Review Reports - Microwaves Induce Histological Alteration of Ovaries and Testis in <i>Rhynchophorus ferrugineus</i> Oliv. (Coleoptera: Curculionidae)

Round 1

Reviewer 1 Report

Thanks to the authors for the opportunity to read more about their work. I think that the presented manuscript is essential, and I appreciate their hard work. The results are really interesting. The data seems promising – with “some and actual” reservations, below – but more effort in writing the manuscript is expected as will be shown below.

Through reading this interesting manuscript, here are a few highlights about issues that would cause concern:

Lines 87-88 How many insects were in each group for each time point? Could the authors explain why so few insects were used in the experiment? Four insects in the control group is a small number that does not eliminate individual variability.

Lines 113-116 Please clarify the procedure for preparing histological sections. What tissue dehydration procedure was used, what microtome was used? The names of the manufacturers of the reagents, paraffin and dyes used are also missing. What microscope was used for the observations? What program was the photo documentation prepared in?

Lines 125-127 What software was used for statistical analysis?

Lines 145-147 There is no information in the manuscript about the statistical method used to investigate significant differences between the groups.

Fig. 2-5 There is no scale in the photos. It is also worth adding in the legend information on which microscope and with what camera the photos were taken. In addition, in my opinion, it is worth marking the changes in the tissues with arrows in the pictures.

General note. Do the authors have any speculations that changes in insects caused by microwaves may contribute to difficulties in reproduction? Did the authors investigate the possibility of conceiving fertile offspring in the test insects?

Author Response

Dear reviewer,

Thank you for the comments and suggestions.

We submitted the revised manuscript (#Agronomy-2147142), keeping in mind all your comments and suggestions that have improved the quality of the paper. All changes were highlighted in yellow, and a point-by-point response is below included.

We hope now the manuscript is suitable for publication.

Manuela Martano

Reviewer 1:

Reviewer’s comment:

Our response : We have evaluated 4 insects in each group for each time point. We are aware that our evaluation has been performed in relatively small groups of weevils. However, this study was performed following a study in a wild environment to evaluate the dose necessary to obtain an increase of temperature inside the insect such as to determine an alteration of the reproductive system. The results obtained on histological examination confirm that no evident changes were observed in the controls, which were used only for study the normal histological aspects of reproductive tissues in RPWs and compare it with irradiated RPWS. Moreover, evident alterations are shown in 30 s irradiated RPWs and the severity of histological lesions increased with increase of irradiation time, suggesting the possibility that microwaves could reduce the reproductive capacity of RPW. We believe that further studies that could reproduce the complexity of the wild environment are necessary.

Reviewer’s comment: Lines 113-116 Please clarify the procedure for preparing histological sections. What tissue dehydration procedure was used what microtome was used? The names of the manufacturers of the reagents, paraffin and dyes used are also missing. What microscope was used for the observations? What program was the photo documentation prepared in?

Our response : We have clarified the histological procedure and added all information required in the main text (lines 116-131)

Reviewer’s comment: Lines 125-127 What software was used for statistical analysis?

Our response : For statistical analysis we used SPC for Excel Software

Reviewer’s comment: Lines 145-147 There is no information in the manuscript about the statistical method used to investigate significant differences between the groups.

Our response : We used t-Test to determine if there is a measured difference between groups exposed to different irradiation time and survival data (line 135).

Reviewer’s comment: 2-5 There is no scale in the photos. It is also worth adding in the legend information on which microscope and with what camera the photos were taken. In addition, in my opinion, it is worth marking the changes in the tissues with arrows in the pictures.

Our response : We have added scales and arrows. I have added information about microscope and camera in material and method (lines 116-131)

Reviewer’s comment: General note. Do the authors have any speculations that changes in insects caused by microwaves may contribute to difficulties in reproduction? Did the authors investigate the possibility of conceiving fertile offspring in the test insects?

Our response: This study leads us to think that the alterations we have observed in the irradiated samples could lead to difficulties in reproduction. To support this hypothesis, the number of eggs laid during the rearing period was also calculated. We observed a dramatic reduction in egg count from the controls to the 5 and 15 second irradiated samples, with a dramatic reduction in egg numbers in the 30 second irradiated samples. We added this data in the manuscript (lines 82-84;160-162) to support our previous results. However, we agree with the reviewer and further studies will focus on the possibility of conceiving fertile offspring.

Author Response File: Author Response.docx

Reviewer 2 Report

#Agronomy-2147142

The manuscript provided evidence about the alteration of anatomical reproductive organs of red palm weevils females and males exposed to a microwave at different times. The authors indicated that such exposition leads to modifications in the insect’s organs and could lead to reproductive impairment, although no data is provided to support the latter affirmation.

