified analytically (e.g. through partition around medioids – pam – analysis) or simply by observing the clustering?

L178 and L232: “Euclidean”

L285: in the text, it is reported that the tested amount of the alcohol preferred by female insects is 10-5 mg, but in Figure 6 the reported amount is 10-4 mg. Please check.

Author Response

Responses to the second reviewer’s comments

Authors. We thank the reviewer for the valuable comments. We accepted the comments and have made corrections. Please, find our explanations point by point.

Reviewer. The manuscript by Mozūraitis and colleagues reports a study with great potential. With this study, the authors analyzed the volatile compounds produced by yeast strains belonging to 11 yeast species isolated at various stages of cherry fruit ripening. Besides observing, as expected, differences among the volatilomes produced by different yeast species, the authors have also assessed the capability of the produced chemicals to modulate the behavior of one of the cherry pests, Rhagoletis cerasi fruit flies. Despite being of great interest, the manuscript severely suffers for lack of experimental details, and potentially for the lack of some controls, as detailed below.

How were the 11 yeast species and strains selected for the analysis? I believe the isolation resulted in more than 11 strains isolated, so I guess the authors had to make a choice on the strains to be tested?

Authors. More than one hundred of yeast strains were isolated from sweet and sour cherries, which were collected for three years. The selection of isolated yeasts for VOC analysis was conditioned to reflect the majority of yeast species found on cherries of different ripeness. The frequency of yeast isolation among cherries samples at different ripening stages are presented in figure S1. Aureobasidium pullulans and Cryptococcus wieringae species were selected as more common on unripe fruits; Hanseniaspora uvarum are observed very often on medium-ripe cherries; while yeasts species from Pichia, Saccharomyces, Torulaspora and Metschnikowia genera are mainly associated with ripe fruits.

Reviewer. Details on the culturing conditions used to produce the quantified volatile compounds should be provided. Which was the substrate (medium)? Were yeasts grown onto solid medium or in liquid medium? At which concentration were yeast cells inoculated? At which temperature were the yeasts grown? At which time were the cultures sampled for GC analysis?

Authors. Details now are provided in section 2.3: “Overnight grown yeast cells (50 μL) at the concentration of about 3-5 x 10⁷ cells/mL were placed on the surface of YPD-agar medium and cultivated for 2 days at 25 °C. For sampling background volatiles, YPD-agar plates without yeast were used as control samples.”

Reviewer. Similarly, further information should be provided on NMDS analysis. E.g. in Figure 2, 3 points are shown for each yeast species. Are these three points biological/technical replicates or profiles obtained from three different isolates of each species? I guess three isolates, as reported in supplementary table 1, but it should be clearly stated. This information, together with the information on culturing conditions, would make a huge difference in how to interpret results.

Authors. Each of yeast species used for VOC sampling were represented by three different isolates. All yeast strains are listed in the Table S1. The information on yeast isolates is included into the Figure 2 and 3 legends.

Reviewer. Furthermore, details on the frequency of isolation of the different yeast species over the different ripening stages of cherries should be provided, especially considering that this characteristic is used to select the yeast species (and strains) analyzed in terms of the production of volatile compounds attracting insects.

Authors. According to your suggestion, a new Figure S1 on the frequency of yeast isolation among cherries samples at different ripening stages was prepared.

Reviewer. Finally, despite the dissection of the contribution of the most abundant volatile metabolites is appreciated, considering the potential applications of the findings of this study, the evaluation of the impact of the entire set of produced metabolites on insect behavior should be evaluated. Indeed, it is likely that even poorly concentrated metabolites contribute to the flavor of the fermented fruit, hence possibly modifying the behavioral response of insects to is. It would be interesting, and surely more useful for the proposed applications, to assess the response of insects to the entire bouquet of aromas produced by the fermenting yeasts. E.g. yeast cultures, rather than isolated volatile compounds produced by them, could be used as a bite for pest biocontrol and make easier and more affordable the approach.

Authors. We completely agree with the reviewer that responses of insects to an entire bouquet of aromas produced by the yeasts have to be tested and such information is useful for the proposed applications. We will continue the study and will test the responses of Rhagoletis cerasi fruit flies to an entire bouquet of aromas produced by the selected yeast species.

The dissection of the behaviour-modifying effect (attractancy or repellence) of the olfactory active volatile metabolites is important for a selection of yeast species producing a desirable blend (composing large or low amounts of repellent or attractants) in respect to the aim of a control method (monitoring, push-pull etc.). Therefore, the current study presents the composition of volatile blends of yeast species associated with cherry fruits at different ripening stages, reveals which volatile yeast metabolites Rhagoletis cerasi fruit flies are able to smell, and reports which behaviour modifying effects the olfactory active compounds elicit.

Reviewer. The title is not clear. I would suggest: “Volatiles produced by yeasts related to Prunus avium and P. cerasus fruits and their potentials to modulate the behavior of the pest Rhagoletis cerasi fruit flies”

Authors. We have changed the title according to the suggestion.

Reviewer. L14: ...little is known on which yeasts…

Authors. We have made the change.

Reviewer. L13-L18: this sentence should be modified to better contextualize the study, possibly reducing the technical aspects (because they are not innovative, one expects the techniques are these actually used by the authors).

Authors. The first sentence (lines 13-15) shows why the current research was carried out, pointing out the increasing importance of yeast produced volatiles in pest management as well as in part revealing the novelty of our study. We did not say that we have implemented these volatiles in pest management programs. We have reduced the technical aspects in the second sentence (lines 15-18). We agree with the reviewer that pointing out GC-MS method used to identify volatiles does not contribute to essential information, therefore, we have removed that part. However, for easier comparison of the data with those already published, it is important already in an abstract briefly to provide information on the methods used, despite that these methods are established (for example solid phase micro extraction method to sample volatiles).

Reviewer. L39: “insects and yeasts…”

Authors. We have made the change.

Reviewer. L224-228: Further details should be provided on the generation of the cluster and the following analyses. First of all, why three volatilome profiles per species have been analyzed through NMDS analysis and only one sample per species is reported in the cluster analysis? Was clustering carried out on the average of quantified volatile compounds? In addition, were the different clusters identified analytically (e.g. through partition around medioids – pam – analysis) or simply by observing the clustering?

Authors. NMDS analysis showed that the isolates were clearly grouped in a species-specific manner, therefore, to make clustering simpler the averages were used.

The clustering was carried out on the average of quantified volatile compounds from three different isolates per species. The different clusters were identified by visually evaluating the clustering. We have added this information in subchapter 2.7 Statistical analysis and in the legend of Fig. 3.

Reviewer. L178 and L232: “Euclidean”

Authors. We have made the changes.

Reviewer. L285: in the text, it is reported that the tested amount of the alcohol preferred by female insects is 10-5 mg, but in Figure 6 the reported amount is 10-4 mg. Please check.

Authors. We have checked the data and corrected the value in the text. The correct amount is 10^-4 mg.

Author Response File: Author Response.docx