Exploring Gut Microbiota in Red Palm Weevil (Rhynchophorus ferrugineus): Effects on Pest Management, Pesticide Resistance, and Thermal Stress Tolerance

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Interesting paper quite well written, with data which look well analyzed and organized. Few notes are reported on the attached file

Comments for author File: Comments.pdf

Comments on the Quality of English Language

 Minor editing of English language required

Author Response

Comment 1: Comments 1: Interesting paper quite well written, with data which look well analyzed and organized. Few notes are reported on the attached file

Response 1: Thank you for the observation. We have made the suggested changes to the text.

Detailed comments from the pdf.

Line 33: the word ferrugineus was removed. Keywords were added.

Line 52: The authority, family, and order were added to the species name.

Line 53: “The” was added to the beginning of the sentence. The authority, family, and order were added to the species name.

Line 61: “The” was added to the beginning of the sentence.

Lines 98-100 were removed.

Line 104: The authority, family, and order were added to the species name.

Line 107: The authority, family, and order were added to the species name.

Line 580: The authority, family, and order were added to the species name.

Line 599: The authority, family, and order were added to the species name.

Line 627: The authority, family, and order were added to the species name. Plutella was changed to P.

Line 640: The authority, family, and order were added to the species name.
Line 651: The authority, family, and order were added to the species name.
Line 661: The authority, family, and order were added to the species name.

Line 683: The authority, family, and order were added to the species name.

Reviewer 2 Report

Comments and Suggestions for Authors

Red palm weevil (RPW) is a destructive pest for palm trees and other important crops and it has caused great economic loss across the world. Although there was much evidence to show that gut bacteria profoundly affect the development and growth of this pest by modulating nutrition metabolism, this manuscript provide some interesting data to indicate that some bacterial species in the gut of RPW were involved in the degradation of petsticides and large carbohydrate molecules in their food. However, the presetation of some data and introduction on the experiment should be significantly improved before being accepted for publication. So this manuscript need major revision before publication. Two major comments on this manuscript were shown as following:

1. Some tables such as Table 3 and 6 do not make sense. Different data was presented in the two tables and it is very confused to be understand. Can F, p-value and LSD be defined as factor? These tables can be removed because the values including F, p-value can be presented following the results that they surpported. 

2. Some experiments should be introduced in detail. For example, the authors mentioned that some pesiticides were used to treat RPW individuals to identify that some gut bacterial species might mediate the pesticide resistance of this pest. However, many important information in these experiments was absent. For instance, How to treat these insects with pesiticides? The content of pesticides was employed? 

Author Response

RPW larvae were treated with chlorpyrifos (Pyriban A®48 % EC) using the dipping food technique (Pieces of sugarcane stem were soaked for 1 min. in different dilutions of the chlorpyrifos insecticide formulations).

As: DOI: 10.21608/asejaiqjsae.2014.2579

DOI: 10.21608/eajbsf.2021.211778

 

Comments 1: Some tables such as Table 3 and 6 do not make sense. Different data was presented in the two tables and it is very confused to be understand. Can F, p-value and LSD be defined as factor? These tables can be removed because the values including F, p-value can be presented following the results that they surpported.

Response 1: Thank you for the suggestion. We have made the suggested changes to the text from line 411 to 565.

Comments 2: Some experiments should be introduced in detail. For example, the authors mentioned that some pesiticides were used to treat RPW individuals to identify that some gut bacterial species might mediate the pesticide resistance of this pest. However, many important information in these experiments was absent. For instance, How to treat these insects with pesiticides? The content of pesticides was employed? 

Response 2: RPW larvae were treated with chlorpyrifos (Pyriban A®48 % EC) using the dipping food technique (Pieces of sugarcane stem were soaked for 1 min. in different dilutions of the chlorpyrifos insecticide formulations) line 270.

Please refer to

DOI: 10.21608/asejaiqjsae.2014.2579

DOI: 10.21608/eajbsf.2021.211778

Reviewer 3 Report

Comments and Suggestions for Authors

Reviewer(s)' Comments to Author: 

Detailed comments as following. 

Ø  Please write authority, family and order for all the organisms scientific names at first mention. Follow this suggestion throughout the manuscript

Ø  Add the in the beginning of a paragraph if it is started with the scientific name.

Ø  The last paragraph of introduction section looks like conclusions. it should only state the objective.

Ø  did the author identify? was it the ferrugineus species?

Ø  from how many trees these larvae were collected? it is a big number

Ø  please provide the farm location coordinates where the RPW larvae were collected.

Ø  give details for the model and manufacturer of all the machines like, microbalance and kits etc.

Note: please see the detailed comments in the attached pdf file.

Comments for author File: Comments.pdf

Author Response

Comments 1: Please write authority, family, and order for all the organisms scientific names at first mention. Follow this suggestion throughout the manuscript.

Response 1: Thank you for the observation. We have made the suggested changes to the text line 53, 55, 119, 122, .

 

Comments 2: Add the in the beginning of a paragraph if it is started with the scientific name.

Response 2: Thank you for the suggestion. We have made the suggested changes to the text lines 55, 63.