However, the manuscript does not come to be innovative, as the group has already made some of the author’s results and further suggestions. For instance, reference 29 (lines 435-436) reports the application of microwaves under semi-conditions (which is a representative condition). It, therefore, does not validate the author’s arguments in lines 353-355 about testing the efficacy of microwaves under field conditions since it was done. More detailed, reference 25 (lines 425-426) provides the first authors’ report regarding the effects of the microwave on reproductive organs and survival, performed under a significative biological condition (palms infested with weevils).

Therefore, the manuscript’s contribution may be related to the specific details of female anatomical changes, which makes it more attractive from an entomological view than in the agronomy topic. Thus, I do not recommend the manuscript for publication in the current journal.

Lines 32, 34, and 311: Scientific name should be in italic

Line 34: Capitalized ‘Palmae’ mentioned as it is a proper noun.

Line 70: The statement ‘they could affect the development of new generation too’ was not tested here.

Line 112: Replace necropsy with dissection since the latter refers to the study of anatomical structure, while the first is more related to determining the cause of death.

Line 121-123: Is there a reference for that scale?

Lines 125-127: The authors mentioned they performed Pearson correlation between the variables. However, in the results, some reports indicate differences between treatments for thermogram (lines 133-134) and survival (lines 145-148). The correlation is stated only in lines 230-233. So, how did the authors compare treatments for the thermogram and survival data? I suggest the authors consider survival analysis to compare the survival data obtained in the manuscript and then compare it between treatments.

Figures 2-5. I suggest the authors include a scale in the figures and arrows to indicate the structures that the authors refer to.

Lines 102: What were the insects’ conditions after the exposition? What was the frequency of survival evaluation?

Lines 342-346: This statement is speculative and not supported by the data, as there is no guarantee that the lesions could lead to the impairments mentioned.

Lines 350-355: This needs to sound conclusion. A study limitation could be moved to the discussion section. Specifically, in lines 353-354, a previous author’s study (references numbers 25, 28 and 29) has shown that.

Author Response

Dear reviewer,

Thank you for the comments and suggestions.

We submitted the revised manuscript (#Agronomy-2147142 ), keeping in mind all your comments and suggestions that have improved the quality of the paper. All changes were highlighted in yellow, and a point-by-point response is below included.

We hope now the manuscript is suitable for publication.

Manuela Martano

Reviewer reports:

Reviewer 2:

Reviewer’s comment: The manuscript provided evidence about the alteration of anatomical reproductive organs of red palm weevils females and males exposed to a microwave at different times. The authors indicated that such exposition leads to modifications in the insect’s organs and could lead to reproductive impairment, although no data is provided to support the latter affirmation.

Our response: Thanks for your comment. This study leads us to think that the alterations we have observed in the irradiated samples could lead to difficulties in reproduction. To support this hypothesis, the number of eggs laid during the rearing period was also calculated. We observed a dramatic reduction in egg count from the controls to the 5 and 15 second irradiated samples, with a dramatic reduction in egg numbers in the 30 second irradiated samples. We added this data in the manuscript (lines 85-87;163-165) to support our previous results. However, we agree with the reviewer and further studies will focus on the possibility of conceiving fertile offspring.

Reviewer’s comment: However, the manuscript does not come to be innovative, as the group has already made some of the author’s results and further suggestions. For instance, reference 29 (lines 435-436) reports the application of microwaves under semi-conditions (which is a representative condition).

Our response: It is true that some authors have already published articles on the effect of microwaves on Rhynchophorus ferrugineus Oliv. (Reference 29) but the highlighted article does not deal in any way with the histopathological aspects, so we believe that the results are original.

Reviewer’s comment: It, therefore, does not validate the author’s arguments in lines 353-355 about testing the efficacy of microwaves under field conditions since it was done. More detailed, reference 25 (lines 425-426) provides the first authors’ report regarding the effects of the microwave on reproductive organs and survival, performed under a significative biological condition (palms infested with weevils).

Our response: Thank you for your comments. Our previous article (reference 25) was carried out, unlike this one, in wild environment conditions on larvae, pupae and adults and their survival data were calculated after only 24 hours (and not for a long period as in this study); it was calculated the temperature of the palm (and not of the insect) after a direct treatment on plant for 30, 40 and 45 minutes (and not 5-15-30 seconds). Furthermore, in the previous study no correlation was made between the data collected and there was no data on fecundity. Moreover, regarding the histological examination, even if it was carried out on samples treated in laboratory conditions, the results obtained were only preliminary, made on a few samples, and no histological gradation or comparison with other data were carried out as has instead been done in the present study. We have specified some of these aspects in discussion (line 272-318).

Reviewer’s comment: Therefore, the manuscript’s contribution may be related to the specific details of female anatomical changes, which makes it more attractive from an entomological view than in the agronomy topic. Thus, I do not recommend the manuscript for publication in the current journal.

Our response: The agronomic consequence of RPW spread is highlighted in the conclusions (lines 338-346)

Reviewer’s comment: Lines 32, 34, and 311: Scientific name should be in italic

Our response: We have corrected as suggested

Reviewer’s comment: Line 34: Capitalized ‘Palmae’ mentioned as it is a proper noun.

Our response: We have corrected as suggested

Reviewer’s comment: Line 70: The statement ‘they could affect the development of new generation too’ was not tested here.

Our response: We have deleted this statement

Reviewer’s comment: Line 112: Replace necropsy with dissection since the latter refers to the study of anatomical structure, while the first is more related to determining the cause of death.

Our response: We have replaced necroscopy with dissection as suggested.

Reviewer’s comment: Line 121-123: Is there a reference for that scale?

Our response: There is not a reference for the scale. An arbitrary scale was applied, ranging from 1 0 to 3 considering the severity of the following alterations: vacuolar degeneration, nuclear necrosis (pyknosis, karyorrhexis), desquamation and loss of germinal cells. The score assigned for each sample was the following: 0- no pathological alterations; 1 mild pathological alteration; 2- moderate pathological alterations; 3- severe pathological alterations.

Reviewer’s comment: Lines 125-127: The authors mentioned they performed Pearson correlation between the variables. However, in the results, some reports indicate differences between treatments for thermogram (lines 133-134) and survival (lines 145-148). The correlation is stated only in lines 230-233. So, how did the authors compare treatments for the thermogram and survival data? I suggest the authors consider survival analysis to compare the survival data obtained in the manuscript and then compare it between treatments.

Our response: Pearson correlation has been performed to compare mean values of survival data and mean values of DT (°C). As reported in results, survival days and DT (°C) showed a moderate, not statistically significant, inverse correlation (R= - 0.1; p 0.2) in all irradiated samples.

Reviewer’s comment: Figures 2-5. I suggest the authors include a scale in the figures and arrows to indicate the structures that the authors refer to.

Our response: We have added scale and arrows in figures.

Reviewer’s comment: Lines 102: What were the insects’ conditions after the exposition? What was the frequency of survival evaluation?

Our response: We have evaluated RPWs clinical conditions every two days. Only 30 seconds irradiated RPWs presented feeding difficulties in the first few days after exposition. Regarding the other groups we have not noticed anything to report.

Reviewer’s comment: Lines 342-346: This statement is speculative and not supported by the data, as there is no guarantee that the lesions could lead to the impairments mentioned.

Our response: We amended the conclusions (lines 338-346).

Reviewer’s comment: Lines 350-355: This needs to sound conclusion. A study limitation could be moved to the discussion section. Specifically, in lines 353-354, a previous author’s study (references numbers 25, 28 and 29) has shown that.

Our response: DONE

Author Response File: Author Response.docx

Reviewer 3 Report

Reviewer’s Comment #:

The manuscript address the interesting concept that “Microwaves induce histological alteration of ovaries and testis in Rhynchophorus ferrugineus Oliv. (Coleoptera: curculionidae)”. Here the author tries to provide practical evidence that microwaves damage reproductory organelles in insects. The manuscript is well written, which I overall appreciate. The work is well performed. The parameters chosen for strategy building are traditional and informative. However, I do suggest certain things, which need addition, improvement, and clarification to support and strengthen the impact of the article.

In general:

The text contains a few typos that need to be corrected.

It could have been better if you put the concept in the form of a graphical abstract.

The material and methods section should be more elaborate for better understanding. Example: Section: 2.4. Macroscopical and microscopical analysis: histological and histochemical staining protocol should be briefly explained with any alterations or followed reference should be added.

Abbreviations needed when first mentioned: (MM, KP and PM).

Results: Section 3.3. Macroscopical and microscopical results: too much elaborative. Concise the data in the form of a table.

My major question will be:

Does this microwave frequency work for the particular insect?

If so how can this microwave frequency reach the larvae or adults which bore deeper into the stem?

How will the heat frequencies of microwaves reach the insect since the steam itself contains a lot of moisture?

Do you try in a field/environmental setup that contains larvae or adults inside the stem?