 

Comments 3: The last paragraph of introduction section looks like conclusions. it should only state the objective.

Response 3: The last paragraph was changed lines 101 to 103.

 

Comments 4: did the author identify? was it the ferrugineus species?

Response 4: 

The collected insects were identified morphologically by keys as referenced in line 120, and then the identification was confirmed in the Insect Classification and Surveying Department and Tree and Wood Product Borers and Termites Department in the Plant Protection Research Institute, Agricultural Research Center.

 

Comments 5:   from how many trees these larvae were collected? it is a big number.

Response 5: about 30 palm trees but it isn't a big number in infested fields.  

 

Comments 6:  please provide the farm location coordinates where the RPW larvae were collected.

Response 6: (30°33'58.1"N 31°55'54.6"E) El Kassasin District, Ismailia Governorate, Egypt in line 118.

 

Comments 7:  give details for the model and manufacturer of all the machines like, microbalance and kits etc.

Response 7: We have made the suggested changes to the text. centrifuged (Centurion VS1283 LED, UK) lines 196 and 251, electronic microbalance (Sartorius R180D, Germany) line 205, rotary water bath shaker (RSB-12 Water Bath Shaker, India) line 246, (all kits for physiological tests, Biodiagnostic, enzymatic colorimetric method, Giza, Egypt) line 217.

 

Note: please see the detailed comments in the attached pdf file.

Response 7: Thank you for the notes. We have made the suggested changes to the text.

Detailed comments from the pdf.

Line 52: The authority, family, and order were added to the species name.

Line 53: “The” was added to the beginning of the sentence. The authority, family, and order were added to the species name.

Line 61: “The” was added to the beginning of the sentence.

Line 70: It was changed to bacteria families.

Lines 98-100 were relocated to the conclusions.

Line 102: breeding was replaced by rearing.

Line 103: The collected insects were identified morphologically by keys as referenced then the identification was confirmed in the Insect Classification and Surveying Department and Tree and Wood Product Borers and Termites Department in the Plant Protection Research Institute, Agricultural Research Center. Samples were collected from about 30 palm trees. The number isn't big for infested fields.  

Line 105: the location is (30°33'58.1"N 31°55'54.6"E) El Kassasin District, Ismailia Governorate, Egypt.

Line 107: because larvae lived and fed in the dark trunk and these conditions were applied in many previous reports as

doi: 10.1002/ps.4485,

doi: 10.3389/fphys.2019.01303,

doi: 10.3389/fmicb.2017.02291

doi: 10.21769/BioProtoc.3456.

Line 111: at -20°C

Line 142: Using Macrogen’s sequencing services.

Line 191: Centurion VS1283 LED, UK for the centrifuge and Satorius R180D, Germany for the balance.

Line 218: all kits for physiological tests.

Lines 220-222 were relocated to the introduction.

Line 650: all spaces were rechecked.

Line 656: all spaces were rechecked.

Line 661: all spaces were rechecked.

Line 860: The reference name was rechecked.

Reviewer 4 Report

Comments and Suggestions for Authors

Reviewing the manuscript Biology-3064646 I found the manuscript  well written and it clearly shows the effort spent by the authors in analyzing several aspects of the gut microbiota of red palm weevil. In particular I found very interesting the experiment addressed to the Chlorpyrifos degradation.

However, I did not understand the reason why the authors invested a lot of energies in studying the effects of thermal stress treatments in a species that -in gregarious conditions- is able to modify the climatic conditions at the interior of the palm trees (independently from what would be the outside climatic conditions). For sure this modification is related to some of symbionts, but I did not find any comment on this aspect in the manuscript. In the attached file I also included a reference of 2013, describing the effects of the RPW infestation on the inside temperatures on the palm trees. 

I also think that a small part of the introduction and a large part of the discussion should be improved.  In the introduction, there is something missing regarding how the gut microbiota can be used in an integrated Pest Management context.  I am inviting the Authors to provide suggestions from literature (eventually on other pests). Actually this part should be more developed in the discussion, emphasizing the use of the microbiota to decrease the fitness of the target insect pest with some speculative hypotheses. 

I also did not like the way how the Discussion Chapter was written: It would be important to better integrate some of the records referred to other pest species in the context. The feeling was a kind of "Copy & Paste" without taking under consideration that should be addressed to the correct target, the red palm weevil. 

Finally, The Bibliography has several mistakes and must revised  and corrected carefully. 

Comments for author File: Comments.pdf

Author Response

Comment1: However, I did not understand the reason why the authors invested a lot of energies in studying the effects of thermal stress treatments in a species that -in gregarious conditions- is able to modify the climatic conditions at the interior of the palm trees (independently from what would be the outside climatic conditions). For sure this modification is related to some of symbionts, but I did not find any comment on this aspect in the manuscript. In the attached file I also included a reference of 2013, describing the effects of the RPW infestation on the inside temperatures on the palm trees. 

Response 1: I agree with you and I’d like to thank you for choosing this paper (doi: 10.5897/JEN2013.0081), because we chose the high temperature (35 °C) in our study according to the RPW threshold and thermal constant (13.1 °C and 40.4 degree days (DD)) and the results of (doi: 10.5897/JEN2013.0081), which indicates the average temperature of infested date palm (32.60 °C) was significantly higher than the average temperature recorded at the same time both inside the healthy trees (29.53 °C) and in the ambient atmosphere (29.35°C), to be sure this makes thermal stress on RPW larvae for a long time. Many studies have studied the RPW threshold temperatures (doi: 10.1603/EN09316, and doi:10.1017/S0007485310000283) and the physiological effect of temperature on the palm weevil larvae, especially at low temperatures (doi.org/10.1016/j.jtherbio.2020.102748, and doi.org/10.3390/insects13020134), apart from its presence inside the palm stem. The study aimed to know the role of symbiotic bacteria (gut bacteria) in the insect’s adaptation. In addition, the results showed that the insects are able to withstand high temperatures. Compared to the AF group, the normal gut bacteria in the CF group were more active and adaptable. This indicates that endosymbiotic gut bacteria have an important role in giving the insect the ability to live at high temperatures efficiently and help it raise the temperatures of the host (palm trees) above its natural temperature as a result of increased insect metabolism, activity, and fermentation processes. This result is consistent with the study referred to (doi: 10.5897/JEN2013.0081, and doi: 10.4039/tce.2015.51). It was added to the text in line 94 (introduction) and from lines 699 to 705 (discussion).

 

 

Comment 2: I also think that a small part of the introduction and a large part of the discussion should be improved.  In the introduction, there is something missing regarding how the gut microbiota can be used in an integrated Pest Management context.  I am inviting the Authors to provide suggestions from literature (eventually on other pests). Actually this part should be more developed in the discussion, emphasizing the use of the microbiota to decrease the fitness of the target insect pest with some speculative hypotheses.

Response 2: gut microbiota can be used in an integrated Pest Management was improved from line 68 to 72. And 529 to 601

 

Comment 3: I also did not like the way how the Discussion Chapter was written: It would be important to better integrate some of the records referred to other pest species in the context. The feeling was a kind of "Copy & Paste" without taking under consideration that should be addressed to the correct target, the red palm weevil

Lines 641,642, 652, 653, 662, 672, 674, 690, 691, 709, 730 – 733, 746, 747, 754, 755,

------

.

Finally, The Bibliography has several mistakes and must revised and corrected carefully.

Line 53: “The” was added to the beginning of the sentence. The authority, family, and order were added to the species name.

Line 56: infected was changed to infested.

Line 106: the species name was added in full.

Line 107: box dimensions were added (30 x 20 x 15 cm). The author and family were added.

Line 110: The number was corrected to 20.

Line 269: 250 larvae in each group.

Figure 11: It was corrected to AF.

Line 252: we studied the presence of Spiroplasma and Rickettsia in RPW collected from 6 different locations in Egypt (https://doi.org/10.1007/s11686-020-00310-2). The 6 samples (4 females and 2 males) of RPW collected from the same location in Ismailia indicate that all 4 females contain Rickettsia while 3 females contain Spiroplasma. (line 608- 612)Line 556: The full genus name was added.

Line 564: The full genus name was added.

Line 567: Spaces were checked.

Line 569: This part provides examples of previous reports on the abilities of the isolated bacterial species to degrade polysaccharides in other insects.

Line 574: The full genus name was added.

Line 577: The full genus name was added.

Line 579: The full genus name was added.

Line 638: I agree with you and I’d like to thank you for choosing this paper (doi: 10.5897/JEN2013.0081), because we chose the high temperature (35 °C) in our study according to the RPW threshold and thermal constant (13.1 °C and 40.4 degree days (DD)) and the results of (doi: 10.5897/JEN2013.0081), which indicates the average temperature of infested date palm (32.60 °C) was significantly higher than the average temperature recorded at the same time both inside the healthy trees (29.53 °C) and in the ambient atmosphere (29.35°C), to be sure this makes thermal stress on RPW larvae for a long time. Many studies have studied the RPW threshold temperatures (doi: 10.1603/EN09316, and doi:10.1017/S0007485310000283) and the physiological effect of temperature on the palm weevil larvae, especially at low temperatures (doi.org/10.1016/j.jtherbio.2020.102748, and doi.org/10.3390/insects13020134), apart from its presence inside the palm stem. The study aimed to know the role of symbiotic bacteria (gut bacteria) in the insect’s adaptation. In addition, the results showed that the insects are able to withstand high temperatures. Compared to the AF group, the normal gut bacteria in the CF group were more active and adaptable. This indicates that symbiotic bacteria have an important role in giving the insect the ability to live at high temperatures efficiently and help it raise the temperatures of the host (palm trees) above its natural temperature as a result of increased insect metabolism, activity, and fermentation processes. This result is consistent with the study referred to (doi: 10.5897/JEN2013.0081, and doi: 10.4039/tce.2015.51).

References were rechecked.

 

Round 2

Reviewer 4 Report

Comments and Suggestions for Authors

The revised manuscript now can be accepted for publication