How do you defend these questions and please address them in your text? Discussion part of the article.

Conclusion:

Since I found some degree of difficulty in reading and understanding certain parts of the manuscript, the article needs some corrections that need to be incorporated for better readability. I do think that the manuscript contains important issues, interesting approaches, and techniques, which can lead to understanding pest control through microwave frequency. Therefore, I consider this manuscript suitable for publication after minor revision in order to publish in Agronomy.

Author Response

Dear reviewer,

Thank you for the comments and suggestions.

We hope now the manuscript is suitable for publication.

Manuela Martano

Reviewer 3:

Reviewer’s comment:

It could have been better if you put the concept in the form of a graphical abstract.

Our response We have performed a graphical abstract as suggested by reviewer

Reviewer’s comment: The material and methods section should be more elaborate for better understanding. Example: Section:4. Macroscopical and microscopical analysis: histological and histochemical staining protocol should be briefly explained with any alterations or followed reference should be added.

Our response Thank you for your suggestion; I agree with you but unfortunately, I can not abbreviate the section 2.4, as reviewer 2 has asked me for additional information on the procedure for histological examination. Regarding the reference requested, there is not a reference for the scale. An arbitrary scale was applied, ranging from 1 0 to 3 considering the severity of the following alterations: vacuolar degeneration, nuclear necrosis (pyknosis, karyorrhexis), desquamation and loss of germinal cells. The score assigned for each sample was the following: 0- no pathological alterations; 1 mild pathological alteration; 2- moderate pathological alterations; 3- severe pathological alterations.

Reviewer’s comment: Abbreviations needed when first mentioned: (MM, KP and PM).

Our response: we have deleted this part, as we have specified it in the part relative to author contribution

Reviewer’s comment Results: Section 3.3. Macroscopical and microscopical results: too much elaborative. Concise the data in the form of a table.

Our response We have revised the entire section 3.3., making it in our opinion shorter and clearer. However, we believe that the reviewer's suggestion to enter the data shown in a table could could make the article confusing and difficult to read

Reviewer’s comment My major question will be: Does this microwave frequency work for the particular insect?If so how can this microwave frequency reach the larvae or adults which bore deeper into the stem?How will the heat frequencies of microwaves reach the insect since the steam itself contains a lot of moisture?Do you try in a field/environmental setup that contains larvae or adults inside the stem?How do you defend these questions and please address them in your text? Discussion part of the article.

Our response

I try to answer to your questions in introduction ( lines 56-58) and discussion sections (lines 272-293)

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

#agronomy-2147142

The authors presented some alterations in the current version of the manuscript. Some minor corrections and suggestions are indicated below.

(i) Scientific names in lines 2 and 294 are not italicized.

(ii) The authors performed a parametric T-test to compare controls versus each treated group (lines 135-136). However, the replicates are unbalanced for that test (8 for each treatment and 4 for the control; Table 1), and assumptions were not tested, or if so, they were not indicated by the authors. Thus, the authors may consider using a non-parametric test to be precise in the statistical results.

(iii) To be clear when reporting the results in lines 143-147 and 156-162, please include the full statistical report (t-value, degrees of freedom, and p-values).

(iv) Lines 338-340 sounds like the introduction. The conclusion starts in line 340.

Based on that, I recommend the publication of the manuscript after a minor review.

Author Response

Dear reviewer,

Thank you for the comments and suggestions.

We submitted the revised manuscript (#Agronomy-2147142 ), keeping in mind all your comments and suggestions that have improved the quality of the paper. All changes were highlighted in yellow, and a point-by-point response is below included.

We hope now the manuscript is suitable for publication.

Manuela Martano

Reviewer 2:

Reviewer’s comment: Scientific names in lines 2 and 294 are not italicized.

Our response: we have corrected as suggested.

Reviewer’s comment: (ii) The authors performed a parametric T-test to compare controls versus each treated group (lines 135-136). However, the replicates are unbalanced for that test (8 for each treatment and 4 for the control; Table 1), and assumptions were not tested, or if so, they were not indicated by the authors. Thus, the authors may consider using a non-parametric test to be precise in the statistical results.

(iii) To be clear when reporting the results in lines 143-147 and 156-162, please include the full statistical report (t-value, degrees of freedom, and p-values).

Our response: we have processed again all data for statistical analysis. It has been reported and highlighted in yellow in the materials and methods and in results sections.

Reviewer’s comment: (iv) Lines 338-340 sounds like the introduction. The conclusion starts in line 340.

Our response: Thank you for the comment. We have revised as suggested the section indicated by reviewer.